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WO2020156363A1 - 一种免疫调节剂 - Google Patents

一种免疫调节剂 Download PDF

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Publication number
WO2020156363A1
WO2020156363A1 PCT/CN2020/073405 CN2020073405W WO2020156363A1 WO 2020156363 A1 WO2020156363 A1 WO 2020156363A1 CN 2020073405 W CN2020073405 W CN 2020073405W WO 2020156363 A1 WO2020156363 A1 WO 2020156363A1
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Prior art keywords
alkyl
independently selected
substituted
membered
halogen
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PCT/CN2020/073405
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English (en)
French (fr)
Inventor
李进
张登友
潘飞
马荣
朱文吉
辛艳飞
李偲
刘卫平
林燕萍
Original Assignee
成都先导药物开发股份有限公司
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Priority to CA3128069A priority Critical patent/CA3128069C/en
Priority to KR1020217027365A priority patent/KR102702618B1/ko
Priority to US17/427,104 priority patent/US20220119379A1/en
Priority to AU2020214477A priority patent/AU2020214477B2/en
Priority to JP2021544657A priority patent/JP7655854B2/ja
Priority to EP20748337.1A priority patent/EP3919488A4/en
Publication of WO2020156363A1 publication Critical patent/WO2020156363A1/zh

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    • A—HUMAN NECESSITIES
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    • C07—ORGANIC CHEMISTRY
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    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5386—1,4-Oxazines, e.g. morpholine spiro-condensed or forming part of bridged ring systems
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    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/08—Antiallergic agents
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    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/048—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being five-membered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/08—Bridged systems
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants

Definitions

  • the invention relates to an immunomodulator, in particular to a class of compounds that activate STING and its use as an immunomodulator in the preparation of medicines.
  • the human immune system can generally be divided into “innate immunity” and “adaptive immunity” systems.
  • the innate immune system plays an important role in fighting infections, inhibiting tumor growth, and the pathogenesis of autoimmune diseases. It mainly recognizes pathogenic microorganisms and cancer cell components through pattern recognition receptors, initiates downstream signaling pathways, and finally induces cytokine expression. Kill pathogenic microorganisms and cancer cell components, and adapt to the immune system to promote the production of antibodies and specific T lymphocytes.
  • STING interferon gene stimulating factor, TMEM173, MITA, etc.
  • TMEM173, MITA interferon gene stimulating factor
  • Compounds that induce human interferon can be used to treat various diseases (including allergic diseases and other inflammatory diseases, allergic rhinitis and asthma, infectious diseases, neurodegenerative diseases, precancerous syndromes and cancer), and can also be used as Immunization composition or vaccine adjuvant. Therefore, the discovery of new compounds that can activate STING is an effective method for treating related type 1 IFN pathway diseases (including inflammatory, allergic and autoimmune diseases, infectious diseases, cancer, and precancerous syndromes).
  • the present invention provides an immunomodulator.
  • the present invention provides a compound represented by formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
  • L is selected from C 2 ⁇ C 6 alkylene, C 2 ⁇ C 6 alkenylene, C 2 ⁇ C 6 alkynylene;
  • R 1 , R 3 , R 1'and R 3' are each independently selected from hydrogen, halogen, C 1 ⁇ C 6 alkyl, cyano, nitro, hydroxyl, amino, C 1 ⁇ C 6 alkoxy, C 1 ⁇ C 6 alkylamino, halogen substituted C 1 ⁇ C 6 alkyl;
  • R 2 ' is independently selected from hydrogen, -C (O) NR a R b, -NR a C (O) R b, -C (O) R a, -C (O) OR a, halo, C 1 ⁇ C 6 alkyl, cyano, nitro, hydroxyl, amino, C 1 ⁇ C 6 alkoxy, C 1 ⁇ C 6 alkylamino, halogen substituted C 1 ⁇ C 6 alkyl;
  • R 4 and R 4' are each independently selected from hydrogen, C 1 ⁇ C 6 alkyl
  • X and X' are independently selected from -CH 2 -, -NH-, -O-, -S-;
  • Ring A and Ring A' are each independently selected from a 5- to 6-membered aromatic heterocyclic ring optionally substituted by 0 to 4 R c ;
  • At least one of the A ring and A'ring is selected from a sulfur-containing 5-6 member optionally substituted by 0-4 R c Aromatic heterocyclic ring or 5-6 membered aromatic heterocyclic ring containing oxygen;
  • R 5 and R 5' are each independently selected from C 1 to C 6 alkyl optionally substituted with 0 to 2 R d ;
  • R a and R b are each independently selected from hydrogen, C 1 ⁇ C 6 alkyl
  • R c are each independently selected from halogen, -CN, -OR a , C 1 -C 6 alkyl, and halogen-substituted C 1 -C 6 alkyl;
  • R d is independently selected from halogen, -OR a, -NR a R b , substituted with 0 to 4 R e is an optionally substituted 3-6 membered cycloalkyl, substituted with 0 to 4 R e 3 ⁇ optionally 6-membered heterocyclic group, substituted with 0 to 4 R e is optionally 7 to 10-membered cycloalkyl, substituted with 0 to 4 R e is optionally substituted 7-10 membered heterocycloalkyl, substituted with 0 to 4 R e is optionally substituted with a 7 to 10-membered spiro cycloalkyl, substituted with 0 to 4 R e is optionally spiro 7 to 10-membered heterocycloalkyl, optionally substituted 0-4 R e.
  • R e are each independently selected from halogen, C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 to C 6 alkyl, and 3 to 6 membered cycloalkyl.
  • L is selected from C 2 ⁇ C 6 alkylene, C 2 ⁇ C 6 alkenylene, C 2 ⁇ C 6 alkynylene;
  • R 1 , R 3 , R 1'and R 3' are each independently selected from hydrogen, halogen, and C 1 ⁇ C 6 alkyl;
  • R 2 is independently selected from hydrogen, -C (O) NR a R b;
  • R 4 and R 4' are each independently selected from hydrogen, C 1 ⁇ C 6 alkyl
  • X and X' are independently selected from -CH 2 -, -NH-, -O-, -S-;
  • Ring A and Ring A' are each independently selected from a 5- to 6-membered aromatic heterocyclic ring optionally substituted by 0 to 4 R c ;
  • At least one of the A ring and A'ring is selected from a sulfur-containing 5-6 member optionally substituted by 0-4 R c Aromatic heterocyclic ring or 5-6 membered aromatic heterocyclic ring containing oxygen;
  • R 5 and R 5' are each independently selected from C 1 to C 6 alkyl optionally substituted with 0 to 2 R d ;
  • R a and R b are each independently selected from hydrogen, C 1 ⁇ C 6 alkyl
  • R c are each independently selected from halogen, -CN, -OR a , C 1 -C 6 alkyl, and halogen-substituted C 1 -C 6 alkyl;
  • R d is independently selected from halogen, -OR a, -NR a R b , substituted with 0 to 4 R e is an optionally substituted 3-6 membered cycloalkyl, substituted with 0 to 4 R e 3 ⁇ optionally 6-membered heterocycloalkyl;
  • R e are each independently selected from halogen, C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 to C 6 alkyl, and 3 to 6 membered cycloalkyl.
  • R 1 , R 3 , R 1'and R 3' are each independently selected from hydrogen;
  • R 2 and R 2' are each independently selected from -C(O)NR a R b ;
  • R 4 and R 4' are each independently selected from hydrogen
  • X and X' are independently selected from -O- and -S-;
  • Ring A and Ring A' are each independently selected from a 5-membered aromatic heterocyclic ring optionally substituted with 0 to 3 R c ;
  • At least one of the A ring and A'ring is selected from a sulfur-containing 5-membered aromatic heterocyclic ring optionally substituted by 0 to 3 R c ;
  • R 5 and R 5' are each independently selected from C 1 to C 6 alkyl groups optionally substituted with 0 to 1 R d ;
  • R c are each independently selected from halogen, C 1 ⁇ C 6 alkyl, and halogen substituted C 1 ⁇ C 6 alkyl;
  • R d is independently selected from substituted with 0 to 2 R e optionally 3 to 6-membered cycloalkyl, substituted with 0 to 2 R e optionally having 3 to 6-membered heterocyclic group;
  • R e are each independently selected from C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 to C 6 alkyl, and 3 to 6 membered cycloalkyl.
  • At least one of X and X' is selected from -S-, A ring and A'ring are each independently selected from those optionally substituted with 0-2 R c or R 5 and R 5'are each independently selected from C 1 ⁇ C 6 alkyl groups, or
  • R c' is selected from C 1 ⁇ C 6 alkyl, halogen substituted C 1 ⁇ C 6 alkyl;
  • R c is selected from halogen, C 1 ⁇ C 6 alkyl, halogen substituted C 1 ⁇ C 6 alkyl;
  • R e' are independently selected from C 1 ⁇ C 6 alkyl, 3 to 6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, and R a is selected from hydrogen, C 1 ⁇ C 6 alkyl.
  • X and X' are selected from -O-, and at least one of A ring and A'ring is selected from those optionally substituted with 0-2 R c R 5 and R 5'are each independently selected from C 1 ⁇ C 6 alkyl groups, or
  • R c is selected from halogen, C 1 ⁇ C 6 alkyl, halogen substituted C 1 ⁇ C 6 alkyl;
  • R e' are independently selected from C 1 ⁇ C 6 alkyl, 3 to 6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, and R a is selected from hydrogen, C 1 ⁇ C 6 alkyl.
  • X and X' are independently selected from -O- and -S-;
  • Ring A and Ring A' are independently selected from those optionally substituted with 0 to 2 R c or And when X and X'are both -O-, at least one of the A ring and A'ring is selected from those optionally substituted by 0-2 R c
  • R c' is selected from C 1 ⁇ C 2 alkyl and halogen substituted C 1 ⁇ C 2 alkyl;
  • R c is selected from halogen, C 1 ⁇ C 2 alkyl, halogen substituted C 1 ⁇ C 2 alkyl; the halogen is preferably F;
  • R 5 and R 5' are each independently selected from C 1 ⁇ C 3 alkyl, or
  • R e' is selected from C 1 ⁇ C 3 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is selected from -OR a' , C 1 -C 3 alkyl, 3 to 4-membered cycloalkyl, and R a'is selected from C 1 to C 3 alkyl.
  • X is selected from -O-, -S-, and X'is selected from -O-;
  • R 5 is selected from C 1 ⁇ C 3 alkyl
  • R 5' is selected from or R e'is selected from C 1 ⁇ C 3 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is selected from -OR a' , C 1 -C 3 alkyl, 3 to 4-membered cycloalkyl, and R a'is selected from C 1 to C 3 alkyl.
  • R 5, R 5 ' are each independently selected from 0 to 2 R d is optionally substituted C 1 ⁇ C 6 alkyl group;
  • R d is independently selected from halogen, -OH, substituted with 0 to 4 R e is optionally 3 to 6-membered cycloalkyl, substituted with 0 to 4 R e is optionally 3 to 6-membered heterocycloalkyl, substituted with 0 to 4 R e is optionally substituted 7-10 membered cycloalkyl, substituted with 0 to 4 substituents R e is optionally 7 to 10-membered heterocycloalkyl, optionally 0-4 R e is 7 to 10-membered spiro cycloalkyl, substituted with 0 to 4 R e is optionally spiro 7 to 10-membered heterocycloalkyl, substituted with 0 to 4 R e 5 to 10-membered optionally bridged cycloalkyl, 5-10 membered bridge heterocycloalkyl optionally substituted by 0-4 R e ;
  • R e are each independently selected from halogen, C 1 ⁇ C 6 alkyl, 3-6 membered cycloalkyl, -C(O)R f ;
  • R f is independently selected from -OR a , C 1 to C 6 alkyl, and 3 to 6 membered cycloalkyl.
  • R 1, R 3, R 1 ', R 3' is independently selected from hydrogen, halogen;
  • Ring A and Ring A' are independently selected from those optionally substituted with 0 to 2 R c or
  • R c' is selected from C 1 ⁇ C 2 alkyl and halogen substituted C 1 ⁇ C 2 alkyl;
  • R c is selected from halogen, C 1 ⁇ C 2 alkyl, and halogen substituted C 1 ⁇ C 2 alkyl;
  • R 5' is selected from C 1 -C 3 alkyl optionally substituted by 0 to 2 R d ;
  • R d is independently selected from -OH, substituted with 0 to 2 substituents
  • R e is optionally 3 to 6-membered cycloalkyl, optionally 0 to 2 R e 3 to 6-membered heterocyclic alkyl, 0 or 2 R e is optionally substituted 7-10 membered cycloalkyl, substituted with 0 to 2 R e, optionally 7 to 10-membered heterocycloalkyl group, 0 to 2 are optionally substituted with R e ⁇ 7 10 yuan spiro cycloalkyl, substituted with 0 to 2 R e, optionally 7 to 10-membered spiro-heterocycloalkyl, substituted 0 to 2 R e 5 to 10-membered optionally bridged cycloalkyl, 0 or 2 R e optionally substituted 5 to 10-membered bridged heterocycloalkyl, substituted with 0 to 2 R e is optionally condensed 7 to 10-membered
  • a ring and A'ring are independently selected from
  • R 5' is selected from C 1 -C 3 alkyl optionally substituted by 0 to 2 R d ;
  • R d are independently selected from -OH,
  • the A ring and the A'ring are each independently preferably a 5-membered nitrogen-containing aromatic heterocyclic ring substituted by 0 to 3 R c , and X and X'are selected In the case of -O-, at least one of ring A and ring A'is selected from sulfur-containing and nitrogen-containing 5-membered aromatic heterocycles.
  • the compound of Formula I, A ring, A 'ring each independently is preferably from 0 to 3 substituents R c pyrrole, substituted with 0 to 3 substituents R c pyrazole, substituted with 0 to 3 R c substituted imidazole, substituted with 0 to 3 R c substituted thiazole, substituted with 0-3 R c isothiazole, substituted with 0 to 3 R a substituted oxazole, substituted with 0 to 3, a substituted isoxazole R c, and X, X 'is selected when -O-, a ring, a' ring has at least one group selected from substituted 0-3 R c thiazole, substituted with 0 to 3 R c Substituted isothiazole.
  • the present invention also provides the use of the above-mentioned compound, or its stereoisomer, or its pharmaceutically acceptable salt in the preparation of activating STING drugs.
  • the present invention also provides the use of the above-mentioned compound, or its stereoisomer, or its pharmaceutically acceptable salt in the preparation of a medicine for treating diseases related to STING activity.
  • the diseases related to STING activity are one or more of diseases related to inflammatory, autoimmune diseases, infectious diseases, cancer, and precancerous syndromes.
  • the present invention also provides the use of the above-mentioned compound, its stereoisomer, or its pharmaceutically acceptable salt in the preparation of a medicine for the treatment of inflammatory, autoimmune diseases, infectious diseases, cancer or precancerous syndrome .
  • the present invention also provides the use of the above-mentioned compound, or its stereoisomer, or its pharmaceutically acceptable salt in the preparation of an immune adjuvant.
  • the present invention also provides a medicament, which is a preparation prepared from the above-mentioned compound, or its stereoisomer, or its pharmaceutically acceptable salt as the active ingredient, plus pharmaceutically acceptable auxiliary materials.
  • the diseases related to STING activity defined in the present invention are diseases in which STING plays an important role in the pathogenesis of the disease.
  • Diseases related to STING activity include inflammatory, allergic and autoimmune diseases, infectious diseases, cancer, and precancerous syndromes.
  • Cancer or “malignant tumor” refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread to other locations locally or through the bloodstream and lymphatic system The body (i.e. metastasis) and any of many characteristic structural and/or molecular characteristics.
  • Cancer cells refer to cells that undergo the early, middle or late stages of tumor progression in multiple steps. Cancers include sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer and prostate cancer.
  • the compound of formula I is used to treat a cancer selected from colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous cell carcinoma.
