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WO2025233676A1 - Compositions and methods related to a combination therapy of sting agonists and immune checkpoint inhibitors - Google Patents

Compositions and methods related to a combination therapy of sting agonists and immune checkpoint inhibitors

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Publication number
WO2025233676A1
WO2025233676A1 PCT/IB2025/000222 IB2025000222W WO2025233676A1 WO 2025233676 A1 WO2025233676 A1 WO 2025233676A1 IB 2025000222 W IB2025000222 W IB 2025000222W WO 2025233676 A1 WO2025233676 A1 WO 2025233676A1
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Prior art keywords
acid sequence
amino acid
seq
compound
antibody
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Pending
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PCT/IB2025/000222
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French (fr)
Inventor
Sook Kyung Chang
Jihye Oh
Yeseul Jang
Ho Young Song
Sung Min Kim
Chul-Woong CHUNG
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Ligachem Biosciences Inc
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Ligachem Biosciences Inc
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Publication of WO2025233676A1 publication Critical patent/WO2025233676A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • STING The stimulator of interferon genes
  • cGAS cyclic GMP-AMP synthase
  • STING is an adapter protein in the cGAS (cyclic GMP-AMP synthase)-STING pathway, which is a sensing pathway that induces activation of type I IFN and other inflammatory cytokines, triggering antiviral and antitumor immune responses
  • cGAS-STING pathway cyclic GMP-AMP synthase
  • STING activates signal transducer and activator of transcription 6 (STAT6) and transcription factor interferon regulatory factor 3 (IRF3) through TANK-binding kinase 1 (TBK1) in antiviral and innate immune responses (Burdette DL, Vance RE, STING and the innate immune response to nucleic acids in the cytosol, 2013, Nature Immunology. 14 (1): 19-26).
  • STAT6 transcription 6
  • IRF3 transcription factor interferon regulatory factor 3
  • TNK1 TANK-binding kinase 1
  • STING agonists can also trigger expression of cytokines, giving rise to a T cell-mediated innate immune response which inhibits the growth of cancer cells.
  • systemic delivery of STING agonists can cause widespread inflammation.
  • CDN cyclic dinucleotide
  • a variety of agonists in the form of CDN (cyclic dinucleotide) compounds (ADU-S100, BISTING, GSK532, JNJ-4412, SB11285, MK-1454, TAK676, etc.), bacterial vectors (SYNB1891, STACT-TREX-1), non-cyclic dinucleotide (CDN), compounds (ALG-031048, JNJ-6196, MK-2118, MSA-1, MSA-2, CRD-5500, etc.), nano vaccines (PC7A NP, cGAMP- NP, etc.) and ADCs (XMT-2056, TAK500, etc.) are under development by various strategies.
  • CDN cyclic dinucleotide
  • ADCs XMT-2056, TAK500, etc.
  • DMXAA vascular disrupting agent
  • STING agonists are disclosed, for example, in WO2021/014365 (a macrocyclic compound as STING agonist), and US2021/0139473 (a heterocyclic amide- containing compound as protein modulator), US 2022/0073509 (a heterocyclic compound as STING activator), and KR 2022-0024467 (a heterocycle-containing STING agonist), each of which is incorporated herein by reference in its entirety.
  • STING agonists appear to exhibit only limited bioavailability and require local administration to tumors due to hyperactivation of cytokine expression. Therefore, there remains a demand for development of STING agonists for effective therapeutic methods.
  • the present disclosure relates to a method of administering a compound to a subject in need thereof, comprising administering the compound conjointly with an antibody-drug conjugate, wherein the compound is represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein:
  • FIG. 1A STING agonist compounds (Compound 214, 221 and 230) induced cytokine production (human CXCL-10).
  • FIG. IB STING agonist compounds (Compound 214, 221 and 230) induced cytokine production (human IFNa).
  • FIG. 2A MDA-MB-468 tumor cells were treated with STING agonist compound (Compound 313).
  • FIG. 2B BxPC3 tumor cells were treated with STING agonist compound (Compound 313).
  • FIG. 3 A Up-regulation of co-stimulatory molecule CD86 when THP-1 was treated with the STING agonist compound (Compound 313).
  • FIG. 3B Up-regulation of MHC class II molecule HLA-DR when THP-1 was treated with the STING agonist compound (Compound 313).
  • FIG. 4A STING agonist compounds (Compound 274 and 277) enhanced CD69 expression on CD8 + T cells.
  • FIG. 4B STING agonist compounds (Compound 274 and TIT) enhanced CD69 expression on NK cells.
  • FIG. 5A STING agonist compounds (Compound 344 and 361) enhanced expansion of activated CD8 + T cells
  • FIG. 5B STING agonist compounds (Compound 344 and 361) enhanced expansion of activated NK cells.
  • FIG. 6A Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 221).
  • FIG. 6B Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 277).
  • FIG. 6C Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 281).
  • FIG. 7A Tumor volume (mm 3 ) following treatment with STING agonist compounds (Compound 214 and 221) in CT26 syngeneic mouse model. When tumor volume reached 70 mm 3 , each compound was given at 1.5 mg/kg intravenously every 3 or 4 days (total three times).
  • FIG. 7B Body weight (%) following treatment with STING agonist compounds (Compound 214 and 221). When tumor volume reached 70 mm 3 , each compound was given at 1.5 mg/kg intravenously every 3 or 4 days (total three times).
  • FIG. 8 Tumor volume (mm 3 ) following treatment with STING agonist compound (Compound 274) in CT26 mouse model at various doses.
  • FIG. 9 Tumor volume (mm 3 ) following treatment with STING agonist compounds (Compound 274 and 281) compared to Comparative compound #1 in CT26 syngeneic mouse model.
  • STING agonist compounds Compound 274 and 281
  • Comparative compound #1 in CT26 syngeneic mouse model.
  • each compound was given at 0.5 mg/kg intravenously once weekly for three weeks.
  • mean tumor growth over time was monitored.
  • FIG. 10A Tumor volume (mm 3 ) following treatment with STING agonist compounds (Compound 274, 313, 344, and 396) compared to Comparative compound #3 in CT26 syngeneic mouse model.
  • STING agonist compounds Compound 274, 313, 344, and 396
  • FIG. 10B Body weight (mm 3 ) following treatment with STING agonist compounds (Compound 274, 313, 344, and 396) compared to Comparative compound #3 in CT26 syngeneic mouse model. When tumor volume reached 80 mm 3 , each compound was given at 0.3 mg/kg intravenously once weekly for 3 weeks. Compared to the comparative compound #3, body weight over time was monitored.
  • STING agonist compounds Compound 274, 313, 344, and 396
  • FIG. 11 Tumor volume (mm 3 ) following treatment with STING agonist compounds (Compound 274, and 313) compared to Comparative compound #1 and #2 in EMT6 syngeneic mouse model.
  • STING agonist compounds Compound 274, and 313
  • each compound was given at 0.125 mg/kg intravenously once weekly for three weeks.
  • mean tumor growth over time was monitored.
  • FIG. 12A Tumor volume (mm 3 ) following treatment with STING agonist compound (Compound 313) at various doses in EMT6 model.
  • FIG. 12B Body weight (%) following treatment with STING agonist compound (Compound 313) at various doses in EMT6 model.
  • FIG. 13 In vivo efficacy evaluation of co-administration anti-PD-Ll Ab_l with Compound 277, 302, or 422 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
  • FIG. 14 In vivo efficacy evaluation of co-administration anti-PD-Ll Ab_l with Compound 274 or 313 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
  • FIG. 15 In vivo efficacy evaluation of co-administration anti-mPD-1 Ab or anti-mPD- L1 with Compound 274 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
  • FIG. 16 In vivo efficacy evaluation of co-administration anti-mPD-1 Ab with Compound 363 in B16F10 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
  • FIG. 17A In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-1 antibody_l
  • FIG. 17B In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-1 antibody_2
  • FIG. 17C In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-Ll antibody_2
  • FIG. 18 In vitro evaluation of enhanced phagocytosis by Compound 363 in the presence of anti-PD-Ll antibody_3.
  • the present disclosure relates to a method of administering a compound to a subject in need thereof, comprising administering the compound conjointly with an antibody-drug conjugate, wherein the compound is represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein:
  • T is a moiety comprising a Stimulator of Interferon Genes (STING) agonist
  • STING Stimulator of Interferon Genes
  • p is 1 or 2
  • each instance of R 1 is independently -CH2OR 11 or -COOR 12
  • each instance of R la , R lb , R lc , and R 11 is independently H or a hydroxyl protecting group
  • each instance of R 12 is independently H or a carboxyl protecting group
  • each instance of R 2 and R 3 is independently H or alkyl, or R 2 and R 3 together with a carbon atom to which they are attached form a cycloalkyl
  • each instance of R 4 is independently selected from halogen, alkyl, CN, and NO2,
  • each instance of k is independently 0, 1, 2, or 3
  • each instance of Y is independently selected from H, -C(O)NHL U U, -C(O)NR'(L U U), -
  • each instance of L u is a first linker
  • each instance of U is independently selected from H, alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, -OH, -DBCO and a saccharide
  • each instance of R’ is independently selected from alkyl, cycloalkyl, alkoxy, alkylthio, mono- or di-alkylamino, heteroaryl, and aryl.
  • each instance of R 1 is independently -CH2OH or -COOH.
  • each instance of R 2 and R 3 is independently H.
  • T is a moiety represented by formula (Ila), or a pharmaceutically acceptable salt thereof: wherein:
  • T is coupled to the -C(O)OCR 2 R 3 - fragment of formula (I) via L 2 ,
  • Q is -X a R a or -X h L' L 2 -, each instance of W 1 and W 2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3,
  • a and B are each independently aryl or heteroaryl
  • R a is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, -(alkylene)guanidino, - (alkylene)NHC(O)CH2guanidino, -(alkylene)O(alkylene)guanidino and O(alkylene)guanidino, each L 1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene, each L 2 is independently selected from a bond, or a linker moiety coupled to L 1 and comprising a nitrogen atom
  • M is C(X b L 1 L 2 -) and/or Q is -XhfJL 2 -.
  • T is a moiety represented by Formula (T 1 ): wherein:
  • T 1 is coupled to the -C(O)OCR 2 R 3 - fragment of formula (I) via the -N(H)- of T 1 ; each Ta and Tb is independently a moiety represented by formula (Ila): wherein:
  • M is N, C(X a R a ) or C(X b L 1 L 2 -),
  • Q is -X a R a or -X h L' L 2 -, each instance of W 1 and W 2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3,
  • Z is selected from alkylene, alkenylene, and alkynylene
  • a and B are each independently aryl or heteroaryl
  • X a and X b are each independently selected from CFb, NH, O, and S,
  • R a is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl,
  • each L 1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene
  • each L 2 is independently selected from a bond between L 1 and -C(0)0(CH2)- fragment of T 1 , or a linker moiety coupled to L 1 and comprising a nitrogen atom coupled to the - C(0)0(CH2)- fragment of T 1 ; wherein either M is CIX' ⁇ ' L 2 -) or Q is -X h L' L 2 -.
  • each instance of W 1 and W 2 is independently amido. In further embodiments, each instance of W 1 and W 2 is -CONH2.
  • n and m are each 1.
  • Z is C2-6 alkenylene. In further embodiments, Z is
  • R a is selected from C1-6 alkyl, -(alkylene)N(H)alkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, and -(alkylene)guanidino.
  • R a is selected from H, C1-6 alkyl, -(alkylene)N(H)alkyl, -(Ci- 6alkylene)carboxylic acid, -(Ci-6alkylene)guanidino, -(Ci-6alkylene)NHC(O)CH2guanidino, - (Ci-6alkylene)O(Ci-6alkylene)guanidino, wherein the C1-6 alkyl is optionally substituted with 5- to 7-membered cycloalkyl or heterocyclyl.
  • R a is selected from H, Methyl, -(CH2)3COOH,
  • X b is O.
  • L 1 is selected from Ci-ealkylene, Ci-ealkenylene, Ci-ealkynylene, cycloalkylene, heterocyclylene, arylene, and heteroaryne.
  • L 1 is selected from Ci-ealkylene, , and
  • L 2 is selected from a bond
  • L 2 is selected from a bond
  • L 2 is selected from N— (alkylene)-(heterocyclyl) y
  • L 2 is selected from
  • Q is -X a R a .
  • M is C(X a R a ).
  • Q is -X a R a or M is C(X a R a ).
  • the alkyl is Ci-6 alkyl, e.g., C3 alkyl.
  • the alkylene is C1-6 alkylene, e.g., C3 alkylene.
  • M is C(X a R a ), e.g., M has the structure selected from: and wherein each I is a point of connection to the remainder of the phenyl ring.
  • Q is -X a R a , e.g., Q has the structure selected from: wherein each I is a point of connection to the phenyl ring.
  • M has the structure: , wherein n represents an integer from 1 to 15, e.g., , wherein each I is a point of connection to the remainder of the phenyl ring.
  • Q has the structure: NH , wherein n
  • H represents an integer from 1 to 15, e.g., NH , wherein I is a point of connection to the phenyl ring.
  • M is CCX 1 ! nowadays?’) or Q is -X b L 1 L 2 -.
  • T is coupled to the -C(O)O(CR 2 R 3 )- fragment of the compound represented by structural formula (I) via L 2 , and T is a moiety represented by formula (lib): (lib), wherein:
  • a and B are each independently 5-membered heteroaryl, and JVW i
  • T is coupled to the -C(O)O(CR 2 R 3 )- fragment of the compound represented by structural formula (I) via L 2 , and T is a moiety represented by formula (lie): wherein: A and B are each independently 5-membered heteroaryl, and «/W i V o
  • I is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
  • L 2 is selected from a bond
  • T is coupled to the -C(O)OCR 2 R 3 - fragment via L 2 , and T is a moiety represented by formula (IIc3): »/VW o wherein l is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
  • T is coupled to the -C(O)OCR 2 R 3 - fragment via L 2 , and T is a moiety represented by formula (IIc4): wherein l is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
  • L 2 is a second linker. Any suitable linker moiety may be used. In some embodiments, L 2 is selected from a bond, ,
  • L 2 is a second linker comprising # OC(O)NR 5 -L 4 -NR 6 , # OC(O)- L 4 -NR 6 , or # OC(O)NR 5 -L 4 -(heterocyclylene), wherein: the heterocyclylene comprises a nitrogen atom connected to the -C(O)O(CR 2 R 3 )- fragment of the compound represented by structural formula (I), and wherein # is the point of connection to L 1 , each instance of L 4 is independently alkylene or arylalkylene, each instance of R 5 is independently selected from H, alkyl, and dialkylaminoalkyl, and each instance of R 6 is independently selected from H, alkyl, and dialkylaminoalkyl.
  • ** indicates the connection point to the -C(O)O(CR 2 R 3 )- fragment of the compound represented by structural formula (I); and # indicates the connection point to L 1 .
  • L 2 is selected from
  • T is coupled to each -C(O)OCR 2 R 3 - fragment via two L 2 , and T is a moiety represented by formula (IIf3): wherein each l is a point of connection to the -C(O)O(CR R )- fragments of the compound represented by structural formula (I).
  • p is 2 and T is a moiety represented by structural formula (lid), wherein T is coupled to each -C(O)OCR 2 R 3 - fragment of formula (I) via two L 2 : (lid).
  • a and B are each independently 5-membered heteroaryl, and i each i is a point of connection to the -C(O)O(CR R )- fragments of the compound represented by structural formula (I).
  • a and B are each independently a 5-membered heteroaryl optionally substituted by 1 to 4 groups independently selected from halogen, OH, CN, NO2, amine, amide, amidine; -(CH2) q NR w R wl ; C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein: each q is independently selected from 0, 1, 2 or 3, and R w and R wl are each independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl.
  • a and B are each independently substituted or unsubstituted oxazole.
  • a and B are each oxazole substituted with two C1-3 alkyls.
  • a and B are each independently substituted or unsubstituted pyrazole or substituted or unsubstituted oxazole.
  • a and B may be each independently represented by one of the following structural formulas:
  • a and B are represented by the following structural formula:
  • each instance of the first linker L u comprises one or more moieties independently selected in each instance from *(alkylene)O(alkylene)**, *(heteroalkylene)**, *(alkylene)**, *(heteroaralkylene)**,
  • each instance of the first linker L u is independently selected from *(CH 2 CH 2 O)tCH2**, *(CH 2 CH2O)2CH2CH 2 N(CH3)CH2**,
  • t represents an integer from 1-6. In certain such embodiments, t represents an integer from 1-3.
  • each instance of U is H or NH2. In some embodiments, each instance of U is -N(H)Me. In some particularly preferred embodiments, each instance of U is - N(Me)2. In other embodiments, each instance of U is independently selected from alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, - DBCO and -OH.
  • the heterocyclyl of U comprises at least one nitrogen which is the point of connection to L u .
  • U is a reactive group. Any suitable reactive group may be used, such that an additional moiety with a complementary reactive group may be coupled the compound of formula (I) through reaction with U.
  • the reactive group is an amino, azido, acetylenyl, -COOH, -P(O)(OH)2, or -OH.
  • U is alkylamino, heterocyclyl, or alkoxy.
  • U is a heterocyclyl comprising at least one nitrogen, which is the point of connection to L u .
  • U is -N(alkyl)2, for example, -N(CH3)2- In some embodiments, U is -(O)alkyl, for example, -OCH3.
  • U is a saccharide.
  • the saccharide is a saccharide.
  • HO A OH is a glucuronide.
  • the glucuronide is 0 CO 2 H
  • the compound is selected from:
  • the compound is selected from:
  • T is selected from:
  • Y is selected from; wherein n represents an integer from 1 to 15.
  • Y is selected from:
  • n represents an integer from 1 to 15.
  • the compound is: pharmaceutically acceptable salt thereof.
  • the compound is selected from:
  • the compound is selected from:
  • the compound is selected from:
  • the immune checkpoint inhibitor is an inhibitor of the PD- 1/PD- L1 pathway.
  • the inhibitor of the PD-1/PD-L1 pathway is an antibody, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, acrixolimab, MGA012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, cosibelimab, or Clone 31E6.
  • pembrolizumab e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizum
  • the antibody is an anti-PD-1 antibody or antigen-binding fragment thereof, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, or AMP-514.
  • the antibody is an antibody in Table 1.
  • the inhibitor of the PD-1/PD-L1 pathway is an anti-mouse PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-L1 antibody is 10F.9G2 or antigen-binding fragment thereof.
  • the inhibitor of the PD-1/PD-L1 pathway is an anti-mouse PD-1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-1 antibody is RPM1-14 or antigen-binding fragment thereof.
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 1;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 2;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 3;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 4;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 11;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 12;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 13;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 14;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 15;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 16;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 19;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 20;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 21;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 22;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 24; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 29:
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 30:
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 31:
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 32;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 33;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 34;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 37
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 38
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 39
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 40;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 41;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 42;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 45;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 46;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 47;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 48;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 49;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 50;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 53;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 54;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 55;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 56;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 57;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 58; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 61;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 62;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 63;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 64;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 65;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 66; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 109;
  • CDRH2 comprises the ammo acid sequence of SEQ ID NO: 110;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 111;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 112;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 113;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 114; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 121;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 122;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 123;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 124;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of:
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 8
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 10
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 18, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 26, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 36
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 44
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 52
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 60
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 68
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68.
  • the antibody is an anti-PD-Ll antibody or antigenbinding fragment thereof, e.g., atezolizumab, avelumab, durvalumab, and cosibelimab. In certain such embodiments, the antibody is an antibody in Table 2.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 69;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 70;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 71;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 72;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 73;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 74;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 79;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 80;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 81;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 82;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 83;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 84; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 87;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 88;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 89;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 90;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 91;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 92; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 97;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 98;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 99;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 100,
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 101;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 102; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 145;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 146;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 147;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 148;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 149;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of:
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 76
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 86
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 94
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 96
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96.
  • the antibody is a monoclonal antibody, a single chain antibody (scAb), a Fab fragment, a F(ab’)2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a multimeric antibody, or a bispecific antibody.
  • scAb single chain antibody
  • Fab fragment fragment
  • F(ab’)2 fragment fragment
  • scFv single chain variable fragment
  • scFv-Fc fragment a multimeric antibody
  • bispecific antibody a bispecific antibody.
  • the antibody is a chimeric, humanized or fully human monoclonal antibody.
  • the antibody is an IgG isotype (e.g., IgGl, IgG2, IgG3, or IgG4 isotype).
  • the antibody is an IgGl isotype.
  • the antibody comprises a N297A substitution according to EU numbering convention.
  • the antibody comprises E234A and E235A substitutions according to EU numbering convention.
  • the antibody comprises a P329G substitution or a P329A substitution according to EU numbering convention.
  • the antibody is an IgG4 isotype.
  • the antibody comprises a S228P substitution according to EU numbering convention.
  • the immune checkpoint inhibitor is selected from:
  • an anti-PD-1 antibody or antigen-binding fragment thereof wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, or antigen-binding fragment thereof;
  • an anti-PD-Ll antibody or antigen-binding fragment thereof wherein the anti-PD-Ll antibody is selected from atezolizumab, Clone 31E6, or antigen-binding fragment thereof; or
  • an anti-mouse PD-1 or PD-L1 antibody or antigen-binding fragment thereof wherein the anti-mouse PD-1 or PD-L1 antibody is selected from 10F.9G2, RPM1-14, or antigen-binding fragment thereof.
  • the compound is selected from: pharmaceutically acceptable salt thereof.
  • the method is a method of preventing or treating a proliferative disease, an infectious disease, an immune-mediated disorder, a central nervous system disease, a peripheral nervous system disease, a neurodegenerative disease, a cerebrovascular disease, a peripheral arterial disease, a cardiovascular disease, or an allergic disease.
  • the proliferative disease is cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, or cirrhosis of the liver.
  • the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
  • the infectious disease is chickenpox, chikungunya, a coronavirus infection, a dengue virus infection, diphtheria, Ebola, influenza, hepatitis, Hib disease, acquired immunodeficiency syndrome (AIDS), a human papillomavirus (HPV) infection, encephalitis, measles, meningococcal disease, Mpox, mumps, a norovirus infection, pneumococcal disease, polio, rabies, respiratory syncytial virus (RSV) infection, rotavirus infection, rubella, shingles, tetanus, whooping cough, or zika virus disease.
  • AIDS acquired immunodeficiency syndrome
  • HPV human papillomavirus
  • encephalitis measles
  • meningococcal disease Mpox
  • mumps a norovirus infection
  • pneumococcal disease polio, rabies, respiratory syncytial virus (
  • the immune-mediated disorder is Crohn’s, ulcerative colitis, uveitis, psoriasis, lupus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, ankylosing spondylitis, hidrosadenitis suppurativa, sarcoidosis, atopic dermatitis, connective tissue disorders, asthma, or multiple sclerosis.
  • the central nervous system disease is catalepsy, encephalitis, epilepsy, meningitis, multiple sclerosis, or myelopathy.
  • the peripheral nervous system disease is lepromatous neuropathy, diabetic neuropathy, Guillain-Barre syndrome, acute motor axonal neuropathy, botulism, Lambert-Eaton syndrome, acute intermittent porphyria, or familial amyloid neuropathy.
  • the neurodegenerative disease is Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barre” syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson’s disease, traumatic brain injury (TBI), epilepsy, or Huntington’s disease (HD).
  • the cerebrovascular disease is aneurysms, arteriovenous malformations (AVM), cerebral cavernous malformations (CCM), arteriovenous fistula (AVF), carotid-cavernous fistula, carotid stenosis, transient ischemic attack (TIA), or stroke.
  • AVM arteriovenous malformations
  • CCM cerebral cavernous malformations
  • AVF arteriovenous fistula
  • TIA transient ischemic attack
  • the cardiovascular disease is arrhythmias, congenital heart disease, coronary artery disease, deep vein thrombosis, pulmonary embolism, heart attack, heart failure, cardiomyopathy, heart valve disease, pericardial disease, peripheral vascular disease, rheumatic heart disease, stroke, or vascular disease.
  • the allergic disease is an allergy, anaphylaxis, aspergillosis, asthma, chronic cough, chronic granulomatous disease, chronic sinusitis, Churg-Strauss syndrome, cold urticaria, common variable immunodeficiency, eosinophilia, eosinophilic esophagitis, esophagitis, hay fever, hypereosinophilic syndrome, nasal congestion, nasal polyps, nonallergic rhinitis, conjunctivitis, pneumonitis, primary immunodeficiency, selective IgA deficiency, systemic mastocytosis, or X-linked agammaglobulinemia.
  • the method is a method of inducing an immune response.
  • the present disclosure relates to a pharmaceutical composition or a kit comprising the compound and the immune checkpoint inhibitor disclosed herein .
  • the composition further comprises a pharmaceutically acceptable excipient.
  • the present disclosure relates to a method of preventing or treating a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof, the method comprising administering to the subject the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.
  • STING stimulator of interferon genes
  • the present disclosure relates to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor for treating or preventing a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof.
  • STING stimulator of interferon genes
  • the present disclosure relates to the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor for use in treating or preventing a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof.
  • STING stimulator of interferon genes
  • the disease mediated by STING is cancer.
  • the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
  • the present disclosure relates to a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor.
  • the present disclosure relates to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof for inducing an immune response in a subject in need thereof conjointly with an immune checkpoint inhibitor.
  • the present disclosure relates to the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof for use in inducing an immune response in a subject in need thereof conjointly with an immune checkpoint inhibitor.
  • the inducing of the immune response is effective to prevent or treat a disease mediated by STING in the subject.
  • the disease mediated by STING is cancer.
  • the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
  • the present disclosure relates to a method of modulating the activity of a STING adaptor protein comprising contacting the STING adaptor protein with the compound of Formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor.
  • the compound increases the activity of the STING adaptor protein.
  • the STING-mediated disease is cancer, bacterial infection disease, viral infection disease, fungal infection disease, immune-mediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, cerebrovascular disease, peripheral Arterial disease, cardiovascular disease, allergic disease or inflammation.
  • the STING-mediated disease is cancer or an infectious disease.
  • the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, and osteosarcoma, Kaposi’ s sarcoma, and melanoma.
  • the pharmaceutical composition may additionally contain a pharmaceutically effective amount of a chemotherapeutic agent.
  • the pharmaceutical composition may include one or more therapeutic co-agents; and a pharmaceutically acceptable excipient may be additionally included.
  • the therapeutic co-agent is an agent that exhibits a preventive, ameliorative, or therapeutic effect on STING-mediated diseases, or an agent that can reduce the expression of side effects that appear when administering a therapeutic agent for STING- mediated diseases, or it may be, but is not limited to, an agent that exhibits an immunity enhancing effect, and when applied in the form of a formulation together with the STING agent represented by the compound, it exhibits a therapeutically useful effect and/or improves the stability of the proteolytic agent, and/or reduce the side effects that may appear when administering the STING agonist represented by the compound, and/or, any agent that exhibits the effect of maximizing the therapeutic effect through the enhancement of immunity can be applied in combination.
  • the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, It may be selected from the group consisting of Kaposi’s sarcoma and melanoma, but any carcinoma for which the STING agonist represented by the compound can exhibit a therapeutic effect can be applied.
  • a method for preventing or treating STING-mediated diseases comprising administering the pharmaceutical composition to a patient.
  • the compound of Formula 1 of the present invention can be used as an adjuvant for the treatment of other infectious diseases, diseases or disorders including cancer, in any one of its tautomers, stereoisomers and pharmaceutically usable salts.
  • the compound disclosed herein, an optical isomer thereof, a stereoisomer thereof, a solvate thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising them may be used alone or in one or more additional therapies (e.g., drug treatment or treatment) can be used in combination with an immune checkpoint inhibitor.
  • additional therapies e.g., drug treatment or treatment
  • Combination therapy may, for example, combine two therapies or may combine three therapies (e.g., a triple therapy of three therapeutic agents) or more.
  • the dosage of one or more of the additional therapies e.g., non-drug treatments or therapeutics
  • the compound, an optical isomer thereof, a stereoisomer thereof, a solvate thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same is administered before, after, or concurrently with one or more such additional therapies.
  • the compound can be administered together, e.g, in a single pharmaceutical composition, or can be administered separately, and when administered separately, this can occur simultaneously or sequentially. Such sequential administration may be close in time or distant.
  • the checkpoint inhibitor is an inhibitor of the PD-1/PD-L1 pathway (Ai L., et al. Drug Des Devel Ther. 2020; 14: 3625-3649).
  • PD-1 Programmed cell death protein-1
  • Pdcdl an inhibitory receptor in the immune response phase
  • PD-1 is a member of the CD28/CTLA-4 family of immunoglobulin (1g) superfamily.
  • PD-1 is a type I transmembrane protein with a size of 50-55 kDa, induced in a variety of hematopoietic cells in the peripheral blood and widely expressed in immune cells (T cells, B cells, macrophages, and certain types of dendritic cells, etc.) and tumor cells by antigen receptor signaling and cytokines.
  • T cells T cells, B cells, macrophages, and certain types of dendritic cells, etc.
  • PD-L1/PD-L2 programmed cell death ligand 1 and 2
  • PD-L1 is a type I transmembrane protein with a size of 40 kDa. It is widely expressed in both lymphoid tissue and non-lymphoid tissue, and in antigen-presenting cells (macrophages, dendritic cells, etc.) and all kinds of tumor cells. Both PD-1 and PD-L1 belong to the immune checkpoint protein family. As co-inhibitors, they can regulate the tolerance of central and peripheral T cells and reduce the proliferation of CD 8+ T cells in lymph nodes by combining and conducting inhibitory signals.
  • PD-1 and PD-L1 inhibitors are important immune checkpoint inhibitors (ICIs) for the treatment of cancer. Local immunosuppression could be eliminated by blocking the binding of PD-1 and PD-L1.
  • ICIs immune checkpoint inhibitors
  • the immune system produces an anti-cancer immune response by executing a cancer immunity cycle that kills cancer cells.
  • the PD-l/PD- L1 pathway is an adaptive immune resistance mechanism of tumor cells to endogenous immune anti-tumor activity. 2
  • PD-l/PD-ligand interaction down-regulates the immune response during the regression of infection or tumor or the development of self-tolerance.
  • PD-L1 is usually overexpressed in tumor cells or untransformed cells in tumor microenvironment and inhibits cytotoxic T cells by binding to PD- 1 receptor on activated T cells, resulting in immune escape.
  • the inhibitors of PD-1 and PD-L1 inhibit the interaction between PD-L1 and PD-1 receptor, preventing cancer cells from evading the immune system in this way and acting as ICIs by reactivating the T-cell-mediated tumor cell death process.
  • checkpoint inhibitors for the PD-1/PD-L1 pathway include anti-PD-1 antibodies (e.g., pembrolizumab, nivolumabcemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, and AMP-514) and anti-PD-Ll antibodies (e.g., atezolizumab, avelumab, durvalumab, and cosibelimab).
  • anti-PD-1 antibodies e.g., pembrolizumab, nivolumabcemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tisleliz
  • the inhibitor of the PD-1/PD-L1 pathway may be an antibody, such as pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, acrixolimab, MGA012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, or cosibelimab.
  • anti-PD-Ll antibodies are disclosed herein and others are known in the art, such as U.S. Pat. Nos 9,567,399 and 9,617,338, both incorporated by reference herein.
  • PD-1 Programmed cell death protein 1
  • CD279 cluster of differentiation 279 is a protein encoded in humans by the PDCD1 gene.
  • PD-1 is a cell surface receptor on T cells and B cells that has a role in regulating the immune system’s response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.
  • PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).
  • PD-1 inhibitors a class of drugs that block PD-1, activate the immune system to attack tumors and are used to treat certain types of cancer.
  • PD-1 binds two ligands, PD-L1 and PD- L2.
  • PD-L1 the ligand for PD1
  • PD-L1 the ligand for PD1
  • Monoclonal antibodies targeting PD-1 that boost the immune system are developed for the treatment of cancer.
  • Many tumor cells express PD-L1, an immunosuppressive PD-1 ligand; inhibition of the interaction between PD-1 and PD-L1 can enhance T-cell responses in vitro and mediate preclinical antitumor activity. This is known as immune checkpoint blockade.
  • nivolumab (Opdivo - Bristol Myers Squibb)
  • nivolumab (Opdivo - Bristol Myers Squibb)
  • Pembrolizumab (Keytruda, MK-3475, Merck)
  • Toripalimab is a humanized IgG4 monoclonal antibody against PD-1.
  • atezolizumab (MPDL3280A, Roche) and avelumab (Merck KGaA, Darmstadt, Germany and Pfizer) target the similar PD-L1 receptor.
  • the anti-PD-1 antibody may be pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, or AMP-514).
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein,
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 1;
  • CDRH2 comprises the ammo acid sequence of SEQ ID NO: 2;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 3;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 4;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 5;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 6;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 11;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 12;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 13;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 14;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 15;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 16;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 19;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 20;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 21;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 22;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 23;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 24;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 29;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 30;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 31;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 32;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 33;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 34;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 37;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 38;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 39;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 40;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 41;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 42;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 45;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 46;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 47;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 48
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 49
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 50;
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 53;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 54;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 55;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 56;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 57;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 58; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 61;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 62;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 63;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 64;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 65;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 66; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 109;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 110;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 111;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 112;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 113;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 114; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 121;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 122;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 123;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 124;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 125;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of:
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 10
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 18, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 26, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 36, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 44, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 52, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 60, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 68, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68.
  • Programmed death-ligand 1 also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene.
  • Programmed death-ligand 1 is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune systems during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis.
  • the anti-PD-Ll antibody may be atezolizumab, avelumab, durvalumab, or cosibelimab.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein,
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 69;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 70;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 71;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 72,
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 73;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 74, or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 79;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 80;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 81;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 82,
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 83;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 84, or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 87;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 88;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 89;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 90,
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 91;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 92, or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 97;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 98;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 99;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 100,
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 101;
  • CDRL3 comprises the amino acid sequence of SEQ ID NO: 102; or
  • CDRH1 comprises the amino acid sequence of SEQ ID NO: 145;
  • CDRH2 comprises the amino acid sequence of SEQ ID NO: 146;
  • CDRH3 comprises the amino acid sequence of SEQ ID NO: 147;
  • CDRL1 comprises the amino acid sequence of SEQ ID NO: 148;
  • CDRL2 comprises the amino acid sequence of SEQ ID NO: 149;
  • CDRL3 comprises the ammo acid sequence of SEQ ID NO: 150.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of: (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76, or
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 86
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 94
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 96
  • variable heavy chain comprising the amino acid sequence of SEQ ID NO:
  • variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94.
  • the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96.
  • the antibody is a monoclonal antibody, a single chain antibody
  • the antibody may be a chimeric, humanized or fully human monoclonal antibody.
  • the antibody is an IgG isotype, such as an IgGl isotype or IgG4 isotype.
  • the antibody is an IgGl isotype.
  • the antibody comprises a N297A substitution according to EU numbering convention.
  • the antibody comprises L234A and L235A substitutions according to EU numbering convention.
  • the antibody comprises a P329G substitution or a P329A substitution according to EU numbering convention. In certain embodiments, the antibody is an IgG4 isotype. In certain embodiments, the antibody comprises a S228P substitution according to EU numbering convention.
  • the inhibitor of the PD-1/PD-L1 pathway is a small molecule.
  • small molecules are described in U.S. Pat. App. Nos. 14/916,290, 17/041,455, 17/259,187, 17/260,547, 17/261,013, 16/963,557, 16/977,374, 15/957,739, 17/098,171, 16/274,106, 16/388,517, 16/388,517, 16/510,647, 17/264,638, and 17/264,657, and U.S. Pat. Nos. 10,568,874, 10,710,986, 11,555,029, 10,669,271, 11,124,511, 10,618,916, 10,906,920, and 11,414,433, all of which are incorporated fully herein by reference.
  • the inhibitor of the PD-1/PD-L1 pathway is a peptide.
  • peptides are described in U.S. Pat. App. No. 14/563,568, and U.S. Pat. Nos. 9,044,442, 8,907,053, 9,783,578, and 10,919,966 all of which are incorporated fully herein by reference.
  • the inhibitor of the PD-1/PD-L1 pathway is a macrocyclic compound. Such compounds are described in U.S. Pat. Nos.
  • the compounds according to the present disclosure may be used in combination with the agents described herein or other suitable agents depending on the condition being treated.
  • one or more compounds of the present disclosure will be coadministered with other therapies as described herein.
  • the compounds described herein may be administered simultaneously or separately with the second agent.
  • Such combined administration can include simultaneous administration of both agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any agent described herein may be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the present invention and any therapy described herein may be administered simultaneously, wherein the two agents are in separate preparations.
  • a compound of the present disclosure may be administered followed by any therapy described herein, or vice versa.
  • the compounds of the present invention and any of the therapies described herein may be administered minutes apart, or hours apart, or days apart.
  • the first therapy e.g., a compound of the invention
  • one or more additional therapies may be administered in any order, simultaneously or sequentially.
  • the compound described above in the pharmaceutical composition of the present invention is contained in a therapeutically effective amount or a prophylactically effective amount.
  • the preferred dosage of the compound according to the present invention varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art.
  • the compound of Formula (I) of the present invention may be administered in an amount of 0.0001 to 1000 mg/kg, preferably 0.01 to 500 mg/kg, divided into once to several times a day.
  • the compound of Formula (I) may be blended in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.
  • compositions and methods of the present disclosure may be utilized to treat an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients.
  • an active compound such as a compound of the disclosure
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient.
  • Compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body.
  • dosage forms can be made by dissolving or dispersing the active compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • parenteral administratio and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are also prepared by entapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • therapeutically effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient’s condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the disclosure.
  • a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.
  • contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the disclosure include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
  • contemplated salts of the disclosure include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, e
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • agent is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Agents include, for example, agents whose structure is known, and those whose structure is not known.
  • a “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
  • Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • preventing is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
  • a condition such as a local recurrence (e.g., pain)
  • a disease such as cancer
  • a syndrome complex such as heart failure or any other medical condition
  • prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • administering or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art.
  • a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • a compound or an agent is administered orally, e.g., to a subject by ingestion.
  • the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
  • the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents).
  • the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • a “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect.
  • the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • the precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not.
  • “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.
  • substituent and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
  • the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O- alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2.
  • alkyl refers to a linear or branched saturated monovalent hydrocarbon.
  • an alkyl group may have 1 to 10 carbon atoms (that is, (Ci-io)alkyl) or 1 to 8 carbon atoms (that is, (Ci-g)alkyl) or 1 to 6 carbon atoms (that is, ( Ci-6 alkyl) or 1 to 4 carbon atoms (that is, (Ci-4)alkyl).
  • alkyl group examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, - CH(CH 3 ) 2 ), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl- 1-propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2 -butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH 2 CH 2 CH3), 3- pen
  • alkyl refers to saturated aliphatic groups, including straightchain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkylsubstituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.
  • alkyl is unsubstituted, except as otherwise specified.
  • alkyl as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
  • alkenyl refers to a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon double bond.
  • an alkenyl group may include 2 to 8 carbon atoms (that is, C2-8 alkenyl), or 2 to 6 carbon atoms (that is, C2-6 alkenyl), or 2 to 4 carbon atoms (that is, C2-4 alkenyl).
  • one terminal hydrogen of the alkenyl group is omitted and may be connected with the next linking group.
  • alkenyl is unsubstituted, except as otherwise specified.
  • alkylene refers to a linear or branched divalent saturated hydrocarbon group having 1 to 6 (Ci-e) carbon atoms.
  • an alkylene having 1 to 4 (C1-4) carbon atoms may be used. Examples thereof include, but are not limited to, methylene, ethylene, trimethylene (propylene), and tetramethylene (n-butylene).
  • alkynyl refers to a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon triple bond.
  • an alkynyl group may include 2 to 8 carbon atoms (that is, C2-8 alkynyl), or 2 to 6 carbon atoms (that is, C2-6 alkynyl), or 2 to 4 carbon atoms (that is, C2-4 alkynyl).
  • alkynyl is unsubstituted, except as otherwise specified.
  • acyl is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
  • acylamino is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.
  • acyloxy is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.
  • alkoxy refers to an alkyl group having an oxygen attached thereto.
  • Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
  • alkoxyalkyl refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
  • alkyl refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.
  • alkyl as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
  • C x -y or “Cx-C y ”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain.
  • Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal.
  • a Ci-6 alkyl group for example, contains from one to six carbon atoms in the chain.
  • alkylene is the divalent moiety of alkyl
  • alkenylene is the divalent moiety of alkenyl
  • alkynylene is the divalent moiety of alkynyl
  • heteroalkylene is the divalent moiety of heteroalkyl
  • hetero alkenylene is the divalent moiety of heteroalkenyl
  • heteroalkynylene is the divalent moiety of heteroalkynyl
  • carbocyclylene is the divalent moiety of carbocyclyl
  • heterocyclylene is the divalent moiety of heterocyclyl
  • arylene is the divalent moiety of aryl
  • heteroarylene is the divalent moiety of heteroaryl.
  • alkylamino refers to an amino group substituted with at least one alkyl group.
  • alkylthio refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.
  • R9 and RIO each independently represent a hydrogen or hydrocarbyl group, or R9 and RIO taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R9, RIO, and RIO’ each independently represent a hydrogen or a hydrocarbyl group, or R 9 and R 10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • aminoalkyl refers to an alkyl group substituted with an amino group.
  • aralkyl or “arylalkyl”, as used herein, refers to an alkyl group substituted with an aryl group.
  • aryl as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
  • carboxylate is art-recognized and refers to a group wherein R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.
  • Carbocyclylalkyl refers to an alkyl group substituted with a carbocycle group.
  • Carbocycle includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings.
  • fused carbocycle refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings.
  • an aromatic ring e.g., phenyl
  • a saturated or unsaturated ring e.g., cyclohexane, cyclopentane, or cyclohexene.
  • Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct- 3-ene, naphthalene and adamantane.
  • Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH- indene and bicyclo[4.1.0]hept-3-ene.
  • Carbocycles may be substituted at any one or more positions capable of bearing a hydrogen atom.
  • carbonate is art -recognized and refers to a group -OCO2-.
  • cycloalkyl includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings.
  • cycloalkyl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R 100 ) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.
  • Non-limiting examples of monocyclic cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, 1 -cyclopent- 3 -enyl, cyclohexyl, 1 -cyclohex- 1-enyl, l-cyclohex-2-enyl, and l-cyclohex-3- enyl.
  • esters refers to a group -C(O)OR 9 wherein R 9 represents a hydrocarbyl group.
  • ether refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.
  • halo and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.
  • heteroarylalkyl refers to an alkyl group substituted with a hetaryl group.
  • heteroaryl refers to a single aromatic ring having at least one non-carbon atom in the ring, wherein the atom may be selected from oxygen, nitrogen, and sulfur, and “heteroaryl” may include a multiple condensed ring system having at least one such aromatic ring. The multiple condensed ring systems will be further described.
  • the “heteroaryl” may include a single aromatic ring having about 1 to 6 carbon atoms and about 1- 4 heteroatoms selected from oxygen, nitrogen and sulfur. Sulfur and nitrogen atoms may also exist in oxidized form, provided that the ring is aromatic.
  • heteroaryl includes, but are not limited to, pyridyl, pyrimidinyl, oxazolyl, or furyl.
  • heteroaryl includes a multiple condensed ring system (for example, a ring system including 2, 3 or 4 rings), and the heteroaryl group as defined above may form a multiple condensed ring system through condensation with at least one ring selected fromheteroaryl (used to form, for example, 1,8-naphthyridinyl), heterocycle (used to form, for example, l,2,3,4-tetrahydro-l,8-naphthyridinyl), carbocycle (used to form, for example, 5, 6, 7, 8 -tetrahydroquinolyl), and aryl (used to form, for example, indazolyl).
  • a heteroaryl (a single aromatic ring or a multiple condensed ring system) may have about 1-20 carbon atoms and about 1-6 heteroatoms in the heteroaryl ring.
  • Such multiple condensed ring systems may be such that the carbocycle or heterocycle portion of the condensed ring may be substituted with one or more (for example, 1, 2, 3, or 4) oxo groups.
  • the rings of a multiple condensed ring system may be linked to one another through fusion, spiro and cross-linking bonds as long as valency requirements are satisfied.
  • the individual rings of the multiple condensed ring system may be linked to one another in any order.
  • the point of attachment for the heteroaryl or the heteroaryl multiple condensed ring system may be any suitable atom of the heteroaryl or the heteroaryl multiple condensed ring system, including carbon atoms and heteroatoms (for example, nitrogen).
  • a particular atom-range member heteroaryl for example, (Cs- Cio) heteroaryl
  • the atomic range is to be understood as being relative to the total number of ring atoms of the heteroaryl and as including a carbon atom and a heteroatom.
  • a Cs heteroaryl may include a thiazolyl and a Cio heteroaryl may include a quinolinyl.
  • heteroaryls include pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, 5,6,7,8-tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3-Z?]pyridinyl, quinazolinyl-4(3/7)-one, triazolyl, 4,5,6,7-tetrahydro- 1/7-indazole, and 3b, 4,4a,5
  • heterocyclyl or “heterocycle” used herein refers to a monosaturated or partially unsaturated non-aromatic compound or non-aromatic multi-ring system in which at least one heteroatom (that is, at least one cyclic heteroatom selected from oxygen, nitrogen and sulfur) is included in the ring.
  • heterocyclyl groups have 5 to about 20 ring atoms, such as 3 to 12 ring atoms, such as 5 to 10 ring atoms.
  • the term includes a single saturated or partially unsaturated ring (for example, 3, 4, 5, 6 or 7-membered rings), having about 1 to 6 cyclic carbon atoms and about 1 to 3 cyclic heteroatoms selected from oxygen, nitrogen and sulfur, in the ring.
  • the rings of a multiple condensed ring system may be linked to one another through fusion, spiro and cross-linking bonds as long as valency requirements are satisfied.
  • heterocycles include azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidinone, tetrahydrofuran, tetrahydro thiophene, dihydropyridine, tetrahydropyridine, quinuclidine, N-bromopyrrolidine, N-chloropiperidine, and the like.
  • Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.
  • hydroxy alkyl refers to an alkyl group substituted with a hydroxy group.
  • lower when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer.
  • acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).
  • polycyclyl refers to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”.
  • Each of the rings of the polycycle can be substituted or unsubstituted.
  • each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.
  • sulfate is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.
  • sulfoxide is art-recognized and refers to the group-S(O)-.
  • sulfonate is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.
  • sulfone is art -recognized and refers to the group -S(O)2-.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • thioalkyl refers to an alkyl group substituted with a thiol group.
  • thioester refers to a group -C(O)SR 9 or -SC(O)R 9 wherein R 9 represents a hydrocarbyl.
  • thioether is equivalent to an ether, wherein the oxygen is replaced with a sulfur.
  • urea is art -recognized and may be represented by the general formula wherein R 9 and R 10 independently represent hydrogen or a hydrocarbyl.
  • hydrozido is art-recognized and may be represented by the general formula 0 alkyl ⁇ y N y linking group H ), wherein R 11 and R 12 independently represent hydrogen or a hydrocarbyl.
  • DBCO dibenzocyclooctyne moiety
  • PDL1 generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which downregulates T cell activation and cytokine secretion after binding to PD-1.
  • P-L1 includes any native PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats).
  • the term covers “full-length,” unprocessed PD-L1 as well as any form of PD-L1 produced by cell processing.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • “PD-L1” includes complete PD-L1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs having at least one common epitope with PD-L1.
  • the basic structure of PD-L1 includes 4 domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain.
  • An exemplary human PD-L1 amino acid sequence can be found under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7.
  • compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
  • pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any base compounds represented by Formula I.
  • Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form.
  • mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sul
  • the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.
  • the selection of the appropriate salt will be known to one skilled in the art.
  • Other non- pharmaceutically acceptable salts e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
  • pharmaceutically acceptable basic addition salt means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates.
  • Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide.
  • Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
  • stereogenic center in their structure.
  • This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30.
  • the disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.
  • Log of solubility is used in the art to quantify the aqueous solubility of a compound.
  • the aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption.
  • LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol/liter.
  • cleavage group refers to a chemical moiety which dissociates when subjected to a stimulus, such as acidic conditions, basic conditions, reducing conditions, oxidizing conditions, light, or heat, or an enzyme, such as an esterase.
  • identity refers to the similarity or relatedness of two or more polypeptide or nucleic acid sequences determined by aligning and comparing the sequences of two or more polypeptides or nucleic acids. Such inter-sequence identity is typically expressed as “percentage identity,” representing the proportion of identical amino acids or nucleotides between the compared molecules, calculated based on the smallest size molecule among the compared molecules. Methods for aligning nucleic acids or polypeptides to calculate identity between them are known in the art and may also be referenced herein.
  • affinity refers to the strength of interaction between an antibody or its antigen-binding fragment and an antigen, determined by characteristics of the antigen such as size, shape, and/or charge, and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining such affinities are known in the art and may also be referenced herein.
  • Antibodies or their antigen-binding fragments used in the present invention are said to “specifically bind” to the target, such as the antigen, when the dissociation constant (KD) is ⁇ 10 -6 M.
  • Antibodies bind “with high affinity” to the target when KD is ⁇ lx 10’ 8 M.
  • antibody-binding fragment of an antibody or immunoglobulin chain (light or heavy) used in the present invention refers to a portion of the antibody that includes some amino acids missing compared to the full-length chain but includes a portion of the antibody that can specifically bind to the target antigen. These fragments can be biologically active in terms of being able to specifically bind to target antigens or compete with other antibodies or antigen-binding fragments for binding to specific epitopes. In some embodiments, such fragments comprise at least one CDR in a full-length light chain or heavy chain, and comprise short heavy chain and/or light chains, or portions thereof in some embodiments.
  • Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFV, scFV-Fc, diabody, minibody, scAb, and dAb and may be derived from any mammal including human, mouse, rat, camelid or rabbit.
  • functional portion of the antibody such as one or more CDRs can be covalently linked to a second protein or small molecule compound and used as a targeted therapeutic agent for a specific target.
  • the “Fc” region is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions and in this invention includes two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. These two heavy chain fragments are linked to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the Cterminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4.
  • the “Fab fragment” in this invention consists of one light chain and one heavy chain containing only the variable region and CHI.
  • the heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.
  • scFab is Fab of two molecules linked by a flexible linker.
  • the “Fab’ fragment” in this invention includes a region between the CHI and CH2 domains of the heavy chain in addition to the Fab fragment, which forms an interchain disulfide bond between the two heavy chains of the two molecules of the Fab’ fragment, thereby forming F (ab ’)2 molecules.
  • the “F(ab’)2 fragment” in this invention includes two light chains and two heavy chains including a variable region, CHI, and a portion of the constant region between the CHI and CH2 domains, which form 2 interchain disulfide bonds are between the heavy chains. Therefore, the F(ab’)2 fragment is composed of two Fab’ fragments, and the two Fab’ fragments are associated with each other by a disulfide bond between them.
  • the “Fv region” in this invention is a fragment of an antibody that includes the variable regions of the heavy chain and light chains, but does not include the constant region.
  • sdFV is where heavy chains and light chains are linked by disulfide bonds.
  • scFv is Fv linked by a flexible linker.
  • scFv-Fc is where Fc is linked to scFV.
  • the minibody is where CH3 is linked to scFV.
  • the diabody contains scFV of two molecules.
  • a “single chain Fv” or “scFv” antibody fragment in this invention comprises the VH and VE domains of an antibody, and these domains are in a single polypeptide chain.
  • the Fv polypeptide may further include a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.
  • a “single chain antibody” (scAb) in this invention is a single polypeptide chain containing one constant region of a heavy chain or a light chain constant region where the heavy chain and light chain variable regions are linked by a flexible linker.
  • scAb single chain antibody
  • a “domain antibody” (dAb) in this invention is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody.
  • the two VH regions of these bivalent domain antibodies can target the same or different antigens.
  • CDR complementarity determining region
  • frame region is variable domain moieties other than CDR moieties.
  • Each variable domain typically has four FRs, identified as FR1, FR2, FR3 and FR4.
  • a “bivalent antigen binding protein” or “bivalent antibody” includes two antigen binding sites.
  • the two antigen binding sites included in such a bivalent antibody may have the same antigen specificity, or may be bispecific antibodies that each bind to different antigens.
  • multispecific antigen binding protein or “multispecific antibody” targets two or more antigens or epitopes.
  • a “chimeric antibody” refers to an antibody (immunoglobulin) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat’l Acad. Sci. USA, 81:6851-55 (1984)).
  • Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like.
  • the number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is one that possesses an amino-acid sequence corresponding to that of an antibody, produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phagedisplay libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Nat’l Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • a “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.
  • an “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s).
  • an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen.
  • Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH- and VL- domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci.
  • conjugates refers to cell binding agents that are covalently bonded to one or more molecules of a cytotoxic compound.
  • cell binding agent is a molecule having affinity for a biological target, and may be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and the binding agent functions to direct a biologically active compound to a biological target.
  • the conjugate may be designed to target tumor cells through cell surface antigens.
  • the antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type.
  • the target antigen may be expressed only on proliferative cells (e.g., tumor cells).
  • the target antigen may be selected on the basis of different expression, usually between proliferative tissues and normal tissues.
  • reaction solution was diluted with ethyl acetate (50 mL), washed with distilled water (30 mL), and then dried with anhydrous magnesium sulfate.
  • the reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography to afford Compound 24 (213 mg, 52.9%).

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Abstract

The disclosure provides combination therapy methods of treating diseases (such as cancer) comprising a compound represented by formula (I), or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.

Description

COMPOSITIONS AND METHODS RELATED TO A COMBINATION THERAPY OF STING AGONISTS AND IMMUNE CHECKPOINT INHIBITORS
Cross-Reference to Related Applications
This application claims the benefit of priority to U.S. Provisional Application No. 63/644,231, filed May 8, 2024, which is hereby incorporated by reference in its entirety.
Background
The stimulator of interferon genes (STING) is a low-molecular-weight protein currently attracting attention as a target for cancer therapies. STING is an adapter protein in the cGAS (cyclic GMP-AMP synthase)-STING pathway, which is a sensing pathway that induces activation of type I IFN and other inflammatory cytokines, triggering antiviral and antitumor immune responses (Chen, Q. et al. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat. Immunol. 2016, 17, 1142-1149; Woo, S.R. et al. STING- dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 2014, 41, 830-842). In addition, STING activates signal transducer and activator of transcription 6 (STAT6) and transcription factor interferon regulatory factor 3 (IRF3) through TANK-binding kinase 1 (TBK1) in antiviral and innate immune responses (Burdette DL, Vance RE, STING and the innate immune response to nucleic acids in the cytosol, 2013, Nature Immunology. 14 (1): 19-26). STING agonists can also trigger expression of cytokines, giving rise to a T cell-mediated innate immune response which inhibits the growth of cancer cells. However, systemic delivery of STING agonists can cause widespread inflammation.
Various STING agonists have been tested in preclinical and clinical environments. A variety of agonists in the form of CDN (cyclic dinucleotide) compounds (ADU-S100, BISTING, GSK532, JNJ-4412, SB11285, MK-1454, TAK676, etc.), bacterial vectors (SYNB1891, STACT-TREX-1), non-cyclic dinucleotide (CDN), compounds (ALG-031048, JNJ-6196, MK-2118, MSA-1, MSA-2, CRD-5500, etc.), nano vaccines (PC7A NP, cGAMP- NP, etc.) and ADCs (XMT-2056, TAK500, etc.) are under development by various strategies.
The most widely used preclinical compound, DMXAA (vascular disrupting agent), was used clinically in combination with paclitaxel and carboplatin, but its lack of efficacy was confirmed in phase 3. Further, ADU-S100, which was first used clinically as the STING agonist, was discontinued from use in 2020. Examples of STING agonists are disclosed, for example, in WO2021/014365 (a macrocyclic compound as STING agonist), and US2021/0139473 (a heterocyclic amide- containing compound as protein modulator), US 2022/0073509 (a heterocyclic compound as STING activator), and KR 2022-0024467 (a heterocycle-containing STING agonist), each of which is incorporated herein by reference in its entirety.
However, existing STING agonists appear to exhibit only limited bioavailability and require local administration to tumors due to hyperactivation of cytokine expression. Therefore, there remains a demand for development of STING agonists for effective therapeutic methods.
Summary
In certain aspects, the present disclosure relates to a method of administering a compound to a subject in need thereof, comprising administering the compound conjointly with an antibody-drug conjugate, wherein the compound is represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein:
T is a moiety comprising a Stimulator of Interferon Genes (STING) agonist, p is 1 or 2, each instance of R1 is independently -CH2OR11 or -COOR12, each instance of Rla, Rlb, Rlc, and R11 is independently H or a hydroxyl protecting group, each instance of R12 is independently H or a carboxyl protecting group, each instance of R2 and R3 is independently H or alkyl, or R2 and R3 together with a carbon atom to which they are attached form a cycloalkyl, each instance of R4 is independently selected from halogen, alkyl, CN, and NO2, each instance of k is independently 0, 1, 2, or 3, each instance of Y is independently selected from H, -C(O)NHLUU, -C(O)NR'(LUU), - C(O)N(LUU)2 and -C(O)OH, each instance of Lu is a first linker, each instance of U is independently selected from H, alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, -OH, -DBCO and a saccharide, and each instance of R’ is independently selected from alkyl, cycloalkyl, alkoxy, alkylthio, mono- or di-alkylamino, heteroaryl, and aryl.
Certain compounds encompassed by structural formula (I) are recited in PCT International Application No. PCT/IB23/00670, filed November 8, 2023, the entire contents of which are hereby incorporated by reference.
Brief Description of the Drawings
FIG. 1A: STING agonist compounds (Compound 214, 221 and 230) induced cytokine production (human CXCL-10).
FIG. IB: STING agonist compounds (Compound 214, 221 and 230) induced cytokine production (human IFNa).
FIG. 2A: MDA-MB-468 tumor cells were treated with STING agonist compound (Compound 313).
FIG. 2B: BxPC3 tumor cells were treated with STING agonist compound (Compound 313).
FIG. 3 A: Up-regulation of co-stimulatory molecule CD86 when THP-1 was treated with the STING agonist compound (Compound 313).
FIG. 3B: Up-regulation of MHC class II molecule HLA-DR when THP-1 was treated with the STING agonist compound (Compound 313).
FIG. 4A: STING agonist compounds (Compound 274 and 277) enhanced CD69 expression on CD8+ T cells.
FIG. 4B: STING agonist compounds (Compound 274 and TIT) enhanced CD69 expression on NK cells.
FIG. 5A: STING agonist compounds (Compound 344 and 361) enhanced expansion of activated CD8+ T cells
FIG. 5B: STING agonist compounds (Compound 344 and 361) enhanced expansion of activated NK cells.
FIG. 6A: Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 221). FIG. 6B: Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 277).
FIG. 6C: Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 281).
FIG. 6D: Graphical representation of the plasma PK in naive Balb/C mouse following single dose of STING agonist compound (Compound 274) compared to Comparative compound #1 and #2.
FIG. 7A: Tumor volume (mm3) following treatment with STING agonist compounds (Compound 214 and 221) in CT26 syngeneic mouse model. When tumor volume reached 70 mm3, each compound was given at 1.5 mg/kg intravenously every 3 or 4 days (total three times).
FIG. 7B: Body weight (%) following treatment with STING agonist compounds (Compound 214 and 221). When tumor volume reached 70 mm3, each compound was given at 1.5 mg/kg intravenously every 3 or 4 days (total three times).
FIG. 8: Tumor volume (mm3) following treatment with STING agonist compound (Compound 274) in CT26 mouse model at various doses.
FIG. 9: Tumor volume (mm3) following treatment with STING agonist compounds (Compound 274 and 281) compared to Comparative compound #1 in CT26 syngeneic mouse model. When tumor volume reached 55 mm3, each compound was given at 0.5 mg/kg intravenously once weekly for three weeks. Compared to the comparative compound #1, mean tumor growth over time was monitored.
FIG. 10A: Tumor volume (mm3) following treatment with STING agonist compounds (Compound 274, 313, 344, and 396) compared to Comparative compound #3 in CT26 syngeneic mouse model. When tumor volume reached 80 mm3, each compound was given at 0.3 mg/kg intravenously once weekly for 3 weeks. Compared to the comparative compound #3, tumor growth over time was monitored.
FIG. 10B: Body weight (mm3) following treatment with STING agonist compounds (Compound 274, 313, 344, and 396) compared to Comparative compound #3 in CT26 syngeneic mouse model. When tumor volume reached 80 mm3, each compound was given at 0.3 mg/kg intravenously once weekly for 3 weeks. Compared to the comparative compound #3, body weight over time was monitored.
FIG. 11: Tumor volume (mm3) following treatment with STING agonist compounds (Compound 274, and 313) compared to Comparative compound #1 and #2 in EMT6 syngeneic mouse model. When tumor volume reached 100 mm3, each compound was given at 0.125 mg/kg intravenously once weekly for three weeks. Compared to the comparative compounds #1 and #2, mean tumor growth over time was monitored.
FIG. 12A: Tumor volume (mm3) following treatment with STING agonist compound (Compound 313) at various doses in EMT6 model.
FIG. 12B: Body weight (%) following treatment with STING agonist compound (Compound 313) at various doses in EMT6 model.
FIG. 13: In vivo efficacy evaluation of co-administration anti-PD-Ll Ab_l with Compound 277, 302, or 422 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
FIG. 14: In vivo efficacy evaluation of co-administration anti-PD-Ll Ab_l with Compound 274 or 313 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
FIG. 15: In vivo efficacy evaluation of co-administration anti-mPD-1 Ab or anti-mPD- L1 with Compound 274 in CT26 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
FIG. 16: In vivo efficacy evaluation of co-administration anti-mPD-1 Ab with Compound 363 in B16F10 syngeneic mouse model. Mean tumor volume (top) and body weight (bottom) over time were monitored.
FIG. 17A: In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-1 antibody_l
FIG. 17B: In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-1 antibody_2
FIG. 17C: In vitro co-culture assay of effector cells and red-labeled SK-BR3 target cells treated with Compound 313 and anti-PD-Ll antibody_2
FIG. 18: In vitro evaluation of enhanced phagocytosis by Compound 363 in the presence of anti-PD-Ll antibody_3.
Detailed Description
In certain aspects, the present disclosure relates to a method of administering a compound to a subject in need thereof, comprising administering the compound conjointly with an antibody-drug conjugate, wherein the compound is represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein:
T is a moiety comprising a Stimulator of Interferon Genes (STING) agonist, p is 1 or 2, each instance of R1 is independently -CH2OR11 or -COOR12, each instance of Rla, Rlb, Rlc, and R11 is independently H or a hydroxyl protecting group, each instance of R12 is independently H or a carboxyl protecting group, each instance of R2 and R3 is independently H or alkyl, or R2 and R3 together with a carbon atom to which they are attached form a cycloalkyl, each instance of R4 is independently selected from halogen, alkyl, CN, and NO2, each instance of k is independently 0, 1, 2, or 3, each instance of Y is independently selected from H, -C(O)NHLUU, -C(O)NR'(LUU), -
C(O)N(LUU)2 and -C(O)OH, each instance of Lu is a first linker, each instance of U is independently selected from H, alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, -OH, -DBCO and a saccharide, and each instance of R’ is independently selected from alkyl, cycloalkyl, alkoxy, alkylthio, mono- or di-alkylamino, heteroaryl, and aryl.
In some embodiments, the compound of formula (I) is a compound of formula (la), or a pharmaceutically acceptable salt thereof:
In some embodiments, each instance of R1 is independently -CH2OH or -COOH.
In some embodiments, each instance of Rla, Rlb, Rlc is independently H.
In some embodiments, each instance of R2 and R3 is independently H.
In some embodiments, k is 0.
In some embodiments, T is a moiety represented by formula (Ila), or a pharmaceutically acceptable salt thereof: wherein:
T is coupled to the -C(O)OCR2R3- fragment of formula (I) via L2,
M is N, C(XaRa) or C(XbL1L2-),
Q is -XaRa or -XhL' L2-, each instance of W1 and W2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3,
Z is selected from alkylene, alkenylene, and alkynylene,
A and B are each independently aryl or heteroaryl,
Xa and Xb are each independently selected from CH2, NH, O, and S,
Ra is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, -(alkylene)guanidino, - (alkylene)NHC(O)CH2guanidino, -(alkylene)O(alkylene)guanidino and O(alkylene)guanidino, each L1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene, each L2 is independently selected from a bond, or a linker moiety coupled to L1 and comprising a nitrogen atom coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I). It will be understood that, according to this embodiment, given that L2 is the moiety that couples Formulas (I) and (Ila), one of both of the following is true M is C(XbL1L2-) and/or Q is -XhfJL2-.
In some embodiments, Xa is O.
In some embodiments, T is a moiety represented by Formula (T1): wherein:
T1 is coupled to the -C(O)OCR2R3- fragment of formula (I) via the -N(H)- of T1; each Ta and Tb is independently a moiety represented by formula (Ila): wherein:
M is N, C(XaRa) or C(XbL1L2-),
Q is -XaRa or -XhL' L2-, each instance of W1 and W2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3,
Z is selected from alkylene, alkenylene, and alkynylene,
A and B are each independently aryl or heteroaryl,
Xa and Xb are each independently selected from CFb, NH, O, and S,
Ra is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl,
-(alkylene)carboxylic acid, -(alkylene)guanidino, -(alkylene)NHC(O)CH2guanidino, -(alkylene)O(alkylene)guanidino and -O(alkylene)guanidino, each L1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene, and each L2 is independently selected from a bond between L1 and -C(0)0(CH2)- fragment of T1, or a linker moiety coupled to L1 and comprising a nitrogen atom coupled to the - C(0)0(CH2)- fragment of T1; wherein either M is CIX'^' L2-) or Q is -XhL' L2-.
In some embodiments, each instance of W1 and W2 is independently amido. In further embodiments, each instance of W1 and W2 is -CONH2.
In some embodiments, n and m are each 1.
In some embodiments, Z is C2-6 alkenylene. In further embodiments, Z is
In some embodiments, Ra is selected from C1-6 alkyl, -(alkylene)N(H)alkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, and -(alkylene)guanidino.
In further embodiments, Ra is selected from H, C1-6 alkyl, -(alkylene)N(H)alkyl, -(Ci- 6alkylene)carboxylic acid, -(Ci-6alkylene)guanidino, -(Ci-6alkylene)NHC(O)CH2guanidino, - (Ci-6alkylene)O(Ci-6alkylene)guanidino, wherein the C1-6 alkyl is optionally substituted with 5- to 7-membered cycloalkyl or heterocyclyl.
In yet further embodiments, Ra is selected from H, Methyl, -(CH2)3COOH,
In some embodiments, Xb is O.
In some embodiments, L1 is selected from Ci-ealkylene, Ci-ealkenylene, Ci-ealkynylene, cycloalkylene, heterocyclylene, arylene, and heteroaryne.
In further embodiments, L1 is selected from Ci-ealkylene, , and
* * is the point of connection to Xb and ** is the point of connection to L2. In some embodiments, L2 is selected from a bond,
— (alkylene) —
H y
Ct N-
N— (alkylene)-(heterocyclyl) ;s'
— (arylene)— —(heteroarylene) — (alkylene)— NH
H — (heterocyclylene)— NH
—(heteroarylene)—
— (heterocyclenylalkylene)— N
CO2H — (alkylene)— N
O — (heteroarylene) — N
H , and *heterocyclylene**, and wherein * is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
In some embodiments, L2 is selected from a bond,
— (alkylene) — ,N— (alkylene)-NH
H (alkylene)— NH
* NH zN— (alkylene)— (heterocyclylene) — * NH2
— (arylene)— N^ —(heteroarylene)— (alkylene)— NH
H — (heterocyclylene)— NH '**
** — (heteroarylene) — / *— (heterocyclenylalkylene)— N
CO2H — (alkylene)— N o
—(heteroary —(heteroarylene)— (alkylene)— NH lene)— N
H
H2N Alkylene Alkylene \ — p
(alkylene)— N
N-H N— (Alkyl) Q
(alkyl)
H / *— 0— (heterocyclylene) — (alkylene) — N \ , a'ky' and *heterocyclylene**; wherein
* is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) and the heterocyclylene of *heterocyclylene** comprises a nitrogen atom connected to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
NH
' NHo
,N— (alkylene)-NH 2
In some embodiments, L2 is selected from N— (alkylene)-(heterocyclyl) y
— (arylene)— N^ — (heteroarylene)— N
H — (heterocyclylene) — NH H
—(heteroarylene)— N
—(heteroarylene) — (alkylene)— NH
CO2H ene , , y y erein
* is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) and the heterocyclylene of *heterocyclylene** comprises a nitrogen atom connected to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I). In yet further embodiments, L2 is selected from
In some embodiments, Q is -XaRa.
In some embodiments, M is C(XaRa).
In some embodiments, Q is -XaRa or M is C(XaRa).
In some embodiments, the alkyl is Ci-6 alkyl, e.g., C3 alkyl.
In some embodiments, the alkylene is C1-6 alkylene, e.g., C3 alkylene.
In some embodiments, M is C(XaRa), e.g., M has the structure selected from: and wherein each I is a point of connection to the remainder of the phenyl ring. In some embodiments, Q is -XaRa, e.g., Q has the structure selected from: wherein each I is a point of connection to the phenyl ring.
In some embodiments, M has the structure: , wherein n represents an integer from 1 to 15, e.g., , wherein each I is a point of connection to the remainder of the phenyl ring.
In some embodiments, Q has the structure: NH , wherein n
H represents an integer from 1 to 15, e.g., NH , wherein I is a point of connection to the phenyl ring.
In some embodiments, M is CCX1!?!?’) or Q is -XbL1L2-.
In some embodiments, T is coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) via L2, and T is a moiety represented by formula (lib): (lib), wherein:
A and B are each independently 5-membered heteroaryl, and JVW i
I is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I). In some embodiments, T is coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) via L2, and T is a moiety represented by formula (lie): wherein: A and B are each independently 5-membered heteroaryl, and «/W i V o
I is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
In some embodiments, L2 is selected from a bond,
— (alkylene) — N— (alkylene) H (alkylene)— NH
NH
Ov N-
N— (alkylene)-(heterocyclyl) N— (alkylene)— i Js'
NH2
— (arylene)— N^ —(heteroarylene) — (alkylene)— NH
H — (heterocyclylene)— NH x**
—(heteroarylene)—
— (heterocyclenylalkylene)— N
CO2H — (alkylene)— N
O — (heteroarylene) — N
H , and *heterocyclylene**, and wherein * is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
In some embodiments, T is coupled to the -C(O)OCR2R3- fragment via L2, and T is a moiety represented by formula (IIc3): »/VW o wherein l is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
In some embodiments, T is coupled to the -C(O)OCR2R3- fragment via L2, and T is a moiety represented by formula (IIc4): wherein l is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
In some embodiments, L2 is a second linker. Any suitable linker moiety may be used. In some embodiments, L2 is selected from a bond, ,
NH
— (alkylene) — N^ zN— (alkylene)-N AH ,
NH
\|— (alkylene)-(heterocyclyl) zN— (alkylene) -(heterocyclylene) — * NH2
**
— (arylene) — N^ / *— (heteroarylene) — (alkylene)— NH
H * — (heterocyclylene) — NH '**
** / *— (heterocyclenylalkylene)— N \
O /** *— (heteroarylene)— N , H , and *heterocyclylene**, and wherein * is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
In some embodiments, L2 is a second linker comprising #OC(O)NR5-L4-NR6, #OC(O)- L4-NR6, or #OC(O)NR5-L4-(heterocyclylene), wherein: the heterocyclylene comprises a nitrogen atom connected to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I), and wherein # is the point of connection to L1, each instance of L4 is independently alkylene or arylalkylene, each instance of R5 is independently selected from H, alkyl, and dialkylaminoalkyl, and each instance of R6 is independently selected from H, alkyl, and dialkylaminoalkyl.
wherein ** indicates the connection point to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I); and # indicates the connection point to L1.
In some embodiments, L2 is selected from
In some embodiments, T is coupled to each -C(O)OCR2R3- fragment via two L2, and T is a moiety represented by formula (IIf3): wherein each l is a point of connection to the -C(O)O(CR R )- fragments of the compound represented by structural formula (I).
In some embodiments, p is 2 and T is a moiety represented by structural formula (lid), wherein T is coupled to each -C(O)OCR2R3- fragment of formula (I) via two L2: (lid). wherein A and B are each independently 5-membered heteroaryl, and i each i is a point of connection to the -C(O)O(CR R )- fragments of the compound represented by structural formula (I).
In some embodiments, each L2 is independently a second linker comprising #OC(O)NR5-L5-NR6, #OC(O)-L4-NR6, or #OC(O)NR5-L5-(heterocyclylene), wherein the heterocyclylene comprises a nitrogen atom connected to the respective -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I), wherein # is the point of connection to the respective L1 , each L5 is independently alkylene or aralkylene, and each R5 and each R6 are each independently selected from H, alkyl, and dialkylaminoalkyl.
In some embodiments, A and B are each independently a 5-membered heteroaryl optionally substituted by 1 to 4 groups independently selected from halogen, OH, CN, NO2, amine, amide, amidine; -(CH2)qNRwRwl ; C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein: each q is independently selected from 0, 1, 2 or 3, and Rw and Rwl are each independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl. For example, A and B may be each independently selected from pyrazole, imidazole, oxazole, isoxazole, thiazole, and isothiazole. In certain preferred embodiments, A and B are each pyrazole. In some embodiments, A and B are each independently substituted or unsubstituted pyrazole. For example, in certain preferred embodiments, A and B are each pyrazole substituted with two C1-3 alkyls.
In some embodiments, A and B are each independently substituted or unsubstituted oxazole. For example, in certain preferred embodiments, A and B are each oxazole substituted with two C1-3 alkyls.
In some embodiments, A and B are each independently substituted or unsubstituted pyrazole or substituted or unsubstituted oxazole.
In some embodiments, A and B are each independently substituted pyrazole or substituted oxazole, wherein the pyrazole and oxazole are each substituted with two C1-3 alkyls.
In some embodiments, A and B are each independently represented by one of the following structural formulas: wherein Rx and Rxl are each independently selected from hydrogen, C1-5 alkyl, C1-5 haloalkyl, halogen, OH, -OP(O)(RyRyl)2, -ORy, -NRyRyl, -OCORy, -CO2Ry, -SORy, -SO2Ry, - CONRyRyl, -SO2NRyRyl, -OCONRyRyl, -NRyCORyl, -NRySORyl, -NRyCO2Ryl, and - NRySO2Ryl, and wherein Ry and Ryl are each independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl.
For example, A and B may be each independently represented by one of the following structural formulas:
In some preferred embodiments, A and B are each represented by the following structural formula: iments, A and B are represented by the following structural formula: iments, A and B are represented by the following structural formula:
In some embodiments, A and B are represented by the following structural formula:
In certain embodiments, each instance of the first linker Lu comprises one or more moieties independently selected in each instance from *(alkylene)O(alkylene)**, *(heteroalkylene)**, *(alkylene)**, *(heteroaralkylene)**,
* (hetero alkylene) (heterocyclylene) * * , *CH2CH2C(O)NHCH* * , * (CH2CH2O)t- * * ,
*(alkylene)O(alkylene)-(amide)-(alkylene)O-**, and
*(alkylene)(heteroarylene)(CH2CH2O)t**, wherein * indicates the point of connection to the -C(O)NH- fragment of the compound represented by structural formula (I), ** indicates the point of connection to U, and t represents an integer from 1-15.
In certain such embodiments, each instance of the first linker Lu is independently selected from *(CH2CH2O)tCH2**, *(CH2CH2O)2CH2CH2N(CH3)CH2**,
*(CH2CH2O)2CH2CH2N(CH3)**, *(CH2CH2O)t**, *(CH2CH2O)tCH2CH2NH**,
*CH2CH2 * * , * (CH2CH2O)tCH2 * * , * (CH2CH2O)tCH2CH2heterocyclylene * * ,
*(CH2CH2O)tCH2**, *(CH2CH2O)tCH2CH2**, *(CH2CH2O)tCH2CH2NHCOC(CH3)2-O- **and *(CH2CH2O)tNH**. In certain such embodiments, t represents an integer from 1-6. In certain such embodiments, t represents an integer from 1-3.
In some embodiments, each instance of U is H or NH2. In some embodiments, each instance of U is -N(H)Me. In some particularly preferred embodiments, each instance of U is - N(Me)2. In other embodiments, each instance of U is independently selected from alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, - DBCO and -OH.
In certain such embodiments, the heterocyclyl of U comprises at least one nitrogen which is the point of connection to Lu.
In other embodiments, U is a reactive group. Any suitable reactive group may be used, such that an additional moiety with a complementary reactive group may be coupled the compound of formula (I) through reaction with U.
In some such embodiments, the reactive group is an amino, azido, acetylenyl, -COOH, -P(O)(OH)2, or -OH. In some embodiments, U is alkylamino, heterocyclyl, or alkoxy. In some embodiments, U is a heterocyclyl comprising at least one nitrogen, which is the point of connection to Lu.
In some embodiments, U is -N(alkyl)2, for example, -N(CH3)2- In some embodiments, U is -(O)alkyl, for example, -OCH3.
In some embodiments,
In some embodiments, U is a saccharide. In certain such embodiments, the saccharide
OH
HO A OH is a glucuronide. In certain such embodiments, the glucuronide is 0 CO2H
In some embodiments, the compound is selected from:
In some embodiments, the compound is selected from:
In some embodiments, T is selected from:
In some embodiments, Y is selected from; wherein n represents an integer from 1 to 15.
In some embodiments, Y is selected from:
and pharmaceutically acceptable salt thereof. wherein n represents an integer from 1 to 15.
In some embodiments, the compound is: pharmaceutically acceptable salt thereof.
In some embodiments, the compound is selected from:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is selected from:
In some embodiments, the compound is selected from:
pharmaceutically acceptable salt thereof, wherein n represents an integer from 1 to 15; and each Ta and Tb is independently a moiety represented by formula (Ila).
In some embodiments, the compound is selected from:
wherein each Ta and Tb is independently a moiety represented by formula (Ila).
In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD- 1/PD- L1 pathway.
In certain such embodiments, the inhibitor of the PD-1/PD-L1 pathway is an antibody, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, acrixolimab, MGA012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, cosibelimab, or Clone 31E6. In certain such embodiments, the antibody is an anti-PD-1 antibody or antigen-binding fragment thereof, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, or AMP-514. In certain such embodiments, the antibody is an antibody in Table 1.
In certain such embodiments, the inhibitor of the PD-1/PD-L1 pathway is an anti-mouse PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-L1 antibody is 10F.9G2 or antigen-binding fragment thereof.
In certain such embodiments, the inhibitor of the PD-1/PD-L1 pathway is an anti-mouse PD-1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-1 antibody is RPM1-14 or antigen-binding fragment thereof.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6; or
(b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16; or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 24; or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29:
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30:
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31:
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34; or
(e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42; or
(f) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50; or
(g) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58; or
(h) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66; or
(i) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109;
(ii) CDRH2 comprises the ammo acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114; or
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of:
(a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18, or
(d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36, or
(f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44, or
(g) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52, or
(h) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60, or
(i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68, or
(j) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68.
In certain such embodiments, the antibody is an anti-PD-Ll antibody or antigenbinding fragment thereof, e.g., atezolizumab, avelumab, durvalumab, and cosibelimab. In certain such embodiments, the antibody is an antibody in Table 2.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74; or
(b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84; or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92; or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102; or
(e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102. In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of:
(a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94, or
(d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
In certain embodiments, the antibody is a monoclonal antibody, a single chain antibody (scAb), a Fab fragment, a F(ab’)2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a multimeric antibody, or a bispecific antibody.
In certain embodiments, the antibody is a chimeric, humanized or fully human monoclonal antibody.
In certain embodiments, the antibody is an IgG isotype (e.g., IgGl, IgG2, IgG3, or IgG4 isotype).
In certain embodiments, the antibody is an IgGl isotype.
In certain embodiments, the antibody comprises a N297A substitution according to EU numbering convention.
In certain embodiments, the antibody comprises E234A and E235A substitutions according to EU numbering convention.
In certain embodiments, the antibody comprises a P329G substitution or a P329A substitution according to EU numbering convention.
In certain embodiments, the antibody is an IgG4 isotype.
In certain embodiments, the antibody comprises a S228P substitution according to EU numbering convention.
In certain embodiments, the immune checkpoint inhibitor is selected from:
(1) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, or antigen-binding fragment thereof;
(2) an anti-PD-Ll antibody or antigen-binding fragment thereof, wherein the anti-PD-Ll antibody is selected from atezolizumab, Clone 31E6, or antigen-binding fragment thereof; or
(3) an anti-mouse PD-1 or PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-1 or PD-L1 antibody is selected from 10F.9G2, RPM1-14, or antigen-binding fragment thereof.
In certain embodiments, the compound is selected from: pharmaceutically acceptable salt thereof.
In certain embodiments, the method is a method of preventing or treating a proliferative disease, an infectious disease, an immune-mediated disorder, a central nervous system disease, a peripheral nervous system disease, a neurodegenerative disease, a cerebrovascular disease, a peripheral arterial disease, a cardiovascular disease, or an allergic disease.
In certain such embodiments, the proliferative disease is cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, or cirrhosis of the liver.
In certain such embodiments, the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
In other embodiments, the infectious disease is chickenpox, chikungunya, a coronavirus infection, a dengue virus infection, diphtheria, Ebola, influenza, hepatitis, Hib disease, acquired immunodeficiency syndrome (AIDS), a human papillomavirus (HPV) infection, encephalitis, measles, meningococcal disease, Mpox, mumps, a norovirus infection, pneumococcal disease, polio, rabies, respiratory syncytial virus (RSV) infection, rotavirus infection, rubella, shingles, tetanus, whooping cough, or zika virus disease.
In certain embodiments, the immune-mediated disorder is Crohn’s, ulcerative colitis, uveitis, psoriasis, lupus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, ankylosing spondylitis, hidrosadenitis suppurativa, sarcoidosis, atopic dermatitis, connective tissue disorders, asthma, or multiple sclerosis.
In certain embodiments, the central nervous system disease is catalepsy, encephalitis, epilepsy, meningitis, multiple sclerosis, or myelopathy.
In certain embodiments, the peripheral nervous system disease is lepromatous neuropathy, diabetic neuropathy, Guillain-Barre syndrome, acute motor axonal neuropathy, botulism, Lambert-Eaton syndrome, acute intermittent porphyria, or familial amyloid neuropathy.
In certain embodiments, the neurodegenerative disease is Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barre" syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson’s disease, traumatic brain injury (TBI), epilepsy, or Huntington’s disease (HD). In certain embodiments, the cerebrovascular disease is aneurysms, arteriovenous malformations (AVM), cerebral cavernous malformations (CCM), arteriovenous fistula (AVF), carotid-cavernous fistula, carotid stenosis, transient ischemic attack (TIA), or stroke.
In certain embodiments, the cardiovascular disease is arrhythmias, congenital heart disease, coronary artery disease, deep vein thrombosis, pulmonary embolism, heart attack, heart failure, cardiomyopathy, heart valve disease, pericardial disease, peripheral vascular disease, rheumatic heart disease, stroke, or vascular disease.
In certain embodiments, the allergic disease is an allergy, anaphylaxis, aspergillosis, asthma, chronic cough, chronic granulomatous disease, chronic sinusitis, Churg-Strauss syndrome, cold urticaria, common variable immunodeficiency, eosinophilia, eosinophilic esophagitis, esophagitis, hay fever, hypereosinophilic syndrome, nasal congestion, nasal polyps, nonallergic rhinitis, conjunctivitis, pneumonitis, primary immunodeficiency, selective IgA deficiency, systemic mastocytosis, or X-linked agammaglobulinemia.
In certain embodiments, the method is a method of inducing an immune response.
In certain aspects, the present disclosure relates to a pharmaceutical composition or a kit comprising the compound and the immune checkpoint inhibitor disclosed herein .
In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.
In some embodiments, the present disclosure relates to a method of preventing or treating a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof, the method comprising administering to the subject the compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.
In some embodiments, the present disclosure relates to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor for treating or preventing a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof.
In some embodiments, the present disclosure relates to the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor for use in treating or preventing a disease mediated by stimulator of interferon genes (STING) in a subject in need thereof.
In some embodiments, the disease mediated by STING is cancer.
In some embodiments, the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
In some embodiments, the present disclosure relates to a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor.
In some embodiments, the present disclosure relates to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof for inducing an immune response in a subject in need thereof conjointly with an immune checkpoint inhibitor.
In some embodiments, the present disclosure relates to the compound of formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof for use in inducing an immune response in a subject in need thereof conjointly with an immune checkpoint inhibitor.
In some embodiments, the inducing of the immune response is effective to prevent or treat a disease mediated by STING in the subject.
In some embodiments, the disease mediated by STING is cancer.
In some embodiments, the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
In some embodiments, the present disclosure relates to a method of modulating the activity of a STING adaptor protein comprising contacting the STING adaptor protein with the compound of Formula (I) or a pharmaceutically acceptable salt thereof conjointly with an immune checkpoint inhibitor. In some embodiments, the compound increases the activity of the STING adaptor protein.
In some embodiments, the STING-mediated disease is cancer, bacterial infection disease, viral infection disease, fungal infection disease, immune-mediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, cerebrovascular disease, peripheral Arterial disease, cardiovascular disease, allergic disease or inflammation. In specific embodiments, the STING-mediated disease is cancer or an infectious disease.
In some embodiments, the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, and osteosarcoma, Kaposi’ s sarcoma, and melanoma.
In some embodiments, the pharmaceutical composition may additionally contain a pharmaceutically effective amount of a chemotherapeutic agent.
In some embodiments, the pharmaceutical composition may include one or more therapeutic co-agents; and a pharmaceutically acceptable excipient may be additionally included.
In some embodiments, the therapeutic co-agent is an agent that exhibits a preventive, ameliorative, or therapeutic effect on STING-mediated diseases, or an agent that can reduce the expression of side effects that appear when administering a therapeutic agent for STING- mediated diseases, or it may be, but is not limited to, an agent that exhibits an immunity enhancing effect, and when applied in the form of a formulation together with the STING agent represented by the compound, it exhibits a therapeutically useful effect and/or improves the stability of the proteolytic agent, and/or reduce the side effects that may appear when administering the STING agonist represented by the compound, and/or, any agent that exhibits the effect of maximizing the therapeutic effect through the enhancement of immunity can be applied in combination.
In some embodiments, the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, It may be selected from the group consisting of Kaposi’s sarcoma and melanoma, but any carcinoma for which the STING agonist represented by the compound can exhibit a therapeutic effect can be applied. In some embodiments, there is provided a method for preventing or treating STING-mediated diseases, comprising administering the pharmaceutical composition to a patient. In addition, the compound of Formula 1 of the present invention can be used as an adjuvant for the treatment of other infectious diseases, diseases or disorders including cancer, in any one of its tautomers, stereoisomers and pharmaceutically usable salts.
Combination Therapy
In some embodiments, the compound disclosed herein, an optical isomer thereof, a stereoisomer thereof, a solvate thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising them may be used alone or in one or more additional therapies (e.g., drug treatment or treatment) can be used in combination with an immune checkpoint inhibitor.
Combination therapy may, for example, combine two therapies or may combine three therapies (e.g., a triple therapy of three therapeutic agents) or more. The dosage of one or more of the additional therapies (e.g., non-drug treatments or therapeutics) may be reduced from the standard dosage when administered alone.
In some embodiments, the compound, an optical isomer thereof, a stereoisomer thereof, a solvate thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same is administered before, after, or concurrently with one or more such additional therapies.
In some embodiments, the compound can be administered together, e.g, in a single pharmaceutical composition, or can be administered separately, and when administered separately, this can occur simultaneously or sequentially. Such sequential administration may be close in time or distant.
In some embodiments, the checkpoint inhibitor is an inhibitor of the PD-1/PD-L1 pathway (Ai L., et al. Drug Des Devel Ther. 2020; 14: 3625-3649).
Programmed cell death protein-1 (PD-1, Pdcdl), an inhibitory receptor in the immune response phase, is a member of the CD28/CTLA-4 family of immunoglobulin (1g) superfamily. PD-1 is a type I transmembrane protein with a size of 50-55 kDa, induced in a variety of hematopoietic cells in the peripheral blood and widely expressed in immune cells (T cells, B cells, macrophages, and certain types of dendritic cells, etc.) and tumor cells by antigen receptor signaling and cytokines. There are two main immunoregulatory ligands of PD-1, programmed cell death ligand 1 and 2 (PD-L1/PD-L2). PD-L1 is a type I transmembrane protein with a size of 40 kDa. It is widely expressed in both lymphoid tissue and non-lymphoid tissue, and in antigen-presenting cells (macrophages, dendritic cells, etc.) and all kinds of tumor cells. Both PD-1 and PD-L1 belong to the immune checkpoint protein family. As co-inhibitors, they can regulate the tolerance of central and peripheral T cells and reduce the proliferation of CD 8+ T cells in lymph nodes by combining and conducting inhibitory signals.
PD-1 and PD-L1 inhibitors are important immune checkpoint inhibitors (ICIs) for the treatment of cancer. Local immunosuppression could be eliminated by blocking the binding of PD-1 and PD-L1.
Under normal circumstances, the immune system produces an anti-cancer immune response by executing a cancer immunity cycle that kills cancer cells. And yet, the PD-l/PD- L1 pathway is an adaptive immune resistance mechanism of tumor cells to endogenous immune anti-tumor activity. 2 PD-l/PD-ligand interaction down-regulates the immune response during the regression of infection or tumor or the development of self-tolerance. PD-L1 is usually overexpressed in tumor cells or untransformed cells in tumor microenvironment and inhibits cytotoxic T cells by binding to PD- 1 receptor on activated T cells, resulting in immune escape. The inhibitors of PD-1 and PD-L1 inhibit the interaction between PD-L1 and PD-1 receptor, preventing cancer cells from evading the immune system in this way and acting as ICIs by reactivating the T-cell-mediated tumor cell death process.
Examples of checkpoint inhibitors for the PD-1/PD-L1 pathway, include anti-PD-1 antibodies (e.g., pembrolizumab, nivolumabcemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, and AMP-514) and anti-PD-Ll antibodies (e.g., atezolizumab, avelumab, durvalumab, and cosibelimab). The inhibitor of the PD-1/PD-L1 pathway may be an antibody, such as pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, acrixolimab, MGA012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, or cosibelimab. Numerous examples of anti-PD-Ll antibodies are disclosed herein and others are known in the art, such as U.S. Pat. Nos 9,567,399 and 9,617,338, both incorporated by reference herein.
Programmed cell death protein 1 (PD-1), (CD279 cluster of differentiation 279) is a protein encoded in humans by the PDCD1 gene. PD-1 is a cell surface receptor on T cells and B cells that has a role in regulating the immune system’s response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).
PD-1 inhibitors, a class of drugs that block PD-1, activate the immune system to attack tumors and are used to treat certain types of cancer. PD-1 binds two ligands, PD-L1 and PD- L2.
PD-L1, the ligand for PD1, is highly expressed in several cancers and hence the role of PD1 in cancer immune evasion is well established. Monoclonal antibodies targeting PD-1 that boost the immune system are developed for the treatment of cancer. Many tumor cells express PD-L1, an immunosuppressive PD-1 ligand; inhibition of the interaction between PD-1 and PD-L1 can enhance T-cell responses in vitro and mediate preclinical antitumor activity. This is known as immune checkpoint blockade.
A number of cancer immunotherapy agents that target the PD-1 receptor have been developed. One such anti-PD-1 antibody drug, nivolumab, (Opdivo - Bristol Myers Squibb), produced complete or partial responses in non-small-cell lung cancer, melanoma, and renalcell cancer. Pembrolizumab (Keytruda, MK-3475, Merck), which also targets PD-1 receptors, was approved to treat metastatic melanoma. It is being used in clinical trials in the US for lung cancer, lymphoma, and mesothelioma. Toripalimab is a humanized IgG4 monoclonal antibody against PD-1. Moreover, atezolizumab (MPDL3280A, Roche) and avelumab (Merck KGaA, Darmstadt, Germany and Pfizer) target the similar PD-L1 receptor.
The anti-PD-1 antibody may be pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, or AMP-514).
In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein,
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (11) CDRH2 comprises the ammo acid sequence of SEQ ID NO: 2;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6; or
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16; or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24; or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34; or
(e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42; or
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50; or
(g) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58; or
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66; or
(i) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114; or
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6. In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 34.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 37; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 38; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 39; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 40; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 41; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 45; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 47; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 48; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 49; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 50.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 53; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 55; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 56; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 57; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 58.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 61; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 63; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 64; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 65; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 66.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of:
(a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18, or
(d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36, or
(f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44, or
(g) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52, or
(h) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60, or
(i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68, or
(j) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60.
In certain such embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68.
Table 1. Anti-PD-1 antibody sequences
Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene. Programmed death-ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune systems during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis.
The anti-PD-Ll antibody may be atezolizumab, avelumab, durvalumab, or cosibelimab. In some embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein,
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74, or
(b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84, or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92, or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102; or
(e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and
(VI) CDRL3 comprises the ammo acid sequence of SEQ ID NO: 150. In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102.
In certain such embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
In some embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of: (a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94, or
(d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
103, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96.
In certain embodiments, the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103, and a variable light chain comprising the amino acid sequence of SEQ ID NO:
104.
Table 2. Anti-PD-Ll antibody sequences
In some embodiments, the antibody is a monoclonal antibody, a single chain antibody
(scAb), a Fab fragment, a F(ab’)2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a multimeric antibody, or a bispecific antibody. The antibody may be a chimeric, humanized or fully human monoclonal antibody. In some embodiments, the antibody is an IgG isotype, such as an IgGl isotype or IgG4 isotype. In certain embodiments, the antibody is an IgGl isotype. In certain embodiments, the antibody comprises a N297A substitution according to EU numbering convention. In certain embodiments, the antibody comprises L234A and L235A substitutions according to EU numbering convention. In certain embodiments, the antibody comprises a P329G substitution or a P329A substitution according to EU numbering convention. In certain embodiments, the antibody is an IgG4 isotype. In certain embodiments, the antibody comprises a S228P substitution according to EU numbering convention.
In some embodiments, the inhibitor of the PD-1/PD-L1 pathway is a small molecule. Such small molecules are described in U.S. Pat. App. Nos. 14/916,290, 17/041,455, 17/259,187, 17/260,547, 17/261,013, 16/963,557, 16/977,374, 15/957,739, 17/098,171, 16/274,106, 16/388,517, 16/388,517, 16/510,647, 17/264,638, and 17/264,657, and U.S. Pat. Nos. 10,568,874, 10,710,986, 11,555,029, 10,669,271, 11,124,511, 10,618,916, 10,906,920, and 11,414,433, all of which are incorporated fully herein by reference.
In some embodiments, the inhibitor of the PD-1/PD-L1 pathway is a peptide. Such peptides are described in U.S. Pat. App. No. 14/563,568, and U.S. Pat. Nos. 9,044,442, 8,907,053, 9,783,578, and 10,919,966 all of which are incorporated fully herein by reference. In some embodiments, the inhibitor of the PD-1/PD-L1 pathway is a macrocyclic compound. Such compounds are described in U.S. Pat. Nos. 10,143,746, 10,143,746, 9,861,680, 9,879,046, 9,308,236, 9,850,283, 9,944,678, 10,633,419, 9,856,292, 11,358,988, 11,066,445, 9,944,678, 10,538,555, 9,944,678, 10,358,463, 10,450,347, 10,988,507, 11,492,375, and 10,538,555, all of which are incorporated fully herein by reference.
The compounds according to the present disclosure may be used in combination with the agents described herein or other suitable agents depending on the condition being treated. Thus, in some embodiments one or more compounds of the present disclosure will be coadministered with other therapies as described herein. When used in combination therapy, the compounds described herein may be administered simultaneously or separately with the second agent. Such combined administration can include simultaneous administration of both agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any agent described herein may be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the present invention and any therapy described herein may be administered simultaneously, wherein the two agents are in separate preparations. In another alternative, a compound of the present disclosure may be administered followed by any therapy described herein, or vice versa. In some embodiments of separate administration protocols, the compounds of the present invention and any of the therapies described herein may be administered minutes apart, or hours apart, or days apart.
In some embodiments of any of the methods described herein, the first therapy (e.g., a compound of the invention) and one or more additional therapies may be administered in any order, simultaneously or sequentially.
The compound described above in the pharmaceutical composition of the present invention is contained in a therapeutically effective amount or a prophylactically effective amount. The preferred dosage of the compound according to the present invention varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the compound of Formula (I) of the present invention may be administered in an amount of 0.0001 to 1000 mg/kg, preferably 0.01 to 500 mg/kg, divided into once to several times a day.
In the composition described above, the compound of Formula (I) may be blended in an amount of 0.0001 to 50% by weight based on the total weight of the total composition. Pharmaceutical Compositions
The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.
A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.
To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
The phrases “parenteral administratio” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are also prepared by entapping the drug in liposomes or microemulsions that are compatible with body tissue.
For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient’s condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
In general, a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present disclosure, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
In certain embodiments, compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.
The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the disclosure include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoro acetic acid, and undecylenic acid acid salts.
The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Definitions
Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.
The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).
Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).
All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.
The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.
A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
“Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.
A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.
It is understood that substituent and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O- alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted. The term “alkyl” used herein refers to a linear or branched saturated monovalent hydrocarbon. For example, an alkyl group may have 1 to 10 carbon atoms (that is, (Ci-io)alkyl) or 1 to 8 carbon atoms (that is, (Ci-g)alkyl) or 1 to 6 carbon atoms (that is, ( Ci-6 alkyl) or 1 to 4 carbon atoms (that is, (Ci-4)alkyl). Examples of the alkyl group include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, - CH(CH3)2), 1 -butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl- 1-propyl (i-Bu, i-butyl, - CH2CH(CH3)2), 2 -butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3- pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-CXCFb^CFFCFb), 3-methyl-2-butyl (- CH(CH3)CH(CH3)2), 3-methyl-l -butyl (-CH2CH2CH(CH3)2), 2-methyl- 1 -butyl (- CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (- CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (- C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (- CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (- CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (- CH(CH3)C(CH3)3, and octyl (-(CFEhCFb), but are not limited thereto.
Furthermore, the term “alkyl” refers to saturated aliphatic groups, including straightchain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkylsubstituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer. In certain embodiments, alkyl is unsubstituted, except as otherwise specified. However, if not specified, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
The term “alkenyl” used herein refers to a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon double bond. For example, an alkenyl group may include 2 to 8 carbon atoms (that is, C2-8 alkenyl), or 2 to 6 carbon atoms (that is, C2-6 alkenyl), or 2 to 4 carbon atoms (that is, C2-4 alkenyl). Examples of the alkenyl group are ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2). 5-hexenyl (-CH2CH2CH2CH2CH=CH2). and 3- hexenyl (-CH2CH2CH=CHCH2CH2), and are not limited thereto. Throughout the present specification, one terminal hydrogen of the alkenyl group is omitted and may be connected with the next linking group. In certain embodiments, alkenyl is unsubstituted, except as otherwise specified.
The term “alkylene" used herein refers to a linear or branched divalent saturated hydrocarbon group having 1 to 6 (Ci-e) carbon atoms. For example, an alkylene having 1 to 4 (C1-4) carbon atoms may be used. Examples thereof include, but are not limited to, methylene, ethylene, trimethylene (propylene), and tetramethylene (n-butylene).
The term “alkynyl” used herein refers to a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon triple bond. For example, an alkynyl group may include 2 to 8 carbon atoms (that is, C2-8 alkynyl), or 2 to 6 carbon atoms (that is, C2-6 alkynyl), or 2 to 4 carbon atoms (that is, C2-4 alkynyl). Examples of alkynyl groups are acetylenyl (- C=CH), propargyl (-CH2C=CH), and -CH2 -C=C-CH3, but are not limited thereto. In certain embodiments, alkynyl is unsubstituted, except as otherwise specified.
The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.
The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.
The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.
Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
The term “Cx-y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci-6 alkyl group, for example, contains from one to six carbon atoms in the chain.
Affixing the suffix “ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, hetero alkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.
The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.
The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.
The term “amido”, as used herein, refers to a group wherein R9 and RIO each independently represent a hydrogen or hydrocarbyl group, or R9 and RIO taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R9, RIO, and RIO’ each independently represent a hydrogen or a hydrocarbyl group, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.
The term “aralkyl” or “arylalkyl”, as used herein, refers to an alkyl group substituted with an aryl group.
The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
The term “carbamate” is art-recognized and refers to a group wherein R9 and R10 independently represent hydrogen or a hydrocarbyl group.
The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.
The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In exemplary embodiments, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct- 3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH- indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom. The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.
The term “carbonate” is art -recognized and refers to a group -OCO2-.
The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.
The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like. Non-limiting examples of monocyclic cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, 1 -cyclopent- 3 -enyl, cyclohexyl, 1 -cyclohex- 1-enyl, l-cyclohex-2-enyl, and l-cyclohex-3- enyl.
The term “ester”, as used herein, refers to a group -C(O)OR9 wherein R9 represents a hydrocarbyl group.
The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.
The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.
The terms “heteroarylalkyl”, “hetaralkyl”, and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.
The term “heteroaryl” used herein refers to a single aromatic ring having at least one non-carbon atom in the ring, wherein the atom may be selected from oxygen, nitrogen, and sulfur, and “heteroaryl” may include a multiple condensed ring system having at least one such aromatic ring. The multiple condensed ring systems will be further described. Thus, the “heteroaryl” may include a single aromatic ring having about 1 to 6 carbon atoms and about 1- 4 heteroatoms selected from oxygen, nitrogen and sulfur. Sulfur and nitrogen atoms may also exist in oxidized form, provided that the ring is aromatic. An example of the heteroaryl ring systems includes, but are not limited to, pyridyl, pyrimidinyl, oxazolyl, or furyl. In some embodiments, “heteroaryl” includes a multiple condensed ring system (for example, a ring system including 2, 3 or 4 rings), and the heteroaryl group as defined above may form a multiple condensed ring system through condensation with at least one ring selected fromheteroaryl (used to form, for example, 1,8-naphthyridinyl), heterocycle (used to form, for example, l,2,3,4-tetrahydro-l,8-naphthyridinyl), carbocycle (used to form, for example, 5, 6, 7, 8 -tetrahydroquinolyl), and aryl (used to form, for example, indazolyl). Thus, a heteroaryl (a single aromatic ring or a multiple condensed ring system) may have about 1-20 carbon atoms and about 1-6 heteroatoms in the heteroaryl ring. Such multiple condensed ring systems may be such that the carbocycle or heterocycle portion of the condensed ring may be substituted with one or more (for example, 1, 2, 3, or 4) oxo groups. The rings of a multiple condensed ring system may be linked to one another through fusion, spiro and cross-linking bonds as long as valency requirements are satisfied. The individual rings of the multiple condensed ring system may be linked to one another in any order. The point of attachment for the heteroaryl or the heteroaryl multiple condensed ring system may be any suitable atom of the heteroaryl or the heteroaryl multiple condensed ring system, including carbon atoms and heteroatoms (for example, nitrogen). Also, when a particular atom-range member heteroaryl (for example, (Cs- Cio) heteroaryl) is referred to, the atomic range is to be understood as being relative to the total number of ring atoms of the heteroaryl and as including a carbon atom and a heteroatom. For example, a Cs heteroaryl may include a thiazolyl and a Cio heteroaryl may include a quinolinyl. Examples of heteroaryls include pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, 5,6,7,8-tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3-Z?]pyridinyl, quinazolinyl-4(3/7)-one, triazolyl, 4,5,6,7-tetrahydro- 1/7-indazole, and 3b, 4,4a,5-tetrahydro-177-cyclopropa[3,4]cyclopenta[l,2-c]pyrazole, and are not limited thereto.