  • the cancer is selected from melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer.
  • the cancer being treated is a metastatic cancer.
  • Inflammatory diseases include a variety of conditions characterized by histopathological inflammation.
  • inflammatory diseases include acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, vasculitis, Airway inflammation and interstitial cystitis caused by house dust mites.
  • Some embodiments of the present invention relate to the treatment of the inflammatory disease asthma.
  • the immune system is usually involved in inflammatory diseases, which are manifested in allergic reactions and some myopathy. Many immune system diseases lead to abnormal inflammation.
  • the compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) naming system.
  • substitution refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.
  • the minimum and maximum content of carbon atoms in a hydrocarbon group is indicated by a prefix.
  • the prefix ( Ca -C b ) alkyl indicates any alkyl group containing "a" to "b” carbon atoms.
  • (C 1 -C 6 )alkyl refers to an alkyl group containing 1 to 6 carbon atoms.
  • alkyl refers to a saturated hydrocarbon chain having a specified number of member atoms.
  • the C 1 to C 6 alkyl group refers to an alkyl group having 1 to 6 carbon atoms.
  • Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein.
  • Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) Base) and hexyl.
  • the alkyl group may also be part of another group, such as a C 1 to C 6 alkoxy group.
  • the C a -C b alkoxy group refers to a group obtained by connecting an alkyl group containing "a" to "b" carbon atoms to the corresponding oxygen atom.
  • C 2 -C 6 alkylene refers to a divalent saturated aliphatic hydrocarbon group having 2 to 6 carbon atoms.
  • Alkylene groups include branched and straight chain hydrocarbyl groups.
  • (C 2 -C 6 )alkylene is meant to include ethylene, propylene, 2-methylpropylene, dimethylethylene, pentylene and the like.
  • C 2 -C 6 alkenylene refers to a divalent aliphatic hydrocarbon group having 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds.
  • Alkenylene groups include branched and straight chain groups.
  • the carbon-carbon double bonds in the alkenylene group include cis double bonds and trans double bonds.
  • C 2 -C 6 alkynylene refers to a divalent aliphatic hydrocarbon group having 2 to 6 carbon atoms and containing one or more carbon-carbon triple bonds.
  • Alkynylene groups include branched and straight chain groups.
  • -C(O)NR a R b -NR a C(O)R b , -C(O)R a , -C(O)OR a , -C(O)R f ⁇ -C( O)- represents a carbonyl group formed by two atoms of carbon and oxygen connected by a double bond
  • halogen refers to a halogen group: fluorine, chlorine, bromine or iodine.
  • the immune adjuvant in the present invention refers to a substance that has the function of enhancing and regulating immunity in the aspects of anti-infection, anti-virus, anti-tumor, anti-allergic reaction, asthma, etc. It is mainly used for inflammation and self Adjuvant treatment of immune diseases, infectious diseases, cancer, and diseases related to precancerous syndrome.
  • pharmaceutically acceptable means that a certain carrier, carrier, diluent, excipient, and/or the formed salt is usually chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form, and physiologically Compatible with the receptor.
  • salts and “pharmaceutically acceptable salts” refer to the above-mentioned compounds or their stereoisomers, acid and/or basic salts formed with inorganic and/or organic acids and bases, and also include zwitterionic salts ( Inner salt), also includes quaternary ammonium salts, such as alkyl ammonium salts. These salts can be directly obtained in the final isolation and purification of the compound. It can also be obtained by mixing the above-mentioned compound, or its stereoisomers, with a certain amount of acid or base appropriately (for example, equivalent).
  • salts may form a precipitate in the solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by freeze-drying after reaction in an aqueous medium.
  • the salt in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, butane Acid salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • one or more compounds of the present invention may be used in combination with each other.
  • the compound of the present invention can be used in combination with any other active agent to prepare drugs or pharmaceutical compositions for regulating cell function or treating diseases. If a group of compounds are used, these compounds can be administered to the subject simultaneously, separately or sequentially.
  • the compound provided by the invention can effectively combine with STING, has a good STING protein agonistic function, shows a good inhibitory effect on various tumors, and can also activate the immune memory mechanism of mice and inhibit tumor re-stimulation. Therefore, the compounds of the present invention can be used as STING agonists and used to treat various related diseases.
  • the compounds of the present invention are used in the preparation of drugs for the treatment of diseases related to STING activity (especially drugs for the treatment of inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer or precancerous syndromes) and in the preparation of immune adjuvants It has very good application prospects and provides a new choice for clinical screening and/or preparation of drugs for diseases related to STING activity.
  • Figure 1 is a graph showing the inhibitory effect of compounds prepared in Examples of the present invention on CT26 tumor model.
  • Figure 2 is a graph showing the inhibitory effect of the compound prepared in the example of the present invention on the CT26 tumor re-excitation model, in which, Example 2 (4.5mg/kg), Example 2 (1.5mg/kg), Example 5 (1.5 mg/kg), Example 15 (3mg/kg), Example 15 (1mg/kg), Example 17 (1.5mg/kg), Example 29 (1mg/kg) the curves overlap.
  • the raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • NMR is measured with (Bruker AvanceIII 400 and Bruker Avance 300) nuclear magnetometer, and the solvent is deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD) ,
  • the internal standard is tetramethylsilane (TMS).
  • Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A) was used for HPLC measurement.
  • Gilson GX-281 reverse-phase preparative chromatography was used for reverse-phase preparative chromatography.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specifications for thin layer chromatography separation and purification products are 0.4mm ⁇ 0.5mm.
  • the known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from Anaiji Chemical, Chengdu Kelong Chemical, Shaoyuan Chemical Technology, Bailingwei Technology and other companies.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the hydrogenation reaction is usually evacuated, filled with hydrogen, and repeated three times.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature.
  • M is mole per liter.
  • Room temperature is the most suitable reaction temperature, ranging from 20°C to 30°C.
  • PE refers to petroleum ether
  • EA refers to ethyl acetate
  • DCM refers to dichloromethane
  • MeOH refers to methanol
  • DMF refers to N,N-dimethylformamide
  • DMSO refers to dimethyl sulfoxide
  • DMAP refers to 4-Dimethylaminopyridine
  • DIPEA refers to diisopropylethylamine
  • Boc refers to tert-butyloxycarbonyl
  • TFA refers to trifluoroacetic acid
  • DBU refers to 1,8-diazabicycloundec- 7-ene
  • HATU refers to 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.
  • 4-ethyl-2-methylthiazole-5-carboxylic acid (2g, 11.7mmol) was dispersed in dry DCM (40ml), and oxalyl chloride (1.9g, 15.1mmol) and Catalytic amount of DMF. After reacting for 1 hour at room temperature, the volatiles were removed by rotary evaporation under reduced pressure. DCM (20ml) was added to the crude product, and the solvent was removed by rotary evaporation to obtain 4-ethyl-2-methylthiazole-5-carboxylic acid chloride (2.2g, 100% yield), which was directly used in the next reaction.
  • Methyl 4-chloro-3-methoxy-5-nitrobenzoate (10g, 40.7mmol) was dispersed in anhydrous dichloromethane (100mL), and boron tribromide ( 40.8g, 162.8mmol), after dripping, slowly rise to room temperature and stir overnight. After the reaction was completed, methanol was slowly added dropwise to quench under an ice bath, and then it was spin-dried. Methanol (100 mL) and concentrated sulfuric acid (0.2 mL) were added thereto, and the reaction solution was heated to 75° C. and stirred overnight. After cooling, the solvent was removed by concentration under reduced pressure, and 150 mL of water was added. After ultrasonic dispersion, filtration, the solid was washed with water again, and the solid was dried to obtain methyl 4-chloro-3-hydroxy-5-nitrobenzoate (8.89g , 38.4mmol).
  • Step 2 Synthesis of 4-(3-(2-chloro-5-(methoxycarbonyl)-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester
  • the reaction solution was poured into water, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated.
  • Step 2 Synthesis of methyl 4-fluoro-3-((3-morpholinopropyl)thio)-5-nitrobenzoate
  • Step 1 Synthesis of (trans)-tert-butyl (4-(4-carbamoyl-2-methoxy-6-nitrophenyl)amino)n-but-2-enyl) carbamate
  • Step 2 Synthesis of (trans)-tert-butyl (4-((2amino-4carbamoyl-6methoxyphenyl)amino)n-but-2-enyl)carbamate
  • Step 3 (trans)-tert-butyl(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1hydro-pyrazole-5carboxamide)-7-methoxy Synthesis of -1H-benzimidazolyl) n-but2-alkenyl) carbamate
  • Step 4 (trans)-1-(4-aminon-but-2-enyl)-2-(1-ethyl-3-methyl-1hydro-pyrazole-5-carboxamido)-7 -Methoxy-1-hydro-benzimidazole-5-carboxamide
  • Step 5 (E)-1-(4-((4-carbamoyl-2-(3-morpholino)-6-nitrophenyl)amino)but-2-en-1-yl)- Synthesis of 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide
  • Step 6 (E)-1-(4-((2-amino-4-carbamoyl-6-(3-morpholino)phenyl)amino)but-2-en-1-yl)-2 Synthesis of -(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide
  • Step 7 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-formyl Amino)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinomethyl)-1H-benzo[ d] Synthesis of imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 2 (E)-3-amino-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-5-methoxybenzoic acid methyl ester synthesis
  • Step 3 (E)-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-methyl Amido)-7-methoxy-1H-methyl-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 4 (E)-1-(4-aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy Synthesis of -1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 5 (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1-(4-((4-(methoxycarbonyl)- Synthesis of 2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 6 (E)-1-(4-((2-amino-4-(methoxycarbonyl)-6-(3-morpholinopropoxy)phenyl)amino)but-2-ene- Synthesis of 1-yl)-2-(4-ethyl)-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 7 (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-3-methyl-1H-pyridine Azole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinomethyl)-1H-benzo[d]imidazol-1-yl)but-2-ene-1 -Yl)-7-methoxy-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 8 (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholino (Methyl)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7- Synthesis of methoxy-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 9 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide Yl)-7-(3-morpholino)-1H--benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-methoxy-1H-benzo[d ]Imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-2-(4-Ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(4-ethyl-2-methylthiazole-5- Carboxamido)-5-(methoxycarbonyl)-7-methyl-(3-morpholinomethyl)-1H-benzo[d]imidazol-1-yl)but-2-ene-1- Yl)-7-methoxy-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinomethyl)- 1H-Benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy Synthesis of -1H-benzo[d]imidazole-5-carboxylic acid
  • Step 3 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7 -(3-morpholinomethyl)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-methoxy-1H-benzo[d]imidazole- Synthesis of 2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 2 (E)-3-amino-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-5-(3-morpholinopropoxy Synthesis of methyl benzoate
  • Step 3 (E)-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-methyl Amido)-7-(3-morpholinomethyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 4 (E)-1-(4-Aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazol-5-carboxamido)-7-(3- Synthesis of morpholinopropoxy)-1H-benzo[d]]imidazole-5-carboxylic acid methyl ester
  • Step 5 (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-((4-(methoxycarbonyl)-2-(methylthio )-6-nitrophenyl)amino)but-2-ketoen-1-yl)-7-(3-morpholinomethyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester Synthesis
  • Step 7 (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-3-methyl-1H-pyridine (Azol-5-carboxamido)-5-(methoxycarbonyl)-7-(methylthio)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)- Synthesis of 7-(3-morpholinomethyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 8 (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)- 1H-Benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3- Synthesis of Morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 9 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide Yl)-7-(methylthio)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinomethyl)-1H-benzene Synthesis of and [d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5--carboxamide
  • Compound 5 is prepared by the same method as compound 4, only the ring-closure materials used in step 3 and step 7 are exchanged, that is, 1-ethyl-3-methyl-1H-pyrazole-5 is used in step 3. -Carbonyl isothiocyanate ring closure, step 7 uses 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate ring closure. The other steps were the same as the experiment method, and compound 5 (67 mg) was obtained.
  • the compound 6 was prepared by the same method as the synthesis of compound 2.
  • the methyl 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzoate used in the synthesis of compound 2 was replaced with 4-fluoro- 3-((3-morpholinopropyl)thio)-5-nitrobenzoic acid methyl ester, other methods are the same.