The term “heterocyclyl” or “heterocycle” used herein refers to a monosaturated or partially unsaturated non-aromatic compound or non-aromatic multi-ring system in which at least one heteroatom (that is, at least one cyclic heteroatom selected from oxygen, nitrogen and sulfur) is included in the ring. Unless otherwise specified, heterocyclyl groups have 5 to about 20 ring atoms, such as 3 to 12 ring atoms, such as 5 to 10 ring atoms. Thus, the term includes a single saturated or partially unsaturated ring (for example, 3, 4, 5, 6 or 7-membered rings), having about 1 to 6 cyclic carbon atoms and about 1 to 3 cyclic heteroatoms selected from oxygen, nitrogen and sulfur, in the ring. The rings of a multiple condensed ring system may be linked to one another through fusion, spiro and cross-linking bonds as long as valency requirements are satisfied. Examples of heterocycles include azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidinone, tetrahydrofuran, tetrahydro thiophene, dihydropyridine, tetrahydropyridine, quinuclidine, N-bromopyrrolidine, N-chloropiperidine, and the like.
The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.
The term “hydroxy alkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.
The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).
The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7. The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.
The term “sulfonamido” is art-recognized and refers to the group represented by the general formulae wherein R9 and R10 independently represents hydrogen or hydrocarbyl.
The term “sulfoxide” is art-recognized and refers to the group-S(O)-.
The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.
The term “sulfone” is art -recognized and refers to the group -S(O)2-.
The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.
The term “thioester”, as used herein, refers to a group -C(O)SR9 or -SC(O)R9 wherein R9 represents a hydrocarbyl.
The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.
The term “urea” is art -recognized and may be represented by the general formula wherein R9 and R10 independently represent hydrogen or a hydrocarbyl.
The term “azido” is art-recognized and may be represented by the general formula
AN3
The term “hydrazido” is art-recognized and may be represented by the general formula 0 alkyl < yNy linking group H ), wherein R11 and R12 independently represent hydrogen or a hydrocarbyl.
The term “guanidino” is art-recognized and may be represented by the general formula wherein R13 and R14 independently represent hydrogen or a hydrocarbyl.
The term “DBCO” used herein refers to an optionally substituted dibenzocyclooctyne moiety, e.g., the following structure:
The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.
The term “PD-L1” or “PDL1” generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which downregulates T cell activation and cytokine secretion after binding to PD-1. “PD-L1” includes any native PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term covers “full-length,” unprocessed PD-L1 as well as any form of PD-L1 produced by cell processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. “PD-L1” includes complete PD-L1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs having at least one common epitope with PD-L1. The basic structure of PD-L1 includes 4 domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. An exemplary human PD-L1 amino acid sequence can be found under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7.
The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
“Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non- pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.
Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.
The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.
The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol/liter.
The term “cleavage group” refers to a chemical moiety which dissociates when subjected to a stimulus, such as acidic conditions, basic conditions, reducing conditions, oxidizing conditions, light, or heat, or an enzyme, such as an esterase.
The term “identity” refers to the similarity or relatedness of two or more polypeptide or nucleic acid sequences determined by aligning and comparing the sequences of two or more polypeptides or nucleic acids. Such inter-sequence identity is typically expressed as “percentage identity,” representing the proportion of identical amino acids or nucleotides between the compared molecules, calculated based on the smallest size molecule among the compared molecules. Methods for aligning nucleic acids or polypeptides to calculate identity between them are known in the art and may also be referenced herein.
The term “affinity” or “avidity” refers to the strength of interaction between an antibody or its antigen-binding fragment and an antigen, determined by characteristics of the antigen such as size, shape, and/or charge, and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining such affinities are known in the art and may also be referenced herein.
Antibodies or their antigen-binding fragments used in the present invention are said to “specifically bind” to the target, such as the antigen, when the dissociation constant (KD) is <10-6 M. Antibodies bind “with high affinity” to the target when KD is <lx 10’8 M.
The term “antigen-binding fragment” of an antibody or immunoglobulin chain (light or heavy) used in the present invention refers to a portion of the antibody that includes some amino acids missing compared to the full-length chain but includes a portion of the antibody that can specifically bind to the target antigen. These fragments can be biologically active in terms of being able to specifically bind to target antigens or compete with other antibodies or antigen-binding fragments for binding to specific epitopes. In some embodiments, such fragments comprise at least one CDR in a full-length light chain or heavy chain, and comprise short heavy chain and/or light chains, or portions thereof in some embodiments. These biologically active fragments may be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFV, scFV-Fc, diabody, minibody, scAb, and dAb and may be derived from any mammal including human, mouse, rat, camelid or rabbit. In this invention, functional portion of the antibody, such as one or more CDRs can be covalently linked to a second protein or small molecule compound and used as a targeted therapeutic agent for a specific target.
The “Fc” region is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions and in this invention includes two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. These two heavy chain fragments are linked to each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The Cterminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4.
The “Fab fragment” in this invention consists of one light chain and one heavy chain containing only the variable region and CHI. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules. scFab is Fab of two molecules linked by a flexible linker.
The “Fab’ fragment” in this invention includes a region between the CHI and CH2 domains of the heavy chain in addition to the Fab fragment, which forms an interchain disulfide bond between the two heavy chains of the two molecules of the Fab’ fragment, thereby forming F (ab ’)2 molecules.
As described above, the “F(ab’)2 fragment” in this invention includes two light chains and two heavy chains including a variable region, CHI, and a portion of the constant region between the CHI and CH2 domains, which form 2 interchain disulfide bonds are between the heavy chains. Therefore, the F(ab’)2 fragment is composed of two Fab’ fragments, and the two Fab’ fragments are associated with each other by a disulfide bond between them.
The “Fv region” in this invention is a fragment of an antibody that includes the variable regions of the heavy chain and light chains, but does not include the constant region. sdFV is where heavy chains and light chains are linked by disulfide bonds. scFv is Fv linked by a flexible linker. scFv-Fc is where Fc is linked to scFV. The minibody is where CH3 is linked to scFV. The diabody contains scFV of two molecules.
A “single chain Fv” or “scFv” antibody fragment in this invention comprises the VH and VE domains of an antibody, and these domains are in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.
A “single chain antibody” (scAb) in this invention is a single polypeptide chain containing one constant region of a heavy chain or a light chain constant region where the heavy chain and light chain variable regions are linked by a flexible linker. For single chain antibodies, see, for example, U.S. Pat. No. 5,260,203, which is disclosed in the present invention for reference.
A “domain antibody” (dAb) in this invention is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. For example, in one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of these bivalent domain antibodies can target the same or different antigens.
As used herein, “complementarity determining region” (CDR; i.e., CDR1, CDR2, and CDR3) refers to amino acid moieties of an antibody variable domain that are required for antigen binding. Generally, antibodies comprise six CDRS; three in the VH (Hl CDR, H2 CDR, H3 CDR), and three in the VL (LI CDR, L2 CDR, L3 CDR). In native antibodies, H3 and L3 display the most diversity of the six CDRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
As used herein, “framework region” (FR) is variable domain moieties other than CDR moieties. Each variable domain typically has four FRs, identified as FR1, FR2, FR3 and FR4.
As used herein, a “bivalent antigen binding protein” or “bivalent antibody” includes two antigen binding sites. The two antigen binding sites included in such a bivalent antibody may have the same antigen specificity, or may be bispecific antibodies that each bind to different antigens.
As used herein, “multispecific antigen binding protein” or “multispecific antibody” targets two or more antigens or epitopes.
As used herein, a “chimeric antibody” refers to an antibody (immunoglobulin) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat’l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
“Humanized” forms of non-human (e.g., murine) antibodies, are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428- 433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.
A “human antibody” is one that possesses an amino-acid sequence corresponding to that of an antibody, produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phagedisplay libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Nat’l Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.
An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In one embodiment, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH- and VL- domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147- 155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
The term “conjugates" as used herein refers to cell binding agents that are covalently bonded to one or more molecules of a cytotoxic compound. In this regard, "cell binding agent" is a molecule having affinity for a biological target, and may be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and the binding agent functions to direct a biologically active compound to a biological target. In certain embodiments of the present disclosure, the conjugate may be designed to target tumor cells through cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen may be expressed only on proliferative cells (e.g., tumor cells). The target antigen may be selected on the basis of different expression, usually between proliferative tissues and normal tissues.
Examples
The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.
Preparation of Compounds
Preparation Example 1: Preparation of Compound 2
Preparation of Compound 1
After dissolving I -cthyl-3-mcthyl- 17/-pyiazolc-5 -carboxylic acid (8 g, 51.9 mmol) in dichloromethane (50 mL), oxalyl chloride (4.97 mL, 57.1 mmol) and A,A-dimethylformamide (0.1 mL) were added at 0 °C, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to afford Compound 1.
Preparation of Compound 2
After dissolving Compound 1 (crude) in acetone (100 mL), potassium thiocyanate (6.5 g, 67.5 mmol) was added at 0 °C. After stirring the reaction solution at room temperature for 30 minutes, hexane (100 mL) was added and the formed solid was filtered. The filtered solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 2 (9.7 g, 95%).
’H-NMR (400 MHz, CDC13), δ 7.78 (d, 1H), 7.41 (s, 1H), 7.38 (d, 1H), 3.88 (s, 3H), 2.57 (s, 3H). ELMS m/z : [M+H]+ 196.00. Preparation Example 2: Preparation of Compound 5
Preparation of Compound 3
To methyl 4-chloro-3-methoxy-5-nitrobenzoate (15 g, 61.1 mmol), aqueous ammonia solution (28-30% ammonia, 200 mL) was added. The reaction solution was stirred at 50 °C for 6 hours, cooled to room temperature, and then washed with water, filtered, and lyophilized to afford Compound 3 (9.51 g, 68%).
’H-NMR (400 MHz, CDCI3) δ 8.29 (s, 1H), 8.04 (d, 1H), 7.87 (d, 1H), 7.78 (s, 1H), 4.01 (s, 3H).
Preparation of Compound 4
Compound 3 (300 mg, 1.30 mmol) was added to dichloromethane (9 mL), and then aluminum chloride (1.04 g, 7.81 mmol) was added at 0 °C. The reaction solution was stirred for 21 hours at room temperature under nitrogen. The reaction solution was added to ice water, and then the resulting solid was filtered and lyophilized to afford Compound 4 (223 mg, 79%).
’H-NMR (400 MHz, CDC13), δ 11.73 (s, 1H), 8.21 (s, 1H), 7.92 (s, 1H), 7.80 (s, 1H), 7.66 (s, 1H).
Preparation of Compound 5
Compound 4 (2 g, 9.23 mmol) and cesium carbonate (3.61 g, 11.08 mmol) were added to MAMimcthylformamidc (15 mL) at 0 °C under nitrogen and then stirred for 5 minutes. Trans- 1 ,4-dibromo-2-butene (5.93 g, 27.70 mmol) was added to the reaction solution at room temperature under nitrogen and stirred for 2 hours. The resulting solution was extracted with ethyl acetate (20 mL x 3) and washed with distilled water (15 mL x 2) and brine (15 mL). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated. After solidification using dichloromethane and hexane, it was filtered and dried to afford Compound 5 (2.53 g, 78%).
’H-NMR (400 MHz, CDCI3), δ 8.23 (s, 2H), 8.02 (d, 7 = 1.8 Hz, 2H), 7.84 (d, 7 = 1.8 Hz, 2H), 7.73 (s, 2H), 6.16-5.98 (m, 4H), 5.70 (s, 1H), 4.83 (d, J = 3.5 Hz, 4H), 4.25 (d, J = 5.4 Hz, 1H), 4.20-4.14 (m, 3H). EI-MS m/z : [M+H]+ 350.98. Preparation Example 3: Preparation of Compound 9
Preparation of Compound 6
After dissolving 4-chloro-3-methoxy-5-nitro-benzamide (11.4 g, 61.57 mmol) in ethanol (100 mL), Compound 3 (10 g, 43.4 mmol) and A,A-diisopropylethylamine (14.9 mL, 86.7 mmol) were added and stirred at 120 °C for 12 hours. The reaction solution was concentrated and diluted with diethyl ether (40 mL), and then the resulting solid was filtered and dried to afford Compound 6 (12.6 g, 76%).
' H-NMR (400 MHz, DMSO-d6), δ 8.18 (d, 1H), 8.01(s, 1H), 7.73 (t, 1H), 7.55 (d, 1H), 7.31 (s, 1H), 6.92 (s, 1H), 5.53 (s, 2H), 4.08 (s, 2H), 3.47 (s, 2H), 1.35 (m, 9H).
Preparation of Compound 7
After dissolving Compound 6 (10 g, 26.28 mmol) in methanol (90 mL), ammonia aqueous solution (28-30% ammonia, 90 mL) and sodium hydrosulfite (Na2S2C>4, 45 g, 262.8 mmol) were sequentially added at 0 °C, and the mixture was stirred at room temperature for 1 hour and 30 minutes. After adding methanol (100 mL) to the reaction solution, the resulting solid was filtered, the filtered solution was concentrated, and then diluted with dichloromethane (100 mL) and washed with distilled water (50 mL), and the organic layer was dried over anhydrous sodium sulfate. After filtration, it was concentrated under reduced pressure to afford Compound 7 (6.9 g, 75%).
1H-NMR(400 MHz, DMSO-d6), δ 7.62 (br s, 1H), 6.98 (br s, 1H), 6.92 (t, 1H), 6.87 (d, 1H), 6.79 (d, 1H), 5.57 (q, 2H), 4.67 (br s, 2H), 3.82 (br s, 1H), 3.76 (s, 3H), 3.51 (dd, 4H), 1.37 (s, 9H).
Preparation of Compound 8
After dissolving Compound 7 (6.9 g, 19.7 mmol) in A,A-dimethylformamide (50 mL), Compound 2 (4.9 g, 25.59 mmol) was added at 0 °C and stirred at room temperature for 30 minutes. Triethylamine (5.4 mL, 39.38 mmol) and A-(3-dimethylaminopropyl)-A'- ethylcarbodiimide (4.2 g, 27.56 mmol) were added at 0 °C, and the mixture was stirred at room temperature for 17 hours. After concentrating the reaction solution, it was diluted with diethyl ether (20 mL) and the resulting solid was filtered to afford Compound 8 (7.7 g, 76%).
’H-NMR (400 MHz, DMSO-d6), δ 12.86 (s, 1H), 8.02 (s, 1H), 7.67 (s, 1H), 7.40 (m, 2H), 6.93 (m, 1H), 6.65 (s, 1H), 5.67 (m, 2H), 4.93 (d, 2H), 4.61 (q, 2H), 3.98 (s, 3H), 3.51 (m, 2H), 2.55 (m, 2H), 2.18 (s, 3H), 1.35 (t, 3H), 1.32 (s, 9H).
Preparation of Compound 9
After dissolving Compound 8 (7.7 g, 15.05 mmol) in dichloromethane (25 mL) and methanol (25 mL), hydrochloric acid (4 M 1,4-dioxane solution, 27 mL) was added and stirred for 2 hours and 30 minutes. The reaction solution was concentrated, diluted with diethyl ether (20 mL), and then the resulting solid was filtered to afford Compound 9 (5.6 g, 76%).
’H-NMR (400 MHz, DMSO-d6), δ 8.02 (s, 1H), 8.67(d, 1H), 7.41 (d, 1H), 7.37 (s, 1H), 6.66 (s, 1H), 6.01 (m, 1H), 5.65 (m, 1H), 4.97 (d, 2H), 4.60 (q, 2H), 3.98 (s, 3H), 2.66 (m, 5H), 2.33 (m, 3H), 1.35 (m, 3H). ELMS m/z : [M+H]+ 823.0.
Preparation of Compound 10
After dissolving Compound 5 (580 mg, 1.66 mmol) in A,A-dimethylformamide (5 mL), morpholine (0.13 mL, 1.01 mmol) and cesium carbonate (590 mg, 1.81 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and dried with anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, diluted with dichloromethane and hexane, and the resulting solid was filtered and dried to afford Compound 10 (504 mg, 70%).
’H-NMR (400 MHz, CDC13), δ 7.73-7.66 (m,lH), 6.03-5.84 (m,lH), 4.75 (dd, 7 = 5.0,
1.2 Hz, 1H), 3.75-3.68 (m, 2H), 3.06 (dd, 7 = 6.0, 1.1 Hz, 1H), 2.46 (t, 7 = 4.7 Hz, 2H). EI-MS m/z : [M+H]+ 356.09.
Preparation of Compound 11 After dissolving Compound 9 (818 mg, 1.68 mmol) and Compound 10 (300 mg, 0.84 mmol) in normal butyl alcohol (13 mL), N, A-diisopropylethylamine (0.74 mL, 4.21 mmol) was added at room temperature, heated to 120 °C, and stirred for 24 hours. After cooling the reaction solution to room temperature, the reaction solution was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layers were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. After dilution with diethyl ether, the resulting solid was filtered and dried to afford Compound 11 (129 mg, 21%). EI-MS m/z : [M+H]+ 731.03.
Preparation of Compound 12
After dissolving Compound 11 (129 mg, 0.17 mmol) in methyl alcohol (2 mL) and distilled water (0.1 mL), aqueous ammonia solution (28-30% ammonia, 0.17 mL) and sodium hydrosulfite (Na2S2C>4, 321 mg, 1.84 mmol) were added to the reaction solution under nitrogen. After stirring at room temperature for 1 hour, methanol (50 mL) was added to the reaction solution, and the resulting solid was filtered, and the filtered solution was concentrated, and then diluted with dichloromethane (100 mL), washed with distilled water (50 mL), and then the organic layer was dried with anhydrous sodium sulfate. After filtration, it was concentrated under reduced pressure to afford Compound 12 (126 mg, crude). EI-MS m/z : [M+H]+ 701.08.
Preparation of Compound 13
After dissolving Compound 12 (126 mg, 0.18 mmol, crude) in N,N- dimethylformamide (2 mL), Compound 2 (39 mg, 0.2 mmol) was dissolved in N,N- dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (40 mg, 0.21 mmol) and triethylamine (0.04 mL, 0.27 mmol) were added, and stirred at room temperature for 16 hours. The resulting material was concentrated under reduced pressure and purified by HPLC to afford Compound 13 (5.6 mg, 4%). EI-MS m/z : [M+H]+ 862.03.
Example 2: Preparation of Compound 18
Preparation of Compound 14
After dissolving Compound 5 (265 mg, 0.76 mmol) in A,A-dimethylformamide (4 mL), t-butyl piperidm-4-ylcarbamate (167 mg, 0.83 mmol) and cesium carbonate (296 mg, 0.91 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and dried with anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, diluted with dichloromethane and hexane, and the resulting solid was filtered and dried to afford Compound 14 (248 mg, 70%). EI-MS m/z : [M+H]+ 469.06.
Preparation of Compound 15
After dissolving Compound 9 (628 mg, 1.3 mmol) and Compound 14 (304 mg, 0.65 mmol) in normal butyl alcohol (5 mL), diisopropylethylamine (0.56 mL, 3.24 mmol) was added at room temperature, heated to 120 °C and stirred for 24 hours. After cooling the reaction solution to room temperature, the reaction solution was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The reaction solution was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, diluted with dichloromethane and hexane, and the resulting solid was filtered and dried to afford Compound 15 (124 mg, 23%). EI-MS m/z : [M+H]+ 844.02.
Preparation of Compound 16
After dissolving Compound 15 (124 mg, 0.15 mmol) in methanol (4 mL) and distilled water (0.5 mL), an aqueous ammonia solution (28-30% ammonia, 0.4 mL) and sodium hydrosulfite (Na2S2C>4, 218 mg, 2.94 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour, diluted with methanol (50 mL), and filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and washed with distilled water (50 mL). The washed material was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 16 (119 mg, crude). EI-MS m/z : [M+H]+ 814.81.
Preparation of Compound 17
After dissolving Compound 16 (119 mg, 0.15 mmol, crude) in N,N- dimethylformamide (1 mL), Compound 2 (32 mg, 0.16 mmol) was dissolved in N,N- dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (35 mg, 0.18 mmol) and triethylamine (0.06 mL, 0.44 mmol) were added, and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 17 (36 mg, 25%). EI-MS m/z : [M+H]+ 975.15.
Preparation of Compound 18
After dissolving Compound 17 (36 mg) in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 2 hours, concentrated, and then purified by HPLC to afford Compound 18 (15.2 mg, 47%). EI-MS m/z : [M+H]+ 875.06.
Example 3: Preparation of Compound 23
Preparation of Compound 19
After dissolving Compound 5 (500 mg, 1.43 mmol) in A,A-dimethylformamide (5 mL), t-butyl piperazine- 1 -carboxylate (320 mg, 1.71 mmol) and cesium carbonate (512 mg, 1.57 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and dried with anhydrous sodium sulfate. The reaction solution was filtered, concentrated under reduced pressure, diluted with dichloromethane and hexane, and the resulting solid was filtered and dried to afford Compound 19 (472 mg, 72%).
’H-NMR (400 MHz, DMSO-d6), δ 8.26 (s, 1H), 8.06 (s, 1H), 7.88 (s, 1H), 7.78 (s, 1H), 5.91-5.83 (m, 2H), 4.84 (d, 1H), 3.29-3.27 (m, 4H), 2.98 (d, 2H), 2.73 (t, 4H), 1.39 (s, 9H). EIMS m/z : [M+H]+ 455.11.
Preparation of Compound 20 After dissolving Compound 9 (495 mg, 1.02 mmol) and Compound 19 (310 mg, 0.68 mmol) in normal butyl alcohol (7 mL), diisopropylethylamine (0.59 mL, 3.41 mmol) was added at room temperature, heated to 120 °C and stirred for 24 hours. The reaction solution was cooled to room temperature, diluted with dichloromethane (100 mL) and methanol (20 mL), and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 20 (336 mg, 46%). EI-MS m/z : [M+H]+ 830.01.
Preparation of Compound 21
After dissolving Compound 20 (336 mg, 0.31 mmol) in methanol (10 mL) and distilled water (1 mL), an aqueous ammonia solution (28-30% ammonia, 0.33 mL) and sodium hydrosulfite (Na2S2C>4, 549 mg, 3.16 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour, diluted with methanol (50 mL), and then filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and then washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to afford Compound 21 (153 mg, 60%, crude). EI-MS m/z : [M+H]+ 800.09.
Preparation of Compound 22
After dissolving Compound 21 (153 mg, 0.19 mmol, crude) in N,N- dimethylformamide (2 mL), Compound 2 (45 mg, 0.23 mmol) was dissolved in N,N- dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (192 mg, 0.28 mmol) and triethylamine (0.1 mL, 0.76 mmol) were added, and stirred at room temperature for 13 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 22 (116 mg, 63%). EI-MS m/z : [M+H]+ 961.07.
Preparation of Compound 23
After dissolving Compound 22 (41 mg) in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 2 hours, concentrated, and then purified by HPLC to afford Compound 23 (26 mg, 46%).
’H-NMR (400 MHz, DMSO-d6), δ 8.66 (br s, 1H), 7.96 (d, 2H), 7.65 (s, 2H), 7.37 (br s, 2H), 7.30 (d, 2H), 6.53 (d, 2H), 5.86-5.63 (m, 4H), 4.93-4.89 (m, 4H), 4.56-4.50 (m, 6H), 3.71 (s, 3H), 2.11 (d, 6H) 1.30-1.25 (m, 6H). EI-MS m/z : [M+H]+ 861.21. Example 4: Preparation of Compound 28
Preparation of Compound 24
/-Butyl 3 -oxopiperazine- 1 -carboxylate (300 mg, 0.86 mmol) was dissolved in tetrahydrofuran (4 mL), and potassium hydroxide (57.7 mg, 1.02 mmol) and TBAB (tetrabutylammonium bromide, 55.3 mg, 0.17 mmol) were sequentially added, and then stirred at room temperature for 30 minutes. Compound 5 (300 mg, 0.858 mmol) was dissolved in THF (2 mL), and then slowly added to the reaction solution, and stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with distilled water (30 mL), and then dried with anhydrous magnesium sulfate. The reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography to afford Compound 24 (213 mg, 52.9%).
’H-NMR (400 MHz, CDC13), δ 8.26 (br s, 1H), 8.06 (s, 1H), 7.87 (s, 1H), 7.78 (s, 1H), 5.90-5.80 (m, 2H), 4.84 (d, J = 4.4 Hz, 1H), 4.00 (d, J = 4.8 Hz, 2H), 3.92 (s, 2H), 3.53-3.51 (m, 2H), 3.27-3.24 (m, 2H), 1.41 (s, 9H). EI-MS m/z : [M+H]+ 469.07.
Preparation of Compound 25
After dissolving Compound 9 (440 mg, 0.91 mmol) and Compound 24 (213 mg, 0.45 mmol) in normal butyl alcohol (4.5 mL), diisopropylethylamine (0.43 mL, 2.49 mmol) was added at room temperature and stirred for 24 hours while heating to 120 °C. The reaction solution was cooled to room temperature and then diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 25 (213 mg, 55.5%). ELMS m/z : [M+H]+ 844.04.
Preparation of Compound 26
After dissolving Compound 25 (210 mg, 0.25 mmol) in methanol (6 mL) and distilled water (1 mL), an aqueous ammonia solution (28-30%, 0.44 mL) and sodium hydrosulfite (Na2S2C>4, 433 mg, 2.48 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour and 45 minutes, diluted with methanol (50 mL), and then filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and then washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 26 (crude). ELMS m/z : [M+H]+ 814.14.
Preparation of Compound 27
After dissolving Compound 26 (0.24 mmol, crude) in A,A-dimethylformamide (2.5 mL), Compound 2 (50.9 mg, 0.26 mmol) was dissolved in A,A-dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, N-(3- dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (68.1 mg, 0.35 mmol) and triethylamine (0.1 mL, 0.71 mmol) were added and stirred at room temperature for 18 hours and 30 minutes. The reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 27 (48 mg, 20%). ELMS m/z : [M+H]+ 975.19.
Preparation of Compound 28
After dissolving Compound 27 (48 mg) in dichloromethane (3.2 mL), trifluoroacetic acid (0.8 mL) was added at 0 °C under nitrogen. The reaction solution was stirred at 0 °C for 40 minutes, concentrated, and then purified by HPLC to afford Compound 28 (10.2 mg, 17%).
’H-NMR (400 MHz, DMSO-d6), δ 1.28 (br s, 2H), 9.13 (br s, 1H), 7.96 (d, J = 14.7 Hz, 2H), 7.65 (s, 2H), 7.37 (br s, 2H), 7.30 (s, 2H), 6.53 (d, J = 6.3 Hz, 2H), 5.86-5.63 (m, 4H), 4.92-4.89 (m, 4H), 4.56-4.50 (m, 4H), 3.31 (s, 5H), 2.11 (d, 7 = 4.9 Hz, 6H) 1.29-1.25 (m, 6H). ELMS m/z : [M+H]+ 875.11. Example 5: Preparation of Compound 33
Preparation of Compound 29
Compound 4 (1.5 g, 6.93 mmol) and cesium carbonate (2.5 g, 7.62 mmol) were dissolved in M A-dimcthylformamidc (6 mL) at 0 °C under nitrogen, and then stirred for 5 minutes. Cis-l,4-dibromo-2-butene (3.7 g, 17.32 mmol) was added to the reaction solution at room temperature under nitrogen and stirred for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (20 mL x 2) and brine (20 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and concentrated. The solid obtained by diluting with dichloromethane and hexane was filtered and dried to afford Compound 29 (1.64 g, 67%). EI-MS m/z : [M+H]+ 351.23.
Preparation of Compound 30
After dissolving Compound 29 (450 mg, 1.29 mmol) in A,A-dimethylformamide (5 mL), morpholine (0.1 mL, 1.17 mmol) and cesium carbonate (417 mg, 1.28 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and then dried with anhydrous sodium sulfate. The reaction solution was filtered and concentrated under reduced pressure, and the solid obtained by diluting with dichloromethane and hexane was filtered and dried to afford Compound 30 (298 mg, 71%). EI-MS m/z : [M+H]+ 356.11.
Preparation of Compound 31 After dissolving Compound 9 (592 mg, 1.22 mmol) and Compound 30 (290 mg, 0.82 mmol) in normal butyl alcohol (6 mL), diisopropylethylamine (0.71 mL, 4.08 mmol) was added at room temperature and stirred for 24 hours while heating to 120 °C. The reaction solution was cooled to room temperature, and then diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The reaction solution was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 31 (174 mg, 29%). EI-MS m/z : [M+H]+ 731.05.
Preparation of Compound 32
After dissolving Compound 31 (173 mg, 0.24 mmol) in methanol (2 mL) and distilled water (0.1 mL), an aqueous ammonia solution (28-30%, 0.34 mL, 4.76 mmol) and sodium hydrosulfite (Na2S2C>4, 412 mg, 2.37 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour, diluted with methanol (50 mL), and filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and then washed with distilled water (50 mL). The washed material was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to afford Compound 32 (48 mg, crude, 29%). EI-MS m/z : [M+H]+ 701.22.
Preparation of Compound 33
After dissolving Compound 32 (48 mg, 0.07 mmol, crude) in A,A-dimethylformamide (1 mL), Compound 2 (16 mg, 0.08 mmol) was dissolved in A,A-dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, N-(3- dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (16 mg, 0.09 mmol) and triethylamine (0.03 mL, 0.2 mmol) were added, and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and purified by HPLC to afford Compound 33 (17.5 mg, 30%). EI-MS m/z : [M+H]+ 862.08.
Preparation of Compound 34
After dissolving Compound 29 (649 mg, 1.86 mmol) in A,A-dimethylformamide (6 mL), t-butyl piperidin-4-ylcarbamate (338 mg, 1.69 mmol) and cesium carbonate (660 mg, 2.03 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and then dried with anhydrous sodium sulfate. The obtained material was filtered and concentrated under reduced pressure, and the solid obtained by diluting with dichloromethane and hexane was filtered and dried to afford Compound 34 (628 mg, 79%).
’H-NMR (400 MHz, DMSO), δ 8.21 (s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 7.73 (s, 1H), 6.16 - 6.01 (m, 2H), 4.82 (d, J = 3.5 Hz, 2H), 4.17 (d, J = 5.5 Hz, 2H). ELMS m/z : [M+H]+ 469.49.
Preparation of Compound 35 After dissolving Compound 9 (413 mg, 0.85 mmol) and Compound 34 (600 mg, 1.28 mmol) in normal butyl alcohol (8 mL), diisopropylethylamine (0.74 mL, 4.27 mmol) was added at room temperature and stirred for 24 hours while heating to 120 °C. The reaction solution was cooled to room temperature, and then diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The washed material was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 35 (147 mg, 20%). EI-MS m/z : [M+H]+ 844.13.
Preparation of Compound 36
After dissolving Compound 35 (147 mg, 0.17 mmol) in methanol (3 mL) and distilled water (0.1 mL), an aqueous ammonia solution (28-30%, 0.25 mL) and sodium hydrosulfite (Na2S2C>4, 303 mg, 1.74 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour, diluted with methanol (50 mL), and then filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and then washed with distilled water (50 mL). The washed material was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to afford Compound 36 (141 mg, crude). EI-MS m/z : [M+H]+ 814.81.
Preparation of Compound 37
After dissolving Compound 36 (141 mg, 0.17 mmol, crude) in N,N- dimethylformamide (1 mL), Compound 2 (37 mg, 0.19 mmol) was dissolved in N,N- dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (50 mg, 0.09 mmol) and triethylamine (0.03 mL, 0.22 mmol) were added and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 37 (48 mg, 28%). EI-MS m/z : [M+H]+ 975.16.
Preparation of Compound 38
After dissolving Compound 37 (48 mg) in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 2 hours, concentrated, and then purified by HPLC to afford Compound 38 (6.4 mg, 14%). EI-MS m/z : [M+H]+ 875.17. Example 7: Preparation of Compound 43
Preparation of Compound 39
/-Butyl 3 -oxopiperazine- 1 -carboxylate (210 mg, 1.05 mmol) was dissolved in N,N- dimethylformamide (6 mL), and potassium hydroxide (66.2 mg, 1.02 mmol) was added and then stirred at room temperature for 30 minutes. After dissolving Compound 29 (350 mg, 1.20 mmol) in A A-di methyl form am ide (4 mL), it was slowly added to the reaction solution and stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with distilled water (30 mL), and then dried with anhydrous magnesium sulfate. The dried material was filtered, then concentrated under reduced pressure, and purified by column chromatography to afford Compound 39 (432 mg, 92.0%).
’H-NMR (400 MHz, CDC13), δ 7.80 (s, 1H), 7.87 (s, 1H), 7.62 (s, 1H), 5.82-5.81 (m, 2H), 4.76, (d, 1H), 4.10-4.05 (m, 4H), 3.65 (t, 2H), 3.34 (t, 2H), 1.47 (s, 9H). EI-MS m/z : [M+H]+ 469.10.
Preparation of Compound 40
After dissolving Compound 9 (542 mg, 1.11 mmol) and Compound 39 (350 mg, 0.746 mmol) in normal butyl alcohol (4.5 mL), diisopropylethylamine (0.65 mL, 3.73 mmol) was added at room temperature and stirred for 24 hours while heating to 120 °C. The reaction solution was cooled to room temperature, and then diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 40 (135 mg, 21.4%). EI-MS m/z : [M+H]+ 844.12.
Preparation of Compound 41
After dissolving Compound 40 (135 mg, 0.16 mmol) in methanol (6 mL) and distilled water (1 mL), an aqueous ammonia solution (28-30%, 0.3 mL) and sodium hydrosulfite (Na2S2C>4, 278 mg, 1.59 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 2 hours, diluted with methanol (50 mL), and then filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to afford Compound 41 (91 mg, 69.8%). EI-MS m/z : [M+H]+ 814.12.
Preparation of Compound 42
After dissolving Compound 41 (91 mg, 0.11 mmol) in A,A-dimethylformamide (2.5 mL), Compound 2 (32.7 mg, 0.16 mmol) was dissolved in A,A-dimethylformamide (1 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, N-(3- dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (34 mg, 0.22 mmol) and triethylamine (0.03 mL, 0.24 mmol) were added and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to afford Compound 42 (125 mg, crude). EI-MS m/z : [M+H]+ 975.17.
Preparation of Compound 43
After dissolving Compound 42 (125 mg, crude) in dichloromethane (3.2 mL), trifluoroacetic acid (0.8 mL) was added at 0 °C under nitrogen. The reaction solution was stirred at 0 °C for 40 minutes, concentrated, and then purified by HPLC to afford Compound 43 (34 mg, 25%).
’H-NMR (400 MHz, DMSO-d6), δ 12.84 (bs, 2H), 9.16 (bs, 2H), 7.97 (d, 2H), 7.65 (s, 2H), 7.36 (s, 2H), 7.30 (s, 2H), 6.53 (d, 2H), 5.79 (s, 2H), 5.73-5.60 (m, 2H), 4.90 (bs, 4H), 4.55-4.50 (m, 6H), 3.84 (d, 2H), 3.73 (s, 2H), 3.69 (s, 4H), 2.11 (d, 6H), 1.29-1.24 (m, 6H). EI- MS m/z : [M+H]+ 875.13. Example 8: Preparation of Compound 48
Preparation of Compound 44 After dissolving Compound 4 (2.0 g, 9.23 mmol) in A,A-dimethylformamide (10 mL),
1 ,4-dibromo-2-butyne (5.8 g, 27.70 mmol) and cesium carbonate (3.6 g, 11.08 mmol) were added under nitrogen. The reaction solution was stirred at room temperature for 2 hours, diluted with ethyl acetate (100 mL), washed with distilled water (50 mL x 2), and dried with anhydrous sodium sulfate. The dried material was filtered and concentrated under reduced pressure, and the solid obtained by diluting with dichloromethane and hexane was filtered and dried to afford Compound 44 (2.0 g, 62%).
’H-NMR (400 MHz, DMSO-d6), δ 8.25 (s, 1H), 8.12 (s, 1H), 7.95 (s, 1H), 7.80 (s, 1H), 5.22 (s, 2H), 4.34 (t, J = 5.5 Hz, 2H). ELMS m/z : [M+H]+ 348.99.
Preparation of Compound 45 After dissolving Compound 44 (300 mg, 0.86 mmol) in A,A-dimethylformamide (3 mL), morpholine (0.09 mL, 1.03 mmol) and cesium carbonate (309 mg, 0.95 mmol) were added under nitrogen. The mixture was stirred at room temperature for 3 hours, diluted with ethyl acetate (50 mL), washed with distilled water (50 mL x 2), and dried with anhydrous sodium sulfate. The dried material was filtered, concentrated under reduced pressure, and purified by column chromatography to afford Compound 45 (230 mg, 75%).
’H-NMR (400 MHz, DMSO-d6) 5 8.25 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.79 (s, 1H), 5.17 (s, 2H), 3.52 (t, J = 11.5 Hz, 4H), 3.29 (s, 2H), 2.36 (t, J = 11.0 Hz, 4H). EI-MS m/z : [M+H]+ 354.13.
Preparation of Compound 46
After dissolving Compound 9 (500 mg, 1.03 mmol) and Compound 45 (215 mg, 0.60 mmol) in normal butyl alcohol (4 mL), diisopropylethylamine (0.53 mL, 3.03 mmol) was added at room temperature and stirred for 24 hours while heating to 120 °C. The reaction solution was cooled to room temperature, and then diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The solid obtained by diluting with dichloromethane and diethyl ether was filtered and dried to afford Compound 46 (303 mg, 68%). EI-MS m/z : [M+H]+ 729.11.
Preparation of Compound 47
After dissolving Compound 46 (303 mg, 0.41 mmol) in methanol (10 mL) and distilled water (1 mL), an aqueous ammonia solution (28-30% ammonia, 0.45 mL) and sodium hydrosulfite (Na2S2O4, 724 mg, 4.16 mmol) were added to the reaction solution under nitrogen. The mixture was stirred at room temperature for 1 hour, diluted with methanol (50 mL), and filtered. The filtrate was concentrated under reduced pressure, diluted with dichloromethane (100 mL) and methanol (20 mL), and then washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to afford Compound 47 (97 mg, 33%, crude). EI-MS m/z : [M+H]+ 699.07.
Preparation of Compound 48
After dissolving Compound 47 (97 mg, 0.14 mmol, crude) in MA-di methyl formamide (1.5 mL), Compound 2 (32 mg, 0.17 mmol) was dissolved in A,A-dimethylformamide (0.5 mL) under nitrogen and added thereto. After stirring at room temperature for 1 hour, N-(3- dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (40 mg, 0.21 mmol) and triethylamine (0.06 mL, 0.42 mmol) were added and stirred at room temperature for 13 hours. The reaction solution was concentrated under pressure and purified by HPLC to afford Compound 48 (38 mg, 32%).
’H-NMR (400 MHz, MeOD-d4), δ 7.60 (d, 7 = 1.3 Hz, 1H), 7.58 (d, 7 = 1.3 Hz, 1H), 7.44 (d, 7 = 1.4 Hz, 1H), 7.31 (d, 7 = 1.4 Hz, 1H), 6.60 (d, 7= 0.6 Hz, 1H), 6.58 (d, 7 = 0.6 Hz, 1H), 5.89-5.86 (m, 4H), 5.04-5.01 (m, 4H), 4.61-4.55 (m, 4H), 4.07(s, 2H), 3.71 (s, 3H), 2.20 (s, 3H), 2.18 (s, 3H), 1.37-1.30 (m, 6H). EI-MS m/z : [M+H]+ 860.08.
Example 9: Preparation of Compound 55
Preparation of Compound 49
To a solution of 4-nitropyrazole (1 g, 6.13 mmol) in methanol (20 mL) were added ammonia solution (28-30% ammonia, 2.2 mL) and sodium hydrosulfite (7.7 g). After stirring at room temperature for 1 hour, the reaction solution was filtered through Celite and the filtrate was evaporated under reduced pressure. Methanol (15 mL) was added to the concentrated filtrate and then di-t-butyl dicarbonate (2.24 mL, 9.73 mmol) and triethylamine (1.86 mL, 13.27 mmol) were added at room temperature. The reaction solution was stirred at room temperature for 16 hours and then diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The reduced was purified by column chromatography to afford Compound 49 (0.7 g, 43%).
’H-NMR (400 MHz, DMSO-d6), δ 9.05 (s, 1H), 7.44 (s, 1H), 1.42 (s, 9H).
Preparation of Compound 50
To a solution of Compound 49 (0.7 g, 3.82 mmol) in N, A-di methyl formamide (15 mL) were added cesium carbonate (3.7 g, 11.46 mmol) and trans- 1 ,4-dibromo-2-butene (2.45 g, 11.46 mmol). After stirring at room temperature for 2 hours, the reaction solution was diluted with ethyl acetate (50 mL) and then washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 50 (867 mg, 71%).
’H-NMR (400 MHz, CDC13), δ 7.66 (s, 1H), 7.33 (s, 1H), 6.24 (s, 1H), 6.00 - 5.90 (m, 1H), 5.87 (td, J = 13.2, 5.9 Hz, 1H), 4.69 (d, J = 5.6 Hz, 2H), 3.94 (d, J = 6.9 Hz, 2H), 1.50 (s, 9H). EI-MS m/z : [M+H]+ 317.26.
Preparation of Compound 51
To a solution of Compound 4 (540 mg, 2.5 mmol) in N, A-dimethylformamide (15 mL) were added cesium carbonate (894 mg, 2.75 mmol) and Compound 50 (867 mg, 2.75 mmol). After stirring at room temperature for 2 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 51 (830 mg, 73%).
’H-NMR (400 MHz, DMSO-d6), δ 9.12 (s, 1H), 8.24 (s, 1H), 8.05 (s, 1H), 7.88 (s, 1H), 7.75 (s, 1H), 7.65 (s, 1H), 7.30 (s, 1H), 6.08 (d, J = 15.4 Hz, 1H), 5.86 (d, J = 15.8 Hz, 1H), 4.82 (d, J = 5.4 Hz, 2H), 4.74 (d, 7 = 5.9 Hz, 2H), 1.44 (d, J = 2.3 Hz, 9H). EI-MS m/z : [M+H]+ 452.31.
Preparation of Compound 52
To a solution of Compound 51 (830 mg, 1.83 mmol) and Compound 9 (1.13 g, 2.76 mmol) in n-butanol (11 mL) were added N, A-diisopropylethylamine (1.6 mL, 9.18 mmol). The reaction solution was stirred at 0 °C for 5 minutes then heated to 120°C for 24 hours. After cooling to room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 52 (420 mg, 28%). EI-MS m/z : [M+H]+ 827.39.
Preparation of Compound 53
To a solution of Compound 52 (420 mg, 0.51 mmol) in methanol (5 mL) were added aqueous ammonia solution (28 ~ 30% ammonia, 1.8 mL, 12.66 mmol) and sodium hydrosulfite (882 mg, 5.06 mmol). After stirring at room temperature for 2.5 hours, the reaction solution was filtered through Celite and washed with methanol. The filtrate was concentrated under reduced pressure and purified by reversed phase column chromatography to afford Compound 53 (400 mg). EI-MS m/z : [M+H]+ 797.48. Preparation of Compound 54
To a solution of Compound 53 (400 mg, 0.51 mmol) in N,N -dimethylformamide (1.5 mL) at 0 °C was added Compound 2 (109 mg, 0.55 mmol) in A,A-dimethylformamide (1 mL). After 30 minutes, A-(3-dimethylaminopropyl)-A’-ethyl carbodiimide (0.1 mL, 0.61 mmol) and triethylamine (0.35 mL, 2.53 mmol) were added to the reaction solution. After stirring at room temperature for 15 hours, the reaction solution was concentrated under reduced pressure and purified by reverse phase chromatography to afford Compound 54 (97 mg, 20%).
Preparation of Compound 55
To a solution of Compound 54 (30 mg, 0.03 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at 0 °C. After stirring at room temperature for 30 minutes, the reaction mixture was concentrated and purified by HPLC to afford Compound 55 (10 mg, 27%).
’H-NMR (400 MHz, CD3OD), δ 7.80 (s, 1H), 7.57 (d, J = 11.4 Hz, 3H), 7.29 (s, 1H), 7.23 (s, 1H), 6.62 (d, 7 = 1.7 Hz, 1H), 6.56 (s, 1H), 5.85 (d, 7 = 18.0 Hz, 3H), 5.70 (d, 7 = 15.6 Hz, 1H), 5.01 (s, 4H), 4.63 (s, 3H), 4.61 - 4.52 (m, 2H), 4.45 (s, 2H), 3.74 (s, 2H), 3.31 (m, 3H), 2.65 (s, 1H), 2.20 (d, J = 12.3 Hz, 6H), 1.34 (dt, J = 21.7, 6.9 Hz, 6H). ELMS m/z : [M+H]+ 858.54.
Example 10: Preparation of Compound 65
Preparation of Compound 56
To a solution of Compound 4 (5 g, 23.08 mmol) in A,A-dimethylformamide (30 mL) was added cesium carbonate (11.2 g, 34.62 mmol) at 0 °C under nitrogen. After 5 minutes, ethyl 4-bromobutyrate (5.4 g, 27.70 mmol) was added to the reaction solution at room temperature under nitrogen. After stirring for 2 hours, the reaction solution was diluted with ethyl acetate (60 mL) and washed with distilled water (15 mL x 2) and brine (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated. After solidification used dichloromethane and hexane, the resulting solid was filtered and dried to afford Compound 56 (4.7 g, 61%), which was used without further purification.
’H-NMR (400 MHz, CDCI3) δ 8.28 (s,lH), 8.04 (s, 1H), 7.86 (s, 1H), 7.73 (s, 1H), 4.25 (m, 2H), 4.09-4.04 (q, J = 7.2 Hz, 2H), 1.19-1.15 (t. 7 = 7.2 Hz, 3H). EI-MS m/z : [M+H]+ 331.20.
Preparation of Compound 57
To a solution of Compound 56 (4.5 g, 13.60 mmol) in ethanol (30 mL) were added t- butyl (E)-(4-aminobut-2-en-l-yl)carbamate (2.5 g, 13.60 mmol) and N,N- diisopropylethylamine (2.37 mL, 27.21 mmol). After stirred at 120 °C for 12 hours, the reaction solution was cooled to room temperature. The reaction mixture was diluted ethyl acetate (60 mL) and washed with distilled water (15 mL x 2) and brine (15 mL). The organic layer was dried over anhydrous sodium sulfate filtered and concentrated. The resulting residue was purified by column chromatography to afford Compound 57 (4.5 g, 68%).
’H-NMR (400 MHz, DMSO-d6), δ 8.10 (s, 1H), 8.01(s, 1H), 7.68 (t, 1H), 7.58 (s, 1H), 7.30 (s, 1H), 6.90 (s, 1H), 5.54 (s, 2H), 4.10 (m, 6H), 3.48 (s, 3H), 2.07 (m, 2H) 1.35 (m, 9H) 1.17 (m, 4H). EI-MS m/z : [M+H]+ 481.28.
Preparation of Compound 58
To a solution of Compound 57 (4.4 g, 9.156 mmol) in methanol (20 mL) were added aqueous ammonia solution (28 ~ 30% ammonia, 10 mL) and sodium hydrosulfite (Na2S2C>4, 15 g, 91.6 mmol) at 0 °C. After stirring at room temperature for 1.5 hours, the reaction solution was filtered through Celite with methanol. The filtrate was concentrated under reduced pressure to afford Compound 58 (4 g, 96%). EI-MS m/z : [M+H]+ 451.31.
Preparation of Compound 59
To a solution of Compound 58 (4.0 g, 8.87 mmol) in N, A-di methyl formamide (30 mL) was added Compound 2 (1.9 g, 9.76 mmol) at 0 °C. After stirring at room temperature for 30 min, triethylamine (3.7 mL, 26.63 mmol) and A-(3-dimethylaminopropyl)-A-ethyl carbodiimide (2.7 g, 17.75 mmol) were added to the reaction solution at 0 °C. The resulting reaction solution was stirred at room temperature for 17 hours. Then, the reaction solution was concentrated, and the resulting residue was purified by column chromatography to afford Compound 59 (3.8 g, 71%). ’H-NMR (400 MHz, DMSO-d6), δ 12.86 (s, 1H), 7.99 (s, 1H), 7.66 (s, 1H), 7.37-7.35 (m, 2H), 6.89 (m, 1H), 6.63 (s, 1H), 5.79-5.72 (d, 7 = 16 Hz, 1H), 5.58-5.54 (d, 7 = 16 Hz, 1H) 4.94 (s, 2H), 4.62 (m, 2H), 4.22 (s, 3H), 4.20 (m, 6H), 2.31 (m, 3H), 2.11 (s, 2H), 1.36 (m, 9H), 1.17 (m, 4H). EI-MS m/z : [M+H]+ 612.31.
Preparation of Compound 60
To a solution of Compound 59 (3.8 g, 6.21 mmol) in dichloromethane (50 mL) was added hydrochloric acid (4 M 1,4-dioxane solution, 11.5 mL). After stirring for 2 hours, the reaction solution was concentrated, and diluted with diethyl ether (20 mL). The resulting solid was filtered to afford Compound 60 (3.9 g, 99%).
’H-NMR (400 MHz, DMSO-d6), δ 8.04 (s, 1H), 7.88 (m, 2H), 7.68 (s, 1H), 7.39 (s, 2H), 6.67 (s, 1H), 6.08-6.04 (d, J = 16 Hz, 1H), 5.59-5.55 (d, J = 16 Hz, 1H), 5.00 (s, 2H), 4.61 (m, 2H), 4.22 (m, 2H), 4.10 (m, 2H), 2.19 (s, 3H), 2.11 (m, 2H), 1.37 (m, 3H), 1.19 (m, 3H). EI-MS m/z : [M+H]+ 512.31.
Preparation of Compound 61
To a solution of Compound 60 (3.8 g, 6.63 mmol) and Compound 51 (2 g, 4.42 mmol) in n-butanol (13 mL) was added A,A-diisopropylethylamine (3.85 mL, 22.13 mmol) at room temperature. After stirring at 100 °C for 21 hours, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The combined organic layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to afford Compound 61 (1.6 g, 38%). EI-MS m/z : [M+H]+ 926.98.
Preparation of Compound 62
To a solution of Compound 61 (1.6 g, 1.83 mmol) in methanol (8 mL) were added ammonia solution (28-30% ammonia, 3.2 mL) and sodium hydrosulfite (Na2S2C>4, 3.1 g, 18.3 mmol) under nitrogen. The reaction solution was stirred at room temperature for 1 hour and then methanol (50 mL) was added, and the resulting solid was filtered out. The filtrate was concentrated and diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford Compound 62 (1.4 g, 85%). EI-MS m/z : [M+H]+ 897.05.
Preparation of Compound 63
To a solution of Compound 62 (1.35 g, 1.51 mmol) in A,A-dimethylformamide (10 mL) was added Compound 2 (324 mg, 1.66 mmol) in A,A-dimethylformamide (1 mL) under nitrogen. The reaction solution was stirred at room temperature for 1 hour and then N-(3- dimethylaminopropyl)-A’-ethylcarbodiimide hydrochloride (465 mg, 3.02 mmol) and triethylamine (0.63 mL, 4.53 mmol) were added. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 63 (560 mg, 35%). EI-MS m/z : [M+H]+ 1057.96.
Preparation of Compound 64
To a solution of Compound 63 (100 mg, 0.094 mmol) in methanol (2 mL) was added lithium hydroxide monohydrate (13.8 mg, 0.28 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction solution was acidified with acetic acid to pH 4-5 and then concentrated and lyophilized to afford Compound 64 (100 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1029.95.
Preparation of Compound 65
To a solution of Compound 64 (100 mg, 0.097 mmol, crude) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated and purified by HPLC to afford Compound 65 (42 mg, 34%).
’H-NMR (400 MHz, DMSO-d6), δ 12.80 (s, 1H), 9.77 (s, 2H), 7.96 (s, 1H), 7.87 (m, 2H), 7.65 (s, 2H), 7.53 (s, 1H), 7.34 (s, 2H), 7.28 (s, 2H), 6.53 (s, 2H), 5.79 (m, 3H), 5.65 (m, 1H), 4.90 (s, 4H), 4.62 (m, 2H), 4.50 (m, 6H), 3.92 (m, 3H), 2.24 (s, 2H), 2.11 (m, 6H), 1.74 (m, 2H), 1.26 (m, 6H). EI-MS m/z : [M+H]+ 929.98.
Example 11: Preparation of Compound 69
Preparation of Compound 67
To a solution of Compound 66 (300 mg, 0.32 mmol, Compound 66 was prepared by the method described in the International Patent Publication No. WO 2022/155518 Al) in dichloroethane (30 mL) was added boron tribromide (1.0 M in dichloromethane, 3.2 mL, 3.16 mmol). After stirring under reflux for 17 hours, the reaction solution was concentrated under reduced pressure and diluted with dichloromethane/diethyl ether (50 mL/50 mL). The resulting solid was filtered to afford Compound 67 (280 mg, 84%). ELMS m/z : [M+H]+ 709.15. Preparation of Compound 68
To a solution of Compound 67 (280 mg, 0.27 mmol) in W/V-dimethylformamide (2 mL) were added cesium carbonate (607 mg, 1.87 mmol) and Compound 50 (101 mg, 0.32 mmol). After stirring at room temperature for 18 hours, the reaction solution was concentrated under reduced pressure and purified by reversed phase column chromatography to afford Compound 68 (158 mg, 62%). ELMS m/z : [M+H]+ 945.00.
Preparation of Compound 69
To a solution of Compound 68 (50 mg) in dichloromethane (1.5 mL) was added trifluoroacetic acid (0.5 mL) under nitrogen at 0°C. After stirring at room temperature for 1 hour, the reaction solution was concentrated and the resulting residue was purified by HPLC to afford Compound 69 (8 mg).
’H-NMR (400 MHz, CD3OD), δ 7.82 (d, 7 = 0.8 Hz, 1H), 7.58 (d, 7 = 0.8 Hz, 1H), 7.53 (d, 7= 1.3 Hz, 1H), 7.42 (d, 7 = 1.5 Hz, 1H), 7.24 (d, 7 = 1.4 Hz, 1H), 7.17 (d, 7 = 1.4 Hz, 1H), 6.61 (d, J = 0.7 Hz, 1H), 6.49 (d, J = 0.7 Hz, 1H), 5.95 - 5.80 (m, 3H), 5.78 - 5.67 (m, 1H), 5.04 (dd, J = 17.4, 3.9 Hz, 4H), 4.67 - 4.57 (m, 4H), 4.57 - 4.46 (m, 4H), 2.20 (s, 3H), 2.16 (s,
3H), 1.36 0. 7 = 7. 1 Hz, 3H), 1.28 (t, 7= 7.1 Hz, 3H). EI-MS m/z : [M+H]+ 845.07.
Example 12: Preparation of Compound 79
Preparation of Compound 70 To a solution of trans- 1 ,4-dibromo-2-butene (10.4 g, 48.7 mmol) in N,N- dimethylformamide (30 mL) was added sodium acetate (2.0 g, 24.4 mmol) at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 17 hours and then diluted with ethyl acetate (100 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 70 (2.98 g, 63%).
’H-NMR (400 MHz, CDCh), δ 6.02-5.82 (m, 2H), 4.59 (d, J = 5.6 Hz, 2H), 3.95 (d, J = 7.2 Hz, 2H), 2.08 (s, 3H).
Preparation of Compound 71
To a solution of Compound 70 (838 mg, 4.34 mmol) in dichloromethane (60 mL) were added triethylamine (1.83 mL, 13.02 mmol) and t-butyl(3-aminopropyl) carbamate (2.27 g, 13.02 mmol) in dichloro methane (40 mL) at 0 °C. The reaction solution was stirred at room temperature for 18 hours and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 71 (380 mg, 30%).
’H-NMR (400 MHz, CDC13), δ 5.87-5.71 (m, 2H), 4.99 (s, 1H), 4.58-4.52 (m, 2H), 3.26 (d, J = 5.7 Hz, 2H), 3.23-3.18 (m, 2H), 2.70-2.65 (m, 2H), 2.07 (d, J = 2.7 Hz, 3H), 1.68 (d, 7 = 6.6 Hz, 2H), 1.45 (d, 7 = 2.6 Hz, 9H).
Preparation of Compound 72
To a solution of Compound 71 (790 mg, 2.76 mmol) in dichloromethane (10 mL) were added fluorenylmethyloxycarbonyl chloride (Fmoc-Cl, 856 mg, 3.31 mmol) and N,N- diisopropylethylamine (0.78 mL, 5.52 mmol). After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 72 (1.4 g, 98%). ELMS m/z : [M+H]+ 509.19.
Preparation of Compound 73
To a solution of Compound 72 (790 mg, 2.76 mmol) in methanol (20 mL) was added potassium carbonate (1.96 g, 14.15 mmol) at 0 °C. After stirring at room temperature for 30 minutes, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was dissolved in dichloromethane (10 mL) and then 4-nitrophenyl(2-(trimethylsilyl)ethyl)carbonate (Teoc-PNP, 962 mg, 3.39 mmol) and AWdiisopropylcthylaminc (0.80 mL, 5.66 mmol) were added and the reaction solution was stirred at room temperature for 18 hours. The reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 73 (596 mg, 54%). ELMS m/z : [M+H]+ 389.28.
Preparation of Compound 74
To a solution of Compound 73 (300 mg, 0.77 mmol) in dichloromethane (5 mL) were added triethylamine (0.33 mL, 2.31 mmol) and methanesulfonyl anhydride (175 mg, 1.00 mmol) at 0 °C. After stirring at room temperature for 1 hour, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure gave Compound 74 (380 mg, crude), which was used without further purification. ELMS m/z : [M+H]+ 467.17.
Preparation of Compound 76
To a solution of Compound 75 (450 mg, 0.62 mmol, Compound 75 was prepared according to the method described in the International Patent Publication No. WO 2022/155518 Al) in A Wdimcthylformamidc (5 mL) were added cesium carbonate (811 mg, 2.49 mmol) and Compound 74 (349 mg, 0.75 mmol) in A Wdimcthylformamidc (2 mL). After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure and diluted with dichloromethane (50 mL) and methanol (10 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 76 (468 mg, 68%). ELMS m/z : [M+H]+ 1093.72.
Preparation of Compound 77
To a solution of Compound 76 (468 mg, 0.43 mmol) in tetrahydrofuran (10 mL) was added tetrabutylammonium fluoride (1.0 M tetrahydrofuran solution, 2.1 mL, 2.14 mmol). After stirring under reflux for 5 hours, the reaction solution was concentrated under reduced pressure and the resulting residue was purified by reversed-phase column chromatography, which afforded Compound 77 (256 mg, 63%). ELMS m/z : [M+H]+ 950.12.
Preparation of Compound 78
To a solution of Compound 77 (256 mg, 0.27 mmol) in AA-dimcthylfoi mamidc (3 mL) were added A/V-bis(t-butoxycarbonyl)-177-pyrazole-l-carboxamidine (126 mg, 0.40 mmol) and triethylamine (0.11 mL, 0.81 mmol). After stirring at 60 °C for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 78 (103 mg, 32%). EI-MS m/z : [M+H]+ 1192.17. Preparation of Compound 79
To a solution of Compound 78 (103 mg, 0.09 mmol) in dichloromethane (3 mL) was added trifluoro acetic acid (1 mL) at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 2 hours. After concentration, the resulting residue was purified by HPLC to afford Compound 79 (48 mg, 45%) ’H-NMR (400 MHz, CD3OD), δ 7.29 (d, 7 = 1.4 Hz, 1H), 7.26 (d, 7 = 1.4 Hz, 1H), 6.59
(d, J = 0.6 Hz, 1H), 6.56 (d, J = 0.6 Hz, 1H), 5.88 - 5.77 (m, 2H), 5.75 - 5.69 (m, 2H), 5.02 (d, J = 3.3 Hz, 4H), 4.64 - 4.50 (m, 6H), 3.94 (d, J = 4.2 Hz, 2H), 3.75 (s, 3H), 3.42 - 3.32 (m, 4H), 3.01 - 2.93 (m, 2H), 2.24 - 2.17 (m, 6H), 1.97 (p. 7 = 7.8 Hz, 2H), 1.34 (dt, 7 = 14.6, 7.1 Hz, 6H). EI-MS m/z : [M+H]+ 891.09.
Example 13: Preparation of Compound 89
Preparation of Compound 80
To a solution of 4-Piperidinethanol (5 g, 38.7 mmol) in dichloromethane (200 mL) were added triethylamine (8.1 mL, 58.05 mmol) and di-t-butyl dicarbonate (9.78 mL, 42.57 mmol) under nitrogen. The reaction solution was stirred at room temperature for 3 hours and then diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 80 (7.25 g, 81.6%).
’H-NMR (CDCI3), δ: 1.04-1.10 (m, 2H), 1.48 (s, 9H), 1.49-1.55 (m, 3H), 1.60-1.66 (m, 2H), 2.27 (s, 1H), 2.64 (t, J = 8.0 Hz, 2H), 3.64 (t, J = 8.0 Hz, 2H), 4.03-4.08 (m, 2H). EI-MS m/z : [M+Na]+ 252.26. Preparation of Compound 81
To a solution of Dimethyl sulfoxide (0.93 mL, 13.08 mmol) in dichloromethane (20 mL) was slowly added oxalyl chloride (0.34 mL, 3.93 mmol) at -78 °C under nitrogen. After stirring for 30 minutes, the reaction solution was added Compound 80 (1 g, 4.36 mmol) in dichloromethane (5 mL). The reaction solution was stirred at -50 °C for 2 hours. The reaction solution was added triethylamine (1.8 mL, 13.1 mmol). After raising to 0 °C and stirring for 30 minutes, the reaction solution was diluted with ethyl acetate (50 mL), washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 81 (990 mg, 99%).
’H-NMR (300 MHz, CDC13), δ (ppm): 9.78 (br s, 1H), 4.08 (br d, 2H), 2.74 (br t, 2H), 2.39 (d, 2H), 2.12-1.89 (m, 1H), 1.79-1.64 (m, 2H), 1.45 (s, 9H), 1.26-1.10 (m, 2H). EI-MS m/z : [M+H]+ 228.23.
Preparation of Compound 82
To a solution of lithium chloride (17.2 g, 40.65 mmol) in acetonitrile (40 mL) was added triethylphosphonoacetate (7.33 mL, 50.81 mmol) at room temperature under nitrogen. After stirring for 5 minutes, the reaction solution was added Triethylamine (5.67 mL 40.65 mmol) at room temperature for 10 minutes. Compound 81 (7.7 g, 33.88 mmol) in acetonitrile (60 mL) was added to the reaction solution. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (150 mL), washed with distilled water (150 mL). The organic layer was dried over anhydrous magnesium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 82 (5.59 g, 58.2%).
’H-NMR (400 MHz, CDCI3), 5 (ppm): 6.96-6.88 (m,lH), 5.85-5.811 (m, 1H), 4.08(s,2H), 3.73(s,3H), 2.07-2.64 (m, 2H), 2.17-2.13 (m, 2H), 1.67(s, 2H), 1.59-1.55 (m, 1H), 1.54 (s, 9H), 1.18-1.11 (m, 2H). EI-MS m/z : [M+H]+284.01.
Preparation of Compound 83
To a solution of Compound 82 (2.5 g, 8.82 mmol) in dichloromethane (30 mL) was added diisobutylaluminium hydride (1.0 M cyclohexane solution, 19 mL, 19.00 mmol) at -78 °C under nitrogen. After stirring at -78 °C for 3 hours, the reaction solution was added methanol (100 mL). The reaction solution was filtered through Celite and washed with methanol. The filtrate was removed under reduced pressure and used without purification to afford Compound 83 (1.91 g, 84.8%). ’H-NMR (400 MHz, CDCI3), δ = 5.70-5.65 (m, 2H), 4.18-4.01 (m, 4H), 2.69 (t, 2H, J = 12.6 Hz), 2.02 (t, 2H, J = 5.6 Hz), 1 .72-1 .62 (m, 3H), 1 .47 (s, 9H), 1 .1 1 (qd, 2H, J = 12.2 Hz, 3.9 Hz). EI-MS m/z : [M+H]+ 256.06.
Preparation of Compound 84
To a solution of Compound 83 (1.91 g, 7.48 mmol) in dichloromethane (20 mL) were added triethylamine (1.6 mL, 11.2 mmol) and methanesulfonyl chloride (0.87 mL, 11.2 mmol) at 0 °C. After stirring at room temperature for 3 hours, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was removed under reduced pressure and used without purification to afford Compound 84 (2.59 g, crude).
Preparation of Compound 85
To a solution of Compound 5 (1 g, 4.62 mmol) in A,A-dimethylformamide (15 mL) were added potassium carbonate (766 mg, 5.54 mmol) and Compound 84 (2.3 g, 6.94 mmol). After stirring at room temperature for 14 hours, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 85 (1.18 g, 56.3%).
’H-NMR (400 MHz, CDCI3), δ = 7.82-7.72, 5.83-5.69 (m, 2H), 4.80-4.71 (m, 2H), 4.04-4.03 (m, 2H), 2.65 (s, 2H), 2.05-1.93 (m, 1H), 1.07-1.04 (m, 2H). EI-MS m/z : [M+H]+ 454.17.
Preparation of Compound 86
To a solution of Compound 85 (550 mg, 1.21 mmol) in n-butanol (5 mL) were added Compound 9 (1.17 g, 2.42 mmol) and N, A-diisopropylethylamine (1.05 mL, 6.06 mmol). After stirring at 0 °C for 5 minutes, the reaction solution was heated to 120 °C and stirred for 24 hours and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure. The concentrated reaction product was purified by column chromatography to afford Compound 86 (157 mg, 15.6%). EI-MS m/z : [M+H]+ 829.22.
Preparation of Compound 87
To a solution of Compound 86 (157 mg, 0.19 mmol) in methanol (3 mL) were added ammonia solution (28-30% ammonia, 0.5 mL, 4.74 mmol) and sodium hydrosulfite (Na2S2C>4, 330 mg, 1.89 mmol). After stirring at room temperature for 1 hour, the reaction solution was passed through Celite filter and washed with methanol. After filtration, the filtrate was removed under reduced pressure and used without purification to afford Compound 87 (151 mg, crude), which was used without further purification.
Preparation of Compound 88
To a solution of Compound 87 (151 mg, 0.19 mmol) in A,A-dimethylformamide (2 mL) was added Compound 2 (40 mg, 0.21 mmol) at 0 °C. The reaction solution was stirred for 30 minutes and then A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide (0.04 mL, 0.24 mmol) and triethylamine (0.11 mL, 0.76 mmol) were added and the reaction solution was stirred at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 88 (100 mg, 54%). EI-MS m/z : [M+H]+ 961.13. Preparation of Compound 89
To a solution of Compound 88 (20 mg, 0.02 mmol) in dichloromethane (2 mL) was added trifluoro acetic acid (0.4 mL) at 0 °C under nitrogen. The reaction solution was stirred at room temperature for 0.5 hours. After concentration, the resulting residue was purified by HPLC to afford Compound 89 (4.8 mg, 27%). EI-MS m/z : [M+H]+ 861.30.
Example 14: Preparation of Compound 95
Preparation of Compound 90
To a solution of 3-Aminophenol (1 g, 9.16 mmol) in tetrahydrofuran (10 mL) was added di-t-butyl dicarbonate (2.52 mL, 10.99 mmol). After stirring for 16 hours at room temperature, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The reaction solution was dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and used without purification to afford Compound 90 (1.8 g, 93%).
’H-NMR (400 MHz, CDCh), δ 9.24 (s, 1H), 9.18 (s, 1H), 6.88(m, 2H), 6.83 (m, 1H), 6.35 (m, 1H), 1.46 (s, 9H). ELMS m/z : [M+H]+ 209.10.
Preparation of Compound 91
To a solution of Compound 90 (300 mg, 1.43 mmol) in A,A-dimethylformamide (5 mL) were added cesium carbonate (560 mg, 1.72 mmol) and Compound 5 (551 mg, 1.57 mmol) at 0 °C under nitrogen. After stirring for 2 hours, the reaction solution was diluted with ethyl acetate (30 mL) and washed with distilled water (15 mL x 2) and brine (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated. The concentrate was solidified by added dichloromethane and diethyl ether, then filtered and dried to afford Compound 91 (474 mg, 69%), which was used without further purification.
’H-NMR (400 MHz, CDC13), δ 9.29 (s, 1H), 8.26 (s, 1H), 8.06 (s, 1H), 7.90 (s, 1H), 7.75 (s, 1H), 7.12 (m, 2H), 7.10 (d, 1H), 6.56 (m, 1H), 6.14 (m, 2H), 4.87 (d, 7 = 3.6 Hz, 2H), 4.57 (d, J = 3.2 Hz, 2H), 1.46 (s, 9H). ELMS m/z : [M+H]+ 478.00.
Preparation of Compound 92
To a solution of Compound 9 (607 mg, 1.47 mmol) and Compound 91 (470 mg, 0.98 mmol) in n-butanol (5 mL) were added diisopropylethylamine (0.68 mL, 3.93 mmol) at room temperature. After heating to 120 °C for 24 hours, the reaction solution was cooled to room temperature. The reaction solution was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated to afford Compound 92 (100 mg, 12%), which was used without further purification. ELMS m/z : [M+H]+ 853.30.
Preparation of Compound 93
To a solution of Compound 92 (100 mg, 0.11 mmol) in methanol (5 mL) were added ammonia solution (28-30% ammonia, 0.209 mL) and sodium hydrosulfite (Na2S2C>4, 204 mg, 1.172 mmol) under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was diluted with methanol (50 mL) and then filtered. The filtrate was concentrated under reduced pressure. The reaction mixture was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated to afford Compound 93 (100 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 823.36.
Preparation of Compound 94
To a solution of Compound 93 (100 mg, 0.12 mmol, crude) in N, A-dimethylformamide (2 mL) were added Compound 2 (26 mg, 0.13 mmol) in N, A-dimethylformamide ( 1 mL) at 0 °C under nitrogen. After stirring at room temperature for 1 hour. The reaction mixture was added A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (28 mg, 0.18 mmol) and triethylamine (0.05 mL, 0.36 mmol) After stirring at room temperature for 13 hours, the reaction solution was concentrated under reduced pressure and solidified by dichloromethane and diethyl ether, then filtered and dried to afford Compound 94 (54 mg, 53%). EI-MS m/z : [M+H]+ 984.84.
Preparation of Compound 95
To a solution of Compound 94 (50 mg, 0.061 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C under nitrogen. After stirring at room temperature for 40 minutes, the reaction solution was concentrated and purified by HPLC to afford Compound 95 (1.7 mg, 3%). EI-MS m/z : [M+H]+ 884.37.
Example 15: Preparation of Compound 104
Preparation of Compound 96
To a solution of methyl 3 -nitrocinnamate (3.5 g, 16.89 mmol) in methanol (20 mL) and distilled water (5 mL) were added cone, hydrochloric acid (0.25 mL) and iron powder (9 g, 161.15 mmol). After heating to reflux with stirring for 17 hours, the reaction solution was filtered through Celite and concentrated under reduced pressure to afford Compound 96 (2.7 g, crude) without purification.
’H-NMR (400 MHz, DMSO-d6), δ 7.48 (d, J= 15.8 Hz, 1H), 7.06 (t, J= 7.8 Hz, 1H), 6.85-6.79 (m, 2H), 6.65-6.61 (m, 1H), 6.41 (d, J= 15.8 Hz, 1H), 5.19 (s, 2H), 3.71 (s, 3H).
Preparation of Compound 97
To a solution of Compound 96 (2.7 g, 15.24 mmol) was dissolved in 1,4-dioxane (10 mL were added di-t-butyl dicarbonate (3.85 mL, 16.76 mmol) and saturated aqueous sodium bicarbonate solution (3.2 g, 38.09 mmol) dissolved in water (50 mL). After stirring at room temperature for 21 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The reaction solution was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by column chromatography to afford Compound 97 (3 g, 71 %).
’H-NMR (CD3OD, 400 MHz), δ 7.65 (s, 1H), 7.62 (d, 1H, J = 16.1 Hz), 7.43 (d, 1H, J = 7.6 Hz), 7.28 (t, 1H, J = 7.6 Hz), 7.20 (d, 1H, J = 7.6 Hz), 6.47 (d, 1H, J = 16.1 Hz), 3.76 (s, 3H), 1.51 (s, 9H).
Preparation of Compound 98
To a solution of Compound 97 (1.3 g, 4.69 mmol) in dichloromethane (20 mL) were added diisobutylaluminum hydride (1.0 M in cyclohexane, 19 mL, 18.75 mmol) at -78 °C under nitrogen. After stirring at -78 °C for 3 hours, the reaction solution was added methanol (100 mL) at room temperature. The reaction solution was filtered through Celite. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 98 (815 mg, 70%). EI-MS m/z : [M+Na]+ 272.05.
Preparation of Compound 99
To a solution of Compound 98 (800 mg, 3.21 mmol) in dichloromethane (16 mL) were added triethylamine (0.7 mL, 4.81 mmol) and methanesulfonyl chloride (0.3 mL, 3.53 mmol) at 0 °C. After stirring at room temperature for 3 hours, the reaction solution diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 99 (1 g, 95%), which was used without further purification.
Preparation of Compound 100
To a solution of Compound 4 (762 mg, 3.52 mmol) in A,A-dimethylformamide (15 mL) were added potassium carbonate (663 mg, 4.8 mmol) and Compound 99 (1.05 g, 3.2 mmol). After stirring at 50 °C for 15 hours, the reaction solution diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 100 (815 mg, 57%), which was used without further purification. EI-MS m/z : [M+H]+ 448.25.
Preparation of Compound 101
To a solution of Compound 100 (500 mg, 1.12 mmol) in n-butanol (6 mL) were added Compound 9 (811 mg, 1.68 mmol) and N,A-diisopropylethylamine (0.8 mL, 4.47 mmol). After stirring at 0 °C for 5 minutes, the reaction mixture was heated to 120 °C and stirred for 23 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 101 (316 mg, 34%). EI-MS m/z : [M+H]+ 823.42.
Preparation of Compound 102
To a solution of Compound 101 (316 mg, 0.38 mmol) in methanol (6 mL) were added aqueous ammonia solution (28-30% ammonia, 0.7 mL, 9.6 mmol) and sodium hydrosulfite (Na2S2C>4, 668 mg, 3.84 mmol). After stirring at room temperature for 1 hour, the precipitate was filtered through Celite and washed with methanol. The filtrate was concentrated under reduced pressure and obtained Compound 102 (304 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 793.51.
Preparation of Compound 103
To a solution of Compound 102 (304 mg, 0.38 mmol) in A,A-dimethylformamide (2 mL) was added Compound 2 (82 mg, 0.42 mmol) at 0 °C. After stirring for 30 minutes, the reaction solution was added A-(3-dimethylaminopropyl)-A’-ethylcarbodiimide (0.08 mL, 0.48 mmol) and triethylamine (0.21 mL, 1.54 mmol) at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 103 (12 mg, 3%). EI-MS m/z : [M+H]+ 954.47.
Preparation of Compound 104
To a solution of Compound 103 (12 mg, 0.001 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C under nitrogen. After stirring at room temperature for 0.5 hours, the reaction solution was concentrated. The resulting residue was purified by HPLC to afford Compound 104 (4.3 mg, 40%). EI-MS m/z : [M+H]+ 855.46.
Example 16: Preparation of Compound 110
Preparation of Compound 105
To a solution of t-butyl prop-2-en-l-ylcarbamate (1.0 g, 6.44 mmol) in N,N- dimethylformamide (9 mL) and methanol (1 mL) were added trimethylsilylazide (1.27 mL, 9.66 mmol) and copper(I) iodide (613 mg, 3.22 mmol). After stirring at 90 °C for 18 hours, the reaction solution was diluted with ethyl acetate (200 mL) and washed with saturated aqueous ammonium chloride solution (50 mL x 2) and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 105 (373 mg, 29%).
’H-NMR (400 MHz, CDC13), δ 7.65 (s, 1H), 5.09 (s, 1H), 4.43 (d, J = 6.0 Hz, 2H), 1.46 (s, 9H).
Preparation of Compound 106
To a solution of Compound 105 (122 mg, 0.62 mmol) and Compound 5 (315 mg, 0.68 mmol) in A,A-dimethylformamide (55 mL) was added potassium carbonate (102 mg, 0.74 mmol) at room temperature under nitrogen. After stirring for 16 hours, the reaction solution was diluted with ethyl acetate (200 mL) and washed with distilled water (150 mLx2) and brine (150 mL). The reaction solution was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 106 (146 mg, 50%). ’H-NMR (400 MHz, DMSO), δ 8.27 (s, 1H), 8.06 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.78 (s, 1H), 7.30 (s, 1H), 6.17 - 6.07 (m, 1H), 5.97 - 5.93 (m, 1H), 5.07 (d, 7 = 6.0 Hz, 2H), 4.84 (d, J = 5.2 Hz, 2H), 4.16 (d, J = 5.9 Hz, 2H), 1.38 (s, 9H).
Preparation of Compound 107
To a solution of Compound 106 (265 mg, 0.57 mmol) and Compound 9 (412 mg, 0.85 mmol) in n-butanol (3 mL) was added A,A-diisopropylethylamine (0.49 mL, 2.84 mmol) at room temperature. After heating to 100 °C and stirred for 21 hours, the reaction solution was cooled to room temperature and diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 107 (352 mg, 73%). EI-MS m/z : [M+H]+ 842.42.
Preparation of Compound 108
To a solution of Compound 107 (352 mg, 0.42 mmol) in methanol (10 mL) and distilled water (2 mL) were added ammonia solution (28-30% ammonia, 0.6 mL) and sodium hydrosulfite (Na2S2C>4, 728 mg, 4.2 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with methanol (50 mL) and then filtered. The filtrate was concentrated under reduced pressure. The reaction mixture was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated to afford Compound 108 (100 mg, 29%), which was used without further purification. EI-MS m/z : [M+H]+ 812.47.
Preparation of Compound 109
To a solution of Compound 108 (100 mg, 0.12 mmol) in A,A-dimethylformamide (3 mL) was added Compound 2 (29 mg, 0.15 mmol) in A,A-dimethylformamide (1 mL) under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was added N-(3- dimethylaminopropyl)-A’-ethylcarbodiimide (0.03 mL, 0.17 mmol) and triethylamine (0.05 mL, 0.05 mmol) at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 109 (80 mg, 66%). EI-MS m/z : [M+H]+ 973.54.
Preparation of Compound 110
To a solution of Compound 109 (80 mg, 0.08 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1 mL) at -78 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 110 (25 mg, 35%).
’H-NMR (400 MHz, DMSO), δ 12.84 (s, 1H), 8.19 (s, 2H), 8.04 - 7.91 (m, 3H), 7.65
(s, 2H), 7.37 (s, 2H), 7.30 (s, 2H), 6.52 (d, J = 2.6 Hz, 2H), 5.96 - 5.79 (m, 3H), 4.95 - 4.87 (m, 6H), 4.58 - 4.48 (m, 6H), 4.10 (q, J = 5.7 Hz, 2H), 3.70 (s, 3H), 2.10 (d, J = 6.5 Hz, 6H),
1.26 (q, 7 = 7.1 Hz, 6H). EI-MS m/z : [M+H]+ 873.55.
Example 17: Preparation of Compound 120 Preparation of Compound 111
To a solution of t-butyl (S)-2-(hydroxymethyl)pyrrolidine-l-carboxylate (2.2 g, 10.93 mmol) in dichloromethane (20 mL) were added dimethyl sulfoxide (5 mL), triethylamine (9.2 mL, 65.6 mmol) and sulfur trioxide pyridine complex (4.3 g, 27.33 mmol) at 0 °C. After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL) and 0.1 N hydrochloric acid solution (50 mL) and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the product was purified by column chromatography to afford Compound 111 (1.65 g, 75%).
’H-NMR (400 MHz, CDCh), δ 9.60 - 9.44 (m, 1H), 4.13 (d, J = 61.6 Hz, 1H), 3.62 - 3.40 (m, 2H), 2.23 - 1.82 (m, 4H), 1.46 (d, J = 19.7 Hz, 9H).
Preparation of Compound 112
To a solution of Lithium chloride (421 mg, 9.94 mmol) and trimethyl phosphonoacetate (2.2 g, 12.42 mmol) in acetonitrile (8 mL) was added triethylamine (1.4 mL, mmol) at 0 °C. The reaction solution was stirred at room temperature for 10 minutes. The reaction solution was added Compound 111 (1.6 g, 8.28 mmol) in acetonitrile (12 mL). After stirring at room temperature for 17 hours, the reaction solution was diluted with diethyl ether (100 mL) and washed with saturated aqueous ammonium chloride solution (50 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 112 (1.43 g, 67%).
’H-NMR (400 MHz, CDCI3) δ 6.14 (t, J = 10.0 Hz, 1H), 5.74 (d, J = 11.4 Hz, 1H), 5.32 - 5.22 (m, 1H), 3.71 (s, 3H), 3.59 - 3.34 (m, 2H), 2.31 (d, J = 12.9 Hz, 1H), 1.84 (ddt, J = 12.5, 8.4, 6.0 Hz, 2H), 1.67 (dt, 7 = 13.2, 6.6 Hz, 1H), 1.42 (d, 7 = 22.5 Hz, 9H).
Preparation of Compound 113
To a solution of Compound 112 (700 mg, 2.74 mmol) in tetrahydrofuran (20 mL) was added sodium hydroxide (219 mg, 5.48 mmol) in distilled water (10 mL) at 0 °C. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (200 mL) and washed with 1 N hydrochloric acid solution (100 mL). The organic layer was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to afford Compound 113 (660 mg, 99%), which was used without further purification.
’H-NMR (400 MHz, CDCI3) δ 6.91 (d, J = 13.0 Hz, 1H), 5.84 (d, J = 15.6 Hz, 1H), 4.46 (d. 7 = 56.8 Hz, 1H), 3.45 (s, 2H), 2.10 (s, 1H), 1.87 (q, 7 = 6.6 Hz, 5H), 1.49 - 1.40 (m, 9H).
Preparation of Compound 114
To a solution of Compound 113 (660 mg, 2.74 mmol) in tetrahydrofuran (10 mL) were added isobutyl chloroformate (0.37 mL, 2.87 mmol) and triethylamine (0.46 mL, 3.28 mmol) at -78 °C. After stirring at room temperature for 1 hour, the reaction solution was added methanol (5 mL) and sodium borohydride (310 mg, 8.21 mmol). The reaction solution was stirred at room temperature for 2 hours and diluted with ethyl acetate (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 114 (494 mg, 79%).
’H-NMR (400 MHz, CDCh), δ 5.66 (s, 1H), 4.31 (d, 7 = 39.6 Hz, 1H), 4.14 (d, 7 = 5.0 Hz, 2H), 3.39 (s, 1H), 2.01 (s, 1H), 1.94 - 1.76 (m, 3H), 1.71 (ddd, 7 = 11.0, 6.7, 3.1 Hz, 4H), 1.45 (d, J = 6.8 Hz, 9H).
Preparation of Compound 115
To a solution of Compound 114 (494 mg, 2.17 mmol) in dichloromethane (20 mL) were added A-methylmorpholine (0.48 mL, 4.34 mmol) and methanesulfonyl anhydride (416 mg, 2.39 mmol) at -78 °C. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated under reduced pressure to afford Compound 115 (632 mg, 95%), which was used without further purification.
’H-NMR (400 MHz, CDCI3), δ 5.82 (s, 1H), 5.67 (s, 1H), 4.72 (d, J = 6.5 Hz, 2H), 4.32 (d, 7 = 39.2 Hz, 1H), 4.17 - 4.09 (m, 1H), 3.40 (s, 1H), 3.02 (s, 3H), 2.05 (s, 1H), 1.84 (p, 7 = 6.4 Hz, 2H), 1.58 (s, 2H), 1.44 (s, 9H).
Preparation of Compound 116
To a solution of Compound 115 (632 mg, 2.07 mmol) and Compound 5 (448 mg, 2.07 mmol) in A,A-dimethylformamide (15 mL) was added potassium carbonate (314 mg, 2.27 mmol) at room temperature under nitrogen. After stirring for 16 hours, the reaction solution was diluted with ethyl acetate (200 mL) and washed with distilled water (100 mL x 2) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 116 (563 mg, 63%).
’H-NMR (400 MHz, DMSO), δ 8.25 (s, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 5.92 - 5.82 (m, 1H), 5.69 - 5.62 (m, 1H), 4.83 (d, J = 5.9 Hz, 2H), 4.21 (s, 1H), 3.29 - 3.19 (m, 2H), 1.77 (q, 7 = 6.7 Hz, 2H), 1.65 (s, 1H), 1.33 (d, 7 = 27.7 Hz, 9H).
Preparation of Compound 117
To a solution of Compound 116 (300 mg, 0.70 mmol) and Compound 9 (511 mg, 0.98 mmol) in n-butanol (4 mL) was added A,A-diisopropylethylamine (0.61 mL, 3.52 mmol) was added at room temperature. After heating to 120 °C and stirring for 20 hours, the reaction solution was cooled to room temperature. The reaction mixture diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 117 (263 mg, 46%). EI-MS m/z : [M+H]+ 801.41.
Preparation of Compound 118
To a solution of Compound 117 (263 mg, 0.32 mmol) in methanol (10 mL) and distilled water (2 mL) were added ammonia solution (28-30% ammonia, 0.5 mL) and sodium hydrosulfite (Na2S2C>4, 572 mg, 3.28 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was added methanol (50 mL). The reaction mixture was filtered through Celite and washed with methanol. The filtrate was concentrated and diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 118 (223 mg, 88%) without purification. EI-MS m/z : [M+H]+ 771.43.
Preparation of Compound 119
To a solution of Compound 118 (223 mg, 0.29 mmol) in A,A-dimethylformamide (2 mL) were added A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide (0.07 mL, 0.43 mmol) and triethylamine (0.12 mL, 0.87 mmol) under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was added Compound 2 (47 mg, 0.24 mmol) in N,N- dimethylformamide (1 mL). After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 119 (99 mg, 36%). EI-MS m/z : [M+H]+ 932.48. Preparation of Compound 120
To a solution of Compound 119 (99 mg, 0.11 mmol) in dichloromethane (1.6 mL) was added trifluoroacetic acid (0.4 mL) at -78 °C under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 120 (56 mg, 64%). Example 18: Preparation of Compound 127
Preparation of Compound 121
To a solution of 5 -nitroindole (1 g, 6.13 mmol) in methanol (15 mL) was added palladium/charcoal (85 mg). After stirring at room temperature under hydrogen balloon for 3 hours, the reaction solution was passed through Celite. The filtrate was concentrated under reduced pressure. The concentrated filtrate was dissolved in A,A-dimethylformamide (15 mL) at room temperature and then di-t-butyl dicarbonate (1.3 g, 6.13 mmol) and diisopropylethylamine (0.79 g, 6.13 mmol) were added. After stirring at room temperature for 5 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 mL x 2) and dried over anhydrous sodium sulfate. The reaction solution was filtered and concentrated under reduced pressure to afford Compound 121 (1.15 g, 80%), which was used without further purification.
’H-NMR (400 MHz, DMSO-d6), δ 12.88 (s, 1H), 9.25 (s, 1H), 7.96 (s, 1H), 7.87 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 1.48 (s, 9H).
Preparation of Compound 122
To a solution of Compound 121 (1 g, 4.28 mmol) in A,A-dimethylformamide (40 mL) were added potassium carbonate (711 mg, 5.14 mmol) and trans- 1 ,4-dibromo-2-butene (2.75 g, 12.86 mmol) at room temperature. After stirring at 60 °C for 18 hours. The reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2) and dried over anhydrous sodium sulfate. The reaction solution was filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 122 (372 mg, 23%).
’H-NMR (400 MHz, DMSO), δ 9.21 (s, 1H), 8.19 (s, 1H), 7.85 (s, 1H), 7.49 (d, J = 8.9 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 6.14 (dd, J = 15.1, 7.1 Hz, 1H), 5.88 (q, J = 7.5 Hz, 1H), 5.05 (d, J = 6.0 Hz, 2H), 4.23 (d, J = 6.8 Hz, 1H), 4.16 (d, J = 7.3 Hz, 2H), 1.48 (s, 9H).
Preparation of Compound 123
To a solution of Compound 122 (369 mg, 1.0 mmol) in A,A-dimethylformamide (4 mL) were added 4-chloro-3-hydroxy-5-nitrobenzamide (182 mg, 0.84 mmol) and potassium carbonate (174 mg, 1.26 mmol) under nitrogen. After stirring at 50 °C for 5 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 123 (312 mg, 74%).
’H-NMR (400 MHz, DMSO-d6), δ 9.23 (s, 1H), 8.24 (d, J = 17.5 Hz, 2H), 8.06 (s, 1H), 7.87 (d, J = 13.6 Hz, 2H), 7.78 (s, 1H), 7.50 (d, J = 9.2 Hz, 1H), 7.22 (d, J = 9.2 Hz, 1H), 6.21 (dt, J = 15.8, 6.2 Hz, 1H), 6.01 - 5.90 (m, 1H), 5.10 (d, J = 6.1 Hz, 2H), 4.86 (d, J = 5.3 Hz, 2H), 1.48 (s, 9H). ELMS m/z : [M+H]+ 502.31.
Preparation of Compound 124
To a solution of Compound 123 (310 mg, 0.62 mmol) and Compound 9 (406 mg, 0.98 mmol) in n-butanol (6 mL) was added diisopropylethylamine (0.21 mL, 1.24 mmol) at room temperature. After heating to 120 °C and stirring for 20 hours, the reaction solution was cooled to room temperature. The reaction mixture was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. After dilution with dichloromethane and diethyl ether, the obtained solid was filtered. The solid was dried to afford Compound 124 (crude), which was used without further purification. EIMS m/z : [M+H]+ 877.59.
Preparation of Compound 125
To a solution of Compound 124 (0.20 mmol, crude) in methanol (7 mL) and distilled water (1 mL) were added ammonia solution (28-30% ammonia, 0.2 mL) and sodium hydrosulfite (Na2S2C>4, 355 mg, 2.04 mmol). After stirring at room temperature for 1 hour, The reaction mixture was diluted with methanol (50 mL) and filtered. The filtrate was concentrated and diluted with acetonitrile (10 mL), and the obtained solid was filtered. The solid was dried to afford Compound 125 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 847.58.
Preparation of Compound 126
To a solution of Compound 125 (0.20 mmol, crude) in N, A-dimethylformamide (4 mL) was added Compound 2 (47 mg, 0.24 mmol) in A, A -dimethylformamide (1 mL) under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was added A-(3- dimethylaminopropyl)-N’-ethylcarbodiimide (47 mg, 0.30 mmol) and triethylamine (0.72 mL, 0.61 mmol) at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 126 (83 mg, 40%). EI-MS m/z : [M+H]+ 1008.64.
Preparation of Compound 127
To a solution of Compound 126 (33 mg, 0.03 mmol) in dichloromethane (1.6mL) was added trifluoroacetic acid (0.4mL) at -78 °C under nitrogen. After stirring at room temperature fori hour, the reaction solution concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 127 (11 mg, 27%).
’H-NMR (400 MHz, DMSO-d6), δ 12.84 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.93 (s, 1H), 7.68 - 7.60 (m, 3H), 7.45 (s, 1H), 7.37 - 7.28 (m, 3H), 7.06 (d, J = 9.2 Hz, 1H), 6.52 (s, 2H), 6.02 (dt, 7 = 14.1, 6.5 Hz, 1H), 5.79 (s, 2H), 4.90 (dd, 7 = 20.3, 7.7 Hz, 5H), 4.52 (dq, 7 = 14.1, 6.3 Hz, 5H), 3.69 (s, 3H), 2.10 (d, 7 = 10.6 Hz, 6H), 1.25 (q, 7 = 7.9 Hz, 6H). EI-MS m/z : [M+H]+ 908.54.
Example 19: Preparation of Compound 133
Preparation of Compound 128
To a solution of t-butyl carbazate (5 g, 37.83 mmol) in A,A-dimethylformamide (30 mL) was added sodium hydride (60%, 3.8 g, 94.6 mmol) at 0 °C. After stirring at 0°C for 0.5 hours, the reaction solution was added 1,3-dibromopropane (3.8 mL, 37.8 mmol) at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate (300 mL) and washed with distilled water (150 mL x 2). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated. The resulting residue was purified by column chromatography to afford Compound 128 (3.9 g, 57%).
’H-NMR (400 MHz, CDC13), δ 3.86 (s, 1H), 3.49 - 3.41 (m, 2H), 3.07 - 2.99 (m, 2H), 2.03 (p, J = 6.8 Hz, 2H), 1.52 - 1.44 (m, 9H).
Preparation of Compound 129
To a solution of Compound 128 (1.2 g, 6.86 mmol) and Compound 5 (2.0 g, 5.72 mmol) in A,A-dimethylformamide (15 mL) was added potassium carbonate (1.1 g, 8.58 mmol) at room temperature under nitrogen. After stirring for 18 hours, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with distilled water (100 mLx2). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated. The resulting residue was purified by column chromatography to afford Compound 129 (2.0 g, 80%). ’H-NMR (400 MHz, DMSO), δ 8.28 (s, 1H), 8.05 (d, J = 2.1 Hz, 1H), 7.88 (s, 1H), 7.78 (s, 1H), 6.02 - 5.83 (m, 2H), 4.81 (d, 7 = 5. 1 Hz, 2H), 3.26 (d, J = 6.0 Hz, 2H), 2.84 (t, J = 6.8 Hz, 2H), 2.00 (t, J = 7.3 Hz, 2H), 1.37 (s, 9H).
Preparation of Compound 130
To a solution of Compound 129 (2 g, 4.54 mmol) and Compound 9 (4.7 g, 9.07 mmol) in n-butanol (35 mL) were added A,A-diisopropylethylamine (5.5 mL, 31.8 mmol) at room temperature and heated to 100 °C. After stirring for 21 hours, the reaction mixture was cooled to room temperature and diluted with dichloromethane and diethyl ether. The resulting solid was filtered and washed ether. The filtered solid was purified by column chromatography to afford Compound 130 (1.0 g, 28%). EI-MS m/z : [M+H]+ 816.52.
Preparation of Compound 131
To a solution of Compound 130 (1.0 g, 1.29 mmol) in methanol (20 mL) and water (4 mL) were added ammonia solution (28-30% ammonia, 1.4 mL) and sodium hydrosulfite (Na2S2C>4, 2.2 g, 12.87 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was added methanol (50 mL). The resulting solid was filtered. The filtrate was concentrated and diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated. The resulting residue was purified by column chromatography to afford Compound 131 (1.0 g, crude). EI-MS m/z : [M+H]+ 786.58.
Preparation of Compound 132
To a solution of Compound 131 (1.0 g, 1.29 mmol) in A,A-dimethylformamide (6 mL) was added Compound 2 (301 mg, 1.54 mmol) in A,A-dimethylformamide (3 mL) under nitrogen at room temperature. After stirring for 1 hour, the reaction mixture was added N-(3- dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (0.34 mL, 1.93 mmol) and triethylamine (0.36 mL, 2.57 mmol) at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 132 (545 mg, 44%). EI-MS m/z : [M+H]+ 947.62.
Preparation of Compound 133
To a solution of Compound 132 (63 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.2 mL) was added at -0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 133 (42 mg, 53%). EI-MS m/z : [M+H]+847.55. Example 20: Preparation of Compound 139
Preparation of Compound 134 To a solution of Compound 5 (300 mg, 0.86 mmol) in M AMimcthylformamidc (5 mL) was added sodium azide (84 mg, 1.29 mmol) at 0 °C. After stirring at 0 °C for 2 hours, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with distilled water (150 mL x 2). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated and diluted with dichloromethane and hexane. The resulting solid was filtered and dried to afford Compound 134 (225 mg, 84%), which was used without further purification.
’H-NMR (400 MHz, DMSO), δ 8.27 (s, 1H), 8.07 (d, J = 1.7 Hz, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 6.11 - 5.94 (m, 2H), 4.88 (d, J = 4.7 Hz, 2H), 3.97 (d, J = 5.5 Hz, 2H).
Preparation of Compound 135 To a solution of Compound 134 (225 mg, 0.72 mmol) in ethanol (3 mL), dichloromethane (2 mL) and water (3 mL) were added t-butyl propa-2-ynylcarbamate (145 mg, 0.94 mmol), copper(II) sulfate pentahydrate (36 mg, 0.14 mmol) and sodium L-ascorbate (57 mg, 0.29 mmol) at 0 °C. After stirring at room temperature for 2 hours, the reaction mixture was diluted with dichloromethane (100 mL) and methanol (10 mL) and washed with saturated aqueous ammonium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated then diluted with dichloromethane and hexane. The resulting solid was filtered and dried to afford Compound 135 (283 mg, 84%), which was used without further purification.
’H-NMR (400 MHz, DMSO), δ 8.27 (s, 1H), 8.06 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.78 (s, 1H), 7.30 (s, 1H), 6.17 - 6.07 (m, 1H), 5.97 - 5.93 (m, 1H), 5.07 (d, 7 = 6.0 Hz, 2H), 4.84 (d, J = 5.2 Hz, 2H), 4.16 (d, J = 5.9 Hz, 2H), 1.38 (s, 9H).
Preparation of Compound 136
To a solution of Compound 135 (243 mg, 0.52 mmol) and Compound 9 (428 mg, 1.04 mmol) in n-butanol (3 mL) were added A,A-diisopropylethylamine (0.45 mL, 2.60 mmol) at room temperature. After heating to 100 °C and stirring for 21 hours, the reaction solution was to room temperature. The reaction solution was diluted with dichloromethane (100 mL), methanol (20 mL) and washed with saturated aqueous ammonium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated. The resulting residue was purified by column chromatography to afford Compound 136 (241 mg, 55%). EI-MS m/z : [M+H]+ 842.54.
Preparation of Compound 137
To a solution of Compound 136 (241 mg, 0.29 mmol) in methanol (10 mL) and water (2 mL) were added ammonia solution (28-30% ammonia, 0.4 mL) and sodium hydrosulfite (Na2S2C>4, 498 mg, 2.86 mmol) to the reaction mixture under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was added methanol (50 mL). The resulting solid was filtered. The filtrate was concentrated and diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated to afford Compound 137 (168 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 812.54.
Preparation of Compound 138
To a solution of Compound 137 (168 mg, 0.21 mmol) in A,A-dimethylformamide (2 mL) was added Compound 2 (48 mg, 0.25 mmol) in A,A-dimethylformamide (1 mL) to the reaction mixture under nitrogen at room temperature. After stirring for 1 hour, the reaction solution was added A-(3-dimethylaminopropyl)-A-ethylcarbodiimide hydrochloride (59 mg, 0.31 mmol) and triethylamine (0.09 mL, 0.62 mmol) at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 138 (175 mg, 87%). EI-MS m/z : [M+H]+ 973.60. Preparation of Compound 139
To a solution of Compound 138 (72 mg, 0.07 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.5 mL) at -0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 139 (51 mg, 56%). EI-MS m/z : [M+H]+ 873.52.
Example 21: Preparation of Compound 147
Preparation of Compound 140
To a solution of di-t-butyl-iminodiacetate (684 mg, 3.15 mmol) in N,N- dimethylformamide (7 mL) was added cesium carbonate (1.12 mg, 3.43 mmol). After stirring at room temperature for 10 minutes, the reaction solution was added Compound 5 (1 g, 2.86 mmol) at room temperature for 17 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous magnesium sulfate and then filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 140 (1.05 g, 71%). ’H-NMR (400 MHz, CDCh), δ 7.79 (d, 7 = 1.9 Hz, 1H), 7.63 (d, 7 = 1.9 Hz, 1H), 6.62 (s, 1H), 5.95 (dt, 7 = 16.0, 5.2 Hz, 1H), 5.81 (dt, 7 = 15.7, 5.6 Hz, 1H), 4.74 (d, J = 5.4 Hz, 2H), 4.24 (d, 7 = 5.1 Hz, 2H), 1.48 (s, 18H).
Preparation of Compound 141
To a solution of Compound 140 (423 mg, 0.87 mmol) in methanol (4 mL) and tetrahydrofuran (10 mL) were added sodium hydroxide (105 mg, 2.61 mmol) in water (0.5 mL). After stirring at room temperature for 1 hour, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous magnesium sulfate and then filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 141 (223 mg, 69%).
’H-NMR (400 MHz, DMSO), δ 8.24 (s, 1H), 8.04 (d, 7 = 2.2 Hz, 1H), 7.88 (s, 1H), 7.76 (s, 1H), 7.04 (s, 1H), 5.95 - 5.72 (m, 2H), 4.80 (d, 7 = 5.5 Hz, 2H), 3.60 (d, 7 = 6.0 Hz, 2H), 1.37 (s, 9H).
Preparation of Compound 142
To a solution of Compound 141 (650 mg, 1.68 mmol) and Compound 9 (831 mg, 2.02 mmol) in n-butanol (8 mL) was added A,A-diisopropylethylamine (1.2 mL, 8.42 mmol) at - 0°C. After stirring for 5 minutes, the reaction mixture was heated to 120°C for 20 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 142 (297 mg, 23%). EI-MS m/z : [M+H]+ 761.45.
Preparation of Compound 143
To a solution of Compound 142 (294 mg, 0.39 mmol) in methanol (6 mL) were added ammonia solution (28-30% ammonia, 0.7 mL, 9.5 mmol) and sodium hydrosulfite (Na2S2C>4, 672 mg, 3.86 mmol). After stirring at room temperature for 1 hour, the resulting solid was filtered through Celite and washed with methanol. The filtrate was concentrated under reduced pressure to afford Compound 143 (202 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 731.5.
Preparation of Compound 144
To a solution of Compound 143 (202 mg, 0.28 mmol) in A,A-dimethylformamide (1 mL) was added Compound 2 (30 mg, 0.15 mmol) in A,A-dimethylformamide (1 mL). After stirring at 0 °C for 30 minutes, the reaction solution was added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide (43.7 mg, 0.28 mmol). The reaction solution was stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 144 (122 mg, 49%). EIMS m/z : [M+H]+ 892.5.
Preparation of Compound 145
To a solution of Compound 144 (50 mg, 0.05 mmol) in dichloromethane (0.8 mL) was added trifluoroacetic acid (0.2 mL) at -0 °C. After reaction mixture was raised to room temperature and stirred under nitrogen for 1 hour, the reaction mixture was concentrated under reduced pressure to afford Compound 145 (63 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 792.53.