  • Step 1 (E)-1-(4-((2-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-4-(methoxycarbonyl)- 6-nitrophenyl)amino)but-2-ketoen-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy Synthesis of 1-H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-1-(4-((2-amino-6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-4-(methoxy Carbonyl)phenyl)amino)but-2 (methylen-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy Synthesis of 1-H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 3 (E)-7-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-2-(4-ethyl-2-methylthiazole-5- Carboxamido)-1-(4-(2)-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-5-(methoxycarbonyl )-1H-Benzo[d]imidazole-1-yl)but-2-en-1-ylmethyl)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 4 (E)-7-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-1-(4-(5-carboxy-2-(1-ethyl 3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2 Synthesis of -(4-Ethyl-2-methylthiazole-5-carboxamido)-1H--benzo[d]imidazole-5-carboxylic acid
  • Step 5 (E)-4-(3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole- 5-carboxamido)-7-tert-butyl methoxy-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2 -Methylthiazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl)piperazine-1-carboxylic acid tert-butyl ester
  • Step 6 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-formyl Amino)-7-methoxy-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy) Synthesis of -1H-Benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 7 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-formyl Amino)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(4-methylpiperazin-1-yl) Synthesis of propoxy)-1H-benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(((2-methoxy-4-(methoxycarbonyl )-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester Synthesis
  • Step 2 (E)-Methyl 1-(4-(((2-amino-6-methoxy-4-(methoxycarbonyl)phenyl)amino)but-2-en-1-yl) -2-(4-Ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester Synthesis
  • Step 3 (E)-Methyl 2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3- Methyl-1H-pyrazole-5-carboxamido)-7-methoxy-5-(methoxycarbonyl)-1H-benzo[d]imidazol-1-yl)but-2-ene-1 -Yl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 4 (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy -1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-( Synthesis of 3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 5 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole- 5-carboxamido)-7-methoxy-1H)-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy) Synthesis of -1H-Benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(4-ethyl-2-methylthiazol-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3- Methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl) Synthesis of but-2-en-1-yl)-7-methoxy-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3 -Morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-methyl Amido)-7-methoxy-1H-benzo[d]imidazole-5-carboxylic acid synthesis
  • Step 3 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole- 5-carboxamido))-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-methoxy Synthesis of -1H-Benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(4-ethyl-2-methylthiazol-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3- Methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(methylthio)-1H-benzo[d]imidazol-1-yl)but-2-ene- Synthesis of 1-yl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole 5-carboxylate
  • Step 2 (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(methyl Thio)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7 Synthesis of -(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 3 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole- 5-carboxamido))-7-(methylthio)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy) Yl)-1H-benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(4-ethyl-2-methylthiazole- 5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl )-7-(Methylthio)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy) -1H-Benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(form Synthesis of thio)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 3 (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy) -1H-Benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(form Synthesis of thio)-1H-benzo[d]imidazole-5-carboxamide
  • Step 1 (E)-Methyl 1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl Synthesis of methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylate
  • Step 2 (E)-Methyl 1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5- Synthesis of Methyl Carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylate
  • Step 3 (E)-Methyl 2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-((4-(methoxy Carbonyl)-2-(methylthio)-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benzo(d ] Synthesis of imidazole-5-carboxylic acid methyl ester
  • Step 5 (E)-Methyl 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl-4- Fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole- Synthesis of 1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 6 (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)- 1H-Benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide Yl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 7 (E)-1-(4-(5-(5-carbamoyl-2-)(1-styrene-3-methyl-1H-pyrazole-5-carboxamido)-7- (Methylthio)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(1-styrene-4-fluoro-3-methyl-1H-pyridine Synthesis of oxazole-5-anthralamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(4-ethyl-2-methylthiazol-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3- Methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)butyl Synthesis of 2-en-1-yl)-7-(methylthio)-1H-benzo[d]imidazole 5-carboxylate
  • Step 2 (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7- (3 -Morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-methyl Synthesis of Amido)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 3 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole- 5-carboxamido))-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(methylsulfide Yl)-1H-benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 1 (E)-Methyl 2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl 4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d ]Imidazol-1-yl)-2-en-1-yl)-7-(methylthio))-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3 -Morpholinopropoxy)-1H-benzo(d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl- Synthesis of 1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 3 (E)-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7- (3-morpholinopropoxy)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl
  • yl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxamide 53b (100mg, 0.111mmol) was dissolved in DMF (5mL ), HATU (102 mg, 0.266 mmol) and DIPEA (88 mg, 0.665 mmol) were added. After 0.5h, ammonium bicarbonate (27mg, 0.333mmol) was added and stirred at room temperature for 2h. The reaction solution was concentrated and purified by preparative HPLC to obtain compound 53 (41 mg,
  • Step 1 (E)-Methyl 1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H- Synthesis of pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 2 (E)-Methyl 1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido) Synthesis of -7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 3 (E)-Methyl 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-((4-(methoxycarbonyl)-2 -(Methylthio)-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5 -Synthesis of methyl carboxylate
  • Step 4 (E)-Methyl 1-(4-(((2-amino-4-(methoxycarbonyl)-6-(methylthio)phenyl)amino)but-2-en-1-yl )-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5 -Synthesis of methyl carboxylate
  • Step 5 (E)-Methyl 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl-3- Methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)butyl Synthesis of 2-en-1-yl)-7-(methylthio)-1H-benzo[d]imidazole 5-carboxylate
  • Step 6 (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholino Propoxy)-1H-benzo(d)imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxy Synthesis of Amido)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxylic acid
  • Step 7 (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-? (Pholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5 -Carboxamido)-7-(methylthio)-1H-benzo[d]imidazole-5-carboxamide
  • Step 1 (E)-1-(4-((2-(3-(((tert-butyldimethylsilyl)oxy)propoxy)-4-carbamoyl-6-nitro Phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzene Synthesis of [d]imidazole-5-carboxamide
  • Step 2 (E)-1-(4-((2-amino-6-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-carbamoylphenyl )Amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[ d] Synthesis of imidazole-5-carboxamide
  • Step 3 (E)-N-(7-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-carbamoyl-1-(4-(5-amino Formyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)butan-2 -En-1-yl)-1H-benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 4 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide (Yl)-7)-methoxy-1H-benzo[d](imidazol-1-yl)but-2-en-1-yl)-7-(3-hydroxypropoxy)-1H-benzo Synthesis of [d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • Step 2 Synthesis of tert-butyl (E)-(4-((4-carbamoyl-5-fluoro-2-methoxyphenyl)amino)but-2-en-1-yl)carbamate
  • Step 5 (E)-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-4-fluoro-7-methoxy Of tert-butyl -1H-benzo[d]imidazol-1-yl)-2-en-1-yl)carbamate
  • Step 6 (E)-1-(4-Aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy Synthesis of -1H-benzo[d]imidazole-5-carboxylic acid methyl ester
  • Step 7 (E)-1-(4-Aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-4 Synthesis of -fluoro-7-methoxy-1H-benzo[d]imidazole-5-carboxamide
  • Step 8 (E)-1-(4-(((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-ene-1- Yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-4-fluoro-7-methoxy-1H-benzo[d]imidazole-5-carboxamide Synthesis
  • Step 9 (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide Yl)-4-fluoro-7-methoxy-1H)-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy) Synthesis of -1H-Benzo[d]imidazol-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide
  • the protein thermal transfer test was used to determine the binding affinity between the compound and the Sting protein.
  • Mix 5X SYPRO Orange dyes measure the dissolution curve of the protein on a qPCR instrument, fit the Tm value with the Protein Thermal Shift Software 1.3 software, calculate the difference between the Tm of the protein when different concentrations of compound and no compound are added, according to ⁇ Tm as the compound concentration
  • the change fitting obtains the dissociation constant Kd.
  • the compound 3 reported in the article (Nature. 2018Dec; 564(7736):439-443) was used as a positive reference compound.
  • the test results are shown in Table 6 below, and the Kd values of each compound are classified according to the following instructions:
  • the function of sting agonist was evaluated by detecting the changes of IFN- ⁇ and CXCL10 (IP10) cytokines produced by THP1 cells (Shanghai Cell Bank) stimulated by the compounds.
  • the ELISA plate was coated according to the IFN- ⁇ (R&D, #DY814-05) and IP10 (BD, #550926) ELISA detection kit instructions.
  • EC 50 is the concentration for 50% of maximal effect (EC50), which refers to the concentration of the drug that can cause 50% of the individual to be effective.
  • Example IFN- ⁇ EC 50 IP10 EC 50