Preparation of Compound 146
To a solution of Compound 145 (35.4 mg, 0.04 mmol) in A,A-dimethylformamide (1 mL) was added A,A-diisopropylethylamine (0.04 mL, 0.22 mmol), carbonyldiimidazole (22 mg, 0.13 mmol), and l-(t-butoxycarbonyl)piper azine (25 mg, 0.13 mmol). After stirring at room temperature for 20 hours, the reaction solution was concentrated under reduced pressure to afford Compound 146 (45 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1004.59.
Preparation of Compound 147
To a solution of Compound 146 (45 mg, 0.04 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at -0 °C. After stirring at room temperature under nitrogen for 1 hour, the reaction mixture was concentrated. The resulting residue was purified by HPLC to afford Compound 147 (5.4 mg, 9.5%).
Example 22: Preparation of Compound 151
Preparation of Compound 148
To a solution of 4-ehyl-2-methyl-oxazole-5-carboxylic acid (100 mg, 0.64 mmol, prepared according to the method described in the Chinese patent publication No. CN 111471056 A) in tetrahydrofuran (1 mL) was added oxalyl chloride (0.82 mL, 0.96 mmol) and lV,lV-dimethylformamide (0.1 mL) at 0 °C. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 148 (crude), which was used without further purification.
Preparation of Compound 149
To a solution of Compound 148 (crude) in acetone (1 mL) was added potassium thiocyanate (125 mg, 1.28 mmol) at 0 °C. After stirring at room temperature for 30 minutes, the reaction solution was added hexane (10 mL). The resulting solid was filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 149 (64 mg, 50%).
’H-NMR (400 MHz, CDC13), δ 2.90 (q, J = 7.6 Hz, 2H), 2.54 (s, 3H), 2.72 (t, J = 7.6 Hz, 3H). ELMS m/z: [M+H]+ 197.21.
Preparation of Compound 150
To a solution of Compound 53 (112 mg, 0.14 mmol) in A,A-dimethylformamide (1.5 mL) was added Compound 149 (30 mg, 0.15 mmol) in A,A-dimethylformamide (1 mL). After stirring at 0°C for 30 min, the reaction solution was added A-(3-dimethylaminopropyl)-M- ethylcarbodiimide (43.7 mg, 0.28 mmol) and triethylamine (0.06 mL, 0.42 mmol) and stirred at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 150 (25 mg, 19%). ELMS m/z : [M+H]+ 959.24.
Preparation of Compound 151
To a solution of Compound 150 (25 mg, 0.03 mmol) in dichloromethane (0.8 mL) was added trifluoroacetic acid (0.2 mL) at -0 °C. After stirring at room temperature for 2 hours. The reaction solution was concentrated. The resulting residue was purified by HPLC to afford Compound 151 (9.3 mg, 42%).
’H-NMR (400 MHz, DMSO-d6), δ 8.00 - 7.83 (m, 2H), 7.65 (s, 1H), 7.54 (s, 1H), 7.36 (s, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.91 (d, 7 = 15.0 Hz, 1H), 5.76 (d, 7 = 14.3 Hz, 2H), 4.89 (s, 3H), 4.66 (d, 7 = 5.9 Hz, 1H), 4.51 (d. 7 = 7.3 Hz, 2H), 3.71 (s, 2H), 2.82 (q, 7 = 7.9 Hz, 1H), 2.40 (s, 2H), 2.10 (s, 2H), 1.25 (t, J = 7.2 Hz, 2H), 1.02 (t, J = 7.7 Hz, 2H). ELMS m/z: [M+H]+ 859.27.
Example 23: Preparation of Compound 155
Preparation of Compound 152
To a solution of 4-ethyl-2-methylthiazole-5-carboxylic acid (100 mg, 0.58 mmol) in tetrahydrofuran (1 mL) were added oxalyl chloride (0.75 mL, 0.87 mmol) and N,N- dimethylformamide (0.1 mL) at 0 °C. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 152 (crude), which was used without further purification.
Preparation of Compound 153
To a solution of Compound 152 (crude) in acetone (1 mL) was added potassium thiocyanate (113 mg, 1.16 mmol) at 0 °C. After stirring at room temperature for 30 minutes, the reaction mixture was added hexane (10 mL). The resulting solid was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 153 (21 mg, 16%). ELMS m/z : [M+H]+ 213.20.
Preparation of Compound 154
To a solution of Compound 53 (166 mg, 0.15 mmol) in A,A-dimethylformamide (1.5 mL) was added Compound 153 (35 mg, 0.16 mmol) dissolved in A,A-dimethylformamide (1 mL) at 0 °C. After stirring for 30 minutes, the reaction solution was added N-(3- dimethylaminopropyl)-A’-ethylcarbodiimide (46 mg, 0.29 mmol) and triethylamine (0.06 mL, 0.44 mmol) at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 154 (30 mg, 21%). ELMS m/z : [M+H]+ 975.26.
Preparation of Compound 155
To a solution of Compound 154 (30 mg, 0.03 mmol) in dichloromethane (0.8 mL) was added trifluoroacetic acid (0.2 mL) at -0 °C. After stirring at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 155 (18 mg, 68%).
’H-NMR (400 MHz, DMSO-d6), δ 7.88 (d, 7 = 14.1 Hz, 1H), 7.82 (s, 1H), 7.56 (d, 7 = 8.4 Hz, 1H), 7.47 (s, OH), 7.30-7.21 (m, 2H), 6.45 (s, 1H), 5.77 (dq, 2H), 4.81 (d, 7 = 17.4 Hz, 2H), 4.60 (d, 7 = 6.0 Hz, 1H), 4.51 (s, 1H), 4.44 (d. 7 = 7.7 Hz, 1H), 3.64 (s, 2H), 3.03 (q, 7 = 7.9 Hz, 2H), 2.66 (s, 2H), 2.02 (s, 2H), 1.19 (t, J = 7.4 Hz, 2H), 1.07 (t, J = 7.7 Hz, 2H). EL MS m/z : [M+H]+ 875.24.
Example 24: Preparation of Compound 160
Preparation of Compound 156
To a solution of Compound 50 (520 mg, 1.64 mmol) in N,N-dimethylformamide (5 mL) were added Potassium phthalimide (335 mg, 1.81 mmol). After stirring at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure to afford Compound 156 (592 mg, 94%), which was used without further purification.
Preparation of Compound 157
To a solution of Compound 156 (592 mg, 1.55 mmol) in methanol (10 mL) was added hydrazine monohydrate (0.2 mL, 4.64 mmol). After stirring at room temperature for 1 hour, the reaction solution was added Dichloromethane and diethyl ether. The resulting solid was filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 157 (352 mg, 90%).
Preparation of Compound 158
To a solution of Compound 157 (350 mg, 1.39 mmol) in dichloromethane (20 mL) were added A, A’ -diisopropylethylamine (1.2 mL, 6.93 mmol) and bis(pentafluorophenyl)carbonate (1.6 g, 4.16 mmol) at -0 °C. After stirring at room temperature for 1 hour, the reaction mixture was concentrated under reduced pressure. The reaction mixture was added dichloromethane and hexane. The resulting solid was filtered and dried to afford Compound 158 (310 mg, 24%), which was used without further purification. Preparation of Compound 159
To a solution of Compound 158 (97 mg, 0.11 mmol) in A,A-dimethylformamide (2 mL) were added Compound 75 (50 mg, 0.05 mmol) and N, A-diisopropylethylamine (0.05 mL, 0.26 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The reaction mixture was added dichloromethane and hexane. The resulting solid was filtered and dried to afford Compound 159 (59 mg, crude), which was used without further purification.
Preparation of Compound 160
To a solution of Compound 159 (59 mg, crude) in dichloromethane (1.5 mL) was added trifluoroacetic acid (0.5 mL) at -0 °C. After stirring at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 160 (30 mg, 45%).
Example 25: Preparation of Compound 164
Preparation of Compound 161 To a solution of methyl 4-nitro- 1 Z7-pyrazole-3-carboxylate (100 mg, 0.54 mmol) in A,A-dimcthylformamidc (3 mL) were added cesium carbonate (285 mg, 0.88 mmol) and trans- 1 ,4-dibromo-2-butene (625 mg, 2.92 mmol) After stirring at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 161 (111 mg, 62%). EI-MS m/z : [M+H]+ 304.11.
Preparation of Compound 162
To a solution of Compound 66 (50 mg, 0.07 mmol) in N, A-dimethylformamide (2 mL) were added cesium carbonate (34 mg, 0.1 mmol) and Compound 161 (23 mg, 0.08 mmol). After stirring at room temperature for 2 hours, the reaction mixture was diluted with dichloromethane (20 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 162 (16 mg, 24%). EI-MS m/z : [M+H]+ 946.27.
Preparation of Compound 163
To a solution of Compound 162 (67 mg, 0.07 mmol) in acetic acid (1 mL) was added zinc powder (46 mg). After stirring at room temperature for 2 hours, the reaction mixture was filtered through Celite then washed with methanol. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 163 (3.2 mg, 3.6%). EI-MS m/z : [M+H]+ 916.03.
Preparation of Compound 164
To a solution of Compound 163 (43 mg, 0.05 mmol) in methanol (1.5 mL) was added lithium hydroxide monohydrate (24 mg, 0.14 mmol) dissolved in water (0.5 mL) at -50 °C. After stirring at 0 °C for 20 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 164. (1 mg, 1.7%). EI-MS m/z : [M+H]+ 902.00. Example 26: Preparation of Compound 166
Preparation of Compound 165
To a solution of Compound 145 (40 mg, 0.04 mmol) in A,A-dimethylformamide (1 mL) were added triethylamine (0.10 mL, 0.745 mmol) and A/,A/-bis(t-butoxycai'bonyl)- l //- pyrazole-l-carboxamidine (17 mg, 0.06 mmol) After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 165 (37 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1035.01.
Preparation of Compound 166 To a solution of Compound 165 (45 mg, 0.04 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.5 mL) at -0 °C. After stirring at room temperature for 1 hour, the reaction mixture was concentrated. The resulting residue was purified by HPLC to afford Compound 166 (23.8 mg, 46%). EI-MS m/z : [M+H]+ 835.09. Example 27: Preparation of Compound 168
Preparation of Compound 167
To a solution of Compound 67 (2.0 g, 2.07 mmol, Compound 67 was prepared according to the method described in the International Patent Publication No. WO 2022/155518 Al) in A Wdimcthylformamidc (20 mL) were added cesium carbonate (5.4 g, 16.54 mmol) and Compound 50 (654 mg, 2.07 mmol) in A Wdimcthylfoi mamidc (5 mL). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure and chloroform (100 mL) and methanol (20 mL) were added and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by reversed-phase column chromatography to afford Compound 167 (958 mg, 38%). EI-MS m/z : [M+H]+ 1179.46.
Preparation of Compound 168
To a solution of Compound 167 (40 mg, 0.03 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at -0 °C. After stirring at room temperature under nitrogen for 1.5 hour, the reaction mixture was concentrated. The resulting residue was purified by HPLC to afford Compound 168 (17 mg, 29%). EI-MS m/z : [M+H]+ 979.29.
Example 28: Preparation of Compound 175
Preparation of Compound 169
To a solution of 4-aminopyrazole (5.89 g, 70.8 mmol) was in tetrahydrofuran (200 mL) were added triethylamine (15 mL, 106.13 mmol) and di-t-butyl dicarbonate (48.8 mL, 212.26 mmol) under nitrogen. After stirring at room temperature for 20 hours, the reaction mixture was added ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 169 (5.9 g, 29%).
’H-NMR (400 MHz, CDC13), δ 8.19 (s, 1H), 7.63 (s, 1H), 6.34 (s, 1H), 1.64 (d, J = 3.9 Hz, 9H), 1.52 (s, 9H).
Preparation of Compound 170
To a solution of Compound 169 (1.1 g, 3.88 mmol) in acetonitrile (30 mL) were added potassium carbonate (590 mg, 4.27 mmol), 18-crown-6 (513 mg, 1.94 mmol) and methyl acrylate (367 mg, 4.27 mmol). After stirring at room temperature for 30 minutes, the reaction solution was added Ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 170. (1.38 g, 96%).
’H-NMR (400 MHz, CDC13), δ 7.77 (s, 1H), 7.27 (d, J = 3.1 Hz, 1H), 3.92 (dt, J = 9.4, 5.8 Hz, 2H), 3.71 - 3.65 (m, 3H), 2.68 - 2.59 (m, 2H), 1.65 (q, J = 2.4 Hz, 9H), 1.54 - 1.48 (m, 9H). ELMS m/z : [M+H]+ 370.32.
Preparation of Compound 171
To a solution of Compound 170 (1.38 g, 3.73 mmol) in methanol (20 mL) was added potassium carbonate (770 mg, 5.6 mmol) at -0°C. After stirring at room temperature for 30 minutes, the reaction solution was added Ethyl acetate (50 mL) was added and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 171 (950 mg, 94%).
’H-NMR (400 MHz, CDC13), δ 7.66 (s, 1H), 7.53 (s, 1H), 3.96 - 3.87 (m, 2H), 3.67 (s, 3H), 2.64 (p, J = 5.0 Hz, 2H), 1.50 (s, 9H).
Preparation of Compound 172
To a solution of Compound 171 (167 mg, 0.62 mmol) in A,A-dimethylformamide (30 mL) were added cesium carbonate (303 mg, 0.93 mmol) and trans- l,4-dibromo-2-butene (397 mg, 1.86 mmol). After stirring at room temperature for 3 hours, the reaction solution was Ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 172 (177 mg, 71%). ELMS m/z : [M+H]+ 402.24.
Preparation of Compound 173 To a solution of Compound 66 (264 mg, 0.37 mmol) in A,A-dimethylformamide (2 mL) were added cesium carbonate (179 mg, 0.43 mmol) and Compound 172 (177 mg, 0.44 mmol). After stirring at room temperature for 12 hours, the reaction solution was added Ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 173 (133 mg, 34%). ELMS m/z : [M+H]+ 1045.34.
Preparation of Compound 174
To a solution of Compound 173 (150 mg, 0.14 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.25 mL) at -0 °C. After stirring at room temperature for 1 hour, the reaction mixture was concentrated under reduced pressure to afford Compound 174 (135 mg, crude), which was used without further purification.
Preparation of Compound 175
To a solution of Compound 174 (135 mg, crude) in methanol (1 mL) was added lithium hydroxide monohydrate (11.7 mg, 0.28 mmol) in water (1 mL) at -45 °C. After stirring at -0°C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 175 (34 mg, 42%). ELMS m/z : [M+H]+ 902.
Example 29: Preparation of Compound 178
Preparation of Compound 176
To a solution of Compound 49 (300 mg, 1.64 mmol) in A,A-dimethylformamide (10 mL) were added cesium carbonate (693 mg, 2.13 mmol) and l,4-dibromo-2-butene (1.04 g, 4.91 mmol). After stirring at room temperature for 1 hours, the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 176 (320 mg, 62%).
Preparation of Compound 177
To a solution of Compound 66 (400 mg, 0.42 mmol, Compound 66 was prepared according to the method described in the International Patent Publication No. WO 2022/155518 Al) in A/iV-dimethylformamide (5 mL) were added cesium carbonate (548 mg, 1.68 mmol) and Compound 176 (158 mg, 0.50 mmol) in A/Wdimethylformamide (2 mL). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure. The reaction mixture was added dichloromethane (50 mL) and methanol (10 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 177 (246 mg, 61%). EI-MS m/z : [M+H]+ 956.52.
Preparation of Compound 178
To a solution of Compound 177 (246 mg, 0.26 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1 mL) at -0 °C. After stirring at room temperature under nitrogen for 1.5 hour, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 178 (92 mg, 30%). EI-MS m/z : [M+H]+ 856.48.
Example 30: Preparation of Compound 187
Preparation of Compound 179
To a solution of 3-bromopropanol (2.0 g, 14.39 mmol) in acetone (30 mL) was added potassium thiocyanate (1.8 g, 15.83 mmol) at -0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 179 (1.26 g, 65%).
’H-NMR (400 MHz, CDC13), δ 3.65 (q, J = 5.6 Hz, 2H), 3.01 (dq, J = 9.3, 3.3 Hz, 2H), 2.39 - 2.30 (m, 3H), 2.04 (t, J = 5.8 Hz. 1H), 1.83 (q, J= 6.0 Hz, 2H).
Preparation of Compound 180
To a solution of Compound 179 (1.26 g, 9.39 mmol) in dichloromethane (30 mL) was added imidazole (958 mg, 14.08 mmol) and triisopropylsilyl chloride (2.0 g, 10.33 mmol) at - 0 °C. After stirring at room temperature for 4 hours, the reaction mixture was diluted with dichloromethane (100 mL), washed with saturated aqueous ammonium chloride solution (70 mL) and distilled water (70 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 180 (2.7 g, 99%).
’H-NMR (400 MHz, CDCh), δ 3.74 (q, J = 5.4 Hz, 2H), 2.99 (q, J = 6.3 Hz, 2H), 2.35 - 2.30 (m, 3H), 1.81 (q, J = 6.2 Hz, 2H), 1.06 (s, 21H).
Preparation of Compound 181
To a solution of Compound 180 (2.7 g, 9.29 mmol) in methanol (30 mL) were added methyl iodide (0.69 mL, 10.2 mmol) and potassium carbonate (4.1 g, 30.25 mmol) at -0 °C. After stirring at room temperature for 30 minutes, the reaction mixture was diluted with dichloromethane (100 mL) and washed with distilled water (70 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 181 (1.73 g, 65%).
’H-NMR (400 MHz, CDCh), δ 3.86 - 3.73 (m, 2H), 2.60 (tt. 7 = 7.0. 2.5 Hz, 2H), 2.11 (q, 7 = 2.3 Hz, 3H), 1.82 (q, 7 = 6.2 Hz, 2H), 1.15 - 1.02 (m, 21H).
Preparation of Compound 182
To a solution of Compound 181 (1.2 g, 4.57 mmol) in methanol (20 mL) were added iodobenzene diacetate (3.7 g, 11.43 mmol) and ammonium carbonate (1.3 g, 13.71 mmol) at - 0 °C. After stirring and refluxing for 2 hours, the reaction mixture was concentrated. The resulting residue was purified by purified by HPLC to afford Compound 182 (1.5 g, crude).
’H-NMR (400 MHz, CDCI3) δ 3.84 (q, J = 5.4 Hz, 2H), 3.28 - 3.21 (m, 2H), 3.03 - 2.97 (m, 3H), 2.11 - 2.03 (m, 2H), 1.06 (d, J = 4.4 Hz, 21H).
Preparation of Compound 183
To a solution of Compound 182 (1.5 g, crude) in dichloromethane (20 mL) were added pyridine (0.71 mL, 8.86 mmol) and ethyl chloroformate (0.51 mL, 5.31 mmol) at 0 °C. After stirring at room temperature for 2 hours, the reaction mixture was diluted with dichloromethane (100 mL), washed with 0.5 N hydrochloric acid solution (70 mL) and distilled water (70 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 183 (1.47 g, 91%).
’H-NMR (400 MHz, CDCh), δ 4.15 (s, 2H), 3.83 (t, 7 = 5.2 Hz, 2H), 3.51 (d, 7 = 10.0 Hz, 2H), 3.25 (s, 3H), 2.10 (s, 2H), 1.29 (t, 7 = 5.8 Hz, 3H), 1.06 (d, 7 = 4.3 Hz, 21H).
Preparation of Compound 184
To a solution of Compound 183 (1.5 g, 4.02 mmol) in dichloromethane (30 mL) was added hydrochloric acid (4 M 1,4-dioxane solution, 12 mL) at 0 °C. After stirring for 2.5 hours, the reaction mixture was concentrated. The reaction mixture was added ethyl acetate (100 mL) and distilled water (70 mL). The obtained aqueous layer was concentrated. The reaction mixture was added dichloromethane (50 mL), methanol (5 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 184 (752 mg, 89%).
’H-NMR (400 MHz, CDCI3), δ 4.14 (tt, J = 8.5, 4.5 Hz, 2H), 3.81 (t, J = 5.4 Hz, 2H), 3.67 - 3.42 (m, 2H), 3.31 - 3.25 (m, 3H), 2.16 (q, J = 6.1 Hz, 2H), 1.29 (dt, J = 8.7, 4.8 Hz, 3H).
Preparation of Compound 185
To a solution of Compound 184 (50 mg, 0.24 mmol) in dichloromethane (3 mL) were added triethylamine (0.07 mL, 0.48 mmol) and methanesulfonyl anhydride (50 mg, 0.29 mmol) at -0 °C. After stirring at room temperature for 2 hours, the reaction mixture was diluted with dichloromethane (50 mL) and washed with distilled water (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to afford Compound 185 (50 mg, 73%), which was used without further purification.
’H-NMR (400 MHz, CDCh), δ 4.41 (d, J = 5.9 Hz, 2H), 4.15 (dq, J = 10.5, 3.7 Hz, 2H), 3.51 (d, J = 53.9 Hz, 2H), 3.30 (q, J = 2.4 Hz, 3H), 3.06 (q, J = 2.4 Hz, 3H), 2.40 (d, J = 8.7 Hz, 2H), 1.35 - 1.25 (m, 3H).
Preparation of Compound 186
To a solution of Compound 66 (100 mg, 0.11 mmol, Compound 66 was prepared according to the method described in the International Patent Publication No. WO 2022/155518 Al) in A,/V-dimethylformamide (2 mL) were added cesium carbonate (113 mg, 0.35 mmol) and Compound 185 (36 mg, 0.13 mmol) dissolved in WN-dimethylformamide (1 mL). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure. The reaction mixture was added dichloromethane (50 mL) and methanol (10 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 186 (48 mg, 50%). EI-MS m/z : [M+H]+ 914.41.
Preparation of Compound 187
To a solution of Compound 186 (48 mg, 0.05 mmol) in ethanol (20 mL) was added sodium ethoxide (21% w/w ethanol, 0.24 mL, 0.64 mmol). After stirring and refluxing for 14 hours, the reaction mixture was concentrated. The resulting residue was purified by HPLC to afford Compound 187 (25 mg, 55%). EI-MS m/z : [M+H]+ 842.39.
Example 31: Preparation of Compound 197
Preparation of Compound 188
To a solution of Compound 4 (400 mg, 1.85 mmol) in A,A-dimethylformamide (5 mL) were added cesium carbonate (782 mg, 2.40 mmol) and Compound 185 (584 mg, 2.03 mmol). After stirring at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 x 2 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 188 (600 mg, 80%).
’H-NMR (400 MHz, CDCh), δ 7.85 (s, 1H), 7.73 (d, J = 4.3 Hz, 1H), 4.38 (q, J = 6.0 Hz, 2H), 4.13 (q, J = 6.5 Hz, 2H), 3.81 - 3.65 (m, 1H), 3.54 (d, J = 14.8 Hz, 1H), 3.37 - 3.25 (m, 3H), 2.56 - 2.48 (m, 2H), 1.29 (q, J = 6.6 Hz, 3H).
Preparation of Compound 189
To a solution of Compound 188 (600 mg, 1.47 mmol) in ethanol (10 mL) were added t-butyl (E)-(4-aminobut-2-en- 1 -yl)carbamate (548 mg, 2.94 mmol) and triethylamine (0.62 mL, 4.41 mmol). After stirring at 120 °C for 20 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 189 (550 mg, 67%). EI-MS m/z : [M+H]+ 558.31.
Preparation of Compound 190
To a solution of Compound 189 (550 mg, 0.99 mmol) in methanol (5 mL) and distilled water (1 mL) were added ammonia solution (28-30% ammonia, 1 mL) and sodium hydrosulfite (Na2S2C>4, 1.7 g, 9.86 mmol) at -0 °C. After stirring at room temperature for 1.5 hour, the reaction solution was added methanol (10 mL). The resulting solid was filtered and washed with methanol. The filtrate was concentrated under reduced pressure. The reaction mixture was added and washed with distilled water (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 190 (339 mg, 65%), which was used without further purification. EI-MS m/z : [M+H]+ 528.39.
Preparation of Compound 191
To a solution of Compound 190 (339 mg, 0.64 mmol) in A,A-dimethylformamide (3 mL) was added Compound 2 (150 mg, 0.77 mmol) at -0 °C. After stirring at room temperature for 30 minutes, the reaction solution was added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide hydrochloride (160 mg, 0.84 mmol) and triethylamine (0.05 mL, 0.38 mmol) at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 191 (390 mg, 88%). EI-MS m/z : [M+H]+ 689.37.
Preparation of Compound 192
To a solution of Compound 191 (390 mg, 0.57 mmol) in dichloromethane (5 mL) and methanol (1 mL) was added hydrochloric acid (4 M 1,4-dioxane solution, 1.5 mL). After stirring for 2 hours, the reaction mixture was concentrated. The reaction mixture was added diethyl ether (20 mL). The resulting solid was filtered and dried to afford Compound 192 (360 mg, 96%). EI-MS m/z : [M+H]+ 589.38.
Preparation of Compound 193
To a solution of Compound 192 (360 mg, 0.54 mmol) in n-butanol (3 mL) were added Compound 51 (170 mg, 0.38 mmol) and triethylamine (0.26 mL, 1.88 mmol) -0 °C. After stirring at 120 °C for 24 hours, the reaction mixture cooled to room temperature. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 193 (127 mg, 34%). EI-MS m/z : [M+H]+ 1004.40.
Preparation of Compound 194
To a solution of Compound 193 (127 mg, 0.13 mmol) in methanol (5 mL) and distilled water (1 mL) were added ammonia solution (28-30% ammonia, 0.2 mL) and sodium hydrosulfite (220 mg, 1.26 mmol) at -0 °C. After stirring at room temperature for 2.5 hours, the reaction mixture was added methanol (10 mL). The resulting solid was filtered and washed with methanol. The filtrate was concentrated under reduced pressure. The reaction mixture was added dichloromethane (60 mL) and washed with distilled water (20 mL). The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated to afford Compound 194 (74 mg, 60%), which was used without further purification. EI-MS m/z : [M+H]+ 974.47.
Preparation of Compound 195
To a solution of Compound 194 (74 mg, 0.08 mmol) in A,A-dimethylformamide (1 mL) was added Compound 2 (18 mg, 0.09 mmol) at -0 °C. After stirring at room temperature for 15 minutes, the reaction mixture was added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide hydrochloride (19 mg, 0.10 mmol) and triethylamine (0.05 mL, 0.38 mmol) at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 195 (42 mg, 49%). EI-MS m/z : [M+H]+ 1135.55.
Preparation of Compound 196
To a solution of Compound 195 (42 mg, 0.04 mmol) in ethanol (2 mL) was added sodium ethoxide (21% w/w ethanol, 0.14 mL, 0.37 mmol). After stirring at reflux for 7 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 196 (50 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1063.53.
Preparation of Compound 197
To a solution of Compound 196 (50 mg, crude) in dichloromethane (3 mL) was added trifluoroacetic acid (1 mL) at -0 °C under nitrogen. After stirring at room temperature for 1 hour, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 197 (23 mg, 49%).
’H-NMR (400 MHz, DMSO), δ 7.96 (d, J = 20.3 Hz, 2H), 7.86 (d, J = 0.8 Hz, 1H), 7.67 (dd, J = 14.8, 1.2 Hz, 2H), 7.41 - 7.36 (m, 2H), 7.28 (dd, J = 7.1, 1.4 Hz, 2H), 6.53 (s, 2H), 5.87 - 5.54 (m, 5H), 4.95 - 4.85 (m, 4H), 4.54 (dq, J = 20.2, 6.5 Hz, 6H), 4.47 - 4.41 (m, 2H), 4.01 (t, J = 6.1 Hz, 3H), 2.12 (d, J = 7.1 Hz, 6H), 2.00 (q, J = 7.1 Hz, 2H), 1.27 (dt, J = 9.3, 7.1 Hz, 6H). ELMS m/z : [M+H]+ 963.53.
Example 32: Preparation of Compound 199
Preparation of Compound 198
To a solution of Compound 64 (90 mg, 0.08 mmol) in iV,/V-dimethylformamide (10 mL). were added alendronic acid (80 mg, 0.32 mmol), A,AWW-tetramethyl-O-(177- benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 91 mg, 0.24 mmol), and triethylamine (0.04 mL, 0.32 mmol) under nitrogen. After stirring at room temperature for 3 days, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 198 (32 mg, 31%). ELMS m/z : [M+H]+ 1262.42.
Preparation of Compound 199
To a solution of Compound 198 (32 mg, 0.02 mmol) in dichloromethane (3 mL) was added trifluoro acetic acid (1 mL) at 0 °C under nitrogen. After stirring at room temperature for 0.5 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 199 (14 mg, 47%). ELMS m/z : [M+H]+ 1162.53.
Preparation Example 4: Preparation of Compound 204
Preparation of Compound 204
To a solution of Compound 203 (2.0 g, 2.74 mmol, Compound 203 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al) in A,/V-dimethylformamide (10 mL) were added bis(pentafluorophenyl)carbonate (881 mg, 2.23 mmol) and A,A-diisopropylethylamine (0.66 mL, 3.72 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 204 (1.38 g, 98%).
’H-NMR (400 MHz, CDCh), δ 8.14 (s, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.4 Hz, 1H), 5.41-5.30 (m, 4H), 4.25-4.18 (m, 1H), 3.73 (s, 4H), 3.59 (s, 3H), 3.43 (d, J = 1.4 Hz, 3H), 2.06 (d, 7 = 2.0 Hz, 9H). EI-MS m/z: [M+H]+ 751.96.
Preparation Example 5: Preparation of Compound 207
Preparation of Compound 206
To a solution of Compound 205 (162 mg, 0.33 mmol, Compound 205 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (3 mL) were added 2, 5, 8,11,14, 17-hexaoxanonadecan-19- amine (100 mg, 0.33 mmol), W/VWW-tetramethyl-(?-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 154 mg, 0.41 mmol), and W/V-diisopropylethylamine (0.12 mL, 0.68 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with ethyl acetate (50 mL) and then washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 206 (185 mg, 72%). EI-MS m/z : [M+Na]+ 784.18, [M+H]+ 762.17.
Preparation of Compound 207
To a solution of Compound 206 (185 mg, 0.24 mmol) in dichloromethane (5 mL) were added bis(pentafluorophenyl)carbonate (115 mg, 0.29 mmol) and A,A-diisopropylethylamine (0.06 ml, 0.36 mmol) at 0 °C under nitrogen. After stirring at room temperature for 5 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 207 (92 mg, 39%). EI-MS m/z : [M+H]+ 972.13.
Example 33: Preparation of Compound 214
Preparation of Compound 208
To a solution of t-butyl piperidin-4-ylcarbamate (1.2 g, 5.99 mmol) in N,N- dimethylformamide (15 mL) were added Compound 5-1 (2.2 g, 6.59 mmol) and then cesium carbonate (2.14 g, 6.59 mmol) at room temperature under nitrogen. After stirring for 16 hours, the reaction solution was added with distilled water (100 mL) and extracted with ethyl acetate (200 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 208 (1.92 g, 70%). ’H-NMR (400 MHz, CDCI3) δ 7.69 (s, 2H), 4.43 (m, 1H), 4.22 (t, J = 6.4 Hz, 2H), 3.46 (m, 1H), 2.85-2.82 (m, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.12-2.01 (m, 4H), 1.94-1.91 (m, 2H), 1.44-1.42 (m, 10H). EI-MS m/z: [M+H]+ 457.52.
Preparation of Compound 209
To a solution of Compound 208 (1.30 g, 2.85 mmol) and Compound 9 (2.75 g, 5.69 mmol) in n-butanol (30 mL) was added A,A-diisopropylethylamine (2.48 mL, 14.22 mmol). After stirring at 120 °C for 48 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The reaction mixture was diluted with acetonitrile and diethyl ether to precipitate a solid and then filtered. The solid was dried to afford Compound 209 (2.48 g, 84%), which was used without further purification. EI-MS m/z : [M+H]+ 832.09, [M/2+H]+ 416.82.
Preparation of Compound 210
To a solution of Compound 209 (2.48 g, 2.39 mmol) in methanol (50 mL) were added sodium hydrosulfite (Na2S2C>4, 4.17 g, 23.95 mmol) in distilled water (30 mL) and ammonia solution (28-30% ammonia, 4.6 mL, 59.9 mmol). After stirring at room temperature for 2.5 hours, the precipitate was filtered through Celite and washed with methanol. The filtrate was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography to afford Compound 210 (1.4 g, 73%). EI-MS m/z : [M+H]+ 802.25.
Preparation of Compound 211
To a solution of Compound 210 (760 mg, 0.95 mmol) in A,A-dimethylformamide (15 mL) was added Compound 2 (241 mg, 1.23 mmol) in A,A-dimethylformamide (5 mL). After stirring at 30 minutes, the reaction solution was added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide (221 mg, 1.42 mmol) and triethylamine (0.26 mL, 1.89 mmol) at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 211 (730 mg, 79%). EI-MS m/z : [M+H]+ 963.04, [M/2+H]+ 482.36.
Preparation of Compound 212
To a solution of Compound 211 (730 mg, 0.76 mmol) in dichloromethane (8 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C under nitrogen. After stirring at room temperature for 0.5 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 212 (830 mg, 90%).
’H-NMR (400 MHz, DMSO-d6), δ 12.85 (s, 1H), 8.00 (s, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.38 (s, 2H), 7.30 (s, 2H), 6.52 (d, J = 5.2 Hz, 2H), 5.75 (d, J = 20.8 Hz, 2H), 4.90 (s, 4H), 4.53 (d, 7 = 7.8 Hz, 4H), 4.01 (s, 2H), 3.69 (s, 3H), 3.06 (s, 3H), 2.92 (d, 7 = 12.5 Hz, 2H), 2.12 (s, 6H), 2.06 (d, 7 = 13.4 Hz, 2H), 1.68 (d, 7 = 13.8 Hz, 2H), 1.28 0. 7 = 7. 1 Hz, 6H). EI-MS m/z: [M+H]+ 863.07, [M/2+H]+ 432.34.
Preparation of Compound 213 To a solution of Compound 212 (68 mg, 0.05 mmol) in MA-dimcthylformamidc (3 mL) was added Compound 204 (38 mg, 0.05 mmol) and IV, IV’ -diisopropylethylamine (0.045 mL, 0.25 mmol). After stirring at room temperature for 15 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 213 (60 mg, 89%). EI-MS m/z: [M+Na]+ 1453.26, [M+H]+ 1431.27, [M/2+H]+ 716.55.
Preparation of Compound 214
To a solution of Compound 213 (60 mg, 0.042 mmol) in methanol (1.5 mL) was added lithium hydroxide monohydrate (7.04 mg, 0.17 mmol) in distilled water (1.5 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 214 (32 mg, 59%).
Example 34: Preparation of Compound 221
Preparation of Compound 215
To a solution of t-butyl 3-oxopiperazine-l -carboxylate (569 mg, 2.84 mmol) and was dissolved in tetrahydrofuran (12 mL), potassium hydroxide (159 mg, 2.84 mmol) and TBAB (tetrabutylammonium bromide, 152 mg, 0.47 mmol) were added and stirred at room temperature for 30 minutes. The reaction mixture was added Compound 5-1 (800 mg, 2.37 mmol) in tetrahydrofuran (4 mL) at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous ammonium chloride solution (50 x 2 mL) and dried over anhydrous sodium sulfate then filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 215 (760 mg, 70.2%). ’H-NMR (400 MHz, DMSO-d6), δ 8.27 (s, 1H), 8.05 (d, J = 1.7 Hz, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.77 (s, 1H), 4.24 (t, 7 = 6.0 Hz, 2H), 3.86 (s, 2H), 3.53 (dt, 7 = 13.4, 5.8 Hz, 4H), 3.37 (t, J = 5.4 Hz, 2H), 2.02 (td, J = 11.2, 4.8 Hz, 2H), 1.41 (d, J = 3.2 Hz, 9H). EI-MS m/z : [M+H]+ 457.08.
Preparation of Compound 216
To a solution of Compound 215 (296 mg, 0.45 mmol) were added Compound 9 (440 mg, 0.91 mmol) and A,A-diisopropylethylamine (0.43 mL, 2.49 mmol) at room temperature. After stirring at 120 °C for 24 hours, the reaction solution was cooled to room temperature and diluted with dichloromethane (100 mL), methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 216 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 832.05, [M/2+H]+ 366.77.
Preparation of Compound 217
To a solution of Compound 216 (crude, 0.45 mmol) in methanol (10 mL) were added sodium hydrosulfite (Na2S2C>4, 433 mg, 2.48 mmol) and ammonia solution (28-30% ammonia, 0.55 mL). After stirring at room temperature for 3 hours, the precipitate was filtered through Celite and washed with methanol. The filtrate was concentrated under reduced pressure. The reaction mixture was diluted with dichloromethane (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 217 (229 mg, 43%), which was used without further purification. EI-MS m/z : [M+H]+ 802.11, [M/2+H]+ 365.25.
Preparation of Compound 218
To a solution of Compound 217 (229 mg, 0.28 mmol) in A,A-dimethylformamide (4 mL) was added Compound 2 (83.6 mg, 0.42 mmol) in N, A-dimethylformamide (1 mL) at -0 °C. After stirring for 30 minutes, the traction solution was added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide (88.6 mg, 0.57 mmol) and triethylamine (0.09 mL, 0.63 mmol) at room temperature for 19 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 218 (110 mg, 40%). EI-MS m/z : [M+H]+ 963.19, [M/2+H]+ 482.31.
Preparation of Compound 219
To a solution of Compound 218 (50 mg) in dichloromethane (1.4 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C under nitrogen. After stirring at room temperature for 40 minutes, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 219 (33 mg, 52%).
’H-NMR (400 MHz, DMSO-d6), δ 12.81 (s, 1H), 9.09 (s, 2H), 7.97 (d, J = 19.3 Hz, 2H), 7.64 (s, 2H), 7.36 (d, 7 = 13.6 Hz, 2H), 7.31 (s, 1H), 7.25 (s, 1H), 6.55 (s, 1H), 6.52 (s, 1H), 5.86 (d, 7 = 15.9 Hz, 1H), 5.76 (d, 7 = 15.8 Hz, 1H), 4.96 (s, 2H), 4.88 (d, 7 = 4.9 Hz, 2H), 4.53 0. 7 = 7.9 Hz, 4H), 3.95 (s, 2H), 3.71 (s, 3H), 3.65 (s, 2H), 2.12 (d, 7 = 5.9 Hz, 6H), 1.75 (s, 2H), 1.27 (q, J = 7.7 Hz, 6H). EI-MS m/z : [M+H]+ 863.09, [M/2+H]+ 432.41.
Preparation of Compound 220
To a solution of Compound 219 (20 mg, 0.017 mmol) in A,A-dimethylformamide (2 mL) was added Compound 204 (12.3 mg, 0.05 mmol) and A,A-diisopropylethylamine (0.014 mL, 0.083 mmol). After stirring at room temperature for 15 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to afford Compound 220 (23 mg, 97%). EI-MS m/z : [M+Na]+ 1453.19, [M+H]+ 1431.19, [M/2+H]+ 716.48.
Preparation of Compound 221
To a solution of Compound 220 (23 mg, 0.016 mmol) in methanol (1 mL) was added lithium hydroxide monohydrate (1.67 mg, 0.06 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 221 (4.4 mg, 21%).
’H-NMR (400 MHz, DMSO-d6), δ 12.80 (s, 1H), 8.31 (s, 1H), 7.95 (d, J = 13.8 Hz, 2H), 7.82 (s, 1H), 7.64 (d, J = 4.1 Hz, 2H), 7.52 (d, J = 8.5 Hz, 1H), 7.37-7.21 (m, 5H), 6.57 (s, 1H), 6.50 (s, 1H), 5.90-5.80 (m, 1H), 5.76 (d, 7 = 15.6 Hz, 1H), 5.15 (d, 7 = 6.8 Hz, 1H), 5.08 (s, 2H), 4.96 (s, 2H), 4.88 (s, 1H), 4.51 (dt, 7= 21.9, 7.3 Hz, 4H), 3.97 (d, 7= 9.5 Hz, 1H), 3.87 (d, 7= 17.5 Hz, 4H), 3.69 (s, 3H), 3.52 (s, 4H), 3.46 (d, 7= 3.7 Hz, 3H), 3.28 (s, 2H), 3.19 (s, 1H), 2.11 (d, J = 15.9 Hz, 6H), 1.68 (s, 2H), 1.27 (dt, J = 21.1, 7.0 Hz, 6H). EI-MS m/z : [M+H]+ 1291.07, [M+H]+ 646.39. Example 35: Preparation of Compound 230
Preparation of Compound 222
To a solution of 3-aminobenzyl alcohol (800 mg, 6.49 mmol) in tetrahydrofuran (9.3 mL) was added di-t-butyl dicarbonate (1.64 mL, 7.14 mmol). After stirring at room temperature for 24 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 222 (1.45 g, 99%).
’H-NMR (400 MHz, CDC13), δ 7.44 (s, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.04 (d. 7 = 7.3 Hz, 1H), 6.50 (s, 1H), 4.66 (s, 2H), 1.52 (s, 9H). Preparation of Compound 223
To a solution of Compound 222 (700 mg, 3.13 mmol) in dichloromethane (5 mL) were added triethylamine (0.65 mL, 4.70 mmol) and methanesulfonyl chloride (0.29 mL, 3.76 mmol) at 0 °C. After stirring at 0 °C for 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 223 (508 mg, 53 %). ’H-NMR (400 MHz, CDCh), δ 9.45 (s, 1H), 7.61 (s, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.29 (d, 7 = 16.0 Hz, 1H), 7.03 (d. 7 = 7.5 Hz, 1H), 5.19 (s, 2H), 3.24-3.19 (m, 3H), 1.48 (s, 9H).
Preparation of Compound 224
To a solution of Compound 4 (300 mg, 1.38 mmol) in A,A-dimethylformamide (3 mL) were added potassium carbonate (287 mg, 2.08 mmol) and Compound 223 (500.9 mg, 1.66 mmol). After stirring at 50 °C for 1 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 224 (487 mg, 83%), which was used without further purification.
’H-NMR (400 MHz, DMSO-d6), δ 9.44 (s, 1H), 8.27 (s, 1H), 8.07 (d, J = 1.7 Hz, 1H), 7.98 (d, 7 = 1.8 Hz, 1H), 7.78 (s, 1H), 7.62 (s, 1H), 7.46-7.39 (m, 1H), 7.30 (t, 7 = 7.8 Hz, 1H), 7.09 (d, 7 = 7.5 Hz, 1H), 5.33 (s, 2H), 1.48 (s, 9H). EI-MS m/z : [M+H]+ 422.13.
Preparation of Compound 225
To a solution of Compound 224 (350 mg, 0.83 mmol) and Compound 9 (683 mg, 1.41 mmol) in n-butanol (6 mL) was added A,A-diisopropylethylamine (0.79 mL, 4.56 mmol) at room temperature. After heating to 120 °C and stirring for 40 hours, the reaction solution was cooled to room temperature. The reaction mixture was diluted with dichloromethane (100 mL), methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and concentrated under reduced pressure. After adding with dichloromethane and diethyl ether, the residue solid was filtered. The residue solid was dried to afford Compound 225 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 797.08.
Preparation of Compound 226
After dissolving Compound 225 (crude, 0.83 mmol) in methanol (14 mL), distilled water (2 mL), aqueous ammonia solution (28-30%, 1.05 mL), and sodium hydrosulfite (Na2S2C>4, 1.05 g, 6.02 mmol) were added under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was diluted with methanol (50 mL) and then filtered. The filtrate was concentrated under reduced pressure. The reaction mixture was diluted with ethyl acetate (100 mL) and methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 226 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 767.08.
Preparation of Compound 227 To a solution of Compound 226 (0.83 mmol, crude) in N, A-dimethylformamide (5 mL) was added Compound 2 (100 mg, 0.52 mmol) in AA-dimethylformamide (1 mL) under nitrogen. After stirring at -0 °C for 1 hour, the reaction mixture was added N-(3- dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (114 mg, 0.65 mmol) and triethylamine (0.18 mL, 1.29 mmol) at room temperature. The reaction mixture was stirred for 18 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 227 (128 mg, 32%). EI-MS m/z : [M+H]+ 928.02.
Preparation of Compound 228
To a solution of Compound 227 (50 mg) in dichloromethane (2.4 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C under nitrogen. After stirring at room temperature for 30 minutes, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 228 (17 mg, 27%).
’H-NMR (400 MHz, DMSO-d6), δ 7.97 (s, 1H), 7.66 (d, J = 9.3 Hz, 2H), 7.43 (s, 1H), 7.37 (s, 1H), 7.28 (s, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.06 (s, 1H), 6.93 (s, 1H), 6.51 (d, J = 7.0 Hz, 2H), 5.85-5.74 (m, 1H), 5.68-5.58 (m, 1H), 5.07 (s, 2H), 4.88 (dd. J = 18.8, 5.6 Hz, 4H), 4.54-4.49 (m, 4H), 3.64 (s, 3H), 2.10 (d, 7 = 4.4 Hz, 6H), 1.25 (q, J = 7.6 Hz, 6H). EI-MS m/z : [M+H]+ 828.07.
Preparation of Compound 229
To a solution of Compound 228 (40 mg, 0.03 mmol) in AA-dimethylformamide (2 mL) was added compound 204 (25 mg, 0.03 mmol), AA-diisopropylethylamine (0.027 mL, 0.15 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 0.9 mg, 0.01 mmol). After stirring at room temperature for 19 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 229 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 1395.83.
Preparation of Compound 230
To a solution of Compound 229 (81 mg, 0.051 mmol) in methanol (0.7 mL) and tetrahydrofuran (0.7 mL) was added lithium hydroxide monohydrate (10.76 mg, 0.25 mmol) in distilled water (0.9 mL) at -45 °C under nitrogen. After stirring at 0 °C for 1 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 230 (3.9 mg, 8.9%).
’H-NMR (400 MHz, DMSO-d6), δ 12.81 (s, 3H), 9.81 (s, 1H), 8.29 (s, 1H), 7.96 (s, 2H), 7.88 (s, 1H), 7.66 (d, 7 = 11.3 Hz, 2H), 7.61 (s, 1H), 7.52 (d, 7 = 8.6 Hz, 1H), 7.44 (s, 1H), 7.34 (t, J = 9.1 Hz, 3H), 7.25 (d, J = 8.6 Hz, 2H), 7.08 (t, J = 7.9 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.49 (d, 7= 16.8 Hz, 2H), 5.82 (d, 7 = 14.8 Hz, 1H), 5.61 (d, 7 = 15.2 Hz, 2H), 5.15 (d, 7 = 6.8 Hz, 1H), 5.08 (d, J = 21.2 Hz, 4H), 4.88 (dd, J = 35.7, 5.6 Hz, 4H), 4.49 (dq, J = 14.0, 7.2 Hz, 4H), 3.96 (d, 7 = 9.5 Hz, 1H), 3.62-3.28 (m, 15H), 2.09 (d, 7 = 9.3 Hz, 6H), 1.24 (dt, J
= 14.2, 7.0 Hz, 6H). EI-MS m/z : [M+H]+ 1445.20.
Example 36: Preparation of Compound 239 Preparation of Compound 231
To a solution of 4-aminophenethyl alcohol (2 g, 14.96 mmol) in tetrahydrofuran (50 mL) was added di-t-butyl dicarbonate (3.8 mL, 16.46 mmol) at 0 °C. After stirring at room temperature for 6 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The reaction solution was concentrated under reduced pressure to afford Compound 231 (3 g, 85%), which was used without further purification.
’H-NMR (400 MHz, CDCh), δ 7.30 (d, 7 = 8.1 Hz, 2H), 7.15 (d, 7 = 8.2 Hz, 2H), 3.83 (q, 7 = 6.4 Hz, 2H), 2.82 (t, 7 = 6.5 Hz, 2H), 1.52 (s, 9H).
Preparation of Compound 232
To a solution of Compound 231 (850 mg, 3.58 mmol) in dichloromethane (15 mL) were added triethylamine (0.75 mL, 5.37 mmol) and methanesulfonyl chloride (0.41 mL, 5.37 mmol) at 0 °C. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The reaction solution was concentrated under reduced pressure to afford Compound 232 (114 mg, crude), which was used without further purification.
Preparation of Compound 233
To a solution of Compound 4 (703 mg, 3.25 mmol) in N, A-dimethylformamide (6 mL) were added potassium carbonate (673 mg, 4.88 mmol) and Compound 232 (1.1 g, 3.57 mmol). After stirring at 50 °C for 22 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (15 mL x 2) and brine (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The reaction solution was concentrated under reduced pressure to afford Compound 233 (1.06 g, 74%).
’H-NMR (400 MHz, DMSO-d6), δ 9.21 (s, 1H), 8.22 (s, 1H), 7.97 (d, 7 = 1.7 Hz, 1H), 7.82 (d, 7 = 1.8 Hz, 1H), 7.71 (s, 1H), 7.34 (d, 7 = 8.2 Hz, 2H), 7.19 (d, 7 = 8.3 Hz, 2H), 4.33 (t, 7 = 6.7 Hz, 2H), 3.00 (t, 7 = 6.7 Hz, 2H), 1.42 (s, 9H). ELMS m/z : [M+H]+436.13.
Preparation of Compound 234
To a solution of Compound 233 (530 mg, 1.22 mmol) and Compound 9 (650 mg, 1.58 mmol) in n-butanol (6 mL) was added A,A-diisopropylethylamine (0.95 mL, 5.47 mmol) at room temperature. After stirring at 120 °C for 21 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous magnesium sulfate and filtered. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 234 (657 mg, 67%). ELMS m/z : [M+H]+436.13.
Preparation of Compound 235
To a solution of Compound 234 (373 mg, 0.46 mmol) in methanol (5 mL) were added aqueous ammonia solution (28-30% ammonia, 0.82 mL) and sodium hydrosulfite (Na2S2C>4, 800 mg, 46 mmol) under nitrogen. After stirring at room temperature for 30 minutes, the reaction solution was added methanol (50 mL). The resulting solid was filtered and washed with methanol. The filtrate was concentrated and diluted with dichloromethane (100 mL), methanol (20 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The reaction solution was concentrated under reduced pressure to afford Compound 235 (360 mg, crude), which was used without further purification. ELMS m/z : [M+H]+ 781.21.
Preparation of Compound 236
To a solution of Compound 235 (360 mg, 0.46 mmol) in A,A-dimethylformamide (2 mL) was added Compound 2 (98 mg, 0.51 mmol) in A,A-dimethylformamide (1 mL) under nitrogen. After stirring at room temperature for 30 minutes, the reaction mixture were added A-(3-dimethylaminopropyl)-A’-ethylcarbodiimide (0.18 mL, 1.01 mmol) and triethylamine (0.2 mL, 1.38 mmol). After stirring at room temperature for 1.5 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 236 (137 mg, 32%). ELMS m/z : [M+H]+ 943.1.
Preparation of Compound 237
To a solution of Compound 236 (50 mg) in dichloromethane (2 mL) was added trifluoroacetic acid (0.2 mL) at 0 °C under nitrogen. After stirring at room temperature for 40 minutes, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 237 (14 mg, 31%).
’H-NMR (400 MHz, DMSO-d6), δ 7.91 (d, J = 12.5 Hz, 1H), 7.60 (d, J = 12.4 Hz, 1H), 7.28 0. 7 = 7.0 Hz, 2H), 7.14 (d, 7 = 8.0 Hz, 1H), 7.05 (d, 7 = 7.9 Hz, 1H), 6.48 (d, 7 = 14.1 Hz, 1H), 5.72 (d, 7 = 9.4 Hz, 2H), 4.85 (s, 1H), 4.65 (s, 1H), 4.46 (p, 7 = 7.1 Hz, 2H), 4.14 (t, 7 = 6.7 Hz, 1H), 3.67 (s, 2H), 2.79 (t, 7 = 6.7 Hz, 1H), 2.46 (s, 2H), 2.05 (d, 7 = 8.9 Hz, 3H), 1.21 (dt, 7 = 10.6, 7.4 Hz, 3H). ELMS m/z : [M+H]+ 842.14.
Preparation of Compound 238
To a solution of Compound 237 (50 mg, 0.06 mmol) in A,A-dimethylformamide (1 mL) was added Compound 204 (50 mg, 0.07 mmol), A,A’-diisopropylethylamine (0.05 mL, 0.3 mmol) and l-Hydroxy-7-azabenzotriazole (HOAt, 1.6 mg, 0.012 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 238 (114 mg), which was used without further purification. ELMS m/z : [M+H]+ 1410.5.
Preparation of Compound 239 To a solution of Compound 238 (114 mg, 0.08 mmol) in methanol (1 mL) and tetrahydrofuran (1 mL) was added lithium hydroxide monohydrate (20 mg, 0.48 mmol) in distilled water (0.75 mL) at -50 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 239 (29 mg, 24%).
’H-NMR (400 MHz, DMSO-d6), δ 9.63 (s, 1H), 8.25 (s, 1H), 7.92 (s, 2H), 7.80 (s, 1H), 7.60 (d, 7 = 11.1 Hz, 2H), 7.46 (d, J = 8.5 Hz, 1H), 7.34-7.24 (m, 5H), 7.20 (d, J = 8.6 Hz, 1H), 7.02 (d, 7 = 8.2 Hz, 2H), 6.47 (s, 2H), 5.10 (d, 7 = 6.7 Hz, 1H), 5.04 (s, 2H), 4.84 (s, 2H), 4.72 (s, 2H), 4.49-4.41 (m, 4H), 4.10 (s, 2H), 3.91 (d, J = 9.3 Hz, 1H), 3.67 (s, 3H), 3.23 (s, 2H),
2.74 (s, 2H), 2.04 (d, 7 = 11.7 Hz, 5H), 1.86 (s, 1H), 1.20 (dt, 7 = 12.8, 7.0 Hz, 6H), 0.81 (d, J = 11.8 Hz, 1H). EI-MS m/z : [M+H]+ 1269.52.
Example 37: Preparation of Compound 252
Preparation of Compound 240
To a solution of diethyl malonate (2 g, 12.49 mmol) in tetrahydrofuran (50 mL) was added sodium hydride (60% dispersion in mineral oil, 0.9 g, 24.93 mmol) at 0 °C. After stirring at 0 °C for 0.5 hours, the reaction solution was added 2-chloro-5-nitropyridine (2.08 g, 13.11 mmol) at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 240 (3.3 g, 94%). ’H-NMR (400 MHz, CDCh), δ 9.37 (d, J = 2.7 Hz, 1H), 8.51 (dt, J = 8.7, 2.4 Hz, 1H), 7.77 (dd, 7 = 8.7, 1.6 Hz, 1H), 5.06 (d, 7 = 1.5 Hz, 1H), 4.35-4.19 (m, 4H), 1.33-1.25 (m, 6H). EI-MS m/z : [M+H]+ 283.41.
Preparation of Compound 241
To a solution of Compound 240 (3.3 g, 11.69 mmol) in Dimethyl sulfoxide (33 mL) was added sodium chloride (0.72 g, 12.27 mmol) in water (0.3 mL). After stirring at 120 °C for 5 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 241 (1.27 g, 51%).
’H-NMR (400 MHz, CDCh), δ 9.38 (d, J = 2.7 Hz, 1H), 8.46 (dd, J = 8.4, 3.0 Hz, 1H), 7.54 (dd, J = 8.5, 2.3 Hz, 1H), 4.26-4.16 (m, 2H), 3.98 (d, J = 2.3 Hz, 2H), 1.28 (td, J = 7.3, 2.3 Hz, 3H). EI-MS m/z : [M+H]+ 211.39.
Preparation of Compound 243
To a solution of Compound 241 (775 mg, 3.68 mmol) in methanol (15 mL) was added palladium/charcoal (10% wt. Pd/C, 77 mg). After stirring at room temperature under hydrogen balloon for 3 hours, the reaction solution was filtered through Celite. The filtrate was concentrated under reduced pressure. The resulting residue was dissolved in 1,4-dioxane (12 mL) at room temperature and then di-t-butyl dicarbonate (885 mg, 4.06 mmol) was added. After stirring at 100 °C for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound
243 (906 mg, 87%).
’H-NMR (400 MHz, CDCh), δ 8.36 (d, 7 = 2.6 Hz, 1H), 7.98-7.93 (m, 1H), 7.29-7.19 (m, 1H), 4.17 (qd, J = 7.2, 2.0 Hz, 2H), 3.78 (d, J = 2.0 Hz, 2H), 1.52 (d, J = 2.3 Hz, 9H), 1.25 (td, 7 = 7.2, 2.1 Hz, 3H). EI-MS m/z : [M+H]+ 281.30.
Preparation of Compound 244
To a solution of Compound 243 (906 mg, 3.23 mmol) in tetrahydrofuran (10 mL) was slowly added Lithium borohydride (1 M in tetrahydrofuran, 9.69 mL, 9.69 mmol) at 0 °C. After stirring at room temperature for 4 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound
244 (294 mg, 38%). ’H-NMR (400 MHz, CDCh), δ 8.65 (s, 1H), 8.16 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.59 (s, 1H), 4.05 (q, J = 5.8 Hz, 2H), 3.40 (t, J = 6.2 Hz, 2H), 2.65 (bs, 1H), 1.53 (s, 9H). EI-MS m/z : [M+H]+ 239.29.
Preparation of Compound 245
To a solution of Compound 244 (294 mg, 1.23 mmol) in dichloromethane (6 mL) were added triethylamine (0.22 mL, 1.60 mmol) and methanesulfonyl chloride (0. 11 mL, 1.36 mmol) at 0 °C under nitrogen. After stirring at room temperature for 1 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 245 (390 mg, 99%).
’H-NMR (400 MHz, CDCh), δ 8.47 (d, J = 2.6 Hz, 1H), 7.99 (s, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.83-6.76 (m, 1H), 4.62 (t, J = 6.6 Hz, 2H), 3.20 (t, J = 6.5 Hz, 2H), 2.92 (s, 3H), 1.53 (s, 9H). EI-MS m/z : [M+H]+ 317.57.
Preparation of Compound 246
To a solution of Compound 4 (220 mg, 1.02 mmol) in A,A-dimethylformamide (3 mL) were added potassium carbonate (210 mg, 1.52 mmol) and Compound 245 (385 mg, 1.22 mmol). After reaction mixture was stirred at 50 °C for 15 hours, then the reaction temperature was raised to 80 °C and stirred for 4 hours. The reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 246 (270 mg, 60%).
’H-NMR (400 MHz, DMSO-d6), δ 9.49 (s, 1H), 8.54 (s, 1H), 8.28 (s, 1H), 8.02 (s, 1H), 7.91 (s, 1H), 7.81 (d, 7 = 8.5 Hz, 1H), 7.76 (s, 1H), 7.30 (d, 7 = 8.5 Hz, 1H), 4.55 (t, 7 = 6.7 Hz, 2H), 3.20 (t, J = 6.6 Hz, 2H), 1.47 (d, 7 = 1.8 Hz, 9H). EI-MS m/z : [M+H]+ 437.26.
Preparation of Compound 247
To a solution of Compound 246 (270 mg, 0.62 mmol) and Compound 9 (508 mg, 1.05 mmol) were dissolved in n-butanol (5 mL) and then N,N -diisopropylethylamine (0.59 mL, 3.40 mmol) was added at room temperature. After stirring at 120 °C for 18 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 247 (200 mg, 39%). EI-MS m/z : [M+H]+ 812.48.
Preparation of Compound 248
To a solution of Compound 247 (213 mg, 0.26 mmol) in methanol (7 mL) and distilled water (1 mL) were added ammonia solution (28-30% ammonia, 0.25 mL) and sodium hydrosulfite (Na2S2C>4, 456 mg, 2.62 mmol) at -0 °C. After stirring at room temperature for 1 hour, the reaction solution was added methanol (50 mL). The resulting solid was filtered and washed with methanol. The filtrate was concentrated under reduced pressure and diluted with acetonitrile. The resulting solid was filtered to afford Compound 248 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 782.35.
Preparation of Compound 249
To a solution of Compound 248 (crude, 0.26 mmol) in N, A-dimethylformamide (3 mL) was added Compound 2 (58 mg, 0.30 mmol) in A, A-dimcthylformamidc (1 mL) at -0 °C. After stirring at room temperature for 1 hour, the reaction solution was added A-(3- dimethylaminopropyl)-A’-ethylcarbodiimide (58 mg, 0.37 mmol) and triethylamine (0.10 mL, 0.75 mmol). After stirring at room temperature for 20 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 249 (116 mg, 49%). EI-MS m/z : [M+H]+ 943.49.
Preparation of Compound 250
To a solution of Compound 249 (50 mg) in dichloromethane (1.6 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C under nitrogen. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 250 (25 mg, 36%).
’H-NMR (400 MHz, DMSO-d6), δ 7.94 (s, 2H), 7.64 (d, J = 10.4 Hz, 2H), 7.54 (q, J = 9.0 Hz, 2H), 7.37 (s, 2H), 7.32 (d, J = 16.5 Hz, 2H), 6.48 (d, J = 26.9 Hz, 2H), 5.69 (s, 2H), 4.86 (s, 2H), 4.76 (s, 2H), 4.48 (p, J = 7.0 Hz, 4H), 4.38 (t, J = 6.5 Hz, 2H), 3.70 (s, 3H), 2.09 (d. 7 = 7.0 Hz, 6H), 1.23 (q, 7= 6.4 Hz, 6H). EI-MS m/z : [M+H]+ 843.41.
Preparation of Compound 251
To a solution of Compound 250 (46 mg, 0.04 mmol) in AA-dimethylformamide (2 mL) was added Compound 204 (29 mg, 0.04 mmol), A,A-diisopropylethylamine (0.03 mL, 0.16 mmol) and HOAt (1 -Hydroxy-7 -azabenzo triazole (0.9 mg, 0.01 mmol). After stirring at room temperature for 21 hours, the reaction mixture was diluted with ethyl acetate (20 mL), washed with distilled water (8 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 251 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 1410.60.
Preparation of Compound 252
To a solution of Compound 251 (crude, 0.04 mmol) in methanol (0.3 mL) and tetrahydrofuran (0.3 mL) was added lithium hydroxide monohydrate (7.45 mg, 0.18 mmol) in distilled water (0.3 mL) at -45 °C under nitrogen. After stirring at 0 °C for 1 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 252 (10.5 mg, 23.4% for 2 steps). ’H-NMR (400 MHz, DMSO), δ 10.04 (s, 1H), 8.60 (s, 1H), 8.28 (s, 1H), 7.95 (s, 1H),
7.87 (s, 2H), 7.63 (d, J = 3.7 Hz, 2H), 7.51 (d, J = 8.6 Hz, 1H), 7.37-7.28 (m, 4H), 7.28-7.20 (m, 2H), 6.50 (d, J = 13.4 Hz, 2H), 5.74 (s, 2H), 5.14 (d, 7 = 11.1 Hz, 3H), 4.87 (s, 2H), 4.74 (s, 2H), 4.49 (d, J = 7.6 Hz, 4H), 4.36 (s, 2H), 3.96 (d, J = 9.4 Hz, 2H), 3.72-3.46 (m, 13H), 3.38 (d, J = 6.3 Hz, 3H), 3.27 (s, 3H), 3.02 (s, 2H), 2.08 (d, J = 5.2 Hz, 6H), 1.23 (d, J = 6.5 Hz, 6H). EI-MS m/z : [M+H]+ 1270.66.
Example 38: Preparation of Compound 254 Preparation of Compound 253
To a solution of Compound 228 (70 mg, 0.06 mmol) in A,A-dimethylformamide (3 mL) was added Compound 207 (45 mg, 0.05 mmol), A,A’-diisopropylethylamine (0.045 mL, 0.25 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 2.5 mg, 0.02 mmol). After stirring at room temperature for 15 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 253 (74 mg, 98%). EI-MS m/z : [M+Na]+ 1637.23, [M+H]+ 1615.23, [M/2+H]+ 808.62.
Preparation of Compound 254
To a solution of Compound 253 (74 mg, 0.045 mmol) in methanol (1.5 mL) was added lithium hydroxide monohydrate (7.68 mg, 0.18 mmol) in distilled water (1.5 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 254 (29 mg, 43%).
’H-NMR (400 MHz, DMSO-d6), δ 9.80 (s, 1H), 8.28 (d, J = 5.8 Hz, 1H), 7.95 (s, 2H), 7.87 (d, J = 2.3 Hz, 1H), 7.66 (d, J = 11.5 Hz, 2H), 7.60 (s, 1H), 7.55-7.48 (m, 1H), 7.44 (s, 1H), 7.32 (d, J = 8.3 Hz, 3H), 7.25 (d, J = 8.5 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.48 (d, 7 = 16.9 Hz, 2H), 5.81 (d, J = 15.3 Hz, 1H), 5.65-5.56 (m, 1H), 5.13 (d, J = 6.9 Hz, 1H), 5.10 (s, 2H), 5.05 (s, 2H), 4.92 (s, 1H), 4.83 (d, 7 = 5.6 Hz, 2H), 4.49 (dd, 7= 13.1, 6.9 Hz, 4H), 3.96 (d, 7= 9.5 Hz, 1H), 3.61 (s, 2H), 3.51 (d, 7 = 22.0 Hz, 20H), 3.22 (d, 7= 1.5 Hz, 3H), 2.08 (d, 7= 9.3 Hz, 6H), 1.23 (dt, 7= 14.2, 7.1 Hz, 6H). EI-MS m/z : [M+H]+ 1476.67, [M/2+H]+ 739.23.
Example 39: Preparation of Compound 262
Preparation of Compound 255
To a solution of 2-(2-(2-azidoethoxy)ethoxy)ethan-l -amine (2 g, 11.48 mmol) in dichloromethane (20 mL) were added di-t-butyl dicarbonate (3.16 mL, 13.78 mmol) and triethylamine (3.16 mL 13.78 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with aqueous IN hydrochloride solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 255 (3 g, crude), which was used without further purification.
’H-NMR (400 MHz, CDCh)), δ 5.02 (br, 1H), 3.69-3.59 (m, 6H), 3.56 (t, J = 4.8 Hz, 2H), 3.42 (t, J = 4.8 Hz, 2H), 3.33 (d, J = 4.4 Hz, 2H), 1.45 (s, 9H).
Preparation of Compound 256 To a solution of Compound 255 (1.9 g, 6.93 mmol) in methanol (20 mL) was added palladium/charcoal (10% wt. Pd/C, 190 mg). After stirring at room temperature under hydrogen balloon for 3 hours, the reaction solution was passed through Celite. The filtrate was concentrated under reduced pressure to afford Compound 256 (1.7 g, crude), which was used without further purification.
’H-NMR (400 MHz, CDCh), δ 5.19 (br, 1H), 3.63-3.54 (m, 6H), 3.32(s, 2H), 2.94 (s, 2H), 2.60 (s, 2H), 1.44 (s, 9H).
Preparation of Compound 257
To a solution of Compound 256 (452 mg, 1.82 mmol) in methanol (10 mL) and distilled water (1 mL) were added paraformaldehyde (175 mg, 3.65 mmol) and sodium cyanoborohydride (251 mg, 4.00 mmol) and Zinc chloride (99 mg, 0.98 mmol). After stirring at room temperature for 16 hours, the reaction mixture was diluted with dichloromethane (20 mL)/methanol (2 mL) and washed with distilled water (10 mL) and dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 257 (207 mg, 41%).
’H-NMR (400 MHz, CDCh), δ 5.20 (br, 1H), 3.61-3.31 (m, 8H), 3.31 (d, J = 4.0 Hz, 2H), 2.52 (t, J = 5.6 Hz, 2H), 2.27 (s, 6H), 1.44 (s, 9H).
Preparation of Compound 258
To a solution of Compound 257 (207 mg, 0.75 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C. The reaction mixture was raised to room temperature and stirred for 1 hour. The reaction mixture was diluted with dichloromethane/methanol (10/1, 20 mL x 2) and washed with aqueous 3N sodium hydroxide solution (5 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 258 (105 mg, 80%), which was used without further purification.
’H-NMR (400 MHz, CDCI3) δ 3.62 (s, 4H), 3.58 (t, J = 4.4 Hz, 2H), 2.86 (t, J = 4.0 Hz, 2H), 2.52 (d, J = 4.0 Hz, 2H), 1.67 (br, 2H).
Preparation of Compound 259
To a solution of Compound 258 (240 mg, 0.49 mmol) in A,A-dimethylformamide (10 mL) was added Compound 205 (105 mg, 0.59 mmol), N,N,N ' ,N ' -Tetramethyl-O-(1H- benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 244 mg, 0.64 mmol), N,N- diisopropylethylamine (0.19 mL, 0.99 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 3 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 259 (380 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 643.41.
Preparation of Compound 260
To a solution of Compound 259 (490 mg, 0.76 mmol) in dichloromethane (10 mL) was added bis(pentafluorophenyl)carbonate (361 mg, 0.92 mmol) and N,N- diisopropylethylamine (0.4 ml, 0.29 mmol) at 0 °C. After raising to room temperature and stirring under nitrogen for 5 hours, the reaction mixture was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 260 (400 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 853.39.
Preparation of Compound 261
To a solution of Compound 237 (60 mg, 0.05 mmol) in A,A-dimethylformamide (3 mL) was added Compound 260 (52 mg, 0.06 mmol), A,A-diisopropylethylamine (0.04 mL, 0.26 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 1.4 mg, 0.01 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 261 (80 mg, crude), which was used without further purification. EI-MS m/z : [1/2M+H]+ 756.82, [M+H]+ 1511.96.
Preparation of Compound 262
To a solution of Compound 261 (crude, 80 mg, 0.053 mmol) in methanol (1 mL) and tetrahydrofuran (1 mL) was added lithium hydroxide monohydrate (22 mg, 0.53 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 262 (12.2 mg, 17%). EI-MS m/z : [M/2+H]+ 686.25. Example 40: Preparation of Compound 270
Preparation of Compound 263
To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-l-ol (10 g, 59.3 mmol) in N,N- dimethylformamide (30 mL) was added sodium azide (4.63 g, 71.2 mmol) at 0 °C. After stirring at 100 °C for 16 hours, the reaction mixture was diluted with chloroform (50 mL) and washed with distilled water (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 263 (10.8 g, crude), which was used without further purification.
’H-NMR (400 MHz, CDC13), δ 3.74 (t, 7 = 5.0 Hz, 2H), 3.71-3.65 (m, 6H), 3.64 - 3.59 (m, 2H), 3.40 (t, 7 = 5.0 Hz, 2H), 2.34 (t, 7 = 6.2 Hz, 1H). ELMS m/z : [M+Na]+ 198.25.
Preparation of Compound 264
To a solution of Compound 263 (2 g, 12.49 mmol) in tetrahydrofuran (50 mL) was added sodium hydride (60% dispersion in mineral oil, 501 mg, 20.9 mmol) at 0 °C. The reaction mixture was stirred at 0°C for 10 minutes, t-butyl dimethylchlorosilane (2.3 ml, 12.6 mmol) was added to the reaction mixture and stirred at room temperature for 15 hours. The reaction mixture was diluted with ethyl acetate (20 mL x 3) and washed with brine (15 mL) and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 264 (3.05 g, 92%). ’H-NMR (400 MHz, CDCh), δ 3.77 (t, 7 = 5.3 Hz, 2H), 3.72-3.62 (m, 6H), 3.57 (t, 7 = 5.4 Hz, 2H), 3.39 (t, 7 = 5.1 Hz, 2H), 0.90 (s, 9H), 0.07 (s, 6H). EI-MS m/z : [M+Na]+ 312.36, [M+H]+ 290.38.
Preparation of Compound 265
To a solution of Compound 264 (3.05 g, 10.5 mmol) in tetrahydrofuran (30 mL) was added triphenylphosphine (4.63 g, 71.2 mmol). After stirring at 100 °C for 16 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 265 (1.5 g, 54%), which was used without further purification.
’H-NMR (400 MHz, CDCh), δ 3.77 (t, 7 = 5.5 Hz, 2H), 3.68-3.65 (m, 2H), 3.64-3.61 (m, 2H), 3.56 (t, 7 = 5.4 Hz, 2H), 3.51 (t, 7= 5.2 Hz, 2H), 2.86 (t, 7= 5.3 Hz, 2H), 0.89 (s, 9H), 0.07 (s, 6H). EI-MS m/z : [M+H]+ 264.38.
Preparation of Compound 266
To a solution of Compound 265 (2.20 g, 4.54 mmol) in A,A-dimethylformamide (12 mL) was added Compound 205 (1.44 g, 5.45 mmol), N,N,N ' ,N ' -Tetramethyl-O-(1H- benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 2.24 g, 5.90 mmol), N,N- diisopropylethylamine (1.6 mL, 9.08 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 3 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium hydrogen carbonate (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 266 (830 mg, crude).
’H-NMR (400 MHz, CDCh), δ 7.96 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 6.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 5.43 - 5.22 (m, 4H), 4.67 (s, 2H), 4.20 (d, J = 9.3 Hz, 1H), 3.77 - 3.72 (m, 6H), 3.69 - 3.65 (m, 6H), 3.55 (t, 7 = 5.5 Hz, 3H), 2.05 (s, 9H), 0.05 (d, 7= 1.4 Hz, 6H). EI-MS m/z : [M+Na]+ 752.41, [M+H]+ 730.44.
Preparation of Compound 267
To a solution of Compound 266 (830 mg, 1.14 mmol) in dichloromethane (5 mL) was added bis(pentafluorophenyl)carbonate (538 mg, 1.36 mmol) and A,A-diisopropylethylamine (0.59 ml, 3.41 mmol) at 0 °C. After raising to room temperature and stirring under nitrogen for 3 hours, the reaction mixture was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 267 (270 mg, 25%).
’H-NMR (400 MHz, CDC13), δ 8.11 (d, J = 2.6 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.09 (d, 7 = 8.5 Hz, 1H), 5.45-5.31 (m, 3H), 5.30-5.27 (m, 3H), 4.22 (d, 7 = 9.1 Hz, 1H), 3.80-3.64 (m, 13H), 3.56 (t, 7 = 5.6 Hz, 3H), 2.06 (s, 9H), 0.91-0.86 (m, 9H), 0.05 (t, 7 = 1.2 Hz, 6H). EI-MS m/z : [M+H]+ 940.43.
Preparation of Compound 268
To a solution of Compound 267 (75.4 mg, 0.06 mmol) in A,A-dimethylformamide (2 mL) was added Compound 237 (65.8 mg, 0.07 mmol), A,A-diisopropylethylamine (0.06 mL, 0.32 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 1.7 mg, 0.01 mmol). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 268, which was used without further purification. EI-MS m/z : [M/2+H]+ 799.81.
Preparation of Compound 269
To a solution of Compound 268 (101 mg, 0.06 mmol) in methanol (1 mL) was added lithium hydroxide monohydrate (13 mg, 0.32 mmol) in distilled water (0.75 mL) at -50 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure to afford Compound 269 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 730.34.
Preparation of Compound 270
To a solution of Compound 269 (0.06 mmol, crude) in dichloromethane (2 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 270 (53.8 mg, 54%; 2 steps). EI-MS m/z : [M+H]+ 1344.70, [M/2+H]+ 673.21.
Example 41: Preparation of Compound 272
Preparation of Compound 271
To a solution of Compound 55 (39 mg, 0.03 mmol) in N, A-dimethylformamide (2 mL) was added Compound 204 (25 mg, 0.03 mmol), A,A’-diisopropylethylamine (0.03 mL, 0.16 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 0.4 mg, 0.003 mmol). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 271 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 1425.67.
Preparation of Compound 272
To a solution of Compound 271 (crude, 0.03 mmol) in methanol (0.5 mL) and tetrahydrofuran (0.5 mL) was added lithium hydroxide monohydrate (13.5 mg, 0.3 mmol) in distilled water (0.7 mL) at -45 °C under nitrogen. After stirring at 0 °C for 1 hour, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 272 (14 mg).
’H-NMR (400 MHz, DMSO-d6), δ 9.51 (s, 1H), 8.29 (s, 1H), 7.95 (d, J = 15.5 Hz, 2H), 7.83 (s, 1H), 7.64 (d, J = 6.9 Hz, 2H), 7.48 (d, J = 8.6 Hz, 1H), 7.36-7.28 (m, 4H), 7.24 (d, J = 8.6 Hz, 1H), 6.51 (s, 2H), 5.91-5.88 (m, 1H), 5.79 (s, 2H), 5.69-5.64 (m, 1H), 5.14 (d, J = 6.7 Hz, 1H), 5.05 (s, 2H), 4.89 (d, 7 = 10.7 Hz, 3H), 4.54-4.49 (m, 6H), 3.96 (d, 7 = 9.5 Hz, 1H), 3.70 (s, 3H), 2.09 (t, J = 2.8 Hz, 6H), 1.25 (q, J = 6.8 Hz, 6H). EI-MS m/z : [M+H]+ 1285.54. Example 42: Preparation of Compound 274
Preparation of Compound 273
To a solution of Compound 55 (34 mg, 0.04 mmol) in N, A-dimethylformamide (2 mL) was added Compound 260 (40 mg, 0.03 mmol), A, A-diisopropylcthylaminc (0.03 mL, 0.17 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 0.9 mg, 0.007 mmol). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 273 (crude), which was used without further purification. ELMS m/z : [1/2M+H]+ 764.28, [M+H]+ 1527.93. Preparation of Compound 274
To a solution of Compound 273 (crude, 0.039 mmol) in methanol (2 mL) was added lithium hydroxide monohydrate (16 mg, 0.39 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 274 (10.6 mg, 19%). ELMS m/z : [1/2M+M]+ 694.16, [M+H]+ 1386.77.
Example 43: Preparation of Compound 277
Preparation of Compound 275
To a solution of Compound 55 (40 mg, 0.033 mmol) in A,A-dimethylformamide (1 mL) was added Compound 267 (34.3 mg, 0.037 mmol), N, A’-diisopropylethylamine (0.03 mL, 0.167 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 0.9 mg, 0.007 mmol). After stirring at room temperature for 4 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 275 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 807.55. Preparation of Compound 276
To a solution of Compound 275 (53.7 mg, 0.033 mmol) in methanol (0.75 mL) and tetrahydrofuran (0.75 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 4 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure to afford Compound 276 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 737.69.
Preparation of Compound 277
To a solution of Compound 276 (crude, 0.033 mmol) in dichloromethane (1 mL) was added trifluoro acetic acid (1 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 277 (13 mg, 29%; 3 steps). EI-MS m/z : [M+H]+ 1359.59, [M/2+H]+ 680.61.
Example 44: Preparation of Compound 281
Preparation of Compound 278
To a solution of Compound 205 (1 g, 2.06 mmol, Compound 205 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al) was dissolved in W/V-dimethylformamide (10 mL) was added P-Alanine ethyl ester hydrochloride (380 mg, 2.48 mmol), 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethylaminium tetrafluoroborate (HBTU, 1.02 g, 2.68 mmol) and W/V’-diisopropylethylamine (0.72 mL, 4.13 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 18 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium hydrogen carbonate (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 278 (398 mg, 32%).
’H-NMR (400 MHz, CDC13), δ 8.02 (d, J = 2.6 Hz, 1H), 7.71 (s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.26 (s, 2H), 7.06 - 6.99 (m, 1H), 5.40 (dt, 7 = 18.9, 7.7 Hz, 3H), 5.29 (d. 7 = 7.3 Hz, 1H), 4.68 (d, J = 2.6 Hz, 2H), 4.26 - 4.14 (m, 3H), 3.79 (d, J = 6.9 Hz, 1H), 3.74 (d, J = 2.6 Hz, 3H), 3.69 - 3.60 (m, 1H), 2.67 (d, J = 7.0 Hz, 2H), 2.09 - 2.03 (m, 9H), 1.29 (td, J = 7.2, 2.5 Hz, 3H).
Preparation of Compound 279
To a solution of Compound 278 (398 mg, 0.68 mmol) in dichloromethane (4 mL) was added bis(pentafluorophenyl)carbonate (323 mg, 0.82 mmol) and A,A-diisopropylethylamine (0.36 ml, 2.04 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 14 hours, the reaction mixture was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 279 (280 mg, 51%). ELMS m/z : [M+H]+ 793.88.
Preparation of Compound 280
To a solution of Compound 55 (300 mg, 0.25 mmol) in A,A-dimethylformamide (5 mL) was added Compound 279 (218 mg, 0.28 mmol) and A,A’-diisopropylethylamine (0.22 mL, 0.13 mmol). After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 280 (370 mg, crude). ELMS m/z : [M+H]+ 1467.76.
Preparation of Compound 281
To a solution of Compound 280 (36 mg, 0.025 mmol) in methanol (1 mL) and tetrahydrofuran (1 mL) was added lithium hydroxide monohydrate (5.24 mg, 0.13 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring at 0 °C for 4 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 281 (13 mg). ELMS m/z : [M+H]+ 1299.88. Example 45: Preparation of Compound 285
Preparation of Compound 282
To a solution of Compound 205 (1 g, 2.06 mmol, Compound 205 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al) was dissolved in dichloromethane (3 mL) was added methyl 17-amino-3,6,9, 12,15- pentaoxaheptadecanoate (766 mg, 2.48 mmol), A-methylmorpholine (0.57 mL, 5.16 mmol), A-Ethyl-A'-(3-dimethylaminopropyl)carbodi imide hydrochloride (EDC-HC1, 415 mg, 2.17 mmol) and 1 -Hydroxybenzotriazole (306 mg, 2.27 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 18 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium hydrogen carbonate (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 282 (673 mg, 42%). EI-MS m/z : [M+H]+ 775.97.
Preparation of Compound 283
To a solution of Compound 282 (670 mg, 0.86 mmol) in dichloromethane (5 mL) was added bis(pentafluorophenyl)carbonate (408 mg, 1.04 mmol) and A,A-diisopropylethylamine (0.45 ml, 2.59 mmol) at -0 °C. After raising to room temperature and stirring under nitrogen for 18 hours, the reaction mixture was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 283 (280 mg, 51%). EI-MS m/z : [M+H]+ 986.17.
Preparation of Compound 284
To a solution of Compound 283 (300 mg, 0.25 mmol) in A,A-dimethylformamide (1.5 mL) were added A,A’-diisopropylethylamine (0.05 mL, 0.28 mmol) and Compound 55 (60 mg, 0.06 mmol) at 0 °C under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 284 (91.9 mg, crude). EI-MS m/z : [1/2M+H]+ 830.331.
Preparation of Compound 285
To a solution of Compound 284 (92 mg, 0.06 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (23 mg, 0.55 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 4 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 285 (35 mg, 42%). EI-MS m/z : [M+H]+ 1505.876.
Example 46: Preparation of Compound 291
Preparation of Compound 286 l-Azido-2-(2-(2-bromoethoxy)ethoxy)ethane (1.5 g, 6.3 mmol) was added morpholine (1.63 mL, 18.9 mmol). After stirring at 50 °C for 5 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 286 (793 mg, 52%).
Preparation of Compound 287
To a solution of Compound 286 (793 mg, 3.25 mmol) in tetrahydrofuran (3 mL) were added triphenylphosphine (937 mg, 3.57 mmol) and distilled water (1 mL) at 0 °C under nitrogen. The reaction mixture was refluxed for 15 hours. After concentrating under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 287 (607 mg, 86%).
’H-NMR (400 MHz, CDCh), δ 3.72 (t, J = 4.6 Hz, 4H), 3.63 (d, J = 2.5 Hz, 5H), 3.55 (d, J = 5.0 Hz, 1H), 2.98 - 2.88 (m, 4H), 2.60 (td, J = 5.9, 2.8 Hz, 2H), 2.51 (t, J = 4.8 Hz, 4H).
Preparation of Compound 288
To a solution of Compound 205 (607 mg, 2.78 mmol, Compound 205 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in dichloromethane (15 mL) were added A-(3-dimethylaminopropyl)-A’-ethylcarbodiimide hydrochloride (EDC-HC1, 560 mg, 2.92 mmol), 1 -hydroxybenzotriazole (HOBt, 413 mg, 3.06 mmol), Compound 287 (1.62 g, 3.34 mmol) and A-methylmorpholine (0.76 mL, 6.95 mmol) at 0 °C under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 288 (1.07 mg, 56%). EIMS m/z : [M+H]+ 685.98.
Preparation of Compound 289
To a solution of Compound 288 (100 mg, 0.15 mmol) in dichloromethane (1 mL) were added bis(pentafluorophenyl)carbonate (73 mg, 0.18 mmol) and A/A’-diisopropylethylamine (0.08 mL, 0.44 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 289 (120 mg), which was used without further purification. EI-MS m/z : [M+H]+ 895.98.
Preparation of Compound 290
To a solution of Compound 55 (40 mg, 0.05 mmol) in A/A-dimethylformamide (0.05 mL) were added A/A’-diisopropylethylamine (0.04 mL, 0.22 mmol) and Compound 289 (54 mg, 0.05 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure to afford Compound 290 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 1568.97.
Preparation of Compound 291
To a solution of Compound 290 (70 mg, 0.05 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (18.76 mg, 0.45 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 291 (42 mg, 53%). EI-MS m/z : [M+H]+ 1429.19.
Preparation of Compound 293
To a solution of Compound 292 (10 g, 17.41 mmol, Compound 292 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in dichloromethane (15 mL) were added imidazole (2.4 g, 34.81 mmol) and t-butyldimethylsilyl chloride (3.94 g, 26.11 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 293 (10.4 g, 87%).
’H-NMR (400 MHz, CDCh), δ 7.70 (s, 1H), 7.47 - 7.30 (m, 6H), 7.15 (d, 7 = 8.7 Hz, 1H), 5.31 (d, J = 17.6 Hz, 5H), 5.14 (d, J = 5.6 Hz, 1H), 4.68 (s, 2H), 4.18 - 4.11 (m, 1H), 3.73 (d, J = 2.9 Hz, 3H), 2.05 (s, 9H), 0.92 (d, J = 3.0 Hz, 9H), 0.08 (d, J = 2.9 Hz, 6H). Preparation of Compound 294
To a solution of Compound 293 (10 g, 15.1 mmol) in tetrahydrofuran (110 mL) and methanol (110 mL) were added 5% palladium/charcoal (520 mg) and distilled water (10 mL) under hydrogen. After stirring at room temperature for 2 hours, the reaction solution was filtered through Celite and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 294 (7.9 g, 87%).
’H-NMR (400 MHz, CDCh), δ 8.01 (d, 7 = 2.5 Hz, 1H), 7.58 (d, 7 = 8.2 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 5.46 - 5.26 (m, 4H), 4.72 (s, 2H), 4.30 (d, J = 8.2 Hz, 1H), 3.69 (d, J = 1.9 Hz, 3H), 2.14 - 1.99 (m, 9H), 0.94 (d, J = 2.0 Hz, 9H), 0.10 (s, 6H).
Preparation of Compound 295
To a solution of Compound 294 (500 mg, 0.84 mmol) in A,A-dimethylformamide (3 mL) were added iodomethane (0.3 mL, 4.18 mmol) and potassium carbonate (144 mg, 1.04 mmol) under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 295 (510 g, 99%). EI-MS m/z : [M+Na]+ 634.976.
Preparation of Compound 296
To a solution of Compound 295 (510 mg, 0.83 mmol) in methanol (3 mL) was added camphorsulfonic acid (39 mg, 0.17 mmol) at 0 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction solution was diluted with ethyl acetate (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 296 (360 g, 87%). EI-MS m/z : [M+Na]+ 521.024.
’H-NMR (400 MHz, CDCh), δ 7.76 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.26 (s, 2H), 7.15 (dd, 7 = 8.9, 2.5 Hz, 1H), 5.40 - 5.30 (m, 3H), 5.15 (d, 7 = 5.9 Hz, 1H), 4.67 (d, 7 = 4.8 Hz, 2H), 4.23 - 4.15 (m, 1H), 3.86 (d, 7 = 2.7 Hz, 3H), 3.74 (d, 7 = 2.8 Hz, 3H), 2.10 - 2.02 (m, 9H).
Preparation of Compound 297
To a solution of Compound 296 (185 mg, 0.24 mmol) in dichloromethane (5 mL) were added bis(pentafluorophenyl)carbonate (342 mg, 0.87 mmol) and A,A-diisopropylethylamine (0.38 ml, 2.17 mmol) at 0 °C under nitrogen. After stirring at room temperature for 14 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 297 (410 mg, 80%). EI-MS m/z : [M+Na]+ 730.794.
Preparation of Compound 298 To a solution of Compound 297 (50 mg, 0.04 mmol) in N, A-dimethylformamide (0.05 mL) were added A,A’-diisopropylethylamine (0.04 mL, 0.21 mmol) and Compound 55 (35 mg, 0.05 mmol) under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was concentrated under reduced pressure to afford Compound 298 (125 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1832.77.
Preparation of Compound 299
To a solution of Compound 298 (58 mg, 0.04 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (17.48 mg, 0.42 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 299 (50 mg, 82%). EI-MS m/z : [M/2+H]+ 614.87.
Example 48: Preparation of Compound 302
Preparation of Compound 300
To a solution of Compound 28 (30 mg, 0.02 mmol) in N, A-dimethylformamide (1 mL) were added Compound 267 (25.5 mg, 0.03 mmol) , A, A’-diisopropylethylamine (0.02 mL, 0.12 mmol) and l-hydroxy-7-azabenzotriazole (HO At, 2 mg, 0.02 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 300 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 759.25. Preparation of Compound 301
To a solution of Compound 300 (36.5 mg, 0.02 mmol) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (5.1 mg, 0.12 mmol) in distilled water (0.5 mL) at -70 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure to afford Compound 301 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 746.27.
Preparation of Compound 302
To a solution of Compound 301 (36.5 mg) in dichloromethane (1 mL) was added trifluoroacetic acid (0.25 mL) at 0 °C under nitrogen. After stirring at room temperature for 40 minutes, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 302 (24.8 mg, 63%). EI-MS m/z : [M+H]+ 1376.63, [M/2+H]+ 689.09.
Example 49: Preparation of Compound 307
Preparation of Compound 304
To a solution of Compound 303 (300 mg, 0.60 mmol, Compound 303 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (3 mL) were added A/,A/,A/’,A/’-tctiamcthyl-O-( l //-bcnzoti iazol- l - yl)uronium hexafluorophosphate (HBTU, 297 mg, 0.78 mmol), Compound 265 (190 mg, 0.72 mmol) and WN’-diisopropylethylamine (0.23 mL, 2.16 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 304 (379 mg, 85%).
’H-NMR (400 MHz, CDCh), δ 8.04 (s, 1H), 7.46 (d, 7 = 8.5 Hz, 1H), 7.38 (s, 1H), 7.06 (d, J = 8.5 Hz, 1H), 5.58 - 5.46 (m, 2H), 5.16 (d, J = 6.6 Hz, 2H), 4.68 (s, 2H), 4.25 - 4.09 (m, 3H), 3.74 (d, J = 6.2 Hz, 3H), 3.67 (d, J = 10.4 Hz, 6H), 3.54 (t, J = 5.8 Hz, 3H), 2.22 (s, 3H), 2.06 (d, J = 2.7 Hz, 6H), 2.02 (s, 3H), 0.89 (d, J = 2.0 Hz, 9H), 0.07 (s, 6H). EI-MS m/z : [M/2+H]+ 744.19.
Preparation of Compound 305
To a solution of Compound 304 (379 mg, 0.51 mmol) in dichloromethane (5 mL) were added bis(pentafluorophenyl)carbonate (241 mg, 0.61 mmol) and A,A-diisopropylethylamine (0.27 ml, 1.53 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 305 (424 mg, 87%). EI-MS m/z : [M+H]+ 954.08.
Preparation of Compound 306
To a solution of Compound 55 (30 mg, 0.03 mmol) in A,A-dimethylformamide (1 mL) were added Compound 305 (26.2 mg, 0.03 mmol) , A,A’-diisopropylethylamine (0.02 mL, 0.13 mmol) and l-hydroxy-7-azabenzotriazole (HO At, 0.7 mg, 0.005 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 306 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 814.51.
Preparation of Compound 307
To a solution of Compound 306 (40 mg, 0.02 mmol) in tetrahydrofuran (0.3 mL) and methanol (0.3 mL) was added lithium hydroxide monohydrate (10.3 mg, 0.25 mmol) in distilled water (0.3 mL) at -70 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The reaction mixture in dichloromethane (2 mL) was added trifluoroacetic acid (0.4 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 307 (24.4 mg, 63%). EI-MS m/z : [M+H]+ 1346.20, [M/2+H]+ 673.38. Example 50: Preparation of Compound 309
Preparation of Compound 308
To a solution of Compound 28 (59 mg, 0.049 mmol) in A,A-dimethylformamide (2 mL) were added Compound 260 (62 mg, 0.073 mmol) , A,A’-diisopropylethylamine (0.04 mL, 0.24 mmol) and l-hydroxy-7-azabenzotriazole (HO At, 1.3 mg, 0.009 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 308 (34 mg, 45%). EI-MS m/z : [M/2+H]+ 772.64.
Preparation of Compound 309
To a solution of Compound 308 (30 mg, 0.019 mmol) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (8.1 mg, 0.19 mmol) in distilled water (0.3 mL) at -70 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 309 (14 mg, 51%). EI-MS m/z : [M/2+H]+ 702.56.
Preparation Example 6: Preparation of Compound 310
Preparation of Compound 310
To a solution of Compound 259 (300 mg, 0.47 mmol) in dichloromethane (60 mL) were added triethylamine (0.13 ml, 0.93 mmol) and 4-nitrophenyl chloroformate (122 mg, 0.61 mmol) at 0 °C under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 310 (342 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 808.22.
Example 51: Preparation of Compound 313
To a solution of Compound 63 (500 mg, 0.09 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (1 mL) under nitrogen at 0°C. After stirring at room temperature for 1.5 hour, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed phase column chromatography to afford Compound 311 (286 mg, 47%). EI-MS m/z : [M+H]+ 958.99. Preparation of Compound 312
To a solution of Compound 311 (286 mg, 0.22 mmol) in A,A-dimethylformamide (3 mL) were added Compound 310 (213 mg, 0.26 mmol) , A,A’-diisopropylethylamine (0.2 mL, 1.10 mmol) and l-hydroxy-7-azabenzotriazole (HO At, 3 mg, 0.02 mmol) at 0 °C under nitrogen. After stirring at room temperature for 21 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed phase column chromatography to afford Compound 312 (294 mg, 68%). EI-MS m/z : [M+H]+ 1626.61.
Preparation of Compound 313
To a solution of Compound 312 (294 mg) in tetrahydrofuran (1.5 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (93 mg, 2.24 mmol) in distilled water (4 mL) at -70 °C under nitrogen. After stirring at 0 °C for 6 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 313 (115 mg, 42%).
’H-NMR (400 MHz, MeOD), δ 7.87 (d, J = 2.3 Hz, 1H), 7.66 (s, 1H), 7.58 (dd, 7 = 6.9, 1.3 Hz, 2H), 7.49 - 7.40 (m, 1H), 7.36 (s, 1H), 7.28 - 7.20 (m, 3H), 6.57 (d, J = 15.2 Hz, 2H), 5.94 - 5.60 (m, 4H), 5.09 (d, J = 4.9 Hz, 2H), 4.96 (t, J = 15.4 Hz, 2H), 4.87 (s, 2H), 4.77 (s, 4H), 4.56 (q, J = 6.4 Hz, 4H), 4.48 (d, J = 5.4 Hz, 2H), 4.05 (d, J = 9.5 Hz, 1H), 3.92 (t, J = 6.4 Hz, 2H), 3.79 (dd, J = 5.7, 4.4 Hz, 2H), 3.70 (s, 2H), 3.69 - 3.48 (m, 4H), 2.84 (s, 6H), 2.31 (t, 7 = 7.3 Hz, 2H), 2.18 (d, 7 = 6.3 Hz, 6H), 1.84 (p, 7 = 6.9 Hz, 2H), 1.33 - 1.29 (m, 6H). EI-MS m/z : [M+H]+ 1459.85.
Example 52: Preparation of Compound 316
Preparation of Compound 314
To a solution of Compound 311 (120 mg, 0.09 mmol) in A,A-dimethylformamide (1 mL) were added Compound 267 (104 mg, 0.11 mmol) , A,A’-diisopropylethylamine (0.1 mL, 0.59 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 314 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 857.39.
Preparation of Compound 315 To a solution of Compound 314 (139 mg, 0.08 mmol) in tetrahydrofuran (1.3 mL) and methanol (1.3 mL) was added lithium hydroxide monohydrate (17 mg, 0.41 mmol) in distilled water (1.3 mL) at -70 °C under nitrogen. After stirring at 0 °C for 6 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure to afford Compound 315 (crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 773.34.
Preparation of Compound 316
To a solution of Compound 315 (121 mg, 0.08 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 316 (58.9 mg, 45%). EI-MS m/z : [M+H] + 1431.89, [M/2+H]+ 716.28.
Example 53: Preparation of Compound 320
Preparation of Compound 318
To a solution of Compound 317 (1.6 g, 2.90 mmol, Compound 317 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (10 mL) were added bis(pentafluorophenyl)carbonate (1.4 g, 3.48 mmol) and W/V-diisopropylethylamine (1.03 ml, 5.80 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (100 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 318 (2.0 g, 90%). EI-MS m/z : [M+H]+ 765.89.
Preparation of Compound 319 To a solution of Compound 318 (25 mg, 0.021 mmol) in MA-dimcthylformamidc (1 mL) were added Compound 55 (17 mg, 0.022 mmol) , MA’-diisopi'opylcthylaminc (0.02 mL, 0.104 mmol) and l-hydroxy-7-azabenzotriazole (HOAt, 0.8 mg, 0.006 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to afford Compound 319 (32 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 1440.26.
Preparation of Compound 320
To a solution of Compound 319 (32 mg, crude) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (9.3 mg, 9.33 mmol) in distilled water (0.5 mL) at -70 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 320 (23 mg). EI-MS m/z : [M+H]+ 1271.95.
Example 54: Preparation of Compound 328
Preparation of Compound 321
To a solution of 3-((t-butylcarbonyl)amino)propanoic acid (500 mg, 2.64 mmol) in dichloromethane (20 mL) were added A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride (EDC-HC1, 760 mg, 3.96 mmol) and A-hydroxysuccinimide (423 mg, 3.96 mmol). After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (50 mL) and then washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 321 (806 mg, crude), which was used without further purification. ’H-NMR (400 MHz, CDCh), δ 5.10 (s, 1H), 3.55 - 3.50 (m, 2H), 2.85 - 2.80 (m, 6H), 1.45 (s, 9H).
Preparation of Compound 322
To a solution of fl-glutamic acid hydrochloride (1 g, 5.45 mmol) in methanol (30 mL) was added thionyl chloride (2 mL, 27.23 mmol) at 0 °C under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure to afford Compound 322 (1.17 g, crude), which was used without further purification.
’H-NMR (400 MHz, MeOD), δ 3.75 (s, 6H), 2.89 - 2.71 (m, 4H).
Preparation of Compound 323
To a solution Compound 322 (493 mg, 2.82 mmol) in dichloromethane (20 mL) were added A,A-diisopropylethylamine (1.47 mL, 8.45 mmol) and Compound 321 (806 mg, 2.82 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (100 mL) and then washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 323 (903 mg, 92%).
’H-NMR (400 MHz, CDCh), δ 5.19 (s, 1H), 4.68 - 4.58 (m, 1H), 3.70 (s, 6H), 3.39 (d, J = 6.6 Hz, 2H), 2.76 - 2.58 (m, 4H), 2.36 (t, J = 6.0 Hz, 2H), 1.44 (s, 9H).
Preparation of Compound 324
To a solution of Compound 323 (903 mg, 2.61 mmol) in dichloromethane (10 mL) was added hydrogen chloride (4M 1,4-dioxane solution, 5 mL). After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 324 (845 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 247.22.
Preparation of Compound 325
To a solution of Compound 205 (1.0 g, 2.06 mmol, Compound 205 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in dichloromethane (20 mL) were added A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (EDC-HC1, 415 mg, 2.27 mmol), 1 -hydroxybenzotriazole (HOBt, 306 mg, 2.27 mmol), Compound 324 (700 mg, 2.48 mmol), and A-methylmorpholine (0.68 mL, 6.19 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 325 (1.03 mg, 70%).
’H-NMR (400 MHz, DMSO-d6), δ 7.98 - 7.88 (m, 2H), 7.59 (s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.11 (d, 7 = 8.5 Hz, 1H), 5.71 (d, 7 = 7.9 Hz, 1H), 5.49 (t, 7 = 9.6 Hz, 1H), 5.28 - 5.18 (m, 2H), 5.09 (t, J = 9.8 Hz, 1H), 4.74 (d, J = 9.9 Hz, 1H), 4.46 (d, J = 5.2 Hz, 2H), 4.39 (d, J = 7.8 Hz, 1H), 3.65 (s, 3H), 3.57 (s, 6H), 2.33 - 2.28 (m, 4H), 2.01 (s, 9H).
Preparation of Compound 326
To a solution of Compound 325 (1.03 g, 1.45 mmol) in dichloromethane (30 mL) were added bis(pentafluorophenyl)carbonate (627 mg, 1.59 mmol) and A,A-diisopropylethylamine (0.38 mL, 2.17 mmol) at 0 °C under nitrogen. After stirring at room temperature for 5 hours, the reaction solution diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 326 (92 mg, 39%). EIMS m/z : [M+H]+ 922.89.
Preparation of Compound 327
To a solution of Compound 55 (50 mg, 0.04 mmol) in N, A-dimethylformamide (1 mL) were added Compound 326 (40 mg, 0.04 mmol), AA’-diisopropylethylamine (0.04 mL, 0.21 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to afford Compound 327 (70 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1596.94.
Preparation of Compound 328
To a solution of Compound 327 (70 mg, crude) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (34.7 mg, 0.83 mmol) in distilled water (1 mL) at -45 °C under nitrogen. After stirring at 0 °C for 1 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 328 (39 mg). EI-MS m/z : [M+H]+ 1428.89. Example 55: Preparation of Compound 330
Preparation of Compound 329
To a solution of Compound 69 (100 mg, 0.08 mmol) in A,A-dimethylformamide (2 mL) were added Compound 260 (82 mg, 0.40 mmol), A,A’-diisopropylethylamine (0.08 mL, 0.42 mmol) and l-hydroxy-7-azabenzotriazole (HOAt, 2.0 mg, 0.02 mmol) at 0 °C under nitrogen. After stirring at room temperature for 20 hours, the reaction solution was concentrated under reduced pressure and purified by reversed phase column chromatography to afford Compound 329 (58 mg). EI-MS m/z : [M+H]+ 1513.05. Preparation of Compound 330
To a solution of Compound 329 (58 mg, crude) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (6.4 mg, 0.15 mmol) in distilled water (0.5 mL) at -70 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 330 (17 mg, 32%). EI-MS m/z : [M+H]+ 1428.89.
Example 56: Preparation of Compound 332 Preparation of Compound 331
To a solution of Compound 79 (35 mg, 0.028 mmol) in W/V-dimethylformamide (1 mL) were added Compound 289 (25 mg, 0.028 mmol), W/V’-diisopropylethylamine (0.02 mL, 0.142 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was concentrated under reduced pressure to afford Compound 331 (45 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1601.84.