  • the above experimental data show that the compound of the present invention has a good stimulating activity of IFN- ⁇ and CXCL10 (IP10) cytokines produced by THP1 cells, and has a good STING protein agonistic function. Especially for compounds 2, 4, 5, 15, 19, 23, 35, 40, 49, 50, 51, 52, 53, etc., compared with the reference compound, the compound of the present invention has comparable or even higher stimulation of immune cells Produce cytokine activity.
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 CT26 cells on the back. After inoculation, tumor-bearing mice with tumor volume in the range of 200-300mm3 were selected for experiments. According to the random method of tumor volume, they were divided into 21 groups with 3 mice in each group. Different doses of drugs (compounds of the present invention) were given via tail vein injection, and the control group was given a control vehicle (5% DMSO, 40% PEG400 formulated in physiological saline). Three hours after administration, blood was collected from the mouse orbital vein, and EDTA ⁇ 2K was used as an anticoagulant. Centrifuge the collected mouse whole blood at 8000g for 5 minutes and then collect the upper plasma for testing.
  • drugs compounds of the present invention
  • mouse tumor tissues were taken, weighed and homogenized with 9 times volume of PBS. After centrifuging the homogenate at 10,000 rpm for 15 minutes, the supernatant was collected for testing. The detection was carried out according to the IFN- ⁇ detection kit (R&D, #DY814-05), the OD450 value was read, and the IFN- ⁇ concentration was converted according to the standard curve.
  • mice can induce the release of IFN- ⁇ downstream of the STING pathway in blood and tumors.
  • the intratumor IFN- ⁇ level is more than 3 times that of the plasma IFN- ⁇ level, indicating that cytokines are relatively enriched in tumor tissues, and blood cytokines are lower, suggesting a higher treatment window.
  • mice After Balb/c mice rested for one week, CT26 cells were inoculated subcutaneously on the back, and each mouse was inoculated with 100uL cell suspension containing 5 ⁇ 10 5 cells. When the average tumor volume grows to about 120mm3, they are randomly grouped according to the tumor size, with 5 in each group. The mice in each group were administered intravenously through the tail vein on the 1, 4, and 8 days after grouping, and the control group was given a control vehicle (5% DMSO, 40% PEG400 formulated in physiological saline).
  • 5% DMSO, 40% PEG400 formulated in physiological saline 5% DMSO, 40% PEG400 formulated in physiological saline.
  • the length and short diameter of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was drawn according to the tumor volume.
  • Kunming rats were inoculated with H22 cells subcutaneously on their backs after a week of resting. Each mouse was inoculated with 100uL cell suspension containing 5 ⁇ 10 6 cells. When the average tumor volume grows to about 200mm3, they are randomly grouped according to the tumor size, with 7 in each group. Respectively on the 1, 4, and 8 days after grouping, the mice in the high, medium and low dose groups were administered intravenously through the tail vein of the mice (compounds of the present invention), or on the 1, 8, and 15 days after the high dose group was administered through the tail vein of mice , The control group was given a control vehicle (5% DMSO, 40% PEG400 formulated in physiological saline).
  • the length and short diameter of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was drawn according to the tumor volume.
  • the compounds of the present invention can effectively inhibit tumor growth, and exhibit a dose-dependent tumor growth inhibitory effect.
  • Example 6 Tumor suppressive effect of the compound of the present invention (tumor re-excitation model)
  • CT26 cells were inoculated subcutaneously on the back. Each mouse was inoculated with 100uL containing 5 ⁇ 10 5 cells. Cell suspension; mice that have not been treated with drugs are used as a control group to inoculate CT26 cells in the same way. Observe the tumor and record the growth of the tumor.
  • the present invention discloses a compound represented by formula I, which can effectively bind to STING, has a good STING protein agonistic function, has a good inhibitory effect on a variety of tumors, and can also activate immune memory in mice. Mechanism to inhibit tumor re-stimulation. Therefore, the compounds of the present invention can be used as STING agonists and used to treat various related diseases.
  • the compounds of the present invention are used in the preparation of drugs for the treatment of diseases related to STING activity (especially drugs for the treatment of inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer or precancerous syndromes) and in the preparation of immune adjuvants It has very good application prospects and provides a new choice for clinical screening and/or preparation of drugs for diseases related to STING activity.

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Abstract

本发明公开了一种免疫调节剂，具体公开了式(I)所示的化合物、或其立体异构体、或其药学上可接受的盐。本发明提供的化合物能够与STING有效结合，具有良好的STING蛋白激动功能，对多种肿瘤表现出良好的抑制效果。所以，本发明的化合物可作为STING激动剂并用于治疗各种相关病症。本发明化合物在制备治疗与STING活性相关疾病的药物(特别是治疗炎性疾病、变应性疾病、自身免疫性疾病、感染性疾病、癌症或癌前期综合征的药物)以及制备免疫佐剂中具有非常好的应用前景，为临床上筛选和/或制备与STING活性相关的疾病的药物提供了一种新的选择。

Description

一种免疫调节剂 技术领域
本发明涉及一种免疫调节剂，具体涉及一类激活STING的化合物及其作为免疫调节剂在制备药物中的用途。
背景技术
人体的免疫系统通常可分为“天然免疫”和“适应免疫”系统。天然免疫系统在抵抗感染、抑制肿瘤生长以及自身免疫疾病的发病过程中起着重要作用，主要通过模式识别受体识别病原微生物和癌细胞组分，启动下游信号通路，最终通过诱导细胞因子表达，杀灭病原微生物和癌细胞组分，以及适应免疫系统促进抗体和特异性T淋巴细胞生成。
STING(干扰素基因刺激因子，TMEM173，MITA等)是胞内应答DNA入侵的关键节点分子，在胞质DNA刺激下，识别胞质DNA受体的信号，对诱导产生干扰素的过程起关键作用。宿主细胞的DNA识别受体识别外源或内源“非己”DNA后，将信号传递给节点分子STING，然后STING迅速二聚化并从内质网转移至核外周小体上。STING的活化导致IRF3和NKκB途径的上调，从而导致干扰素-β和其它细胞因子的诱导。
诱导人干扰素的化合物可用于治疗各种病症(包括过敏性疾病和其它炎性病症、过敏性鼻炎和哮喘、感染性疾病、神经退行性疾病、癌前期综合症和癌症)，也可以用作免疫组合物或疫苗佐剂。所以，研究出能够激活STING的新化合物是治疗有关1型IFN途径疾病(包括炎性、变应性和自身免疫性疾病、感染性疾病、癌症、癌前期综合征)的有效方法。
发明内容
为了解决上述问题，本发明提供了一种免疫调节剂。
本发明提供了式I所示的化合物、或其立体异构体、或其药学上可接受的盐：
Figure PCTCN2020073405-appb-000001
其中，
L选自C 2～C 6亚烷基、C 2～C 6亚烯基、C 2～C 6亚炔基；
R 1、R 3、R 1’、R 3’分别独立选自氢、卤素、C 1～C 6烷基、氰基、硝基、羟基、氨基、C 1～C 6烷氧基、C 1～C 6烷胺基、卤素取代的C 1～C 6烷基；
R 2、R 2’分别独立选自氢、-C(O)NR aR b、-NR aC(O)R b、-C(O)R a、-C(O)OR a、卤素、C 1～C 6烷基、氰基、硝基、羟基、氨基、C 1～C 6烷氧基、C 1～C 6烷胺基、卤素取代的C 1～C 6烷基；
R 4、R 4’分别独立选自氢、C 1～C 6烷基；
X、X’分别独立选自-CH 2-、-NH-、-O-、-S-；
A环、A’环分别独立选自被0～4个R c任选取代的5～6元芳杂环；
且X、X’选自-CH 2-、-NH-、-O-时，A环、A’环至少有一个选自被0～4个R c任选取代的含硫的5～6元芳杂环或含氧的5～6元芳杂环；
R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
R a、R b分别独立选自氢、C 1～C 6烷基；
R c分别独立选自卤素、-CN、-OR a、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
R d分别独立选自卤素、-OR a、-NR aR b、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基、被0～4个R e任选取代的7～10元环烷基、被0～4个R e任选取代的7～10元杂环烷基、被0～4个R e任选取代的7～10元螺环烷基、被0～4个R e任选取代的7～10元螺杂环烷基、被0～4个R e任选取代的5～10元桥环烷基、被0～4个R e任选取代的5～10元桥杂环烷基、被0～4个R e任选取代的7～10元稠环烷基、被0～4个R e任选取代的7～10元稠杂环烷基；
R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
进一步地，所述化合物的结构如式I所示：
Figure PCTCN2020073405-appb-000002
其中，
L选自C 2～C 6亚烷基、C 2～C 6亚烯基、C 2～C 6亚炔基；
R 1、R 3、R 1’、R 3’分别独立选自氢、卤素、C 1～C 6烷基；
R 2、R 2’分别独立选自氢、-C(O)NR aR b；
R 4、R 4’分别独立选自氢、C 1～C 6烷基；
X、X’分别独立选自-CH 2-、-NH-、-O-、-S-；
A环、A’环分别独立选自被0～4个R c任选取代的5～6元芳杂环；
且X、X’选自-CH 2-、-NH-、-O-时，A环、A’环至少有一个选自被0～4个R c任选取代的含硫的5～6元芳杂环或含氧的5～6元芳杂环；
R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
R a、R b分别独立选自氢、C 1～C 6烷基；
R c分别独立选自卤素、-CN、-OR a、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
R d分别独立选自卤素、-OR a、-NR aR b、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基；
R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
进一步地，R 1、R 3、R 1’、R 3’分别独立选自氢；
R 2、R 2’分别独立选自-C(O)NR aR b；
R 4、R 4’分别独立选自氢；
X、X’分别独立选自-O-、-S-；
A环、A’环分别独立选自被0～3个R c任选取代的5元芳杂环；
且X、X’选自-O-时，A环、A’环至少有一个选自被0～3个R c任选取代的含硫的5元芳杂环；
R 5、R 5’分别独立选自被0～1个R d任选取代的C 1～C 6烷基；
R c分别独立选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
R d分别独立选自被0～2个R e任选取代的3～6元环烷基、被0～2个R e任选取代的3～6元杂环烷基；
R e分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
进一步地，X、X’至少有一个选自-S-，A环、A’环分别独立选自被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000003
或
Figure PCTCN2020073405-appb-000004
R 5、R 5’分别独立选自C 1～C 6烷基、
Figure PCTCN2020073405-appb-000005
Figure PCTCN2020073405-appb-000006
或
Figure PCTCN2020073405-appb-000007
R c’选自C 1～C 6烷基、卤素取代的C 1～C 6烷基；R c选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
R e’分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基，R a选自氢、C 1～C 6烷基。
进一步地，X、X’选自-O-，A环、A’环至少有一个选自被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000008
R 5、R 5’分别独立选自C 1～C 6烷基、
Figure PCTCN2020073405-appb-000009
或
Figure PCTCN2020073405-appb-000010
R c选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
R e’分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基，R a选自氢、C 1～C 6烷基。
进一步地，所述化合物的结构如式II所示：
Figure PCTCN2020073405-appb-000011
其中，X、X’分别独立选自-O-、-S-；
A环、A’环分别独立选自被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000012
或
Figure PCTCN2020073405-appb-000013
且当X、X’均为-O-时，A环、A’环至少有一个选自被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000014
R c’选自C 1～C 2烷基、卤素取代的C 1～C 2烷基；
R c选自卤素、C 1～C 2烷基、卤素取代的C 1～C 2烷基；所述卤素优选为F；
R 5、R 5’分别独立选自C 1～C 3烷基、
Figure PCTCN2020073405-appb-000015
或
Figure PCTCN2020073405-appb-000016
R e’选自C 1～C 3烷基、3～6元环烷基、-C(O)R f；
R f选自-OR a’、C 1～C 3烷基、3～4元环烷基，R a’选自C 1～C 3烷基。
进一步地，X选自-O-、-S-，X’选自-O-；
R 5选自C 1～C 3烷基；
R 5’选自
Figure PCTCN2020073405-appb-000017
或
Figure PCTCN2020073405-appb-000018
R e’选自C 1～C 3烷基、3～6元环烷基、-C(O)R f；
R f选自-OR a’、C 1～C 3烷基、3～4元环烷基，R a’选自C 1～C 3烷基。
进一步地，所述被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000019
为
Figure PCTCN2020073405-appb-000020
或
Figure PCTCN2020073405-appb-000021
所述被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000022
为
Figure PCTCN2020073405-appb-000023
进一步地，所述化合物为：
Figure PCTCN2020073405-appb-000024
Figure PCTCN2020073405-appb-000025
Figure PCTCN2020073405-appb-000026
Figure PCTCN2020073405-appb-000027
Figure PCTCN2020073405-appb-000028
Figure PCTCN2020073405-appb-000029
Figure PCTCN2020073405-appb-000030
Figure PCTCN2020073405-appb-000031
Figure PCTCN2020073405-appb-000032
进一步地，所述化合物中，R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
R d分别独立选自卤素、-OH、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基、被0～4个R e任选取代的7～10元环烷基、被0～4个R e任选取代的7～10元杂环烷基、被0～4个R e任选取代的7～10元螺环烷基、被0～4个R e任选取代的7～10元螺杂环烷基、被0～4个R e任选取代的5～10元桥环烷基、被0～4个R e任选取代的5～10元桥杂环烷基；
R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
进一步地，所述化合物如式III所示：
Figure PCTCN2020073405-appb-000033
其中，R 1、R 3、R 1’、R 3’分别独立选自氢、卤素；
A环、A’环分别独立选自被0～2个R c任选取代的
Figure PCTCN2020073405-appb-000034
或
Figure PCTCN2020073405-appb-000035
R c’选自C 1～C 2烷基、卤素取代的C 1～C 2烷基；
R c选自卤素、C 1～C 2烷基、卤素取代的C 1～C 2烷基；
R 5’选自被0～2个R d任选取代的C 1～C 3烷基；
R d分别独立选自-OH、被0～2个R e任选取代的3～6元环烷基、被0～2个R e任选取代的3～6元杂环烷基、被0～2个R e任选取代的7～10元环烷基、被0～2个R e任选取代的7～10元杂环烷基、被0～2个R e任选取代的7～10元螺环烷基、 被0～2个R e任选取代的7～10元螺杂环烷基、被0～2个R e任选取代的5～10元桥环烷基、被0～2个R e任选取代的5～10元桥杂环烷基、被0～2个R e任选取代的7～10元稠环烷基、被0～2个R e任选取代的7～10元稠杂环烷基；R e分别独立选自卤素、C 1～C 3烷基。
进一步地，A环、A’环分别独立选自
Figure PCTCN2020073405-appb-000036
R 5’选自被0～2个R d任选取代的C 1～C 3烷基；
R d分别独立选自-OH、
Figure PCTCN2020073405-appb-000037
Figure PCTCN2020073405-appb-000038
进一步地，所述化合物为：
Figure PCTCN2020073405-appb-000039
Figure PCTCN2020073405-appb-000040
在本发明的一些实施方案中，对于式I所示的化合物，A环、A’环分别独立优选为被0～3个R c取代的5元含氮芳杂环，且X、X’选自-O-时，A环、A’环至少有一个选自含硫且含氮的5元芳杂环。
在本发明的一些实施方案中，对于式I所示的化合物，A环、A’环分别独立优选为被0～3个R c取代的吡咯、被0～3个R c取代的吡唑、被0～3个R c取代的咪唑、被0～3个R c取代的噻唑、被0～3个R c取代的异噻唑、被0～3个R a取代的恶唑、被0～3个R c取代的异恶唑，且X、X’选自-O-时，A环、A’环至少有一个选自被0～3个R c取代的噻唑、被0～3个R c取代的异噻唑。
本发明还提供了上述的化合物、或其立体异构体、或其药学上可接受的盐在 制备激活STING类药物中的用途。
本发明还提供了上述的化合物、或其立体异构体、或其药学上可接受的盐在制备治疗与STING活性相关的疾病的药物中的用途。
进一步地，所述与STING活性相关的疾病是与炎性、自身免疫性疾病、感染性疾病、癌症、癌前期综合征相关的疾病中的一种或几种。
本发明还提供了上述的化合物、或其立体异构体、或其药学上可接受的盐在制备治疗炎性、自身免疫性疾病、感染性疾病、癌症或癌前期综合征的药物中的用途。
本发明还提供了上述的化合物、或其立体异构体、或其药学上可接受的盐在制备免疫佐剂中的用途。
本发明还提供了一种药物，它是以上述的化合物、或其立体异构体、或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
本发明所定义的STING活性相关的疾病是STING在该疾病的病理发生中起重要作用的疾病。
STING活性相关的疾病包括炎性、变应性和自身免疫性疾病、感染性疾病、癌症、癌前期综合征。