Preparation of Compound 332
To a solution of Compound 331 (45 mg, crude) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (12 mg, 0.28 mmol) in distilled water (0.5 mL) at -70 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 332 (32 mg). EI-MS m/z : [M+H]+ 1461.96.
Example 57: Preparation of Compound 337
Preparation of Compound 334
To a solution of Compound 333 (300 mg, 0.60 mmol, Compound 333 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (3 mL) were added A/,A/,A/’,A/’-tctiamcthyl-O-( l //-bcnzoti'iazol- l - yl)uronium hexafluorophosphate (HBTU, 297 mg, 0.78 mmol), Compound 258 (127 mg, 0.72 mmol) and A,A’-diisopropylethylamine (0.20 mL, 1.20 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 334 (190 mg, 48%). EI-MS m/z : [M+H]+ 657.14.
Preparation of Compound 335
To a solution of Compound 334 (57 mg, 0.09 mmol) in dichloromethane (3 mL) were added bis(pentafluorophenyl)carbonate (34 mg, 0.09 mmol) and A,A-diisopropylethylamine (0.01 mL, 0.26 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to afford Compound 335 (crude), which was used without further purification. EI-MS m/z : [M+H]+ 867.76.
Preparation of Compound 336
To a solution of Compound 335 (50 mg, 0.06 mmol) in dichloromethane (3 mL) were added Compound 55 (60 mg, 0.07 mmol), A,A’-diisopropylethylamine (0.03 mL, 0.17 mmol) and l-hydroxy-7-azabenzotriazole (HO At, 1.6 mg, 0.01 mmol) at 0 °C under nitrogen. After stirring at room temperature for 18 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed phase column chromatography to afford Compound 336 (36 mg, 40%). EI-MS m/z : [M+H]+ 1450.92.
Preparation of Compound 337
To a solution of Compound 336 (36 mg, crude) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (10 mg, 0.24 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 337 (10 mg, 30%). EI-MS m/z : [M+H]+ 1372.04.
Example 58: Preparation of Compound 344
Preparation of Compound 339
To a solution of Compound 338 (300 mg, 0.755 mmol, Compound 338 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in dichloromethane (5 mL) were added Molecular Sieve (500 mg), t-butyl (2-(2-(2- hydroxyethoxy)ethoxy)ethyl)carbamate (226 mg, 0.91 mmol) and trifluoromethanesulfonate (233 mg, 0.91 mmol) at 0 °C under nitrogen. After stirring at room temperature for 40 minutes, the reaction solution was diluted with dichloromethane (100 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 339 (150 mg, 35%). ELMS m/z : [M+H]+ 566.19.
Preparation of Compound 340
To a solution of Compound 339 (150 mg, 0.265 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.1 mL) at 0 °C under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure to afford Compound 340 (153 mg, crude), which was used without further purification. ELMS m/z : [M+H]+ 466.19.
Preparation of Compound 341 To a solution of Compound 340 (154 mg, 0.32 mmol) in A,A-dimethylformamide (1 mL) were added A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (EDC-HC1, 53.38 mg, 0.28 mmol), 1 -hydroxybenzotriazole (HOBt, 39.4 mg, 0.29 mmol), Compound 205 (153 mg), and A-methylmorpholine (0.07 mL, 0.66 mmol) at 0 °C under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 341 (139 mg, 56%). ELMS m/z : [M+H]+ 932.01.
Preparation of Compound 342
To a solution of Compound 341 (130 mg, 0.14 mmol) in dichloromethane (1 mL) were added bis(pentafluorophenyl)carbonate (66 mg, 1.17 mmol) and A,A-diisopropylethylamine (0.07 ml, 0.42 mmol) at 0 °C under nitrogen. After stirring at room temperature for 20 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 342 (159 mg, 99%). ELMS m/z : [M+H]+ 1141.92.
Preparation of Compound 343
To a solution of Compound 55 (184 mg, 0.15 mmol) in A,A-dimethylformamide (1 mL) were added Compound 342 (159 mg, 0.14 mmol) and A,A’-diisopropylethylamine (0.12 mL, 0.7 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 343 (90 mg, 36%). ELMS m/z : [M+H]+ 908.59.
Preparation of Compound 344
To a solution of Compound 343 (90 mg, 0.05 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (20.8 mg, 0.5 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 344 (13.8 mg, 18%). ELMS m/z : [M+H]+ 1535.25. Preparation of Compound 346
To a solution of Compound 345 (2 g, 3.84 mmol; Compound 345 was prepared by the method described in U.S. Patent No. 11,654,197 B2) in dichloromethane (20 mL) were added bis(pentafluorophenyl)carbonate (1.65 g, 4.99 mmol) and N,N-diisopropylethylamine (2 ml, 11.51 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 346 (2 g, 71%).
’H-NMR (400 MHz, DMSO), δ 7.96 (d, J = 6.4 Hz, 1H), 7.74 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 5.79 (d, J = 7.9 Hz, 1H), 5.49 (t, J = 9.8 Hz, 1H), 5.39 (s, 2H), 5.22 (t, J = 8.9 Hz, 1H), 5.08 (t, J = 9.7 Hz, 1H), 4.75 (d, J = 10.4 Hz, 1H), 4.12 (d, J = 2.5 Hz, 2H), 4.08 - 3.96 (m, 1H), 3.64 (d, J = 2.2 Hz, 4H), 3.54 (q, J = 5.1 Hz, 10H), 2.06 - 1.96 (m, 10H). ELMS m/z : [M+H]+ 731.84. Preparation of Compound 347
To a solution of Compound 55 (108 mg, 0.09 mmol) in A,iV-dimethylformamide (1 mL) were added Compound 346 (26.2 mg, 0.03 mmol) and A,iV’-diisopropylethylamine (0.02 mL, 0.13 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 347 (128 mg, crude), which was used without further purification. ELMS m/z : [M+H]+ 1405.24.
Preparation of Compound 349
To a solution of Compound 347 (124 mg, 0.091 mmol) in ethanol (2 mL), dichloromethane and distilled water (2 mL) were added Compound 348 (37 mg, 0.091 mmol, Compound 348 was prepared by the method described in the United States patent publication No. US 2010-0323973 Al), Tris(3-hydroxypropyltriazolymethyl)amine (THPTA, 4.7 mg, 0.04 mmol), Copper(II) sulfate pentahydrate (18.2 mg, 0.073 mmol) and ascorbic acid (24.1 mg, 0.136 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 349 (164 mg, crude), which was used without further purification. ELMS m/z : [M+H]+ 1808.89.
Preparation of Compound 350
To a solution of Compound 349 (164 mg, 0.09 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (17 mg, 0.41 mmol) in distilled water (1 mL) at -70 °C under nitrogen. After stirring at 0 °C for 1 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 350 (16 mg, 11%). ELMS m/z : [M/2+H]+ 1529.044. Example 60: Preparation of Compound 352
Preparation of Compound 351
To a solution of Compound 69 (100 mg, 0.08 mmol) and Compound 310 (82 mg, 0.10 mmol) in A,A-dimethylformamide (2 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 2 mg, 0.02 mmol) and A,A’-diisopropylethylamine (0.08 mL, 0.42 mmol) at 0 °C under nitrogen. After stirring at room temperature for 20 hours under nitrogen, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography to afford Compound 351 (58 mg). EI-MS m/z : [M+H]+ 1513.05. Preparation of Compound 352
To a solution of Compound 351 (58 mg) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (38 mg, 0.91 mmol) in distilled water (1 mL) at -70 °C. After stirring for 6 hours at room temperature, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 352 (17 mg, 2-steps 15%). EIMS m/z : [M+H]+ 1372.54.
Example 61: Preparation of Compound 357
Preparation of Compound 354
To a solution of Compound 353 (300 mg, 0.52 mmol; Compound 353 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in dichloromethane (10 mL) were added bis(pentafluorophenyl)carbonate (417 mg, 1.06 mmol) and A/A’-diisopropylethylamine (0.27 mL, 1.58 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with dichloromethane (30 mL) and washed with distilled water (30 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 354 (402 mg, 97%). EIMS m/z : [M+H]+ 777.54.
Preparation of Compound 355
To a solution of Compound 55 (100 mg, 0.11 mmol) and Compound 354 (108 mg, 0.08 mmol) in A A-dimcthylformamidc (1 mL) was added A,A’-diisopropylethylamine (0.06 mL, 0.35 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was diluted with ethyl acetate (30 mL) and washed with saturated aqueous sodium bicarbonate solution (30 mL) and distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate. After being filtered and concentrated under reduced pressure to afford Compound 355 (128 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1450.47.
Preparation of Compound 356
To a solution of Compound 355 (64 mg) and but-3-yn-l-yl dihydrogen phosphate (299 mg, 1.10 mmol) in ethanol (2 mL), dichloromethane (2 mL) and distilled water (2 mL) were added tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 4.4 mg, 0.02 mmol), copper(II) sulfate pentahydrate (18.2 mg, 0.073 mmol) and sodium ascorbate (3.5 mg, 0.02) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtered and concentrated under reduced pressure to afford Compound 356 (72 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1601.37.
Preparation of Compound 357
To a solution of Compound 356 (72 mg) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (70.8 mg, 0.49 mmol) in distilled water (1 mL) at -45 °C. After stirring for 1 hour at room temperature, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 357 (10 mg). EI-MS m/z : [M+H]+ 1461.43. Example 62: Preparation of Compound 359
Preparation of Compound 358
To a solution of Compound 168 (100 mg, 0.08 mmol) and Compound 310 (152 mg, 0.19 mmol) in A A-di methyl formamide (3 mL) were added l-hydroxy-7-azabenzotriazole
(HO At, 4 mg, 0.03 mmol) and A,A’-diisopropylethylamine (0.07 mL, 0.38 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography to afford Compound 358 (168 mg, 80%). ELMS m/z : [1/2M+H]+ 1158.97, [1/3M+H]+ 772.95.
Preparation of Compound 359
To a solution of Compound 358 (168 mg, 0.073 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL) was added lithium hydroxide monohydrate (92 mg, 2.18 mmol) in distilled water (3 mL) at -70 °C. After stirring for 6 hours at room temperature, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 359 (22 mg, 15%). ELMS m/z : [M+H]+ 2036.20, [1/2M+H]+ 1018.64.
Example 63: Preparation of Compound 361
Preparation of Compound 360
To a solution of Compound 311 (45 mg, 0.034 mmol) and Compound 289 (31 mg, 0.034 mmol) in M A-dimcthylformamidc (1 mL) was added N, A’-di isopropylethylamine (0.02 mL, 0.14 mmol) at 0 °C under nitrogen. After stirring at room temperature for 6 hours, the reaction solution was concentrated under reduced pressure to afford Compound 360 (51 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1668.24.
Preparation of Compound 361 To a solution of Compound 360 (51 mg) in tetrahydrofuran (0.5 mL) and methanol
(0.5 mL) was added lithium hydroxide monohydrate (12 mg, 0.28 mmol) in distilled water (0.5 mL) at -70 °C. After stirring for 3 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 361 (22 mg, 15%). EI-MS m/z : [M+H]+ 1500.26.
Example 64: Preparation of Compound 363
Preparation of Compound 362
To a solution of Compound 174 (178 mg, 0.19 mmol) and Compound 310 (183 mg, 0.23 mmol) in MA-dimcthylformamidc (2 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 5 mg, 0.04 mmol) and A,A’-diisopropylethylamine (0.13 mL, 0.94 mmol) at 0 °C under nitrogen. After stirring at room temperature for 40 hours under nitrogen, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 362 (65 mg, 21%). ELMS m/z : [M/2+H]+ 807.37.
Preparation of Compound 363
To a solution of Compound 362 (72 mg, 0.05 mmol) in tetrahydrofuran (0.5 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (9.4 mg, 0.22 mmol) in distilled water (1 mL) at -45 °C. After stirring at 0 °C for 1 hours, the reaction solution was added lithium hydroxide monohydrate (20 mg, 0.48 mmol) in distilled water (5 mL) at 0 °C. After stirring for 4 hours at room temperature, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 363 (23 mg, 35%). ELMS m/z : [M+H]+ 1458.92.
Example 65: Preparation of Compound 367
Preparation of Compound 364
To a solution of Compound 205 (350 mg, 0.72 mmol, Compound 205 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (2 mL) were added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide hydrochloride (EDC-HC1, 411 mg, 1.08 mmol), 1 -hydroxybenzotriazole (HOBt, 140 mg, 1.08 mmol), 2-(2-(2-azidoethoxy)ethoxy)ethanamine (151 mg, 0.86 mmol), and A/A’-diisopropylethylamine (0.23 mL, 2.16 mmol) at 0 °C under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 364 (250 mg, 54%).
’H-NMR (400 MHz, CDCh), δ 8.03 (m, 2H), 7.47 (m, 1H), 7.39 (m, 1H), 7.04 (m, 1H), 5.40 (m, 3H), 5.24 (s, 1H), 4.69 (s, 2H), 4.20 (m, 1H). 3.74 (m, 13H), 3.57 (m, 4H), 3.37 (m, 3H), 2.95 (s, 4H), 2.89 (s, 4H), 2.07 (s, 9H). ELMS m/z : [M+H]+ 762.17.
Preparation of Compound 365
To a solution of Compound 364 (250 mg, 0.39 mmol) in dichloromethane (2 mL) were added bis(pentafluorophenyl)carbonate (307 mg, 0.78 mmol) and W/V’-diisopropylethylamine (0.2 mL, 1.17 mmol) at 0 °C under nitrogen. After stirring at room temperature for 14 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 365 (230 mg, 69%). ELMS m/z : [M+H]+ 851.25.
Preparation of Compound 366
To a solution of Compound 55 (223 mg, 0.26 mmol) and Compound 365 (150 mg, 0.17 mmol) in A A-di methyl formamide (3 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 2.3 mg, 0.017 mmol) and A,A’-diisopropylethylamine (0.12 mL, 0.69 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 366 (240 mg, 90%). ELMS m/z : [M/2+H]+ 763.14
Preparation of Compound 367
To a solution of Compound 366 (210 mg, 0.13 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (26 mg, 0.61 mmol) in distilled water (2 mL) at -50 °C. After stirring for 3 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 367 (80 mg, 41%).
’H-NMR (400 MHz, DMSO), δ 9.50 (s, 1H), 8.20 (t, 7 = 8.0 Hz, 1H), 7.92 (d, 7 = 16.0 Hz, 2H), 7.95 (s,lH), 7.83 (m, 3H), 7.64 (m, 1H), 7.30 (m, 4H), 7.20 (m, 1H), 6.50 (s, 2H), 5.92 (m, 1H), 5.79 (m, 2H), 5.69 (m,2H), 5.42 (m, 2H), 5.12 (d, 7 = 8.0 Hz, 1H), 2.05 (s, 2H), 4.90 (m, 4H), 3.97 (d, J = 16.0 Hz, 1H), 3.69 (s, 3H). 3.61 (m, 8H), 2.68 (s, 2H), 2.33(s, 2H), 2.09 (m, 6H), 1.27 (m, 8H). ELMS m/z : [M/2+H]+ 693.12.
Example 66: Preparation of Compound 372
Preparation of Compound 368
To a solution of Compound 205 (169 mg, 0.68 mmol, Compound 205 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in W/V-dimethylformamide (5 mL) were added A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide hydrochloride (EDC-HC1, 178 mg, 0.92 mmol), 1 -hydroxybenzotriazole (HOBt, 125 mg, 0.92 mmol), Compound 256 (300 mg, 0.61 mmol), and N,N’- diisopropylethylamine (0.2 mL, 1.85 mmol) at 0 °C under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 368 (390 mg, 88%).
’H-NMR (400 MHz, CDCh), δ 7.89 (t, J = 5.7 Hz, 1H), 7.59(d, J = 2.3 Hz, 1H), 7.39 (dd, J = 8.5. 2.3 Hz, 1H), 7.12 (d, 7 = 8.6 Hz, 1H), 6.74 (s, 1H), 5.77-5.70 (m, 1H), 5.49 (t, 7 = 9.6 Hz, 1H), 5.20(dd, J = 9.7, 7.9 Hz, 2H), 5.07 (t, J = 9.7 Hz, 1H), 4.73 (d, J = 9.9 Hz, 2H), 4.46 (d, 7 = 4.8 Hz, 2H), 3.63 (s, 1H), 3.59-3.48 (m, 6H), 3.43-3.37 (m, 4H), 3.06 (q, 7 = 6.0 Hz, 2H), 2.04-1.96 (m, 9H), 1.37 (s, 10H). EI-MS m/z : [M+Na]+ 715.44.
Preparation of Compound 369
To a solution of Compound 368 (150 mg, 0.20 mmol) in dichloromethane (3 mL) were added bis(pentafluorophenyl)carbonate (124 mg, 0.31 mmol) and A,A’-diisopropylethylamine (0.1 mL, 0.62 mmol) at 0 °C under nitrogen. After stirring at room temperature for 14 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 369 (180 mg, 92%).
’H-NMR (400 MHz, DMSO), δ 7.98 (t, 7 = 5.7 Hz, 1H), 7.74 (d, 7 = 2.3 Hz, 1H), 7.59 (dd, 7 = 8.6, 2.4 Hz, 1H), 7.22 (d, 7 = 8.7 Hz, 1H), 6.73 (s, 1H), 5.84-5.73 (m, 3H), 5.49 (t, 7 = 9.6 Hz, 1H), 5.39 (s, 2H), 5.22 (dd, J = 9.7, 7.8 Hz, 1H), 5.08 (t, J = 9.7 Hz, 1H), 4.75 (d, J = 10.0 Hz, 1H), 3.64 (s, 3H), 3.53 (dt, J = 10.8, 5.4 Hz, 6H), 3.39 (q, J = 5.9 Hz, 6H), 3.06 (q, J = 6.0 Hz, 2H), 2.69-2.65 (m, 1H), 2.35-2.30 (m, 1H), 1.36 (s, 9H). EI-MS m/z : [M+H]+ 925.42. Preparation of Compound 370
To a solution of Compound 55 (70 mg, 0.08 mmol) and Compound 369 (90 mg, 0.10 mmol) in A,A-dimethylformamide (3 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 1.1 mg, 0.01 mmol) and A,A’-diisopropylethylamine (0.07 mL, 0.40 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 370 (120 mg, 91%).
’H-NMR (400 MHz, DMSO), δ 12.82 (s, 2H), 9.49 (s, 1H), 8.48 (s, 3H), 7.92 (s, 3H), 7.63 (d, J = 8.7 Hz, 3H), 7.46 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 7.4 Hz, 4H), 7.15 (d, J = 8.6 Hz, 1H), 6.72 (s, 1H), 6.50 (d, 7 = 2.8 Hz, 2H), 5.85-5.63 (m, 4H), 5.48 (t, 7 = 9.6 Hz, 1H), 5.21 (d, J = 8.1 Hz, 1H), 5.12-5.00 (m, 3H), 4.89 (d, J = 10.2 Hz, 3H), 4.72 (d, J = 9.9 Hz, 1H), 4.52 (t, 7 = 8.5 Hz, 6H), 3.69 (s, 2H), 3.62 (s, 7H), 3.56-3.47 (m, 5H), 3.20-3.11 (m, 6H), 3.05 (d, 7 = 6.0 Hz, 5H), 2.09 (d, 7 = 3.8 Hz, 4H), 2.05-1.98 (m, 6H), 1.36 (s, 9H). EI-MS m/z : [M/2+H]+ 801.00.
Preparation of Compound 371
To a solution of Compound 370 (120 mg, 0.075 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (12 mg, 0.30 mmol) in distilled water (2 mL) at -50 °C. After stirring for 3 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 371 (80 mg, 73%). EI-MS m/z : [M/2+H]+ 730.32.
Preparation of Compound 372 To a solution of Compound 371 (80 mg, 0.075 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (0.45 mL) at 0 °C under nitrogen. After stirring at room temperature for 1.5 hours under nitrogen, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography to afford Compound 372 (90 mg, 96%). EI-MS m/z : [M/2+H]+ 680.30.
Example 67: Preparation of Compound 374
Preparation of Compound 373
To a solution of Compound 259 (38 mg, 0.06 mmol) in dichloromethane (2 mL) were added A,A’-diisopropylethylamine (0.02 mL, 0.13 mmol) and triphosgene (8 mg, 0.03 mmol) at -70 °C under nitrogen. After stirring for 2 hours at -70 °C under nitrogen, the reaction solution was added Compound 178 (40 mg, 0.03 mmol) and A,A’-diisopropylethylamine (0.023 mL, 0.17 mmol) in A,A-dimethylformamide (3 mL) at -70 °C. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure to afford Compound 373 (60 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1524.75.
Preparation of Compound 374
To a solution of Compound 373 (60 mg) in tetrahydrofuran (0.4 mL) and methanol (0.2 mL) was added lithium hydroxide monohydrate (20 mg, 0.49 mmol) in distilled water (1.2 mL) at -70 °C. After stirring for 6 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 374 (22 mg, 39%). EI-MS m/z : [M+H]+ 1384.70, [1/2M+H]+ 693.18. Example 68: Preparation of Compound 376
Preparation of Compound 375
To a solution of Compound 259 (30 mg, 0.045 mmol) in dichloromethane (1 mL) were added A,A’-diisopropylethylamine (0.01 mL, 0.08 mmol) and triphosgene 5 mg, 0.017 mmol) at -70 °C under nitrogen. After stirring for 2 hours at -70 °C, the reaction solution was added Compound 197 (15 mg, 0.011 mmol) and A,A’-diisopropylethylamine (0.008 mL, 0.056 mmol) in A,A-dimethylformamide (3 mL) at -70 °C. After stirring at room temperature for 16 hours under nitrogen, the reaction solution was concentrated under reduced pressure to afford Compound 375 (40 mg, crude), which was used without further purification. EI-MS m/z : [1/2M+H]+ 1151.05, [1/3M+H]+ 767.81.
Preparation of Compound 376
To a solution of Compound 375 (40 mg) in tetrahydrofuran (0.4 mL) and methanol (0.2 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol) in distilled water (1.2 mL) at -70 °C. After stirring for 6 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 376 (5 mg, 19%). EI-MS m/z : [M+H]+ 2020.33, [1/2M+H]+ 1011.01, [1/3M+H]+ 674.39.
Example 69: Preparation of Compound 381
Preparation of Compound 377
To a solution of 3-(2-(2-azidoethoxy)ethoxy)prop-l-yne (260 mg, 1.53 mmol) in tetrahydrofuran (3 mL) were added triphenylphosphine (443 mg, 1.68 mmol) and distilled water (0.27 mL, 15.3 mmol) at 0 °C under nitrogen. The reaction mixture was refluxed for 16 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 377 (160 mg, 72%).
’H-NMR (400 MHz, CDC13), δ 4.22 (s, 2H), 4.14-3.93 (m, 4H), 3.66-3.59 (m, 2H), 2.95-3.93 (m, 2H). EI-MS m/z : [M+H]+ 144.28.
Preparation of Compound 378
To a solution of Compound 205 (141 mg, 0.99 mmol Compound 205 was prepared by the method described in the International Patent Publication No. WO 2018/182341 Al) in N,N- dimethylformamide (5 mL) were added A-(3-dimethylaminopropyl)-A’ -ethylcarbodiimide hydrochloride (EDC-HC1, 189 mg, 0.99 mmol), 1 -hydroxybenzotriazole (HOBt, 133 mg, 0.99 mmol), Compound 377 (320 mg, 0.66 mmol), and A,A’-diisopropylethylamine (0.21 mL, 1.98 mmol) at 0 °C under nitrogen. After stirring at room temperature for 15 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 378 (330 mg, 81%).
’H-NMR (400 MHz, DMSO), δ 7.95 (s, 1H), 7.89 (t, J = 5.6 Hz, 2H), 7.60 (d, J = 2.3 Hz, 1H), 7.39 (dd, 7 = 8.6, 2.3 Hz, 2H), 7.11 (d. 7 = 8.5 Hz, 2H), 5.78-5.70 (m, 2H), 5.49 (t, J = 9.6 Hz, 2H), 5.20 (dd, J = 9.7, 7.9 Hz, 3H), 5.07 (t, J = 9.8 Hz, 2H), 4.73 (d, J = 9.9 Hz, 2H), 4.46 (s, 3H), 4.15 (d, J = 2.4 Hz, 4H), 3.63 (s, 5H), 3.58 (s, 8H), 2.89 (s, 3H), 2.73 (s, 3H), 2.05-1.98 (m, 12H). EI-MS m/z : [M+H]+ 610.34.
Preparation of Compound 379
To a solution of Compound 378 (110 mg, 0.18 mmol) in dichloromethane (3 mL) were added bis(pentafluorophenyl)carbonate (71 mg, 0.18 mmol) and A,A’-diisopropylethylamine (0.1 mL, 0.62 mmol) at 0 °C under nitrogen. After stirring at room temperature for 14 hours, the reaction solution was diluted with dichloromethane (15 mL) and washed with distilled water (15 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 379 (140 mg, 94%). EI-MS m/z : [M+H]+ 820.34.
Preparation of Compound 380
To a solution of Compound 55 (44 mg, 0.05 mmol) and Compound 379 (50 mg, 0.04 mmol) in A A-di methyl formamide (2 mL) was added A,A’-diisopropylethylamine (0.036 mL, 0.20 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 380 (62 mg, 99%). EI-MS m/z : [M/2+H]+ 747.67.
Preparation of Compound 381
To a solution of Compound 380 (60 mg, 0.04 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (7.5 mg, 0.18 mmol) in distilled water (2 mL) at -50 °C. After stirring for 3 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 381 (40 mg, 73%).
’H-NMR (400 MHz, DMSO), δ 12.82 (s, 1H), 9.51 (s, 1H), 8.29 (s, 1H), 7.94 (d, J = 15.6 Hz, 2H), 7.83 (d, J = 2.3 Hz, 1H), 7.64 (d, J = 6.5 Hz, 2H), 7.54 - 7.43 (m, 1H), 7.37-7.27 (m, 3H), 7.24 (d, J = 8.6 Hz, 1H), 6.50 (s, 2H), 5.88 (s, 1H), 5.79 (s, 2H), 5.70 (s, 2H), 5.13 (d. 7 = 7.3 Hz, 1H), 5.05 (s, 2H), 4.89 (d, 7 = 10.7 Hz, 3H), 4.53 (d, 7 = 9.5 Hz, 5H), 4.14 (d, J = 2.4 Hz, 1H), 3.96 (d, J = 9.5 Hz, 2H), 3.69 (s, 2H), 3.57 (s, 3H), 3.52 (d, J = 6.1 Hz, 2H), 2.67 (p. 7 = 1.9 Hz, 4H), 2.33 (p, J = 1.9 Hz, 3H), 2.09 (d, 7 = 3.8 Hz, 5H), 1.91 (s, 1H), 1.25 (q, J = 6.8 Hz, 5H). EI-MS m/z : [M/2+H]+ 677.68.
Example 70: Preparation of Compound 396
Preparation of Compound 395
To a solution of Compound 311 (150 mg, 0.16 mmol) and Compound 342 (150 mg, 0.13 mmol) in A A-di methyl formamide (5 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 3.6 mg, 0.026 mmol) and A,A’-diisopropylethylamine (0.11 mL, 0.65 mmol) at 0 °C under nitrogen. After stirring at room temperature for 13 hours, the reaction solution was diluted with ethyl acetate (50 mL) and washed with distilled water (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 395 (274 mg, 98%). EI-MS m/z : [M/2+H]+ 958.92.
Preparation of Compound 396
To a solution of Compound 395 (274 mg, 0.14 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL) was added lithium hydroxide monohydrate (60 mg, 1.43 mmol) in distilled water (2 mL) at -45 °C. After stirring for 0.5 hours at 0 °C, the reaction solution was added lithium hydroxide monohydrate (60 mg, 1.43 mmol) in distilled water (12 mL) at 0 °C. After stirring for 1 hour at room temperature, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 396 (48 mg, 21%). EI-MS m/z : [M+H]+ 1608.92. Example 71: Preparation of Compound 400
399 400
Preparation of Compound 397
To a solution of trans-2-butene-l,4-diol (1.5 g, 11.94 mmol) in dichloromethane (100 mL) were added trimethylamine (2.2 mL, 15.92 mmol) and t-butyldimethylsilyl chloride (1.0 g, 7.96 mmol) at 0 °C under nitrogen. After stirring at room temperature for 4 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 397 (1.2 g, 74%).
’H-NMR (400 MHz, MeOD), δ 5.88 - 5.71 (m, 2H), 4.19 (dt, J = 4.3, 1.4 Hz, 2H), 4.09 - 4.02 (m, 2H), 0.92 (s, 9H).
Preparation of Compound 398
To a solution of Compound 397 (200 mg, 0.99 mmol) in dichloromethane (10 mL) were added trimethylamine (0.28 mL, 1.98 mmol) and methanesulfonyl anhydride (206 mg, 1.19 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 398 (140 mg, crude), which was used without further purification. ’H-NMR (400 MHz, CDCI3) δ 6.05 - 5.83 (m, 2H), 4.74 (dq, J = 6.3, 1.2 Hz, 2H), 4.21 (dt, 7 = 4.2, 1.6 Hz, 2H), 3.01 (s, 3H), 0.91 (s, 9H), 0.08 (s, 6H).
Preparation of Compound 399
To a solution of Compound 66 (400 mg, 0.42 mmol, Compound 66 was prepared by the method described in the International Patent Publication No. WO 2022/155518 Al) and Compound 398 (130 mg, 0.46 mmol) in A/Wdimethylformamide (3 mL) was added cesium carbonate (685 mg, 2.10 mmol) were added at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure and diluted with dichloromethane (50 mL) and methanol (10 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 399 (128 mg, 33%). ELMS m/z : [M+H]+ 907.58.
Preparation of Compound 400
To a solution of Compound 399 (20 mg, 0.02 mmol) in dichloromethane (0.4 mL) and methanol (0.2 mL) was added hydrochloric acid (4.0 M in 1,4-dioxane solution, 0.03 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 400 (10 mg, 42%). ELMS m/z : [M+H]+ 794.27.
Example 72: Preparation of Compound 408
Preparation of Compound 401
To a solution of Compound 397 (500 mg, 2.47 mmol) in dichloromethane (10 mL) were added trimethylamine (1.04 mL, 7.41 mmol), pyridine (0.60 mL, 7.41 mmol) and 4- nitrophenyl chloroformate (747 mg, 3.70 mmol) at 0 °C under nitrogen. After stirring at room temperature for 20 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and 2% sodium hydroxide aqueous solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 401 (729 mg, 80%). ’H-NMR (400 MHz, CDCh), δ 8.32 - 8.24 (m, 2H), 7.42 - 7.34 (m, 2H), 6.04 - 5.84 (m, 2H), 4.78 (dq, 7 = 5.9, 1.1 Hz, 2H), 4.23 (dq, 7 = 4.2, 1.4 Hz, 2H), 0.92 (s, 9H), 0.08 (s, 6H).
Preparation of Compound 402
To a solution of Compound 401 (729 mg, 1.98 mmol) in dichloromethane (10 mL) were added trimethylamine (0.56 mL, 3.97 mmol) and tert-butyl methyl(2- (methylamino)ethyl)carbamate (485 mg, 2.58 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and 2% sodium hydroxide aqueous solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 402 (825 mg, 99%).
’H-NMR (400 MHz, CDCh), δ 5.85 - 5.78 (m, 2H), 4.58 (s, 2H), 4.18 (d, J = 2.6 Hz, 2H), 3.37 (s, 4H), 2.94 (s, 3H), 2.88 (s, 3H), 1.45 (s, 9H), 0.91 (s, 9H), 0.07 (s, 6H).
Preparation of Compound 403
To a solution of Compound 402 (825 mg, 0.43 mmol) in tetrahydrofuran (10 mL) was added tetrabutylammonium fluoride solution (1.0 M in tetrahydrofuran, 3 mL, 2.97 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and saturated aqueous ammonium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 403 (514 mg, 86%).
’H-NMR (400 MHz, CDCh), δ 5.96 - 5.73 (m, 2H), 4.58 (d, J = 5.9 Hz, 2H), 4.15 (d, 7 = 12.5 Hz, 2H), 3.45 - 3.27 (m, 4H), 2.94 (d, 7 = 5.0 Hz, 3H), 2.87 (d, 7 = 11.5 Hz, 3H), 1.45 (s, 9H).
Preparation of Compound 404
To a solution of Compound 403 (150 mg, 0.50 mmol) in dichloromethane (5 mL) were added trimethylamine (0.14 mL, 0.99 mmol) and methanesulfonyl anhydride (103 mg, 0.59 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was diluted with dichloromethane (50 mL) and washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate concentrated under reduced pressure to afford Compound 404 (164 mg, crude), which was used without further purification.
’H-NMR (400 MHz, CDCh), δ 6.06 - 5.77 (m, 2H), 4.73 (dt, 7 = 6.2, 1.1 Hz, 2H), 4.63 (s, 2H), 3.38 (s, 4H), 3.03 (s, 3H), 2.95 (s, 3H), 2.87 (d. 7 = 7.8 Hz, 3H), 1.45 (s, 9H).
Preparation of Compound 405
To a solution of Compound 66 (350 mg, 0.37 mmol, Compound 66 was prepared by the method described in the International Patent Publication No. WO 2022/155518 Al) and Compound 404 (154 mg, 0.40 mmol) in A/Wdimethylformamide (2 mL) was added cesium carbonate (600 mg, 1.84 mmol) were added at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure and diluted with dichloromethane (50 mL) and methanol (10 mL) and washed with distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 405 (207 mg, 56%). ELMS m/z : [M+H]+ 1007.74.
Preparation of Compound 406
To a solution of Compound 405 (75 mg, 0.12 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 406 (93 mg. crude), which was used without further purification. ELMS m/z : [M+H]+ 908.38.
Preparation of Compound 407
To a solution of Compound 204 (50 mg, 0.067 mmol) and Compound 406 (93 mg) in W/V-dimethylformamide (2 mL) was added A,A’-diisopropylethylamine (0.039 mL, 0.280 mmol) at 0 °C under nitrogen. After stirring at room temperature for 19 hours, the reaction solution was concentrated under reduced pressure. The solid was triturated with dichloromethane and diethyl ether. The reaction mixture was filtered and dried to afford Compound 407 (77 mg, 74%), which was used without further purification. ELMS m/z : [M+H]+ 1475.87.
Preparation of Compound 408
To a solution of Compound 407 (77 mg) in tetrahydrofuran (1 mL) and methanol (0.5 mL) was added lithium hydroxide monohydrate (14 mg, 0.33 mmol) in distilled water (1.5 mL) at -70 °C. After stirring for 4 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 408 (42 mg, 75%). ELMS m/z : [M+H]+ 1335.77. Example 73: Preparation of Compound 415
Preparation of Compound 409
To a solution of Compound 6 (741 mg, 1.95 mmol) in methanol (2 mL) was added hydrochloric acid (4.0 M in 1 ,4-dioxane solution, 4.2 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction mixture was triturated with dichloromethane and diethyl ether. The reaction mixture was filtered and dried to afford Compound 409 (614 mg, 89%), which was used without further purification. EI-MS m/z : [M+H]+ 281.41.
Preparation of Compound 410 To a solution of Compound 409 (469 mg, 1.33 mmol) and Compound 51 (300 mg,
0.66 mmol) in normal butyl alcohol (3 mL) was added triethylamine (0.65 mL, 4.65 mmol) at room temperature. After stirring 120 °C for 60 hours under sealed. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 410 (140 mg, 30%). EI-MS m/z : [M+H]+ 697.06.
Preparation of Compound 411
To a solution of Compound 410 (140 mg, 0.30 mmol) in methanol (4 mL) and distilled water (1 mL) were added aqueous ammonia solution (28 ~ 30% ammonia, 0.4 mL) and sodium hydrosulfite (700 mg, 4.02 mmol). After stirring at room temperature for 2 hours, the reaction solution was diluted with methanol (10 mL) and filtered. The filtrate was concentrated under reduced pressure to afford Compound 411 (120 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 637.04.
Preparation of Compound 412
To a solution of Compound 411 (120 mg) in A,A-dimethylformamide (2 mL) was added Compound 149 (148 mg, 0.75 mmol) in A,A-dimethylformamide (1 mL) at 0 °C under nitrogen. After stirring at room temperature for 1 hour, the reaction solution triethylamine (0.19 mL, 1.32 mmol) and A-(3-dimethylaminopropyl)-M-ethyl carbodiimide (144 mg, 0.75 mmol) were added at 0 °C. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 412 (25 mg, 14%). EI-MS m/z : [M+H]+ 961.42.
Preparation of Compound 413
To a solution of Compound 412 (25 mg, 0.07 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.5 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 413 (10 mg, 56%). EI-MS m/z : [M+H]+ 861.31.
Preparation of Compound 414
To a solution of Compound 204 (5.5 mg, 0.007 mmol) and Compound 413 (8 mg, 0.007 mmol) in A,A-dimethylformamide (0.3 mL) was added A,A’-diisopropylethylamine (0.006 mL, 0.033 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to afford Compound 414 (9 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1428.79.
Preparation of Compound 415
To a solution of Compound 414 (9 mg) in tetrahydrofuran (0.2 mL) and methanol (0.1 mL) was added lithium hydroxide monohydrate (4 mg, 0.095 mmol) in distilled water (0.3 mL) at -70 °C. After stirring for 4 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 415 (6.8 mg, 71%). EI-MS m/z : [M+H]+ 1288.80.
Example 74: Preparation of Compound 417
Preparation of Compound 416
To a solution of Compound 28 (56 mg, 0.046 mmol) in A,A-dimethylformamide (2 mL) were added Compound 204 (35 mg, 0.046 mmol), A,A’-diisopropylethylamine (0.064 mL, 0.370 mmol) and l-hydroxy-7-azabenzotriazole (HOAt, 0.6 mg, 0.005 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 416 (66 mg, crude), which was used without further purification. EI-MS m/z : [1/2M+H]+ 722.10.
Preparation of Compound 417
To a solution of Compound 416 (66 mg) in methanol (0.5 mL) and tetrahydrofuran (0.5 mL) was added lithium hydroxide monohydrate (39 mg, 0.92 mmol) in distilled water (0.7 mL) at -50 °C under nitrogen. After stirring for 4 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 417 (30 mg, 50 %). EI-MS m/z : [1/2M+H]+ 652.10, [M+H]+ 1302.65. Example 75: Preparation of Compound 419
Preparation of Compound 418
To a solution of Compound 133 (30 mg, 0.025 mmol) in A,A-dimethylformamide (3 mL) were added Compound 204 (22 mg, 0.030 mmol), A,A’-diisopropylethylamine (0.02 mL, 0.12 mmol) and l-hydroxy-7-azabenzotriazole (HOAt, 1 mg, 0.01 mmol) under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The reaction mixture was added acetonitrile. The resulting solid was filtered and dried to afford Compound 418 (35 mg, crude), which was used without further purification. EI-MS m/z : [1/2M+H]+ 708.16, [M+H]+ 1414.67.
Preparation of Compound 419
To a solution of Compound 418 (35 mg) in methanol (1 mL) was added lithium hydroxide monohydrate (9 mg, 0.22 mmol) in distilled water (1 mL) at -50 °C under nitrogen. After stirring for 3 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 419 (10 mg, 32 %). EI-MS m/z : [1/2M+H]+ 638.09, [M+H]+ 1274.53.
Example 76: Preparation of Compound 422
Preparation of Compound 420 To a solution of Compound 267 (122 mg, 0.13 mmol) in A,A-dimethylformamide (1 mL) was added Compound 133 (140 mg, 0.12 mmol), A,A-diisopropylethylamine (0.1 mL, 0.59 mmol) and HOAt (l-Hydroxy-7-azabenzotriazole, 3 mg, 0.02 mmol). After stirring at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 420 (189 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 801.90.
Preparation of Compound 421
To a solution of Compound 420 (189 mg, crude) in methanol (0.5 mL) and tetrahydrofuran (0.5 mL) was added lithium hydroxide monohydrate (49 mg, 1.17 mmol) in distilled water (0.5 mL) at -50 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure to afford Compound 421 (172 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 731.84.
Preparation of Compound 422
To a solution of Compound 421 (172 mg, crude) in dichloromethane (3 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 422 (70 mg, 38%). EI-MS m/z : [1/2M+H]+ 674.80, [M+H]+ 1347.93.
Example 77: Preparation of Compound 424
Preparation of Compound 423
To a solution of Compound 133 (42 mg, 0.05 mmol) in A,A-dimethylformamide (2 mL) were added Compound 260 (63 mg, 0.07 mmol), A,A’-diisopropylethylamine (0.04 mL, 0.25 mmol) and l-hydroxy-7-azabenzotriazole (HOAt, 1 mg, 0.01 mmol) at 0 °C under nitrogen. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 423 (40 mg, crude). EI-MS m/z : [1/2M+H]+ 758.66, [M+H]+ 1515.77.
Preparation of Compound 424 To a solution of Compound 423 (40 mg) in tetrahydrofuran (0.75 mL) and methanol
(0.75 mL) was added lithium hydroxide monohydrate (11 mg, 0.26 mmol) in distilled water (1 mL) at -40 °C under nitrogen. After stirring at 0 °C for 3 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 424 (9.7 mg, 26%). EI-MS m/z : [1/2M+H]+ 688.56, [M+H]+ 1376.62.
Example 78: Preparation of Compound 446
Preparation of Compound 442 To a solution of tert-butyl (2-hydroxyethyl)(methyl)carbamate (880 mg, 5.0 mmol) in dichloromethane (5 mL) were added pyridine (0.97 mL, 11.7 mmol) and phosgene solution (15 wt% in toluene, 7.2 mL, 10.8 mmol) in dichloromethane (15 mL) at -78 °C under nitrogen. After stirring at room temperature for 0.5 hours, the reaction solution Compound 441 (50 mg, 0.054 mmol, Compound 441 was prepared by the method described in the International Patent Publication No. WO 2020/092617 Al) in pyridine (2.5 mL) was added at 0 °C. After stirring at room temperature for 4 hours, the reaction solution was diluted with dichloromethane (5 mL) and washed with and distilled water (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue to afford Compound 442 (120 mg), which was used without further purification. EIMS m/z : [M+H]+ 1498.75.
Preparation of Compound 443
To a solution of Compound 442 (120 mg) in methanol (2 mL) was added methylamine (33% in ethanol, 2 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The resulting solid obtained by diluted with diethyl ether was filtered and dried to afford Compound 443 (48 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 1097.25.
Preparation of Compound 444
To a solution of Compound 443 (48 mg, crude) in dichloromethane (1 mL) was added trifluoroacetic acid (0.25 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure. The resulting solid obtained by diluted with methanol and acetonitrile was filtered and dried to afford Compound 444 (59 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 896.94.
Preparation of Compound 445
To a solution of Compound 204 (78 mg, 0.104 mmol) and Compound 444 (49 mg, 0.043 mmol) in A/A-dimethylformamide (2 mL) were added l-hydroxy-7-azabenzotriazole (HOAt, 0.4 mg, 0.003 mmol) and A,A’-diisopropylethylamine (0.04 mL, 0.217 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to afford Compound 445 (50 mg, crude), which was used without further purification. EI-MS m/z : [M+H]+ 2031.53.
Preparation of Compound 446
To a solution of Compound 445 (50 mg, crude) tetrahydrofuran (1 mL) and methanol (1 mL) was added tetramethylammonium hydroxide solution (10 wt% in water, 0.03 mL) and lithium hydroxide monohydrate (8.2 mg, 0.19 mmol) in distilled water (1 mL) at -45 °C. After stirring at 0 °C for 2 hours, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 446 (4.8 mg, 11%). EI-MS m/z : [M+H]+ 1751.32. Example 79: Preparation of Compound 447
372 447
Preparation of Compound 447
To a solution of Compound 372 (70 mg, 0.04 mmol) and 5-carboxyfluorescein N- succinimidyl ester (21 mg, 0.044 mmol) in A,A-dimethylformamide (2 mL) was added triethylamine (0.03 mL, 0.21 mmol) at 0 °C under nitrogen. After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 447 (47 mg).
’H-NMR (400 MHz, DMSO), δ 10.15 (s, 2H), 9.50 (s, 1H), 8.89 (s, 1H), 8.46 (s, 1H), 8.30 (s, 1H), 8.27 - 8.22 (m, 1H), 7.94 (d, J = 15.4 Hz, 2H), 7.83 (d, J = 2.3 Hz, 1H), 7.67 -
7.60 (m, 2H), 7.47 (d, J = 10.8 Hz, 1H), 7.40 - 7.28 (m, 5H), 7.24 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6.58 (s, 1H), 6.55 (d, J = 2.3 Hz, 1H), 6.50 (s, 1H), 5.78 (s, 2H), 5.14 (d, J = 7.0 Hz, 2H), 5.05 (s, 2H), 4.89 (d, J = 10.4 Hz, 3H), 4.52 (d, J = 15.8 Hz, 6H), 3.97 (d, J = 9.5 Hz, 1H), 3.69 (s, 3H), 3.56 (d, J = 15.7 Hz, 7H), 2.11 - 2.03 (m, 5H), 1.91 (s, 1H), 1.24 (q, J = 6.8 Hz, 5H).
Example 80: Preparation of Compound 453
Preparation of Compound 451
To a solution of Compound 450 (25.5 mg, 0.03 mmol, Compound 450 was prepared by the method described in the Korean Patent Application No. 10-2023-0099038) and Compound 55 (30 mg, 0.02 mmol) in MN-dimcthylformamidc (1 mL) were added 1-hydroxy- 7-azabenzotriazole (HOAt, 0.7 mg, 0.005 mmol) and A,A’-diisopropylethylamine (0.02 mL, 0.12 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Compound 451 (36 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 759.25.
Preparation of Compound 452
To a solution of Compound 451 (36 mg, crude) in methanol (0.5 mL) was added lithium hydroxide monohydrate (5.1 mg, 0.12 mmol) in distilled water (0.5 mL) at -50 °C. After stirring for 2 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure to afford Compound 452 (36 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H] 746.27. Preparation of Compound 453
To a solution of Compound 452 (36 mg) in dichloromethane (1 mL) was added trifluoroacetic acid (0.35 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours under nitrogen, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 453 (25 mg, 63%). ELMS m/z : [M+H]+ 1376.63, [M/2+H]+ 689.09.
Example 81: Preparation of Compound 458 Preparation of Compound 454
To a solution of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)(methyl)carbamate (2.84 g, 10.82 mmol) in dichloromethane (50 mL) were added Compound 205 (162 mg, 0.33 mmol, Compound 205 was prepared according to the method described in the International Patent Publication No. WO 2018/182341 Al), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC-HC1, 2.18 g, 11.36 mmol), 1 -hydroxybenzotriazole (HOBt, 1.61 g, 11.91 mmol) and A-methylmorpholine (3.57 mL, 32.47 mmol) under N2. After stirring at room temperature for 17 hours, the reaction mixture was washed with saturated aq. sodium bicarbonate solution (30 mL) and saturated aq. ammonium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 454 (6.4 g, 81%) as a colorless oil. EI-MS m/z : [M+H]+ 729.30.
Preparation of Compound 455
To a solution of Compound 454 (2 g, 2.74 mmol) in dichloromethane (20 mL) were added bis(pentafluorophenyl)carbonate (1.4 g, 3.57 mmol) and A/A-diisopropylethylamine (0.96 mL, 5.49 mmol) at 0 °C under N2. After stirring at room temperature for 6 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL), 0.5 N aq. HC1 solution (50 mL), and 2% aq. NaOH solution (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 455 (2.2 g, 85%) as a colorless oil. EI-MS m/z : [M+H]+ 938.99.
Preparation of Compound 456
To a solution of Compound 455 (525 mg, 0.56 mmol) and Compound 174 (600 mg, 0.47 mmol) in A/iV-dimethylformamide (5 mL) were added l-hydroxy-7-azabenzotriazole (HOAt, 12.7 mg, 0.09 mmol) and AW-diisopropylethylamine (0.4 mL, 2.33 mmol) at 0 °C under nitrogen. After stirring at room temperature for 18 hours, the reaction mixture was diluted with dichloromethane (50 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 456 (751 mg, 94%) as a brown solid. EI-MS m/z : [M/2+H]+ 850.00.
Preparation of Compound 457
To a solution of Compound 456 (751 mg, 0.44 mmol) in methanol (3 mL) and tetrahydrofuran (6 mL) was added lithium hydroxide monohydrate (92 mg, 2.21 mmol) in distilled water (9 mL) at -50 °C. After stirring for 2 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction solution was concentrated under reduced pressure to afford Compound 457 (682 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 772.86.
Preparation of Compound 458 To a solution of 457 (682 mg, 0.44 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (2 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours under nitrogen, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 458 (608 mg, 77%). EI-MS m/z : [M+H]+ 1444.95, [M/2+H]+ 722.85.
Example 82: Preparation of Compound 461
Preparation of Compound 459
To a solution of Compound 455 (175 mg, 1.87 mmol) and Compound 311 (200 mg, 0.16 mmol) in A,A-dimethylformamide (3 mL) was added A,A’-diisopropylethylamine (0.14 mL, 0.78 mmol) at 0 °C under nitrogen. After stirring at room temperature for 5 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 459 (240 mg, 91%) as a brown solid. EI-MS m/z : [M/2+H]+ 850.24. Preparation of Compound 460
To a solution of Compound 459 (223 mg, 0.13 mmol) in tetrahydrofuran (3 mL) was added lithium hydroxide monohydrate (26.2 mg, 0.62 mmol) in distilled water (2 mL) at -50 °C. After stirring for 4 hours at 0 °C, the reaction mixture was adjusted to pH 4~5 with acetic acid. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 465 (132 mg, 65%). EI-MS m/z : [M+H]+ 1616.79.
Preparation of Compound 461
To a solution of Compound 460 (18 mg, 0.012 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2 mL) at 0 °C under nitrogen. After stirring at room temperature for 2 hours under nitrogen, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 461 (2 mg, 12%). EI-MS m/z : [M+H]+ 1444.29, [M/2+H]+ 723.05.
275 Preparation of Compound 462
To a solution of Compound 4 (888 mg, 4.11 mmol) in A,A-dimethylformamide (20 mL), potassium carbonate (850 mg, 6.15 mmol) and methyl 4-bromobutanoate (817 mg, 4.51 mmol) were added. After stirring at 60 °C for 4 hours, the mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 465 (1.23 g, 95%). ’H-NMR (400 MHz, DMSO-d6), δ 8.29 (s, 1H), 8.05 (d, 7 = 1.8 Hz, 1H), 7.86 (d, 7 = 1.9 Hz, 1H), 7.77 (s, 1H), 4.26 (t, J = 6.2 Hz, 2H), 3.61 (s, 3H), 2.55 (d, 7 = 7.3 Hz, 2H), 2.12-2.01 (m, 2H). ELMS m/z : [M+H]+ 317.59.
Preparation of Compound 463
To a solution of Compound 462 (1.23 g, 3.87 mmol) in methanol (30 mL) were added tert-butyl (E)-(4-aminobut-2-en-l-yl)carbamate (868 mg, 4.65 mmol) and triethylamine (2.73 mL, 19.37 mmol) at 70 °C under N2. After stirring for 63 hours, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (60 mL), and washed with distilled water (2 x 15 mL) and brine (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 463 (1.81 g, crude). 1 H-NMR (400 MHz, CDCh) 5 8.15 (d, 7 = 1.9 Hz, 1H), 8.10 (s, 1H), 7.50 (d, J = 2.0 Hz, 1H), 5.71 (dt, J = 4.7, 2.8 Hz, 2H), 4.73 (s, 1H), 4.27 (ddd, 7 = 5.8, 4.1, 1.5 Hz, 2H), 4.09 (t, 7 = 6.4 Hz, 2H), 3.75 (s, 2H), 3.72 (s, 3H), 2.52 (t, J = 7.2 Hz, 2H), 2.18 (p, 7 = 6.8 Hz, 2H), 1.44 (s, 9H). ELMS m/z : [M+H]+ 467.79.
Preparation of Compound 464
To a solution of Compound 463 (1.24 g, 2.41 mmol) in dichloromethane (5 mL) and methanol (5 mL) was added HC1 (4 M in 1,4-dioxane, 3.0 mL, 12 mmol) at 0 °C under N2. After 4 hours, the reaction mixture was concentrated under the reduced pressure to afford Compound 464 (885 mg, 83%) as a brown oil, which was used for the next step without further purification. ELMS m/z : [M+H]+ 467.63.
Preparation of Compound 465
To a solution of Compound 4 (771 mg, 3.56 mmol) in A,A-dimethylformamide (20 mL) were added cesium carbonate (1.72 g, 4.28 mmol) and Compound 172 (1.72 g, 4.28 mmol). After stirring at room temperature for 24 hours, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with distilled water (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 465 (1.15 g, 60 %). EI-MS m/z : [M+H]+ 538.17.
Preparation of Compound 466
To a solution of Compound 465 (5 g, 9.29 mmol) and Compound 464 (6.12 g, 13.94 mmol) in dimethyl sulfoxide (32 mL) and A,A-diisopropylethylamine (8.1 mL, 46.47 mmol) was added at room temperature. After stirring at 100 °C for 93 hours, the reaction mixture was cooled to room temperature, diluted with dichloromethane (100 mL) and methanol (20 mL). Then the mixture was washed with distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 466 (4.53 g, 56%). EIMS m/z : [M+H]+ 868.13.
Preparation of Compound 467
To a solution of Compound 466 (3.59 g, 4.13 mmol) in methanol (60 mL) were added ammonia solution (28-30%, 2.4 mL) and sodium hydrosulfite (Na2S2C>4, 10.8 g, 123.9 mmol) under N2. After stirring at room temperature for 4 hours, the mixture was diluted with dichloromethane (100 mL) and filtered. The filtrate was concentrated under reduced pressure, then diluted with dichloromethane (100 mL) and methanol (50 mL), and washed with distilled water (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to afford Compound 467 (3.4 g, crude), which was used without further purification. EI-MS m/z : [M+H] + 808.23.
Preparation of Compound 468
To a solution of Compound 467 (884 mg, 1.1 mmol) in A,A-dimethylformamide (4 mL) was added Compound 2 (470 mg, 2.41 mmol) in A,A-dimethylformamide (1 mL) under N2. After stirring at room temperature for 1 hour, A-(3-dimethylaminopropyl)-A’- ethylcarbodiimide (0.43 mL, 2.41 mmol) and triethylamine (0.77 mL, 5.47 mmol) were added. The mixture was stirred at room temperature for 16 hours, then concentrated under reduced pressure. After diluting with dichloromethane and diethyl ether, the resulting solid was filtered and dried to afford Compound 468 (1.24 g, crude). EI-MS m/z : [M+H]+ 1131.06.
Preparation of Compound 469
To a solution of Compound 468 (1.24 g, crude) in dichloromethane (15 mL), trifluoroacetic acid (5 mL) was added at 0 °C under N2. After stirring at 0 °C for 2 hours, the reaction mixture was concentrated under reduced pressure to afford crude Compound 469 (1.08 g), which was used without further purification. EI-MS m/z : [M+H]+ 1030.14. Preparation of Compound 470
To a solution of Compound 260 (24.2 mg, 0.03 mmol) and Compound 469 (20.6 mg, 0.02 mmol) in MA-dimcthylformamidc (1 mL) were added l-hydroxy-7-azabenzotriazole (HO At, 0.7 mg, 0.005 mmol) and A,A’-diisopropylethylamine (0.02 mL, 0.12 mmol) at 0 °C under nitrogen. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to afford Compound 470 (36 mg, crude), which was used without further purification. EI-MS m/z : [M/2+H]+ 850.25.
Preparation of Compound 471
To a solution of Compound 470 (36 mg, crude) in methanol (0.5 mL) was added lithium hydroxide monohydrate (5.1 mg, 0.12 mmol) in distilled water (0.5 mL) at -50 °C. After stirring for 2 hours at 0 °C, the reaction mixture was adjusted to pH 4-5 with acetic acid. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 471 (16.5 mg, 54%). [M/2+H]+ 766.27.
Example 84: Preparation of Compounds 472-514
Compounds 472-514, as shown in Table A below, were prepared according to procedures analogous to those outlined above using the appropriate monomers described as Examples above, or as obtained from commercial sources, in the coupling and deprotected step.
Example 85: Preparation of Comparative Compound #1
The compound of Comparative Compound #1 has the following structure and was purchased from ChemScene (#CS-0077291) and used. Example 86: Preparation of Comparative Compound #2
The compound of Comparative Compound #2 has the following structure and was prepared by the method described in the United States patent publication No. 2021-0032269 Al.
Example 87: Preparation of Comparative Compound #3
The compound of Comparative Compound #3 has the following structure and was prepared by the method described in the United States patent publication No. 2022-0267364 Al.
Example 88: Preparation of Exemplified Antibodies
Preparation of Exemplified PD-L1 Specific Antibodies
An exemplified anti-PD-Ll antibody_l (Atezolizumab), that specifically binds to PD- Ll, was produced by the methods described in U.S. Patent No. 8,217,149 B2, the entirety of which is incorporated herein by reference. The Fc region of Atezolizumab contains an N297A mutation. Additionally, LALA mutations (L235A/L236A) were introduced into the heavy chain constant region of Atezolizumab. The amino acid sequences of PD-L1 antibody_l are shown in Table C below.
[Table C] PD-L1 Antibody_l Sequences
|PD-L1 Antibody_ 1 Heavy Chain |SEQ ID NO: |
An exemplified anti-PD-Ll antibody_2 (Clone 31E6), that specifically binds to PD- Ll, was produced by the methods described in U.S. Patent No. 10,919,966 B2, the entirety of which is incorporated herein by reference. Additionally, LALA mutations (L236A/L237A) were introduced into the heavy chain constant region of Clone 31E6. The amino acid sequences of PD-L1 antibody_2 are shown in Table D below.
[Table D] PD-L1 Antibody_l Sequences
An exemplified anti-PD-Ll antibody_3 (Avelumab), that specifically binds to PD-L1, was produced by the methods described in U.S. Patent No. 9,624,298 B2, the entirety of which is incorporated herein by reference. The amino acid sequences of PD-L1 antibody_3 are shown in Table E below.
[Table E] PD-L1 Antibody_l Sequences
|PD-L1 Antibody_3 Heavy Chain |SEQ ID NO: ~|
An exemplified anti-mouse PD-L1 antibody (10F.9G2), that specifically binds to mouse PD-E1, was purchased from MedChemExpress (Cat. No. HY-P990172). Preparation of Exemplified PD-1 Specific Antibodies
An exemplified anti-PD-1 antibody_l (Pembrolizumab), that specifically binds to PD-
1, was purchased from MedChemExpress (Cat. No. HY-P9902A). The Fc region of Pembrolizumab contains an S228P mutation. The amino acid sequences of PD-1 antibody_l are shown in Table F below. [Table F] PD-L1 Antibody_l Sequences
An exemplified anti-PD-1 antibody_2 (Nivoluamb), that specifically binds to PD-1, was produced by the methods described in U.S. Patent No. 8,008,449 B2, the entirety of which is incorporated herein by reference. The IgG4 constant region of the heavy chain of Nivolumab was converted to an IgGl constant region. Additionally, LALA mutations (L230A/L231A) were introduced into the heavy chain constant region of Nivolumab. The amino acid sequences of PD-1 antibody_2 are shown in Table G below.
[Table G] PD-1 Antibody_l Sequences
An exemplified anti-mouse PD-1 antibody (RMP1-14), that specifically binds to mouse PD-L1, was purchased from MedChemExpress (Cat. No. HY-P99144).
Testing of Compounds
Experimental Example 1: Assessment of STING activation and direct STING binding
To assess potency of STING agonist compounds described herein, THPl-Dual™ Cells (NF-KB-SEAP and IRF-Lucia luciferase reporter monocytes, InvivoGen) were used. The reporter cells were knocked-in either of hSTING-WT (R232, #thpd-r232), HAQ variant (#thpd- nfis), or H232 variant (#thpd-h232). All cells were cultured at 37°C in a humidified incubator at 5% CO2 and maintained in RPMI 1640 medium (Gibco, #22400097) supplemented with 10% heat-inactivated FBS and 1 x Pen-Strep (Gibco, #15140122). To measure STING agonist- mediated IRF3 activity, the cells (9.0 x 104 cells per well) were seeded in a flat-bottomed 96- well plate. The next day, the cells were treated with compounds in a serial dilution for 24 hours. Subsequently, 20 p L of the cell culture supernatant and 50 p L of the QUANTI-Luc™ reagent (InvivoGen, #rep-qlc) were mixed in a 96-well white plate and then, luciferase activity was measured using an EnVision Xcite multilabel reader. The data were analyzed using Prism8 software (4-parameter). The potency of STING agonist compounds was evaluated by the activation of Type I Interferon pathway using THP-1 IRF3 reporter assay. Although the activities varied among the STING agonist compounds, half maximal effective concentration (ECso) values for all IRF3 activation were below 1 pM and were comparable to Comparative compound #1 or #2, as shown in Table 1 below. In addition, the STING agonist compounds activated human STING variants, such as HAQ and H232, as shown in Table 2. Collectively, these results indicate that the STING agonist compounds strongly stimulate STING signaling pathway.
Table 1. STING activity using hSTING-R232 cells
Table 2. STING activity using hSTING-HAQ or H232 cells
Additionally, for analysis of direct binding potency of STING agonist compounds described herein to human STING, HUMAN STING WT BINDING KIT (Cisbio, #64BDSTGPEG) was used according to the manufacture’s instruction. It was confirmed that the STING agonist compounds directly bind to human STING protein in vitro, and summary result data were presented as EC50 in Table 3. Table 3. Binding potency
Experimental Example 2: Cytokine production induction in vitro assay For analysis of STING agonist-induced cytokine production, human peripheral blood mononuclear cells (PBMCs, STEMCELL, #70025.3) and human cancer cell lines (BxPC-3 (ATCC, #CRL-1687) and MDA-MB-468 (DSMZ, #ACC 738)) were cultured in RPMI 1640 medium (Gibco, #22400097) supplemented with 10% heat-inactivated FBS and lx Antibiotic- Antimycotic (Gibco, #15240062). The cells (2.0 x 104 cells per well) were seeded in a flat- bottomed 96-well plate. PBMCs were stimulated with STING agonist compounds described herein (Compound 214, 221, and 230) at 1 pM for 24 hours. BxPC-3 and MDA-MB-468 tumor cells were treated with STING agonist compounds described herein (Compound 313) in a 5- fold serial dilution from 3 pM for 24 hours, respectively. The levels of cytokines in the culture supernatants were measured using the ELISA kit (R&D systems, #DY266, #DY9345-05) according to the manufacture”s instruction.
Briefly, each well of a 96-well plate was coated with 100 pL of the diluted capture antibody in PBS and incubated overnight at room temperature (RT). Next day, each well was washed with wash buffer (0.05% Tween 20 in PBS) three times using an auto-washer. The plates were blocked with 300 pL of Reagent Diluent (1% BSA in PBS) for 1 hour at RT. The aspiration/wash step was repeated. 100 pL of diluted samples or standards in Reagent Diluent was added to each well and incubated for 2 hours at RT. After wash, 100 pL of the Detection Antibody pre-diluted in Reagent Diluent was added into each well and incubated for 2 hours at RT. After wash, 100 pL of the working diluted Streptavidin-HRP in Reagent Diluent was added and incubated under subdued light conditions for 20 minutes, followed by repeated aspiration/wash. 100 pL of substrate solution (mixed H2O2 and Tetramethylbenzidine in a 1:1 ratio) was added and incubated for 20 minutes at RT in avoiding direct light exposure. The reaction was stopped by adding 50 pL of stop solution (2N H2SO4). The optical density was determined using a microplate reader (Molecular Devices VersaMax Microplate Reader). The optical density (O.D.) of each well was normalized by subtracting the reading at 570 nm from the reading at 450 nm.
Since the STING is expressed in both tumor and immune cells, the STING agonist compounds induced cytokine production through activation of STING pathway in those cells. Specifically, as shown in FIGS. 1 and 2, the STING agonist compounds strongly mediated the production of human CXCL-10 and human IFNa, which are key downstream cytokines of STING pathway.
Experimental Example 3: Immune cells activation in vitro assay
To test the effect of STING agonist compounds described herein in the activation of innate and adaptive immune cells, flow cytometry (FACS) was performed. Human THP-1 cells (Korean Cell Line Bank, #40202) and PBMCs were seeded at 1.2 x 105 cells per well in a round-bottomed 96-well plate in RPMI 1640 medium (Gibco, #22400097) supplemented with 10% heat-inactivated FBS and lx Antibiotic-Antimycotic (Gibco, #15240062) and rested for 24 hours at 37°C. The cells were stimulated with the STING agonist compounds as described; For THP-1 stimulation, the cells were treated with the STING agonist compound (Compound 313) in a 5-fold serial dilution from 1 pM for 48 hours. The induction of cell surface activation markers, CD86 (co-stimulatory molecule) and HLA-DR (MHC-class II), was measured by flow cytometry. For PBMCs, the cells were stimulated with the STING agonist compounds, Compound 274 and 277 at 100 nM for 48 hours or Compound 344 and 361 at 3 pM for 24 hours, respectively. For flow cytometry analysis, the stimulated cells were centrifuged at 300g for 5 minutes at 4°C, and then resuspended in FACS buffer (2% FBS, 2 mM EDTA in PBS). After staining cells with human Fc blocker (BD Biosciences, #564220) in FACS buffer for 10 minutes at 4°C. Subsequently, the cells were stained with fluorescence-conjugated antibodies against CD8, CD3, CD56, and CD69 for CD8 T and NK cells activation and against CD86 and MHC-II for THP-1 naive cells activation for 40 minutes at 4°C. After washing three times with FACS buffer, the cells were resuspended in FACS buffer and analyzed by FACSlyric (BD bioscience). The data were analyzed with Flowjo 9.0 software (BD bioscience).
Table 4. Antibody used in FACS
As macrophages and monocytes express high levels of STING protein, the human monocytic cell line THP-1 also shows high STING expression. As shown in FIGS. 3A and 3B, when THP-1 was treated with the STING agonist compounds, co-stimulatory molecule CD86 and MHC class II molecule HLA-DR were up-regulated on cell surface in a dose-dependent manner. These suggest that the STING agonist compounds activate myeloid cells, such as macrophages and dendritic cells, and may enhance their tumor antigen presentation through up-regulation of the antigen presentation machinery.
After treating human PBMCs with the STING agonist compounds, it was analyzed the activation of CD8+ T cells and NK cells by measuring CD69, an activation marker, on their cell surface by flow cytometry. The STING agonist compounds greatly enhanced CD69 expression on both CD8+ T and NK cells (FIGS. 4A and 4B), and the expansion of those activated cells (FIGS. 5A and 5B). Thus, the results suggest that the STING agonist compounds effectively induce innate and adaptive immune cell responses.
Experimental Example 4: Assessment of pharmacokinetics
For in vivo evaluation of STING agonist compounds described herein in naive Balb/C mouse, single doses of STING agonist compounds were given at 1.5 mg/kg intravenously into female BALB/c mice (Orientbio, South Korea) at 6-8 weeks of age. Pharmacokinetics were studied following injection of the STING agonist compounds into Balb/C mice. Plasma samples were taken at various time points and stored frozen for analysis. The plasma concentrations of the STING agonist compounds at the indicated time points were measured using a LC-MS/MS analysis method.
Briefly, 250 pL of acetonitrile (ACN) solution was added in both 50 pL of sample and 50 pL of plasma containing 10 nM Dextromethorphan (internal standard), and the solutions were mixed vigorously using a vortex mixer for 5 minutes. The samples were then spun down at 14,000 rpm, 4 °C for 5 minutes. 100 pL of supernatants were combined with 100 pL of mobile phase A (0.1% formic acid in water with 5% ACN) and mixed thoroughly. The samples were measured for the STING agonist compounds using a LC-MS/MS (Nexera LC40 (SHIMADZU) and QTRAP4500 (SCIEX)).
The PK profiles of Compound 221, 277, 281, 274, Comparative #1 and #2 were summarized in Tables 5 to 10. Graphical representation of the plasma PK was shown in FIGS. 6A to 6D. Compared to the Comparative #1 and 2, the STING agonist compounds showed significantly stable pharmacokinetic profiles in mice.
Table 5. Compound 221
Table 6. Compound 277
Table 7. Compound 281
Table 8. Compound 274
Table 9. Comparative #1
Table 10. Comparative #2 Experimental Example 5: In vitro Assessment of normal cell cytotoxicity
PBMCs were also purchased from STEMCELL™ (# 700025.2). The cells (8.0 x 104 cells per well) were seeded in a flat-bottomed 96- well plate in RPMI 1640 medium (Gibco, #22400097) with 10% heat-inactivated FBS and 1 x Antibiotic-Antimycotic (Gibco, #15240062) and rested for 24 hours at 37°C. The cells were treated with the STING agonist compounds described herein (Compound 277, 302, 422, 274, 309, 424, 316, 313, 281, Comparative #1 and #2) in a serial dilution. After 72 hours, the cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega, #G7573) according to the manufacture’s instruction. The signal was detected using an EnVision Xcite multilabel reader and data were analyzed using GraphPad Prism 8 software.
To verify the effect of the STING agonist compounds in normal cell cytotoxicity, the PBMCs were treated with the STING agonist compounds for 3 days and measured the percentage of cell death. As shown in 50% cytotoxic concentration (CCso) values in Table 11, compared to the Comparative #1 and #2, all STING agonist compounds had higher CCso values, implying lower cytotoxicity in normal immune cells.
Table 11. In vitro cell cytotoxicity in PBMCs
The CD34+ hematopoietic stem cells (HSCs) were purchased from STEMCELL™ (# 70002.3). The cells (2 x 104 cells per well) were cultured in StemSpan™ SFEM II medium supplemented with StemSpan™ CD34+ Expansion Supplement (#02691) in a 6-well plate at 37°C for 7 days. On day 3 or 4, an equal volume of fresh complete medium was added into the cell culture. On day 7, the cells (4 x 104 cells per well) were seeded in a 96-well white plate and rested for 24 hours in the same condition.
To verify the effect of STING agonist compounds described herein in normal cell cytotoxicity, the HSCs were treated with STING agonist compounds described herein (Compound 277, 302, 422, 424, 313, 363, 396 and Comparative #1) in a serial dilution. After 72 hours, the cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega, # G7573) according to the manufacture’s instruction. The signal was detected using an EnVision Xcite multilabel reader and data were analyzed using GraphPad Prism 8 software. As shown in 50% cytotoxic concentration (CCso) values in Table 12, compared to the Comparative #1, all STING agonist compounds had higher CCso values, implying lower cytotoxicity in HSCs.
Table 12. In vitro cell cytotoxicity in CD34+ HSC
Experimental Example 6: In vivo efficacy in syngeneic mouse models
Female BALB/c mice (KOTECH, South Korea) with 6 weeks of age, were used for all studies that were completed under the approval of the Legochembio Science’s Institutional Animal Care and Use Committee (IACUC). CT26 or EMT6 cells (American Type Culture Collection (ATCC), #CRL-2638, #CRL-2755) were maintained in RPMI 1640 medium (Gibco, #22400097) supplemented with 10% heat-inactivated FBS and 1 x Antibiotic- Antimycotic (Gibco, #15240062) at 37°C with 5% CO2. The mycoplasma-negative cells were used for all experiments and mycoplasma test was done regularly using e-Myco™ VALiD Mycoplasma PCR Detection Kit (iNtRON biotechnology, #25239). CT26 cells (2xl05 cells/mouse) or EMT6 cells (5xl05 cells/mouse) in PBS were implanted subcutaneously into the shaved right flank. Tumor volumes and body weights were measured every 3 to 4 days, and the tomor volumes were calculated according to the formula 0.5 x (length) x (width)2.
Specifically, to determine in vivo efficacy of STING agonist compounds (Compound 214 and 221) in CT26 syngeneic mouse model, when tumor volume reached 70 mm3, each compound was given at 1.5 mg/kg intravenously every 3 or 4 days (total three times). To determine in vivo efficacy of STING agonist compound (Compound 274) in CT26 syngeneic mouse model, when tumor volume 55 mm3, the compound was given at a variety of doses intravenously once weekly for 3 weeks except a single dose of 1 mg/kg. To compare in vivo efficacy of STING agonist compounds (Compound 274 and 281) in CT26 syngeneic mouse model to the Comparative Compound #1, when tumor volume 55 mm3, each compound was given at 0.5 mg/kg intravenously once weekly for three weeks. To compare in vivo efficacy of STING agonist compounds (Compound 274, 313, 344 and 396) in CT26 syngeneic mouse model to the Comparative Compound #3, when tumor volume 80 mm3, each compound was given at 0.3 mg/kg intravenously once weekly for 3 weeks. To compare in vivo efficacy of STING agonist compounds (Compound 274 and 313) in EMT6 syngeneic mouse model to the Comparative Compounds #1 and #2, when tumor volume 100 mm3, each compound was given at 0.125 mg/kg intravenously once weekly for three weeks. To determine in vivo efficacy of STING agonist compound (Compound 313) in EMT6 syngeneic mouse model, when tumor volume 100 mm3, the compound was given at the indicated doses intravenously once weekly for 3 weeks except for the dose at 0.05 mg/kg twice a week.
A syngeneic system was used to assess the ability of the STING agonist compounds to induce immune responses and drive an anti-tumor immune response. In both CT26 and EMT6 syngeneic models, the STING agonist compounds remarkably controlled tumor growth at various doses (FIGS. 7 to 12).
Particularly, Compound 274 completely eradicated tumors in a group of 0.5 mg/kg (2 out of 5 mice) and in single-dose group of Img/kg (2 out of 5 mice) in CT26 model (FIG. 8). Compound 313 also eliminated tumors in all groups (1 out of 5 for 0.05 mg/kg, 2 out of 5 for 0.1 mg/kg, 5 out of 5 for both 0.3 and 0.6 mg/kg) in EMT6 model (FIG. 12).
Consistent with in vitro effectiveness, the STING agonist compounds described herein showed the superior in vivo efficacy in different syngeneic mouse models.
Taken together, the STING agonist compounds described herein have strongly competitive profiles of high anti-tumor activity, but low toxicity.
Experimental Example 7: In vivo efficacy assessment of STING agonists in combination with immune checkpoint inhibitors (ICIs) in syngeneic mouse models
To evaluate in vivo effects of combination treatment of STING agonists with immune checkpoint inhibitors (ICI), such as anti-PD-1 or anti-PD-Ll antibodies, in vivo studies in syngeneic mouse tumor models were conducted using commercially available murine cancer cell lines of CT26 or B16F10.
Female BALB/c mice (KOTECH, South Korea) or C57BL/6 mice (Shanghai Lingchang BioTech Co. Ltd.) at seven weeks of age were used for CT26 or B16F10 mouse syngeneic model, respectively. All experiments were conducted under the approval of the LigaChem Biosciences Institutional Animal Care and Use Committee (IACUC).
CT26 cells, murine colon cancer cell line (American Type Culture Collection; ATCC), were maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning) and lx Antibiotic-Antimycotic (Gibco™) at 37°C with 5% CO2. B16F10 cells, murine melanoma cell line, were maintained with DMEM medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2.
Mycoplasma-negative cells were used for all experiments and tested regularly using the e-Myco™ VALiD Mycoplasma PCR Detection Kit (iNtRON biotechnology, cat# 25239). CT26 cells (2 x 105 cells/mouse) or B16F10 cells (2 x 105 cells/mouse) were inoculated subcutaneously into the shaved right lower flank of each mouse.
Three separate treatment experiments were conducted in the CT26 syngeneic mouse model, with treatments initiated when the tumor volumes reached approximately 60 mm3. In the first experiment, a single intravenous dose of PD-L1 Antibody_l at 5 mg/kg was administered to the monotherapy group. For the combination groups, PD-L1 Antibody_l was administered intravenously at the same dose and schedule, along with either Compound 277, 302, or 422, each administered intravenously at 1.5 mg/kg twice a week for a total of three doses. In the second experiment, Compound 313 was administered intravenously at 0.25 mg/kg once a week for a total of three doses to the monotherapy group. For the combination groups, either Compound 274 or 313 was administered intravenously at 0.25 mg/kg once a week for a total of three doses, along with a single intravenous dose of PD-L1 Antibody_l at 5 mg/kg. In the third experiment, a single intravenous dose of either mPD-1 antibody or mPD-Ll antibody at 10 mg/kg was administered intravenously to the monotherapy group. For the combination groups, either mPD-1 antibody or mPD-Ll antibody was administered intravenously at the same dose and schedule, along with Compound 274 administered intravenously at 0.25 mg/kg once a week for a total of three doses.
In the Bl 6F 10 syngeneic mouse model, treatments were initiated when tumor volumes reached approximately 100 mm3. mPD-1 antibody was administered intravenously at 10 mg/kg every 3 or 4 days for a total of two doses. Compound 363 was administered at 3 mg/kg once a week for a total of three doses. In the combination group, mPD-1 antibody was co-administered with Compound 363, following the respective dosing schedules.
Tumor volume was monitored every 3 to 4 days for CT26 and three times per week for Bl 6F 10 by measuring perpendicular tumor diameters and calculated using the formula : Tumor volume (mm3) = 0.5 x (length) x (width) 2. Percent tumor growth inhibition (%TGI) was determined using the formula : 100 -[AT/AC*100], where AT is the volume change for treated group and AC is the volume change for control group. The results are represented in Figures 1 to 4.
As shown in FIG. 13, combination therapy of Compound 277, 302, or 422 with PD- L1 Antibody_l showed superior anti-tumor efficacy compared to the PD-L1 Antibody_l monotherapy in a CT26 mouse syngeneic model. Of note, combination treatment of PD-L1 Antibody_l with either Compound 277 or 302 induced complete tumor regression in all mice. Compound 422 treatment with PD-L1 Antibody_l resulted in tumor regression in four out of five mice.
As shown in FIG. 14, Compound 313 or Compound 274 with PD-L1 Antibody_l also showed significant tumor growth suppression including the complete tumor regression in the combination group of Compound 274 (three out of five mice).
Furthermore, as shown in FIG. 15, Compound 274 enhanced anti-tumor effects when administered in a combination with mPD-1 antibody or mPD-Ll antibody compared to antibody monotherapy with either antibody alone. Importantly, as shown in FIG. 16, these effects were reproducible even in ICI-resistant B 16F10 model where the combination treatment of STING agonist with mPD-1 antibody exhibited stronger tumor growth suppression compared to weak activity of mPD- 1 antibody alone.
Collectively the combination of STING agonists with PD-L1 antibody or PD-1 antibody significantly suppressed tumor growth compared to antibody monotherapy, which suggested that STING agonist described herein can be a great combination partner with an immune checkpoint inhibitor, such as PD-1 or PD-L1 blockade.
Experimental Example 8: In vitro assessment of the combination efficacy of a STING agonist with anti-PD-1 or anti-PD-Ll antibody
To evaluate the synergistic cytotoxic effects of STING agonist in combination with anti-PD-1 or anti-PD-Ll antibody, an in vitro co-culture assay was conducted using human immune effector cells and commercially available human breast cancer cell line, SK-BR3.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and CD14+ monocytes were depleted using a CD14+ cell isolation kit (Stemcell Technologies, cat#17858) according to the manufacturer’s instructions. The resulting lymphocyte-enriched CD 14 cells were used as effector cells. SK-BR3 cells was infected with Incucyte Nuclight Red lentivirus (Sartorius, #4625) and stable cell population expressing RFP (RFP+SK-BR3) was generated under puromycin selection. Red-labeled SK-BR3 target cells (5 x 103 cells/well) were seeded into 96-well plates and incubated overnight to allow for cell attachment. On the following day, effector cells (2.5 x 104 cells/well) were added into the tumor cells at an effector- to-target (E:T) ratio of 5:1 in the presence of CD3/CD28 activation beads (Gibco, cat#11132D) and IL-2 (PeproTech, cat#200-02-50UG) at a final concentration of 5 ng/ml.
Compound 313 (50 nM) and PD-1 antibody_l, PD-1 antibody_2 or PD-L1 antibody_2 were added simultaneously in the beginning of the co-culture. Live-cell imaging analysis was performed using the Incucyte® system (Satorius). Images of the red and phase channels were captured every 4 hours using a lOx objective. Red object counts were automatically acquired and analyzed over 72 hours to evaluate treatment - mediated changes in cell growth. To assess relative changes over time, the red object count per well was normalized by the count at 0 hour for each group. The results were presented as mean ± standard deviation (SD) with individual replicates overlaid as dots to visualize variability across samples.
As shown in FIGS. 17A to 17C, the combination of Compound 313 with PD-1 antibody_l, PD-1 antibody_2 or PD-L1 antibody_2 inhibited cancer cell growth over time compared to the single treatment groups. These in vitro results also supported that STING agonist described herein can serve as an effective combination partner with ICIs, such as PD- 1 or PD-L1 blockade.
Experimental Example 9: In vitro evaluation of macrophage activity enhanced by STING agonist and anti-PD-Ll antibody Macrophage-mediated anti-tumor activity can be achieved by increased phagocytosis of tumor cells. To test whether STING agonists enhance phagocytic function of macrophages, human monocyte-derived macrophages (Mo-Mac) were co-cultured with hPD-Ll- overexpressed Raji cells as target cells in the presence of an anti-PD-1 antibody (full human IgGl, intact Fc) in vitro.
For generation of Mo-mac, freshly isolated CD14+ cells from PBMCs were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), lx antibiotic-antimycotic solution, and 50 ng/mL macrophage colony- stimulating factor (M-CSF; PeprotTech cat#300-25-100UG). The cells were seeded at a density of 5 x 105 cells per well in a 12-well low-attachment plate containing 0.5 mL of differentiation medium and incubated at 37°C in a humidified 5% CO2 incubator.
On day 3, the medium was replaced with fresh differentiation medium. On day 6, fully differentiated Mo-Mas were harvested by gentle pipetting and were seeded at 5 x 104 cells/well in flat-bottomed ultra-low attachment 96-well plates in serum-free media. CFSE-labeled Raji cells (5 x 104 cells/well) were then added into the wells at an effector-to-target (E:T) ratio of 1:1.
Compound 363 (untreated, 0.037 pM, 0.33 pM, and 3 pM) and 0.37 pg/mL PD-L1 antibody_3 were added into the culture. Two hours later, cells were harvested and stained with LIVE/DEAD fixable Near-IR (780) viability dye (Invitrogen cat#L34994) per the manufacturer’s protocol. After washing, cells were blocked with human Fc receptor blocker for 10 minutes and then stained with APC-conjugated anti-human CDllb antibody (Biolegend cat#301350) for 1 hour on ice in FACS buffer. Cells were washed and fixed with 4% paraformaldehyde (PFA) for 20 minutes on ice. Samples were then washed and analyzed by flow cytometry (BD FACS lyric). The phagocytosis rate was calculated as the proportion of CFSE-positive cells within the CDllb-positive macrophages.
As shown in FIG. 18 Compound 363 enhanced phagocytosis by Mo-mac in the presence of PD-L1 antibody_3. These data indicated that STING agonists described herein effectively boost phagocytosis in the presence of anti-PD-Ll antibody with intact Fc function.
Incorporation by Reference
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
While specific embodiments of the disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

We claim:
1. A method of administering a compound to a subject in need thereof, comprising administering the compound conjointly with an immune checkpoint inhibitor, wherein the compound is represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein:
T is a moiety comprising a Stimulator of Interferon Genes (STING) agonist, p is 1 or 2, each instance of R1 is independently -CH2OR11 or -COOR12, each instance of Rla, Rlb, Rlc, and R11 is independently H or a hydroxyl protecting group, each instance of R12 is independently H or a carboxyl protecting group, each instance of R2 and R3 is independently H or alkyl, or R2 and R3 together with a carbon atom to which they are attached form a cycloalkyl, each instance of R4 is independently selected from halogen, alkyl, CN, and NO2, each instance of k is independently 0, 1, 2, or 3, each instance of Y is independently selected from H, -C(O)NHLUU, -C(O)NR'(LUU), -
C(O)N(LUU)2 and -C(O)OH, each instance of Lu is a first linker, each instance of U is independently selected from H, alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, -COOH, -P(O)(OH)2, -OH, -DBCO and a saccharide, and each instance of R' is independently selected from alkyl, cycloalkyl, alkoxy, alkylthio, mono- or di-alkylamino, heteroaryl, and aryl.
2. The method of claim 1 , wherein the compound of formula (I) is a compound of formula (la), or a pharmaceutically acceptable salt thereof:
3. The method of claim 1 or 2, wherein T is a moiety represented by formula (Ila) or a pharmaceutically acceptable salt thereof: wherein:
T is coupled to the -C(O)OCR2R3- fragment of formula (I) via L2,
M is N, C(XaRa) or C(XbL1L2-),
Q is -XaRa or -XhL‘ L2-, each instance of W1 and W2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3,
Z is selected from alkylene, alkenylene, and alkynylene,
A and B are each independently aryl or heteroaryl,
Xa and Xb are each independently selected from CH2, NH, O, and S,
Ra is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, -(alkylene)guanidino, - (alkylene)NHC(O)CH2guanidino, -(alkylene)O(alkylene)guanidino and -O(alkylene)guanidino, each L1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene, each L2 is independently selected from a bond, or a linker moiety coupled to L1 and comprising a nitrogen atom coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I), wherein M is C(XbL1L2-) and/or Q is -XhL’ L2-.
4. The method of claim 3, wherein Xa is O.
5. The method of claim 3 or 4, wherein Ra is selected from Ci-6 alkyl, - (alkylene)N(H)alkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, and - (alkylene)guanidino.
6. The method of any one of claims 3-5, wherein L2 is selected from a bond, * NH2 _
J* NH NH2
HN ** K /
\=NI-I /* / II i ' ML NH2 HN^ N
/^NH *— (alkylene)-N zN-(alkylene)-NH I /
* v y ’ H * (alkylene)— NH
** **\ NH
*\-(alkylene)-(heterocyclyl) ^N-OlkyleneJ-lheterocyclylene)— {
* •> •> NH2 •>
**
*_ (aryiene-j-bf 7* (heteroarylene)— (alkylene)— NH
'H * — (heterocyclylene) — NH 1 '** 1
*— (heteroarylene)—
** / *— (heterocyclenylalkylene)— N /
CO2H * — (alkylene)-N
O
^NH |_i M * — (heteroarylene)— N
2 , H ; and *heterocyclylene**, and wherein * is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
7. The method of any one of claims 3-6, wherein T is coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) via L2, and T is a moiety represented by formula (lib): (lib), wherein:
A and B are each independently 5-membered heteroaryl, and x/X/X i/X/ /") Q l is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
8. The method of any one of claims 3-6, wherein T is coupled to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I) via L2, and T is a moiety represented by formula (lie): wherein: A and B are each independently 5-membered heteroaryl, and «/VW i
I is the point of connection to the -C(O)O(CR R )- fragment of the compound represented by structural formula (I).
9. The method of any one of claim 7 or 8, wherein L2 is selected from a bond, * NH2 ,
**x NH
C> N—
N— (alkylene)-(heterocyclyl) 's' zN— (alkylene)-(heterocyclylene) —
* NH2
— (arylene)— 7* *— (heteroarylene)— (alkylene)— NH
H * — (heterocyclylene)— NH '**
*— (heteroarylene)— N
** I
*— (heterocyclenylalkylene)— o
(alkylene)— NH
I )=NH y*
^NH u * — (heteroarylene) — N
2 , H , and *heterocyclylene**, and wherein * is the point of connection to L1 and ** is the point of connection to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I).
10. The method of claim 7 or 8, wherein L2 is a second linker comprising #OC(O)NR5-L4- NR6, #OC(O)-L4-NR6, or #OC(O)NR5-L4-(heterocyclylene), wherein: the heterocyclylene comprises a nitrogen atom connected to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I), and wherein # is the point of connection to L1 , each instance of L4 is independently alkylene or arylalkylene, and each instance of R5 is independently selected from H, alkyl, and dialkylaminoalkyl, and each instance of R6 is independently selected from H, alkyl, and dialkylaminoalkyl. wherein ** indicates the connection point to the -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I); and # indicates the point of connection to L1.
12. The method of any one of claims 3-6, wherein p is 2 and T is a moiety represented by structural formula (lid), wherein T is coupled to each -C(O)OCR2R3- fragment of formula (I) via two L2:
wherein:
A and B are each independently 5-membered heteroaryl, and «/VW i each I is a point of connection to the -C(O)O(CR R )- fragments of the compound represented by structural formula (I).
13. The method of claim 12, wherein each L2 is independently a second linker comprising #OC(O)NR5-L5-NR6, #OC(O)-L4-NR6, or #OC(O)NR5-L5-(heterocyclylene), wherein the heterocyclylene comprises a nitrogen atom connected to the respective -C(O)O(CR2R3)- fragment of the compound represented by structural formula (I), wherein # is the point of connection to the respective L1 , each L4 is independently alkylene or arylalkylene, each L5 is independently alkylene or aralkylene, and each R5 and each R6 are each independently selected from H, alkyl, and dialkylaminoalkyl.
14. The method of claim 1 or 2, wherein, T is a moiety represented by formula (T1) or a pharmaceutically acceptable salt thereof:
wherein:
T1 is coupled to the -C(O)OCR2R3- fragment of formula (I) via the -N(H)- of T1; each Ta and Tb is independently a moiety represented by formula (Ila); wherein:
M is N, C(XaRa) or C(XbL1L2-);
Q is -XaRa or -XVL2-; each instance of W1 and W2 is independently selected from alkyl, amino, amido, carboxylic acid, ester, and hydrazido; n and m are each independently 0, 1, 2, or 3;
Z is selected from alkylene, alkenylene, and alkynylene;
A and B are each independently aryl or heteroaryl;
Xa and Xb are each independently selected from CH2, NH, O, and S;
Ra is selected from H, alkyl, alkenyl, alkynyl, heteroalkyl (e.g., -(alkylene)N(H)alkyl), cycloalkyl, heterocyclyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)carboxylic acid, -(alkylene)guanidino, - (alkylene)NHC(O)CH2guanidino, -(alkylene)O(alkylene)guanidino and -O(alkylene)guanidino; each L1 is independently selected from alkylene, heteroalkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene, and heteroarylene; each L2 is independently selected from a bond between L1 and -C(0)0(CH2)- fragment of T1, or a linker moiety coupled to L1 and comprising a nitrogen atom coupled to the - C(0)0(CH2)- fragment of T1; wherein either M is C(XbL1L2-) or Q is -XhL'L2-.
15. The method of any one of claims 3-14, wherein A and B are each independently substituted or unsubstituted pyrazole or substituted or unsubstituted oxazole.
16. The method of claim 15, wherein A and B are each independently substituted pyrazole or substituted oxazole, wherein the pyrazole and oxazole are each substituted with two Ci-3 alkyls.
17. The method of any one of claims 1-16, wherein each instance of the first linker Lu comprises one or more moieties independently selected in each instance from *(alkylene)O(alkylene)**, *(heteroalkylene)**, *(alkylene)**, *(heteroaralkylene)**, *(heteroalkylene)(heterocyclylene)**, *CH2CH2C(O)NHCH**, *(CH2CH2O)t-**, *(alkylene)O(alkylene)-(amide)-(alkylene)O-**, and *(alkylene)(heteroarylene)(CH2CH2O)t**, wherein * indicates the point of connection to the -C(O)NH- fragment of the compound represented by structural formula (I), ** indicates the point of connection to U, and t represents an integer from 1-15.
18. The method of claim 17, wherein each instance of the first linker Lu is independently selected from *(CH2CH2O)tCH2**, *(CH2CH2O)2CH2CH2N(CH3)CH2**, *(CH2CH2O)2CH2CH2N(CH3)**, *(CH2CH2O)t**, *(CH2CH2O)tCH2CH2NH**, *CH2CH2**, *(CH2CH2O)tCH2**, *(CH2CH2O)tCH2CH2heterocyclylene**, *(CH2CH2O)tCH2**, *(CH2CH2O)tCH2CH2**, *(CH2CH2O)tCH2CH2NHCOC(CH3)2-O-**and *(CH2CH2O)tNH**.
19. The method of claim 17 or 18, wherein t represents an integer from 1-6.
20. The method of any one of claims 17-19, wherein t represents an integer from 1-3.
21. The method of any one of claims 1-20, wherein each instance of U is H or NH2.
22. The method of any one of claims 1-20, wherein each instance of U is -N(Me)2.
23. The method of any one of claims 1-20, wherein each instance of U is independently selected from alkyl, alkynyl, amino, azido, acetylenyl, alkylamino, heterocyclyl, alkoxy, - COOH, -P(O)(OH)2, -DBCO and -OH.
24. The method of claim 23, wherein the heterocyclyl of U comprises at least one nitrogen which is the point of connection to Lu.
25. The method of claim 1, wherein the compound is pharmaceutically acceptable salt thereof.
26. The method of any one of claims 1-19, wherein each instance of U is a saccharide, e.g. a glucuronide.
27. The method of claim 26, wherein the saccharide is a glucuronide.
OH
28. The method of claim 27, wherein the glucuronide is .
29. The method of claim 28, wherein the compound is selected from
30. The method of any one of claims 1-29, wherein the immune checkpoint inhibitor is an inhibitor of the PD-1/PD-L1 pathway.
31. The method of any one of claims 30, wherein the inhibitor of the PD-1/PD-L1 pathway is an antibody, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, acrixolimab, MGA012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, or cosibelimab.
32. The method of claim 31 , wherein the antibody is an anti-PD- 1 antibody or antigenbinding fragment thereof, e.g., pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, MGA012, AMP-224, or AMP-514. 33. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6, or
(b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 11;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 12;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 13;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 14,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 15; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 16, or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 19;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 20;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 21;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 22,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 23; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 24, or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 29;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 30;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 31;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 32,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 33; and (vi) CDRL3 comprises the ammo acid sequence ofSEQ ID NO: 34, or
(e) (i) CDRH1 comprises the amino acid sequence ofSEQIDNO: 37;
(ii) CDRH2 comprises the amino acid sequence of SEQIDNO: 38;
(iii) CDRH3 comprises the amino acid sequence ofSEQ ID NO: 39;
(iv) CDRL1 comprises the amino acid sequence ofSEQ ID NO: 40,
(v) CDRL2 comprises the amino acid sequence ofSEQ ID NO: 41; and
(vi) CDRL3 comprises the amino acid sequence ofSEQ ID NO: 42, or
(f) (i) CDRH1 comprises the amino acid sequence ofSEQ ID NO: 45;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 46;
(iii) CDRH3 comprises the amino acid sequence ofSEQ ID NO: 47;
(iv) CDRL1 comprises the amino acid sequence ofSEQ ID NO: 48,
(v) CDRL2 comprises the amino acid sequence ofSEQ ID NO: 49; and
(vi) CDRL3 comprises the amino acid sequence ofSEQ ID NO: 50, or
(g) (i) CDRH1 comprises the amino acid sequence ofSEQ ID NO: 53;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 54;
(iii) CDRH3 comprises the amino acid sequence ofSEQ ID NO: 55;
(iv) CDRL1 comprises the amino acid sequence ofSEQ ID NO: 56,
(v) CDRL2 comprises the amino acid sequence ofSEQ ID NO: 57; and
(vi) CDRL3 comprises the amino acid sequence ofSEQ ID NO: 58, or
(h) (i) CDRH1 comprises the amino acid sequence ofSEQ ID NO: 61;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 62;
(iii) CDRH3 comprises the amino acid sequence ofSEQ ID NO: 63;
(iv) CDRL1 comprises the amino acid sequence ofSEQ ID NO: 64,
(v) CDRL2 comprises the amino acid sequence ofSEQ ID NO: 65; and
(vi) CDRL3 comprises the amino acid sequence ofSEQ ID NO: 66; or
(i) (i) CDRH1 comprises the amino acid sequence ofSEQIDNO: 109;
(ii) CDRH2 comprises the amino acid sequence ofSEQIDNO: 110;
(iii) CDRH3 comprises the amino acid sequence ofSEQIDNO: 111;
(iv) CDRL1 comprises the amino acid sequence ofSEQIDNO: 112;
(v) CDRL2 comprises the amino acid sequence ofSEQIDNO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114; or
(j) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 121 ;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 122;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 123;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 124;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 125; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 126.
34. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 2; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 3; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 4; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 5; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 6.
35. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 109; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 110; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 111; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 112; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 113; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 114.
36. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of:
(a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 18, or
(d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 26, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36, or
(f) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 44, or
(g) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 52, or
(h) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 60, or
(i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 68, or j) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 27, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 28.
37. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8. 38. The method of claim 32, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.
39. The method of claim 31, wherein the antibody is an anti-PD-Ll antibody or antigenbinding fragment thereof, e.g., atezolizumab, avelumab, durvalumab, or cosibelimab.
40. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(a) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74, or
(b) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84, or
(c) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 87;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 88;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 89; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 90,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 91; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 92, or
(d) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100,
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101 ; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102; or
(e) (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 145;
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 146;
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 147;
(iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 148;
(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 149; and
(vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 150.
41. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 69; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 70; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 71; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 72; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 73; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 74.
42. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 79; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 80; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 81; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 82; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 83; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 84.
43. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a variable heavy chain complementarity determining region 1 (CDRH1), a variable heavy chain complementarity determining region 2 (CDRH2), a variable heavy chain complementarity determining region 3 (CDRH3), a variable light chain complementarity determining region 1 (CDRL1), a variable light chain complementarity determining region 2 (CDRL2), and a variable light chain complementarity determining region 3 (CDRL3); wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 97; (ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 98; (iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 99; (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 100; (v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 101; and (vi) CDRL3 comprises the amino acid sequence of SEQ ID NO: 102.
44. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of:
(a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76, or
(b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86, or
(c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 93, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 94, or (d) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 95, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 96, or
(e) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
45. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 76.
46. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 85, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 86.
47. The method of claim 39, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a combination of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 103, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 104.
48. The method of any one of claims 31-47, wherein the antibody is a monoclonal antibody, a single chain antibody (scAb), a Fab fragment, a F(ab’)2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a multimeric antibody, or a bispecific antibody.
49. The method of any one of claims 31-48, wherein the antibody is a chimeric, humanized or fully human monoclonal antibody.
50. The method of any one of claims 31-49, wherein the antibody is an IgG isotype.
51. The method of claim 50, wherein the antibody is an IgGl isotype. 52. The method of claim 50 or 51, wherein the antibody comprises L234A and L235A substitutions according to EU numbering convention.
53. The method of claim 52, wherein the antibody comprises a P329G substitution or a P329A substitution according to EU numbering convention.
54. The method of any one of claims 50-53, wherein the antibody comprises a N297A substitution according to EU numbering convention.
55. The method of claim 50, wherein Ab is an IgG4 isotype.
56. The method of claim 55, wherein the antibody comprises a S228P substitution according to EU numbering convention.
57. The method of claim 30, wherein the immune checkpoint inhibitor is selected from:
(1) an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, or antigen-binding fragment thereof;
(2) an anti-PD-Ll antibody or antigen-binding fragment thereof, wherein the anti-PD-Ll antibody is selected from atezolizumab, Clone 31E6, avelumab or antigen-binding fragment thereof; or
(3) an anti-mouse PD-1 or PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-mouse PD-1 or PD-L1 antibody is selected from 10F.9G2, RPM1-14, or antigenbinding fragment thereof.
58. The method of claim 1, wherein the compound is selected from:
pharmaceutically acceptable salt thereof.
59. The method of any one of claims 1-58, wherein the method is a method of preventing or treating a proliferative disease, an infectious disease, an immune-mediated disorder, a central nervous system disease, a peripheral nervous system disease, a neurodegenerative disease, a cerebrovascular disease, a peripheral arterial disease, a cardiovascular disease, or an allergic disease.
60. The method of claim 59, wherein the proliferative disease is cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, or cirrhosis of the liver.
61. The method of claim 60, wherein the cancer is selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi sarcoma, and melanoma.
62. The method of claim 59, wherein the infectious disease is chickenpox, chikungunya, a coronavirus infection, a dengue virus infection, diphtheria, Ebola, influenza, hepatitis, Hib disease, acquired immunodeficiency syndrome (AIDS), a human papillomavirus (HPV) infection, encephalitis, measles, meningococcal disease, Mpox, mumps, a norovirus infection, pneumococcal disease, polio, rabies, respiratory syncytial virus (RSV) infection, rotavirus infection, rubella, shingles, tetanus, whooping cough, or zika virus disease.
63. The method of claim 59, wherein the immune-mediated disorder is Crohn’s, ulcerative colitis, uveitis, psoriasis, lupus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, ankylosing spondylitis, hidradenitis suppurativa, sarcoidosis, atopic dermatitis, connective tissue disorders, asthma, or multiple sclerosis.
64. The method of claim 59, wherein the central nervous system disease is catalepsy, encephalitis, epilepsy, meningitis, multiple sclerosis, or myelopathy.
65. The method of claim 59, wherein the peripheral nervous system disease is lepromatous neuropathy, diabetic neuropathy, Guillain-Barre syndrome, acute motor axonal neuropathy, botulism, Lambert-Eaton syndrome, acute intermittent porphyria, or familial amyloid neuropathy.
66. The method of claim 59, wherein the neurodegenerative disease is Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barre" syndrome (GBS), Myasthenia Gravis, Bullous Pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson’s disease, traumatic brain injury (TBI), epilepsy, or Huntington’s disease (HD).
67. The method of claim 59, wherein the cerebrovascular disease is aneurysms, arteriovenous malformations (AVM), cerebral cavernous malformations (CCM), arteriovenous fistula (AVF), carotid-cavernous fistula, carotid stenosis, transient ischemic attack (TIA), or stroke.
68. The method of claim 59, wherein the cardiovascular disease is arrhythmias, congenital heart disease, coronary artery disease, deep vein thrombosis, pulmonary embolism, heart attack, heart failure, cardiomyopathy, heart valve disease, pericardial disease, peripheral vascular disease, rheumatic heart disease, stroke, or vascular disease.
69. The method of claim 59, wherein the allergic disease is an allergy, anaphylaxis, aspergillosis, asthma, chronic cough, chronic granulomatous disease, chronic sinusitis, Churg- Strauss syndrome, cold urticaria, common variable immunodeficiency, eosinophilia, eosinophilic esophagitis, esophagitis, hay fever, hyper eosinophilic syndrome, nasal congestion, nasal polyps, nonallergic rhinitis, conjunctivitis, pneumonitis, primary immunodeficiency, selective IgA deficiency, systemic mastocytosis, or X-linked agammaglobulinemia.
70. The method of any one of claims 1-69, wherein the method is a method of inducing an immune response.
71. A pharmaceutical composition or a kit comprising the compound and the immune checkpoint inhibitor of any one of claims 1-70.
72. The pharmaceutical composition or a kit of claim 71, wherein the composition further comprises a pharmaceutically acceptable excipient.
PCT/IB2025/000222 2024-05-08 2025-05-08 Compositions and methods related to a combination therapy of sting agonists and immune checkpoint inhibitors Pending WO2025233676A1 (en)

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WO2022155518A1 (en) * 2021-01-15 2022-07-21 Seagen Inc. Immunomodulatory antibody-drug conjugates
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