“癌症”或“恶性肿瘤”是指以不受控制的细胞异常增殖为特征的多种疾病中的任何一种，受影响的细胞在局部或通过血流和淋巴系统扩散到其他部位的能力的身体(即转移)以及许多特征结构和/或分子特征中的任何一个。“癌细胞”是指经历多步骤肿瘤进展的早期，中期或晚期阶段的细胞。癌症包括肉瘤、乳腺癌、肺癌、脑癌、骨癌、肝癌、肾癌、结肠癌和前列腺癌。在一些实施方案中，式I的化合物用于治疗选自结肠癌、脑癌、乳腺癌、纤维肉瘤和鳞状细胞癌的癌症。在一些实施方案中，癌症选自黑素瘤、乳腺癌、结肠癌、肺癌和卵巢癌。在一些实施方案中，所治疗的癌症是转移性癌症。
炎性疾病包括以组织病理性炎症为特征的多种病症。炎性疾病的例子包括寻常性痤疮、哮喘、腹腔疾病、慢性前列腺炎、肾小球性肾炎、炎症性肠病、盆腔炎、再灌注损伤、类风湿性关节炎、结节病、血管炎、房尘螨引起的气道炎症和间质性膀胱炎。炎性疾病与自身免疫性疾病之间存在显著重叠。本发明的一些实施方案涉及炎性疾病哮喘的治疗。免疫系统通常涉及炎症性疾病，在过敏反应和一些肌病中都有表现，许多免疫系统疾病导致异常炎症。
本发明中提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或CAS(化学文摘服务社，Columbus，OH)命名系统命名。
关于本发明的使用术语的定义：除非另有说明，本文中基团或者术语提供的初始定义适用于整篇说明书的该基团或者术语；对于本文没有具体定义的术语，应该根据公开内容和上下文，给出本领域技术人员能够给予它们的含义。
“取代”是指分子中的氢原子被其它不同的原子或分子所替换。
碳氢基团中碳原子含量的最小值和最大值通过前缀表示，例如，前缀(C a～C b)烷基表明任何含“a”至“b”个碳原子的烷基。因此，例如，(C 1～C 6)烷基是指 包含1～6个碳原子的烷基。
本发明中“烷基”是指具有指定数目的成员原子的饱和烃链。例如，C 1～C 6烷基是指具有1至6个碳原子的烷基。烷基基团可以是直链或支链的。代表性的支链烷基基团具有一个、两个或三个支链。烷基基团可任选地被一个或多个如本文所定义的取代基取代。烷基包括甲基、乙基、丙基(正丙基和异丙基)、丁基(正丁基、异丁基和叔丁基)、戊基(正戊基、异戊基和新戊基)和己基。烷基基团也可以是其他基团的一部分，所述其他基团为例如C 1～C 6烷氧基。
本发明中C a～C b烷氧基指含有“a”至“b”个碳原子的烷基与对应的氧原子相连得到的基团。
本发明中“C 2～C 6亚烷基”指具有2至6个碳原子的二价饱和脂族烃基。亚烷基基团包括支链和直链烃基基团。例如，“(C 2～C 6)亚烷基”意在包括亚乙基、亚丙基、2-甲基亚丙基、二甲基亚乙基、亚戊基等。
同样的，“C 2～C 6亚烯基”指具有2～6个碳原子的、含一个或多个碳碳双键的二价脂族烃基。亚烯基基团包括支链和直链基团。亚烯基基团中的碳碳双键包括顺式双键和反式双键。
同样的，“C 2～C 6亚炔基”指具有2～6个碳原子的、含一个或多个碳碳三键的二价脂族烃基。亚炔基基团包括支链和直链基团。
本发明中-C(O)NR aR b、-NR aC(O)R b、-C(O)R a、-C(O)OR a、-C(O)R f的-C(O)-表示由碳和氧两种原子通过双键连接而成的羰基
Figure PCTCN2020073405-appb-000041
本发明中“卤素”是指卤素基团：氟、氯、溴或碘。
本发明中免疫佐剂即免疫调节剂，是指在抗感染、抗病毒、抗肿瘤、抗变态反应、哮喘等方面具有增强及调节免疫的功能的物质，在临床上主要用于炎性、自身免疫性疾病、感染性疾病、癌症、癌前期综合征相关疾病的辅助治疗。术语“药学上可接受的”是指某载体、运载物、稀释剂、辅料，和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容。
术语“盐”和“药学上可接受的盐”是指上述化合物或其立体异构体，与无机和/或有机酸和碱形成的酸式和/或碱式盐，也包括两性离子盐(内盐)，还包括季铵盐，例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物，或其立体异构体，与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集，或在溶剂蒸发后回收而得到，或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。
在某些实施方式中，本发明的一种或多种化合物可以彼此联合使用。也可选择将本发明的化合物与任何其它的活性试剂结合使用，用于制备调控细胞功能或 治疗疾病的药物或药物组合物。如果使用的是一组化合物，则可将这些化合物同时、分别或有序地对受试对象进行给药。
本发明提供的化合物能够与STING有效结合，具有良好的STING蛋白激动功能，对多种肿瘤表现出良好的抑制效果，而且还能激活小鼠的免疫记忆机制，抑制肿瘤再激发。所以，本发明的化合物可作为STING激动剂并用于治疗各种相关病症。本发明化合物在制备治疗与STING活性相关疾病的药物(特别是治疗炎性疾病、变应性疾病、自身免疫性疾病、感染性疾病、癌症或癌前期综合征的药物)以及在制备免疫佐剂中具有非常好的应用前景，为临床上筛选和/或制备与STING活性相关的疾病的药物提供了一种新的选择。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明实施例制得的化合物对CT26肿瘤模型的抑制效果图。
图2为本发明实施例制得的化合物对CT26肿瘤肿瘤再激发模型的抑制效果图，其中，实施例2(4.5mg/kg)、实施例2(1.5mg/kg)、实施例5(1.5mg/kg)、实施例15(3mg/kg)、实施例15(1mg/kg)、实施例17(1.5mg/kg)、实施例29(1mg/kg)的曲线重合。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品，通过购买市售产品获得。
化合物的结构是通过核磁共振(NMR)或(和)质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的的单位给出。NMR的测定是用(Bruker AvanceIII 400和Bruker Avance 300)核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)，氘代氯仿(CDCl 3),氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
LC-MS的测定用岛津液质联用仪(Shimadzu LC-MS 2020(ESI))。
HPLC的测定使用岛津高压液相色谱仪(Shimadzu LC-20A)。
反相制备色谱使用Gilson GX-281反相制备色谱仪。
薄层层析硅胶板用烟台黄海HSGF254或青岛GF254硅胶板，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买于安耐吉化学、成都科龙化工、韶远化学科技、百灵威科技等公司。
氢气氛围是指反应瓶连接一个约1L容积的氢气球。
氢化反应通常抽真空，充入氢气，反复操作3次。
实施例中无特殊说明，反应在氮气氛围下进行。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温。
实施例中无特殊说明，M是摩尔每升。
室温为最适宜的反应温度，为20℃～30℃。
过夜为12±1h。
PE是指石油醚；EA是指乙酸乙酯；DCM是指二氯甲烷；MeOH是指甲醇；DMF是指N,N-二甲基甲酰胺；DMSO是指二甲基亚砜；DMAP是指4-二甲氨基吡啶；DIPEA是指二异丙基乙基胺；Boc是指叔丁基氧羰基；TFA是指三氟乙酸；DBU是指1,8-二氮杂二环十一碳-7-烯；HATU是指2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯。
以下为中间体化合物的制备：
1、中间体化合物：4-氯-3-甲氧基-5-硝基苯甲酰胺的合成
Figure PCTCN2020073405-appb-000042
将4-氯-3-甲氧基-5-硝基苯甲酯(18.5g，75.3mmol)加入到含有氨水(200mL)的单口瓶中，在60℃搅拌3h。反应液浓缩至100mL，冷却后过滤，固体用冰水洗涤，经干燥后得到4-氯-3-甲氧基-5-硝基苯甲酰胺(12.5g，54.1mmol)，褐色固体。
MS(ESI)m/z＝231[M+H] +,
1HNMR(400MHz,DMSO-d6):δ8.29(s,1H),8.04(d,1H),7.87(d,1H),7.78(s,1H),4.01(s,3H)。
2、中间体化合物：4-氯-3-羟基-5-硝基苯甲酰胺的合成
Figure PCTCN2020073405-appb-000043
在冰浴下，将4-氯-3-甲氧基-5-硝基苯甲酰胺(7.5g，32.5mmol)分散于干燥的DCM(90mL)中，再把三溴化硼(120mL，1M)慢慢滴加到其中。滴加完后撤掉冰浴，氮气保护，室温下反应过夜。反应完全后，反应液倒入冰水中，剧烈搅拌30min后，过滤，并用水洗涤滤饼，滤饼经干燥后得到4-氯-3-羟基-5-硝基苯甲酰胺(6.00g，27.7mmol，85.3％收率)，淡黄色固体。
MS(ESI)m/z＝217[M+H] +
1H NMR(400MHz,DMSO-d6):δ11.73(s,1H),8.21(s,1H),7.92(s,1H),7.80(s,1H),7.66(s,1H)。
3、中间体化合物：1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯的合成
Figure PCTCN2020073405-appb-000044
将1-乙基-3-甲基-1H-吡唑-5-羧酸(4.00g，25.9mmol)分散于干燥DCM(80mL)中，在冰浴下，向其中滴加草酰氯(3.9g，31.1mmol)和催化量的DMF。在室温下反应1h后，将可挥发物减压旋蒸除去。向粗品中加入DCM(20mL)，再旋蒸除去溶剂后得到1-乙基-3-甲基-1H-吡唑-5-碳酰氯(4.46g，100％收率)，直接用于下一步反应。
在0℃下，将1-乙基-3-甲基-1H-吡唑-5-碳酰氯(4.46g，25.9mmol)溶于干燥丙酮(20mL)并滴加到硫氰酸钾(5.0g，51.5mmol)的丙酮(100ml)溶液中，室温下搅拌3h，反应体系经过滤除去无机盐，滤液浓缩后的粗品经硅胶柱纯化(洗脱剂：乙酸乙酯/石油醚(v/v)＝1/15)得到1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(4.0g，20.4mmol，78.7％)，澄清的棕黄色液体。
MS(ESI)m/z＝196[M+H] +。
4、中间体化合物：4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯的合成：
Figure PCTCN2020073405-appb-000045
步骤1：4-乙基-2-甲基噻唑-5-甲酰氯的合成
将4-乙基-2-甲基噻唑-5-羧酸(2g，11.7mmol)分散于干燥DCM(40ml)中，在冰浴下，向其中滴加草酰氯(1.9g，15.1mmol)和催化量的DMF。在室温下反应1h后，将可挥发物减压旋蒸除去。向粗品中加入DCM(20ml)，再旋蒸除去溶剂后得到4-乙基-2-甲基噻唑-5-甲酰氯(2.2g，100％收率)，直接用于下一步反应。
步骤2：4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯的合成
在0℃下，将4-乙基-2-甲基噻唑-5-甲酰氯(2.2g，11.7mmol)溶于干燥丙酮(10ml)并滴加到硫氰酸钾(2.3g，23.4mmol)的丙酮(50ml)溶液中，室温下搅拌3h，反应体系经过滤除去无机盐，滤液浓缩后的粗品经硅胶柱纯化(洗脱剂乙酸乙酯/石油醚,v/v＝1/15)得到4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(2.15g，10.2mmol，收率87％)，澄清的棕黄色液体。
MS(ESI)m/z＝213[M+H] +。
5、中间体化合物：4-氯-3-(3-吗啉代)-5-硝基苯甲酰胺的合成
Figure PCTCN2020073405-appb-000046
向3-吗啉代丙-1-醇(1.0g，9.79mmol)的二氯甲烷(20mL)溶液中加入TEA(2.7mL，19.58mmol)和对甲苯磺酰氯(2.0g，10.77mmol)，室温下反 应2h，加水和二氯甲烷萃取，有机相分别用水、饱和食盐水洗涤，用无水硫酸钠干燥、过滤并浓缩后得到4-吗啉代丙基4-甲基苯磺酸酯，直接用于下一步反应。
向4-氯-3-羟基-5-硝基苯甲酰胺(845mg，3.9mmol)的DMF(10mL)溶液中加入碳酸钾(1.08g，7.8mmol)、碘化钾(0.12g，0.72mmol)和4-吗啉代丙基4-甲基苯磺酸酯(1.17g，3.9mmol)，在75℃下反应24h。反应完后过滤除掉无机盐，将滤液倒入水中并用乙酸乙酯萃取，有机相依次用水、饱和食盐水洗，再干燥浓缩后，得到4-氯-3-(3-吗啉代)-5-硝基苯甲酰胺(1.0g，收率75.5％)。
6、中间体化合物：4-(3-(2-氯-5-(甲氧基羰基)-3-硝基苯氧基)丙基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2020073405-appb-000047
步骤1：4-氯-3-羟基-5-硝基苯甲酸甲酯的合成
将4-氯-3-甲氧基-5-硝基苯甲酸甲酯(10g，40.7mmol)分散于无水二氯甲烷(100mL)中，在冰浴下慢慢滴加三溴化硼(40.8g,162.8mmol),滴加完后慢慢升至室温搅拌反应过夜。反应完全后，在冰浴下缓慢滴加甲醇淬灭，然后将其旋干。在向其中加入甲醇(100mL)，浓硫酸(0.2mL),反应液加热至75℃，搅拌过夜。冷却后，减压浓缩除掉溶剂，再加入150mL水，超声分散后，过滤，固体再用水洗涤，将固体干燥后得到4-氯-3-羟基-5-硝基苯甲酸甲酯(8.89g，38.4mmol)。
步骤2：4-(3-(2-氯-5-(甲氧基羰基)-3-硝基苯氧基)丙基)哌嗪-1-羧酸叔丁酯的合成
将4-氯-3-羟基-5-硝基苯甲酸甲酯(10g，47.2mmol)、4-(3-羟丙基)哌嗪-1-羧酸叔丁酯(10.6g，47.2mmol)和三苯基膦(20.4g，77.7mmol)溶于无水THF(200mL)中，氮气保护。在冰浴下，向上述反应液中滴加DIAD(15.8g，77.7mmol)，滴加完毕后反应液升至室温搅拌16h。反应液倒入水中，用乙酸乙酯萃取，有机相用饱和食盐水洗涤并用无水硫酸镁干燥后浓缩。得到的粗品经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝2/1)得到目标化合物(13g，收率61％)，淡黄色固体。
7、中间体化合物：4-(3-(2-氯-5-(甲氧基羰基)-3-硝基苯氧基)丙基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2020073405-appb-000048
将4-氯-3-羟基-5-硝基苯甲酸甲酯(36.8g，159.3mmol)溶于DMF(300mL) 中，再加入4-吗啉代丙基4-甲基苯磺酸酯(74g，247.5mmol)和K 2CO 3(44.5g，318.6mmol)。该反应液在75℃下搅拌16h，过滤出掉无机盐，DMF减压蒸馏除去DMF。将粗品溶于乙酸乙酯中，分别用水、饱和食盐水洗涤，无水硫酸镁干燥后，过滤浓缩至剩余乙酸乙酯(30mL)时，过滤得到目标化合物，黄色固体(48.7g，收率86％)。
8、中间体化合物：4-氟-3-(甲硫基)-5-硝基苯甲酸甲酯的合成
Figure PCTCN2020073405-appb-000049
步骤1：3-溴-4-氟-5-硝基苯甲酸的合成
将4-氟-3-硝基苯甲酸(50g，270mmol)分散于浓硫酸(200ml)中，再加入NBS(47.5g，270mmol)。升温至75℃，搅拌过夜。冷至室温后，慢慢倒入冰水中并搅拌，有淡黄色固体析出，过滤出固体并干燥后得到目标化合物(66g)。
步骤2：3-溴-4-氟-5-硝基苯甲酸甲酯的合成
在冰浴下，向3-溴-4-氟-5-硝基苯甲酸(66g，249mmol)的甲醇(400ml)溶液中滴加氯化亚砜(44.5g，373.5mmol)。升至75℃，搅拌过夜。浓缩至剩余溶剂约100ml，冷却后固体继续析出，过滤并干燥固体得到3-溴-4-氟-5-硝基苯甲酸甲酯(56g)。
步骤3：4-氟-3-(甲硫基)-5-硝基苯甲酸甲酯的合成
向3-溴-4-氟-5-硝基苯甲酸甲酯(80mg,0.14mmol)的甲苯()和叔丁醇(mL)溶液中依次加入甲硫醇乙酸酯(11mg，0.14mmol)，Pd 2(dba) 3(13mg,0.014mmol)，xantphos(8mg,0.014mmol)，K 3PO 4(36mg，0.166mmol)，氮气保护下110℃反应过夜，反应完成。冷至室温，将反应液浓缩，得到粗品用正相柱纯化(洗脱剂：PE/EA＝3/1，v/v)分离得到目标化合物化合物(g，收率45％)。
9、中间体化合物：4-氟-3-((3-吗啉代丙基)硫代)-5-硝基苯甲酸甲酯的合成
Figure PCTCN2020073405-appb-000050
步骤1：3-吗啉代丙基硫醇乙酸酯的合成
向4-吗啉代丙基4-甲基苯磺酸酯(9g，30.1mmol)的DMF(100mL)溶液中加入硫代乙酸钾(6.86g，60.2mmol)，碳酸钾(12.5g，90.3mmol)，室温搅拌过夜。过滤除掉无机盐旋干溶剂，粗品经硅胶柱纯化(洗脱剂：二氯甲烷/甲醇＝20/1)得到目标化合物(5g，收率81％)
步骤2：4-氟-3-((3-吗啉代丙基)硫代)-5-硝基苯甲酸甲酯的合成
向3-溴-4-氟-5-硝基苯甲酸甲酯(3.4g，12.3mmol)的甲苯(50mL)和叔丁 醇(5mL)溶液中依次加入3-吗啉代丙基硫醇乙酸酯(3g，14.8mmol)，Pd 2(dba) 3(1.13g，1.23mmol)，xantphos(0.72g，1.23mmol)，K 3PO 4(7.83g，36.9mmol)，氮气保护下110℃反应过夜，反应完成。冷至室温，将反应液浓缩，得到粗品用正相柱纯化(洗脱剂：PE/EA＝1/1，v/v)分离得到目标化合物化合物(1.8g，收率41％)。
10、中间体化合物：1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯的合成：
Figure PCTCN2020073405-appb-000051
步骤1：1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基氯的合成
将1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酸(2g，11.7mmol)分散于干燥DCM(40ml)中，在冰浴下，向其中滴加草酰氯(1.9g，15.1mmol)和催化量的DMF。在室温下反应1h后，将可挥发物减压旋蒸除去。向粗品中加入DCM(20ml)，再旋蒸除去溶剂后得到4-乙基-2-甲基噻唑-5-甲酰氯(2.2g，100％收率)，直接用于下一步反应。
步骤2：1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯的合成
在0℃下，将1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基氯(2.2g，11.7mmol)溶于干燥丙酮(10ml)并滴加到硫氰酸钾(2.3g，23.4mmol)的丙酮(50ml)溶液中，室温下搅拌3h，反应体系经过滤除去无机盐，滤液浓缩后的粗品经硅胶柱纯化(洗脱剂乙酸乙酯/石油醚,v/v＝1/15)得到4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(2.15g，10.2mmol，收率87％)，澄清的棕黄色液体。
MS(ESI)m/z＝214[M+H] +
以下为本发明化合物的制备：
实施例1
Figure PCTCN2020073405-appb-000052
Figure PCTCN2020073405-appb-000053
步骤1：(反式)-叔丁基(4-(4-氨基甲酰-2-甲氧基-6-硝基苯基)氨基)正丁-2-烯基)氨基甲酸酯的合成
室温下将4-氯-3-甲氧基-5-硝基苯酰胺(500mg，2.17mmol)和(4-氨基丁-2-烯-1-基)氨基甲酸叔丁酯(966mg，4.34mmol)溶解在二甲亚砜(10mL)中，三乙胺(1.1g，10.84mmol)滴入反应液。反应混合液升温至115℃，并在此温度下搅拌过夜。冷却至室温后，加水稀释，大量橙色固体析出，过滤，滤饼用水洗涤(15mL×3)，真空泵旋转蒸发仪干燥得到(反式)-叔丁基(4-(4-氨基甲酰-2-甲氧基-6-硝基苯基)氨基)正丁-2-烯基)氨基甲酸酯(化合物1b)(450mg，收率57％)，橙黄色固体。
MS(ESI)m/z＝381[M+H] +。
步骤2：(反式)-叔丁基(4-((2氨基-4氨基甲酰-6甲氧苯基)氨基)正丁-2-烯基)氨基甲酸酯的合成
化合物1b(450mg，1.18mmol)溶解于甲醇(15mL)，溶液冷却到0℃，然后依次加入氨水(1.62mL，11.80mmol)、连二亚硫酸钠(1.22g，7.01mmol)的水溶液(6mL)。反应混合液在0℃下搅拌1h，反应液颜色由橙红色变为白色。反应液旋除甲醇，然后加水稀释，加乙酸乙酯萃取(20mL×4)，分离得到的有机相用饱和食盐水洗(20mL×2)，无水硫酸钠干燥，然后旋干得到(反式)-叔丁基(4-((2氨基-4氨基甲酰-6甲氧苯基)氨基)正丁-2-烯基)氨基甲酸酯(化合物1c)(260mg，收率58％)，白色固体。
MS(ESI)m/z＝351[M+H] +。
步骤3：(反式)-叔丁基(4-(5-氨基甲酰-2-(1-乙基-3-甲基-1氢-吡唑-5甲酰胺)-7-甲氧基-1H-苯并咪唑基)正丁2-烯基)氨基甲酸酯的合成
化合物1c(260mg，0.742mmol)和化合物1-乙基-3-甲基-1H-吡唑-5-甲酰基异硫氰酸酯(145mg，0.74mmol)溶解在N,N-二甲基甲酰胺(5mL)中，并于室温下搅拌1h，然后HATU(338mg，0.89mmol)和1,8-二氮杂双环[5.4.0]十一碳-7-烯(147mg，0.96mmol)加入反应液，继续室温搅拌1h，反应液用水稀释，过滤收集产生的白色固体，并用水(5mL×3)洗涤，然后反相柱分离(洗脱剂：乙腈/水＝1/3,v/v)得到(反式)-叔丁基(4-(5-氨基甲酰-2-(1-乙基-3-甲基-1氢-吡唑-5甲酰胺)-7-甲氧基-1H-苯并咪唑基)正丁2-烯基)氨基甲酸酯(化合物1d)(313mg，收率82％)，白色固体。
MS(ESI)m/z＝512[M+H] +
1H NMR(400MHz,DMSO-d 6)δ12.86(s,1H),8.02(s,1H),7.67(s,1H),7.48–7.32(m,2H),7.00–6.87(m,1H),6.65(s,1H),5.85–5.50(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)。
步骤4：(反式)-1-(4-氨基正丁-2-烯基)-2-(1-乙基-3-甲基-1氢-吡唑-5-甲酰胺基)-7-甲氧基-1氢-苯并咪唑-5-甲酰胺的合成
化合物1d(290mg，0.57mmol)溶解在甲醇(10mL)中，盐酸(6N水溶液，3mL，18mmol)逐滴加入。反应液室温搅拌30分钟后，升温至40℃并继续搅拌3h。旋干反应液，得到的粗品用反相柱分离纯化(洗脱剂：水/乙腈＝50/50,v/v)得到化合物(反式)-1-(4-氨基正丁-2-烯基)-2-(1-乙基-3-甲基-1氢-吡唑-5-甲酰胺基)-7-甲氧基-1氢-苯并咪唑-5-甲酰胺(化合物1e)(230mg，收率98％)，浅黄色固体。
MS(ESI)m/z＝412.0[M+H] +。
步骤5：(E)-1-(4-((4-氨基甲酰基-2-(3-吗啉代)-6-硝基苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺的合成
化合物1e(150mg，0.37mmol)、4-氯-3-(3-吗啉代)-5-硝基苯甲酰胺(127mg，0.37mmol)和DIPEA(220mg，1.85mmol)混溶于正丁醇(5mL)中，反应混合液升温至120℃并在此温度下搅拌48h，降温至室温后，加水稀释，乙酸乙酯(20mL×3)萃取，合并的有机相以饱和食盐水洗涤(15mL×2)，无水硫酸钠干燥，旋干溶剂得到的粗品经反相柱层析分离(洗脱剂：乙腈/水＝30/70，v/v)得到(E)-1-(4-((4-氨基甲酰基-2-(3-吗啉代)-6-硝基苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺(化合物1f)(130mg，收率48.9％)。
MS(ESI)m/z＝719.3[M+H] +。
步骤6：(E)-1-(4-((2-氨基-4-氨基甲酰基-6-(3-吗啉代)苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d] 咪唑-5-甲酰胺的合成
在冰浴下，氨水(0.35mL)滴加入化合物1f(130mg，0.18mmol)的甲醇(5mL)溶液中，反应液在零度下搅拌5分钟后，将连二亚硫酸钠(154mg，0.28mmol)水溶液(2mL)缓慢加入。反应混合液缓慢升至室温，继续搅拌3h。加水稀释，过滤，滤液旋干，所得粗品用反相柱层析(洗脱剂：乙腈/水＝50/50，v/v)分离得到(E)-1-(4-((2-氨基-4-氨基甲酰基-6-(3-吗啉代)苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺1g(91mg，收率74％)，白色固体。
MS(ESI)m/z＝688.3[M+H] +。
步骤7：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
化合物1g(91mg，0.13mmol)和4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(28mg，0.13mmol)混溶于DMF(3mL)中，室温下搅拌0.5h，然后依次加入HATU(49mg,0.13mmol)和DIPEA(34mg，0.26mmol)，室温下继续搅拌2h。反应液经反相HPLC纯化得到化合物1(21mg,收率19％)，白色固体。
MS(ESI)m/z＝867.4[M+H] +
1H NMR(400M,DMSO-d6)δ12.80-12.92(m,1H),9.64-9.73(m,1H),7.93-8.01(m,1H),7.64-7.67(m,2H),7.28-7.43(m,3H),6.52-6.57(m,2H),5.76-5.82(m,2H),4.85-4.94(m,4H),4.51-4.58(m,2H),3.99-4.05(m,3H),3.90-3.97(m,4H),3.71(s,3H),3.24-3.32(m,4H),3.11-3.17(m,4H),2.92-3.06(m,4H),2.65-2.70(m,4H),2.54-2.55(m,4H),2.31-2.36(m,4H),2.11(s,3H),1.86-1.95(m,2H),1.26-1.33(m,3H),1.14-1.21(m,3H).
实施例2
Figure PCTCN2020073405-appb-000054
Figure PCTCN2020073405-appb-000055
步骤1：(E)-4-((4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)氨基)-3-甲氧基-5-硝基苯甲酸甲酯的合成
将4-氯-3-甲氧基-5-硝基苯甲酸甲酯(30g，122mmol)分散于正丁醇(500mL)中，再加入(E)-(4-氨基丁-2-烯-1-基)氨基甲酸叔丁酯(22.8g，122mmol)和DIPEA(78.9g，609mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，冰浴下加入适量稀盐酸(0.5M)，调pH至中性，分离得有机相分别用水、饱和食盐水洗涤，无水硫酸镁干燥后旋干得到2b(48.3g，收率99％)。
MS(ESI)m/z＝396.3[M+H]
步骤2：(E)-3-氨基-4-((4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)氨基)-5-甲氧基苯甲酸甲酯的合成
在冰浴下，向2b(48.3g，122mmol)的甲醇(400mL)溶液中加入氨水(120mL)，10分钟后加入连二亚硫酸钠(106.3g，611mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，在用硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/1)得到化合物2c(28.6g，收率64％)
MS(ESI)m/z＝366.3[M+H]
步骤3：(E)-1-(4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-甲基-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2c(15g，41.1mmol)的DMF(200mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(8.7g，41.1mmol)。反应0.5h后，加入DIPEA (15.9g，123.3mmol)和HATU(18.8g，49.3mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到2d(14.4g，收率64％)。
MS(ESI)m/z＝544.3[M+H]
步骤4：(E)-1-(4-氨基丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2d(6g，11mmol)的DCM(200mL)中加入TFA(6mL)，然后升至室温反应2h。减压旋干溶剂，加入乙酸乙酯后再减压旋干除去游离的TFA，得到化合物2e(4.9g，收率99％)。
MS(ESI)m/z＝444.3[M+H]
步骤5：(E)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1-(4-((4-(甲氧基羰基)-2-(3-吗啉代丙氧基)-6-硝基苯基)氨基)丁-2-烯-1-基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
将2e(4.9g，11mmol)分散于正丁醇(100mL)中，再加入4-氯-3-(3-吗啉代丙氧基)-5-硝基苯甲酸甲酯(4.0g，11mmol)和DIPEA(7.2g，55mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，分别用水、饱和食盐水洗涤，无水硫酸镁干燥后旋干，在经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/2)得到2f(8.4g，收率99％)。
MS(ESI)m/z＝766.3[M+H]
步骤6：(E)-1-(4-((2-氨基-4-(甲氧基羰基)-6-(3-吗啉代丙氧基)苯基)氨基)丁-2-烯-1-基)-2-(4-乙基)-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2f(8.4g，11mmol)的甲醇(50mL)溶液中加入氨水(11mL)，10分钟后加入连二亚硫酸钠(9.6g，55mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，再用硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/1)得到化合物2g(5.1g，收率63％)
MS(ESI)m/z＝736.3[M+H]
步骤7：(E)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧基羰基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2g(5.1g，6.94mmol)的DMF(100mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(1.4g，6.94mmol)。反应0.5h后，加入DIPEA(2.7g，20.82mmol)和HATU(3.2g，8.33mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到2h(4.8g，收率77％)。
MS(ESI)m/z＝897.3[M+H]
步骤8：(E)-1-(4-(5-羧基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5- 甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸的合成
将2h(4.8g，5.38mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液(20ml)中，加入氢氧化锂(2.3g 53.8mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物2i(4.65g，收率99％)。
MS(ESI)m/z＝869.3[M+H]
步骤9：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-吗啉代)-1H--苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将2i(4.65g，5.38mmol)溶于DMF(100mL)中，加入HATU(4.9g，12.86mmol)和DIPEA(4.2g，32.14mmol)。0.5h后，加入碳酸氢铵(1.3g，16.07mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物2(2.5g，收率54％)
MS(ESI)m/z＝867.3[M+H]
1H NMR(400M,DMSO-d6)δ12.79-12.96(m,1H),11.30-11.46(m,1H),7.92-8.14(m,2H),7.63-7.69(m,2H),7.33-7.45(m,3H),7.26-7.32(m,1H),6.53-6.59(m,1H),5.81-5.92(m,1H),5.68-5.79(m,1H),4.51-4.59(m,2H),3.97-4.04(m,2H),3.85-3.93(m,2H),3.81(s,3H),3.72-3.79(m,2H),3.02-3.24(m,6H),2.85-2.98(m,2H),2.58(s,3H),2.13(s,3H),1.90-2.01(m,2H),1.30(t,J＝7.08Hz,3H),1.17(t,J＝7.44Hz,3H).
实施例3
Figure PCTCN2020073405-appb-000056
步骤1：(E)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-5-(甲氧基羰基)-7-甲酯-(3-吗啉代甲基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2g(0.5g，0.69mmol)的DMF(5mL)溶液中加入4-乙基-2- 甲基噻唑-5-羰基异硫氰酸酯(0.18g，0.83mmol)。反应0.5h后，加入DIPEA(0.27g，2.08mmol)和HATU(0.32g，0.83mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到3a(623mg，收率99％)。
MS(ESI)m/z＝914.3.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸的合成
将3a(623mg，0.68mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入水合氢氧化锂(0.17g 4.08mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物3b(610mg，收率99％)。
MS(ESI)m/z＝886.3[M+H]
步骤3：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将3b(610mg，0.68mmol)溶于DMF(10mL)中，加入HATU(4.9g，1.36mmol)和DIPEA(0.53g，4.08mmol)。0.5h后，加入碳酸氢铵(0.16g，2.04mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物3(206mg，收率34％)。
MS(ESI)m/z＝884.3[M+H]
1H NMR(400M,DMSO-d6)δ12.80-12.91(m,1H),11.10-11.23(m,1H),7.91-8.12(m,2H),7.60-7.69(m,2H),7.26-7.48(m,4H),5.71-5.95(m,2H),3.98-4.11(m,2H),3.85-3.95(m,2H),3.71-3.83(m,5H),3.04-3.29(m,8H),2.88-3.01(m,2H),2.56-2.60(m,4H),1.95-2.04(m,2H),1.12-1.24(m,6H).
实施例4
Figure PCTCN2020073405-appb-000057
Figure PCTCN2020073405-appb-000058
步骤1：(E)-4-((4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)氨基)-3-(3-吗啉代丙氧基)-5-硝基苯甲酸甲酯的合成
将4-氯-3-(3-吗啉代丙氧基)-5-硝基苯甲酸甲酯(6.8g，19mmol)分散于正丁醇(100mL)中，再加入(E)-(4-氨基丁-2-烯-1-基)氨基甲酸叔丁酯(3.5g，19mmol)和DIPEA(12.3g，95mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，冰浴下加入适量稀盐酸(0.5M)，调pH至中性，分离得有机相分别用水、饱和食盐水洗涤，无水硫酸镁干燥后旋干得到4b(7.3g，收率76％)。
MS(ESI)m/z＝509.3[M+H]
步骤2：(E)-3-氨基-4-((4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)氨基)-5-(3-吗啉代丙氧基)苯甲酸甲酯的合成
在冰浴下，向4b(7.3g，14.4mmol)的甲醇(100mL)溶液中加入氨水(15mL)，10分钟后加入连二亚硫酸钠(12.5g，71.9mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，在用硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/2)得到化合物4c(4.5g，收率65％)
MS(ESI)m/z＝479.3[M+H]
步骤3：(E)-1-(4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-)吗啉代甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4c(1.8g，3.76mmol)的DMF(30mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(0.96g，4.52mmol)。反应0.5h后，加入DIPEA(1.46g，11.3mmol)和HATU(1.72g，4.52mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到4d(1.9g，收 率77％)。
MS(ESI)m/z＝657.3[M+H]
步骤4：(E)-1-(4-氨基丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]〕咪唑-5-羧酸甲酯的合成
在冰浴下，向4d(1.9g，2.9mmol)的DCM(40mL)中加入TFA(3mL)，然后升至室温反应2h。减压旋干溶剂，加入乙酸乙酯后再减压旋干除去游离的TFA，得到化合物4e(1.6g，收率99％)。
MS(ESI)m/z＝557.3[M+H]
步骤5：(E)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-((4-(甲氧基羰基)-2-(甲硫基)-6-硝基苯基)氨基)丁-2-酮烯-1-基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
向(E)-1-(4-氨基丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]〕咪唑-5-羧酸甲酯(556mg，1mmol)的DMF(10mL)溶液中加入DIPEA(387mg，3mmol)和4-氟-3-甲硫基-5-硝基苯甲酸甲酯(245mg，1mmol)，室温反应3h。反应液倒入水中，用乙酸乙酯萃取，饱和食盐水洗涤并无水硫酸钠干燥，减压旋干溶剂得到化合物4f(616mg，收率79％)
MS(ESI)m/z＝782.3[M+H]
步骤6：(E)-1-(4-((2-氨基-4-(甲氧基羰基)-6-(甲硫基)苯基)氨基)丁-2-烯-1-基)-2-(4-乙基-2)甲酯-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4f(616mg，0.79mmol)的甲醇(5mL)溶液中加入氨水(1mL)，10分钟后加入连二亚硫酸钠(686mg，3.94mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，再用硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/1)得到化合物4g(435mg，收率73％)
MS(ESI)m/z＝752.3[M+H]
步骤7：(E)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧基羰基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4g(145mg，0.193mmol)的DMF(5mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(46mg，0.232mmol)。反应0.5h后，加入DIPEA(75mg，0.579mmol)和HATU(89mg，0.232mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到4h(150mg，收率85％)。
MS(ESI)m/z＝913.3[M+H]
步骤8：(E)-1-(4-(5-羧基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸的合成
将4h(150mg，0.16mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入氢氧化锂(69mg，1.64mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物4i(126mg，收率86％)。
MS(ESI)m/z＝885.3[M+H]
步骤9：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5--甲酰胺的合成
将4i(126mg，0.143mmol)溶于DMF(5mL)中，加入HATU(131mg，0.342mmol)和DIPEA(110mg，0.855mmol)。0.5h后，加入碳酸氢铵(34mg，0.428mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物4(54mg，收率43％)
MS(ESI)m/z＝883.3[M+H]
1H NMR(400M,DMSO-d6)δ12.79-12.88(m,1H),11.10-11.12(m,1H),7.95-8.12(m,2H),7.85-7.89(m,1H),7.62-7.68(m,2H),7.33-7.46(m,3H),6.52(s,1H),5.71-5.78(m,2H),5.11-5.18(m,2H),4.85-4.94(m,2H),4.47-4.55(m,2H),4.05-4.10(m,4H),3.87-3.91(m,4H),3.71-3.81(m,4H),3.22-3.29(m,2H),3.08-3.18(m,8H),2.89-3.01(m,2H),2.57(s,3H),2.46(s,3H),2.10(s,3H),2.00-2.07(m,2H),1.28(t,J＝7.08Hz,3H),1.17(t,J＝7.48Hz,3H).
实施例5
Figure PCTCN2020073405-appb-000059
采用与合成化合物4相同的方法制备得到化合物5，只需将步骤3和步骤7使用的关环原料互换，即步骤3中使用1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯关环，步骤7中使用4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯关环。其它步骤实验方法相同，得到化合物5(67mg)。
MS(ESI)m/z＝883.3[M+H]
1H NMR(400M,DMSO-d6)δ12.90-13.04(m,1H),10.92-11.05(m,1H),7.93-8.14(m,2H),7.83-7.87(m,1H),7.62-7.70(m,2H),7.34-7.48(m,2H),7.27-7.32(m,1H),6.51-6.67(m,1H),5.66-5.83(m,4H),5.06-5.12(m,2H),4.89-4.96(m,2H),4.50-4.59(m,2H),3.99-4.05(m,2H),3.86-3.94(m,2H), 3.70-3.80(m,2H),3.19-3.27(m,2H),3.06-3.15(m,4H),2.88-3.00(m,2H),2.49(s,3H),2.12(s,3H),1.95-2.03(m,2H),1.30(t,J＝7.08Hz,3H),1.17(t,J＝7.48Hz,3H).
实施例6
Figure PCTCN2020073405-appb-000060
采用与合成化合物2相同的方法制备得到化合物6，将合成化合物2使用的4-氯-3-(3-吗啉代丙氧基)-5-硝基苯甲酸甲酯替换为4-氟-3-((3-吗啉代丙基)硫代)-5-硝基苯甲酸甲酯，其它方法一致。
MS(ESI)m/z＝883.3[M+H]
1H NMR(400M,DMSO-d6)δ12.73-12.84(m,1H),10.59-10.82(m,1H),7.98-8.14(m,2H),7.91-7.98(m,1H),7.75-7.83(m,1H),7.59-7.67(m,1H),7.31-7.49(m,3H),6.51-6.59(m,1H),5.87-5.96(m,1H),5.69-5.78(m,1H),5.18-5.29(m,2H),4.83-4.90(m,4H),4.51-4.58(m,4H),3.88-3.97(m,2H),3.81(s,3H),3.67-3.79(m,2H),3.27-3.37(m,2H),3.04-3.15(m,4H),2.91-3.03(m,4H),2.55(s,3H),2.11(s,3H),1.77-1.88(m,2H),1.29(t,J＝7.04Hz,3H),1.14(t,J＝7.48Hz,3H).
实施例7
Figure PCTCN2020073405-appb-000061
步骤1：(E)-1-(4-((2-(3-(4-(叔丁氧基羰基)哌嗪-1-基)丙氧基)-4-(甲氧基羰基)-6-硝基苯基)氨基)丁-2-酮烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
将(E)-1-(4-氨基丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]〕咪唑-5-羧酸甲酯(0.65g，1.4mmol)分散于正丁醇(15mL)中，再加入4-(3-(2-氯-5-(甲氧基羰基)-3-硝基苯氧基)丙基)哌嗪-1-羧酸叔丁酯(630mg，1.4mmol)和DIPEA(1.8g，14mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，分别用水、饱和食盐水洗涤，无水硫酸镁干燥后旋干，在经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/2)得到7a(312mg，收率26％)。
MS(ESI)m/z＝865.2[M+H]+
步骤2：(E)-1-(4-((2-氨基-6-(3-(4-(叔丁氧基羰基)哌嗪-1-基)丙氧基)-4-(甲氧基羰基)苯基)氨基)但-2的甲基烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向7a(285mg，0.36mmol)的甲醇(10mL)溶液中加入氨水(0.5mL)，10分钟后加入连二亚硫酸钠(311mg，1.8mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，化合物7b(296mg，纯度90％,收率96％)
MS(ESI)m/z＝835.0[M+H]+
步骤3：(E)-7-(3-(4-(叔丁氧基羰基)哌嗪-1-基)丙氧基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2)-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7- 甲氧基-5-(甲氧基羰基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向7b(296mg，0.34mmol)的DMF(3mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(66mg，0.34mmol)。反应0.5h后，加入HATU(155mg,0.41mmol)和DIPEA(88mg，0.68mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到7c(346mg，收率97％)。
MS(ESI)m/z＝996.4[M+H]+
步骤4：(E)-7-(3-(4-(叔丁氧基羰基)哌嗪-1-基)丙氧基)-1-(4-(5-羧基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1H--苯并[d]咪唑-5-羧酸的合成
将7c(340mg，0.34mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/0.5)的混合溶液(5.5ml)中，加入氢氧化锂(142mg,3.4mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，粗品溶于水中，加乙酸乙酯萃取,分层所得水相,冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物7d(220mg,收率67％)。
MS(ESI)m/z＝968.4[M+H]+
步骤5：(E)-4-(3-((5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-叔丁基酯甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1H-苯并[d]咪唑-7-基)氧基)丙基)哌嗪-1-羧酸叔丁酯的合成
将7d(158mg，0.16mmol)溶于DMF(2mL)中，加入HATU(182mg，0.48mmol)和DIPEA(103mg，0.8mmol)。0.5h后，加入碳酸氢铵(64.5mg,0.82mmol)，室温搅拌2h。反应液加水析出固体，经过滤，纯水洗涤，干燥后得到化合物7e(140mg，收率90％)
MS(ESI)m/z＝966.5[M+H]+
步骤6：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
冰浴下,向化合物7e(140mg,0.14mmol)的DCM(5mL)溶液中加入TFA(2mL)，缓慢升至室温后,反应1h后旋干溶剂。并用油泵减压除去游离TFA，得到化合物7f(130mg,80％纯度)。
MS(ESI)m/z＝866.4[M+H]
步骤7：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(4-甲基哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
向化合物7f(10mg,0.011mmol)的甲醇(1mL)溶液中加入30％甲醛溶液(2滴)，0.5h后加入乙酸调节pH至4左右，再加入硼氢化钠(1.3mg)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物7(8.5mg，收率87％)
1H NMR(400MHz,DMSO- d6+D 2O)δ7.59(s,1H),7.58(s,1H),7.31(s,1H),7.26(s,1H),6.46(s,1H),5.87–5.67(m,2H),4.99–4.78(m,4H),4.53–4.38(m,2H),4.10(t,J＝6.1Hz,2H),3.70(s,3H),3.48(s,8H),3.17(dt,J＝13.6,5.6Hz,2H),3.04(dd,J＝15.0,7.7Hz,2H),2.86(s,3H),2.07(s,3H),2.06–1.97(m,2H),1.21(t,J＝7.1Hz,3H),1.09(t,J＝7.5Hz,3H).
MS(ESI)m/z＝880.4[M+H] +。
实施例8
Figure PCTCN2020073405-appb-000062
向化合物7f(60mg，0.07mmol)的甲醇溶液中加入乙醛(18mg)，0.5h后加入乙酸调节pH至4左右，再加入氰基硼氢化钠(13mg，0.21mmol)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物8(31mg，收率49.5％)
1H NMR(400MHz,MeOD)δ7.67(s,1H),7.63(s,1H),7.38(s,2H),6.76(s,1H),6.07–5.96(m,1H),5.93–5.79(m,1H),5.19(d,J＝5.5Hz,2H),5.07(d,J＝5.1Hz,2H),4.68(q,J＝7.1Hz,2H),4.21(t,J＝5.9Hz,2H),3.87(s,7H),3.64(s,4H),3.51–3.40(m,3H),3.40–3.34(m,2H),3.29–3.22(m,2H),2.85(s,3H),2.28(s,3H),2.27–2.17(m,2H),1.44(t,J＝7.2Hz,6H),1.33(t,J＝7.6Hz,3H).
MS(ESI)m/z＝894.41[M+H] +。
实施例9
Figure PCTCN2020073405-appb-000063
向化合物7f(50mg，0.058mmol)的甲醇溶液中加入丙酮(3mL)，再乙酸调节pH至4左右，加热至50℃反应4h，反应液冷却至0℃，然后加入氰基硼氢化钠(11mg，0.17mmol)，室温下反应过夜，加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物9(23mg，收率43％)
1H NMR(400MHz,DMSO -d6+D 2O)δ7.61(s,1H),7.57(s,1H),7.33(s,1H),7.26(s,1H),6.48(s,1H),5.87–5.62(m,2H),4.90(s,4H),4.45(q,J＝6.7Hz,2H),4.13(t,J＝5.7Hz,2H),3.69(s,3H),3.67–3.32(m,10H),3.32–3.23(m,2H),3.04(q,J＝7.3Hz,2H),2.58(s,3H),2.18–2.09(m,2H),2.07(s,3H),1.29(d,J＝6.6Hz,6H),1.21(t,J＝7.1Hz,3H),1.10(t,J＝7.5Hz,3H).
MS(ESI)m/z＝908.43[M+H] +。
实施例10
Figure PCTCN2020073405-appb-000064
向化合物7f(60mg，0.055mmol)的甲醇溶液中加入1-乙氧基-1-三甲硅氧基环丙烷(48mg，0.277mmol)，再乙酸调节pH至4左右，然后加入醋酸硼氢化钠(47mg，0.22mmol)，加热至50℃反应过夜，LCMS显示全部转化为中间体亚胺，冷却至室温加入氰基硼氢化钠(10mg，0.16mmol)。加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物10(27.7mg，收率55％)。
1H NMR(400MHz,DMSO-d6+D2O)δ7.57(s,2H),7.23(s,1H),7.21(s,1H),6.46(s,1H),5.83–5.63(m,2H),4.85(d,J＝10.9Hz,4H),4.45(dd,J＝13.3,6.5Hz,2H),3.64(s,4H),3.27–2.91(m,10H),2.87(s,3H),2.05(s,3H),2.01(s,1H),1.85(s,2H),1.21(t,J＝7.1Hz,3H),1.09(t,J＝7.5Hz,3H),0.55(d,J＝6.5Hz, 2H),0.43(s,2H).
MS(ESI)m/z＝906.41[M+H] +。
实施例11
Figure PCTCN2020073405-appb-000065
向7f(10mg，0.011mmol)的DMF(1mL)溶液中加入TEA(5.5mg，0.055mmol)和乙酸酐(3.3mg，0.033mmol)，室温下反应2h，将反应液经制备HPLC纯化得到化合物11(6.5mg,收率62％)
1H NMR(400MHz,DMSO-d6+D2O)δ7.61(d,J＝1.6Hz,2H),7.27(s,2H),6.48(s,1H),5.84–5.58(m,2H),4.87(d,J＝4.9Hz,4H),4.47(dd,J＝13.8,6.8Hz,2H),4.01(s,2H),3.68(s,3H),3.33(s,4H),3.17–3.04(m,5H),2.87(s,4H),2.54(s,3H),2.07(s,3H),2.02(s,3H),1.97(s,2H),1.23(t,J＝7.1Hz,3H),1.12(t,J＝7.5Hz,3H).
MS(ESI)m/z＝908.39[M+H] +。
依照实施例7的合成方法还制备了实施例12～23的化合物12～23。
实施例12
Figure PCTCN2020073405-appb-000066
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(40mg,0.044mmol)的甲醇(2mL)溶液中加入30％甲醛溶液(2滴)，0.5h后加入乙酸调节pH至4左右，再加入硼氢化钠(5.2mg)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物12(29mg，收率75％)。
1H NMR(400MHz,DMSO)δ12.79(s,1H),11.85(s,2H),8.03(s,1H),7.97(s,1H),7.65(s,1H),7.62(s,1H),7.36(s,2H),7.34(s,2H),6.49(s,1H),5.97–5.84(m, 1H),5.79–5.65(m,1H),4.96(d,J＝3.9Hz,2H),4.86(d,J＝5.3Hz,2H),4.49(q,J＝7.0Hz,3H),4.15–4.08(m,4H),3.83(s,8H),3.64(s,5H),3.44(s,4H),3.22(s,2H),3.09(q,J＝7.5Hz,2H),2.86(d,J＝20.8Hz,3H),2.55(s,3H),2.09(s,5H),1.25(t,J＝7.1Hz,3H),1.14(t,J＝7.5Hz,3H).
MS(ESI)m/z＝880.4[M+H] +。
实施例13
Figure PCTCN2020073405-appb-000067
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(50mg,0.058mmol)的甲醇溶液中加入乙醛(18mg)，0.5h后加入乙酸调节pH至4左右，再加入氰基硼氢化钠(13mg，0.21mmol)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物13(30mg，收率58％)。
1H NMR(400MHz,DMSO)δ12.80(s,1H),11.80(s,1H),8.03(s,1H),7.96(s,1H),7.65(s,1H),7.63(s,1H),7.36(s,3H),7.34(s,1H),6.50(s,1H),5.94–5.89(m,1H),5.78–5.70(m,1H),4.96(d,J＝3.8Hz,2H),4.86(d,J＝5.3Hz,2H),4.50(q,J＝6.9Hz,2H),4.09(t,J＝5.5Hz,2H),3.68(s,4H),3.38(s,4H),3.20(s,4H),3.09(q,J＝7.5Hz,2H),2.54(s,3H),2.09(s,5H),1.26(q,J＝6.9Hz,6H),1.14(t,J＝7.5Hz,3H).
MS(ESI)m/z＝894.41[M+H] +。
实施例14
Figure PCTCN2020073405-appb-000068
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1- 基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(50mg,0.058mmol)的甲醇溶液中加入丙酮(3mL)，再乙酸调节pH至4左右，加热至50℃反应4h，反应液冷却至0℃，然后加入氰基硼氢化钠(11mg，0.17mmol)，室温下反应过夜，加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物14(23mg，收率43.7％)。
1H NMR(400MHz,DMSO)δ12.80(s,1H),11.86(s,1H),8.03(s,1H),7.96(s,1H),7.65(s,1H),7.63(s,1H),7.36(s,3H),7.34(s,1H),6.50(s,1H),5.99–5.87(m,1H),5.79–5.70(m,1H),4.96(d,J＝4.2Hz,2H),4.86(d,J＝5.5Hz,2H),4.50(q,J＝6.9Hz,2H),4.10(t,J＝5.5Hz,2H),3.83(s,3H),3.71–3.37(m,8H),3.22(s,2H),3.09(q,J＝7.4Hz,2H),2.54(s,3H),2.09(s,5H),1.30(d,J＝6.5Hz,5H),1.25(t,J＝7.1Hz,3H),1.14(t,J＝7.5Hz,3H).
MS(ESI)m/z＝908.43[M+H] +。
实施例15
Figure PCTCN2020073405-appb-000069
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(50mg,0.058mmol)的甲醇溶液中加入1-乙氧基-1-三甲硅氧基环丙烷(48mg，0.277mmol)，再乙酸调节pH至4左右，然后加入醋酸硼氢化钠(47mg，0.22mmol)，加热至50℃反应过夜，LCMS显示全部转化为中间体亚胺，冷却至室温加入氰基硼氢化钠(10mg，0.16mmol)。加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物15(9mg，收率17％)。
1H NMR(400MHz,DMSO)δ12.82(s,1H),8.03(s,1H),7.96(s,1H),7.66(s,1H),7.64(s,1H),7.45–7.26(m,3H),6.52(s,1H),5.98–5.84(m,1H),5.79–5.68(m,1H),4.95(s,2H),4.85(d,J＝5.4Hz,2H),4.52(dd,J＝14.2,7.2Hz,2H),4.03(s,1H),3.79(s,2H),3.66–3.14(m,6H),3.10(dd,J＝15.0,7.5Hz,2H),2.53(s,2H),2.11(s,2H),1.99(s,1H),1.27(t,J＝7.1Hz,2H),1.15(t,J＝7.5Hz,2H),0.99(s,2H),0.74(s,2H).
MS(ESI)m/z＝906.41[M+H] +。
实施例16
Figure PCTCN2020073405-appb-000070
向化合物(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(20mg,0.023mmol)的甲醇溶液中加入30％甲醛溶液(2滴)，0.5h后加入乙酸调节pH至4左右，再加入硼氢化钠(2.6mg,0.069mmol)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物16(10mg,收率48％).
MS(ESI)m/z＝896.37[M+H] +。
实施例17
Figure PCTCN2020073405-appb-000071
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(60mg，0.07mmol)的甲醇溶液中加入乙醛(18mg)，0.5h后加入乙酸调节pH至4左右，再加入氰基硼氢化钠(13mg，0.21mmol)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物17(26mg，收率41％)
1H NMR(400MHz,DMSO)δ12.80(s,1H),11.77(s,1H),8.06(s,1H),7.98(s,1H),7.85(s,1H),7.63(s,2H),7.40(s,1H),7.36(s,2H),6.51(s,1H),5.92–5.79(m,1H),5.79–5.68(m,1H),5.15(d,J＝2.6Hz,2H),4.91(d,J＝3.9Hz,2H),4.51(dd,J＝14.1,7.0Hz,4H),4.14(t,J＝5.3Hz,2H),3.70(s,4H),3.39(s,5H),3.20(s,4H),3.09(q,J＝7.4Hz,2H),2.53(s,3H),2.45(s,3H),2.13(s,2H),2.09(s,3H),1.33– 1.19(m,7H),1.14(t,J＝7.5Hz,3H).
MS(ESI)m/z＝910.39[M+H] +。
实施例18
Figure PCTCN2020073405-appb-000072
向((E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(60mg，0.07mmol)的甲醇溶液中加入丙酮(3mL)，再乙酸调节pH至4左右，加热至50℃反应4h，反应液冷却至0℃，然后加入氰基硼氢化钠(11mg，0.17mmol)，室温下反应过夜，加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物18(17mg，26％)
1H NMR(400MHz,DMSO)δ12.81(s,1H),12.12(s,1H),11.84(s,1H),8.06(s,1H),7.98(s,1H),7.85(s,1H),7.63(s,2H),7.41(s,1H),7.37(s,2H),6.52(s,1H),5.91–5.67(m,2H),5.15(d,J＝2.5Hz,2H),4.91(d,J＝3.5Hz,2H),4.51(q,J＝7.0Hz,2H),4.16(t,J＝5.6Hz,2H),3.81–3.39(m,10H),3.24(s,2H),3.09(q,J＝7.4Hz,2H),2.54(s,3H),2.45(s,4H),2.14(s,2H),2.09(s,3H),1.30(d,J＝6.5Hz,6H),1.26(t,J＝7.1Hz,3H),1.14(t,J＝7.5Hz,3H).
MS(ESI)m/z＝924.40[M+H] +。
实施例19
Figure PCTCN2020073405-appb-000073
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3- (哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(60mg，0.07mmol)的甲醇溶液中加入1-乙氧基-1-三甲硅氧基环丙烷(48mg，0.277mmol)，再乙酸调节pH至4左右，然后加入醋酸硼氢化钠(47mg，0.22mmol)，加热至50℃反应过夜，LCMS显示全部转化为中间体亚胺，冷却至室温加入氰基硼氢化钠(10mg，0.16mmol)。加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物19(16mg，收率25％)。
MS(ESI)m/z＝922.39[M+H] +。
实施例20
Figure PCTCN2020073405-appb-000074
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(10mg,0.011mmol)的甲醇溶液中加入30％甲醛溶液(2滴)，0.5h后加入乙酸调节pH至4左右，再加入硼氢化钠(1.3mg,0.033mmol)，反应3h后加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物20(5mg,收率51％).
MS(ESI)m/z＝896.37[M+H] +。
实施例21
Figure PCTCN2020073405-appb-000075
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(60mg，0.07mmol)的甲醇溶液中加入乙醛(18mg)，0.5h后加入乙酸调节pH至4左右，再加入氰基硼氢化钠(13mg，0.21mmol)，反应3h后加水淬灭反 应。将反应液浓缩后，经制备HPLC纯化得到化合物21(8mg,收率12％)
1H NMR(400M,MeOD-d4)δ7.83-7.90(m,1H),7.74-7.79(m,1H),7.68-7.74(m,1H),7.34-7.45(m,1H),6.75-6.83(m,1H),5.82-6.02(m,2H),5.18-5.34(m,4H),4.67-4.79(m,2H),4.17-4.28(m,2H),3.54-3.99(m,8H),3.36-3.47(m,4H),3.26-3.30(m,2H),2.89(s,3H),2.58(s,3H),2.32(s,3H),2.17-2.27(m,2H),1.42-1.53(m,6H),1.32-1.39(m,3H).
MS(ESI)m/z＝910.39[M+H] +。
实施例22
Figure PCTCN2020073405-appb-000076
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(60mg，0.07mmol)的甲醇溶液中加入丙酮(3mL)，再乙酸调节pH至4左右，加热至50℃反应4h，反应液冷却至0℃，然后加入氰基硼氢化钠(11mg，0.17mmol)，室温下反应过夜，加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物22(17mg,26％)。
1H NMR(400M,MeOD-d4)δ7.85-7.90(m,1H),7.74-7.79(m,1H),7.70-7.74(m,1H),7.37-7.41(m,1H),6.78-6.83(m,1H),5.82-6.01(m,2H),5.18-5.34(m,4H),4.68-4.78(m,2H),4.19-4.26(m,2H),3.66-3.87(m,8H),3.40-3.47(m,2H),3.28-3.32(m,2H),2.89(s,3H),2.58(s,3H),2.32(s,3H),2.18-2.28(m,2H),1.44-1.53(m,9H),1.31-1.40(m,3H).
MS(ESI)m/z＝924.40[M+H] +。
实施例23
Figure PCTCN2020073405-appb-000077
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺(40mg，0.045mmol)的甲醇溶液中加入1-乙氧基-1-三甲硅氧基环丙烷(39mg，0.225mmol)，再乙酸调节pH至4左右，然后加入醋酸硼氢化钠(47mg，0.22mmol)，加热至50℃反应过夜，加水淬灭反应。将反应液浓缩后，经制备HPLC纯化得到化合物23(10mg，收率24％)。
MS(ESI)m/z＝922.39[M+H] +。
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰氨基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的DMF溶液中加入TEA和对应的酰氯，室温下反应2h，反应液经制备HPLC纯化得到化合物24,25,26,27,28。化合物结构和表征如下表1所示：
表1部分实施例化合物结构和表征
Figure PCTCN2020073405-appb-000078
Figure PCTCN2020073405-appb-000079
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的DMF溶 液中加入TEA和相应的酰氯，室温下反应2h，反应液经制备HPLC纯化得到化合物29,30,31,32,33,34。化合物结构和表征如下表2所示：
表2部分实施例化合物结构和表征
Figure PCTCN2020073405-appb-000080
Figure PCTCN2020073405-appb-000081
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的DMF溶液中加入TEA和相应的酰氯，室温下反应2h，反应液经制备HPLC纯化得到化合物35,36,37,38,39,40。化合物结构和表征如下表3所示：
表3部分实施例化合物结构和表征
Figure PCTCN2020073405-appb-000082
Figure PCTCN2020073405-appb-000083
Figure PCTCN2020073405-appb-000084
向(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-(哌嗪-1-基)丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的DMF溶液中加入TEA和相应的酰氯，室温下反应2h，反应液经制备HPLC纯化得到化合物41,42,43,44,45,46。化合物结构和表征如下表4所示：
表4部分实施例化合物结构和表征
Figure PCTCN2020073405-appb-000085
Figure PCTCN2020073405-appb-000086
实施例47
Figure PCTCN2020073405-appb-000087
步骤1：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(((2-甲氧基-4-(甲氧羰基)-6-硝基苯基)氨基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
将4e(2.1g，3.8mmol)分散于正丁醇(20mL)中，再加入4-氯-3-(3-吗啉代丙氧基)-5-硝基苯甲酸甲酯(0.93g，3.8mmol)和DIPEA(2.5g，19mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，别用水、饱和食盐水洗涤，无水硫酸镁干燥后旋干，在经硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/2)得到47a(2.0g，收率70％)。
MS(ESI)m/z＝766.2[M+H]
步骤2：(E)-甲基1-(4-(((2-氨基-6-甲氧基-4-(甲氧基羰基)苯基)氨基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向47a(600mg，0.78mmol)的甲醇(5mL)溶液中加入氨水(1mL)，10分钟后加入连二亚硫酸钠(679mg，3.9mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到化合物47b(459mg，收率80％)。
MS(ESI)m/z＝736.3[M+H]
步骤3：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-5-(甲氧基羰基)-1H-苯并[d]咪唑-1-基)但是-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向47b(200mg，0.27mmol)的DMF(5mL)溶液中加入1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(58mg，0.27mmol)。反应0.5h后，加入DIPEA(70mg，0.54mmol)和HATU(102mg，0.27mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到47c(185mg，收率75％)。
MS(ESI)m/z＝915.3[M+H]
步骤4：(E)-1-(4-(5-羧基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸的合成
将47c(185mg，0.2mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液(5mL)中，加入氢氧化锂(42mg，1.0mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物47d(141mg，收率80％)。
MS(ESI)m/z＝887.3[M+H]
步骤5：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H)-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将47d(141mg，0.16mmol)溶于DMF(5mL)中，加入HATU(152mg，0.4mmol)和DIPEA(82mg，0.64mmol)。0.5h后，加入碳酸氢铵(38mg，0.48mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物47(30mg，收率43％)
MS(ESI)m/z＝885.4[M+H]
1H NMR(400M,DMSO- d6)δ12.81-12.98(m,2H),9.69-9.77(m,1H),7.93-8.02(m,2H),7.61-7.69(m,2H),7.37-7.43(m,2H),7.26-7.34(m,2H),5.71-5.90(m,4H),4.80-4.94(m,4H),4.46-4.54(m,2H),3.90-4.00(m,4H),3.75(s,3H),3.58-3.64(m,4H),3.21-3.26(m,2H),3.05-3.20(m,6H),2.93-3.00(m,2H),2.64-2.70(m,2H),2.31-2.36(m,2H),2.12(s,3H),1.80-1.94(m,4H),1.29(t,J＝7.04Hz,3H),1.16(t,J＝7.48Hz,3H).
实施例48
Figure PCTCN2020073405-appb-000088
Figure PCTCN2020073405-appb-000089
步骤1：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧基羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向2g(0.51g，0.69mmol)的DMF(5mL)溶液中加入1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(0.15g，0.69mmol)。反应0.5h后，加入DIPEA(0.27g，2.07mmol)和HATU(0.32g，0.83mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到48a(0.48g，收率77％)。
MS(ESI)m/z＝915.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸的合成
将48a(0.48g，0.52mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液(20mL)中，加入水合氢氧化锂(0.22g，5.2mmoL)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物48b(0.45g，收率97％)。
MS(ESI)m/z＝886.3[M+H]
步骤3：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基))-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-甲氧基-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将48b(0.45g，0.51mmol)溶于DMF(5mL)中，加入HATU(0.46g，1.22mmol)和DIPEA(0.33g，2.55mmol)。0.5h后，加入碳酸氢铵(0.2g，2.55mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物48(0.26g，收率57％)
MS(ESI)m/z＝884.4[M+H]
1H NMR(400M,DMSO- d6)δ12.82-12.97(m,2H),9.55-9.85(m,1H),7.92-8.02(m,2H),7.61-7.67(m,2H),7.35-7.45(m,2H),7.28-7.35(m,3H),5.77-5.89(m,2H),4.86-4.93(m,4H),4.44-4.52(m,2H),4.03-4.07(m,2H),3.91-3.98(m,2H),3.73(s,3H),3.59-3.67(m,4H),3.26-3.32(m,4H),3.12-3.18(m,4H),2.95-3.04(m,2H),2.53(s,3H),2.09(s,3H),1.86-1.99(m,2H),1.26(t,J＝7.04Hz,3H),1.17(t,J＝7.48Hz,3H).
实施例49
Figure PCTCN2020073405-appb-000090
步骤1：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧羰基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑5-羧酸甲酯的合成
在冰浴下，向4g(145mg，0.19mmol)的DMF(5mL)溶液中加入入1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(49mg，0.23mmol)。反应0.5h后，加入DIPEA(75mg，0.58mmol)和HATU(89mg，0.23mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到49a(116mg，收率64％)。
MS(ESI)m/z＝931.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸的合成
将49a(116mg，0.12mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入水合氢氧化锂(54mg,12.4mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物49b(102mg，收率90％)。
MS(ESI)m/z＝903.3[M+H]
步骤3：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基))-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将49b(102mg，0.11mmol)溶于DMF(5mL)中，加入HATU(103mg，0.27mmol)和DIPEA(88mg，0.68mmol)。0.5h后，加入碳酸氢铵(27mg，0.34mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物49(46mg，收率45％)。
MS(ESI)m/z＝901.3[M+H]
1H NMR(400M,DMSO- d6)δ12.77-13.08(m,1H),10.91-10.99(m,1H),7.94-8.14(m,2H),7.83-7.90(m,1H),7.61-7.68(m,2H),7.31-7.48(m,3H),5.72-5.84(m,2H),5.09-5.17(m,2H),4.87-4.91(m,2H),4.41-4.49(m,2H),4.03-4.13(m,2H),3.86-3.97(m,2H),3.69-3.81(m,2H),3.22-3.33(m,2H),3.07-3.20(m,4H),2.91-3.04(m,2H),2.56(s,3H),2.47(s,3H),2.08(s,3H),1.99-2.07(m,2H),1.25(t,J＝7.04Hz,3H),1.16(t,J＝7.48Hz,3H).
实施例50
Figure PCTCN2020073405-appb-000091
步骤1：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-5-(甲氧羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4g(145mg，0.19mmol)的DMF(5mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(49mg，0.23mmol)。反应0.5h后，加入DIPEA(75mg，0.58mmol)和HATU(89mg，0.23mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到50a(116mg，收率64％)。
MS(ESI)m/z＝930.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-甲酸的合成
将50a(116mg，0.12mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入水合氢氧化锂(54mg,12.4mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物50b(103mg，收率91％)。
MS(ESI)m/z＝902.3[M+H]
步骤3：(E)-1-(4-(5-羧基-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-甲酰胺的合成
将50b(103mg，0.11mmol)溶于DMF(5mL)中，加入HATU(103mg，0.27mmol)和DIPEA(88mg，0.68mmol)。0.5h后，加入碳酸氢铵(27mg，0.34mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物50(36mg，收率35％)。
MS(ESI)m/z＝900.3[M+H]
1H NMR(400M,DMSO- d6)δ12.79-12.99(m,1H),10.99-11.08(m,1H),7.89-8.16(m,2H),7.83-7.87(m,1H),7.63-7.67(m,2H),7.29-7.48(m,3H),5.75-5.81(m,2H),5.08-5.12(m,4H),4.88-4.92(m,4H),4.01-4.10(m,2H),3.86-3.95(m,2H),3.70-3.81(m,2H),3.21-3.30(m,2H),3.07-3.17(m,6H),2.90-3.01(m,2H),2.56(s,3H),1.97-2.09(m,2H),1.17(dt,J＝7.44,3.00Hz,6H).
实施例51
Figure PCTCN2020073405-appb-000092
步骤1：(E)-甲基1-(4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4c(0.74g，1.55mmol)的DMF(5mL)溶液中加入1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(0.34g，1.55mmol)。反应0.5h后，加入DIPEA(0.6g，4.65mmol)和HATU(0.71g，1.86mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到51a(0.88g，收率86％)。
MS(ESI)m/z＝658.3[M+H]
步骤2：(E)-甲基1-(4-氨基丁-2-烯-1-基)-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向51a(0.88g，1.34mmol)的DCM(40mL)中加入TFA(3mL)，然后升至室温反应2h。减压旋干溶剂，加入乙酸乙酯后再减压旋干除去游离的TFA，得到化合物51b(0.66g，收率88％)。
MS(ESI)m/z＝558.3[M+H]
步骤3：(E)-甲基2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-1-(4-((4-(甲氧羰基)-2-(甲硫基)-6-硝基苯基)氨基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
向51b(557mg，1mmol)的DMF(10mL)溶液中加入DIPEA(387mg，3mmol)和4-氟-3-甲硫基-5-硝基苯甲酸甲酯(245mg，1mmol)，室温反应3h。反应液倒入水中，用乙酸乙酯萃取，饱和食盐水洗涤并无水硫酸钠干燥，减压旋干溶剂得到化合物51c(451mg，收率58％)
MS(ESI)m/z＝783.3[M+H]
步骤4：(E)-1-(4-((2-氨基-4-(甲氧基羰基)-6-(甲硫基)苯基)氨基)丁-2-烯-1-基)-2-(4-乙基-2)甲酯-甲基噻唑-5-甲酰胺基)-7-(3-吗啉代甲基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向51c(451mg，0.58mmol)的甲醇(5mL)溶液中加入氨水(1mL)，10分钟后加入连二亚硫酸钠(502mg，2.88mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，再用硅胶柱纯化(洗脱剂：石油醚/乙酸乙酯＝1/1)得到化合物51d(417mg，收率94％)
MS(ESI)m/z＝753.3[M+H]
步骤5：(E)-甲基2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧基羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向51d(139mg，0.193mmol)的DMF(5mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(46mg，0.232mmol)。反应0.5h后，加入DIPEA(75mg，0.579mmol)和HATU(89mg，0.232mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到51e(120mg，收率71％)。
MS(ESI)m/z＝914.3[M+H]
步骤6：(E)-1-(4-(5-羧基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸的合成
将51e(120mg，0.13mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入氢氧化锂(55mg，1.31mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物51f(112mg，收率96％)。
MS(ESI)m/z＝886.3[M+H]
步骤7：(E)-1-(4-(5-(5-氨基甲酰基-2-)(1-苯乙烯-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-苯乙烯-4-氟-3-甲基-1H-吡唑-5-蒽酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-甲酰胺的合成
将51f(112mg，0.126mmol)溶于DMF(5mL)中，加入HATU(116mg，0.304mmol)和DIPEA(98mg，0.759mmol)。0.5h后，加入碳酸氢铵(30mg，0.380mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物51(51mg，收率46％)
MS(ESI)m/z＝884.3[M+H]
1H NMR(400M,DMSO-d6)δ12.76-13.12(m,1H),11.07-11.17(m,1H),7.95-8.13(m,2H),7.85-7.90(m,1H),7.61-7.70(m,2H),7.31-7.46(m,3H),6.51(s,1H),5.71-5.76(m,2H),5.09-5.16(m,2H),4.89-4.96(m,2H),4.42-4.47(m,4H),4.00-4.07(m,2H),3.85-3.94(m,2H),3.70-3.81(m,2H),3.18-3.28(m,2H),3.04-3.13(m,2H),2.87-3.00(m,2H),2.45(s,3H),2.10(s,6H),1.95-2.04(m,2H),1.23-1.32(m,6H).
实施例52
Figure PCTCN2020073405-appb-000093
步骤1：(E)-甲基2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑5-羧酸甲酯的合成
在冰浴下，向51d(139mg，0.193mmol)的DMF(5mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(49mg，0.232mmol)。反应0.5h后，加入DIPEA(75mg，0.579mmol)和HATU(89mg，0.232mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到52a(118mg，收率68％)。
MS(ESI)m/z＝931.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7- (3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酸的合成
将52a(118mg，0.13mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入氢氧化锂(55mg，1.31mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物52b(110mg，收率96％)。
MS(ESI)m/z＝903.3[M+H]
步骤3：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基))-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
将52b(110mg，0.122mmol)溶于DMF(5mL)中，加入HATU(112mg，0.293mmol)和DIPEA(95mg，0.732mmol)。0.5h后，加入碳酸氢铵(29mg，0.366mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物52(32mg，收率29％)
MS(ESI)m/z＝901.3[M+H]
1H NMR(400M,DMSO-d6)δ12.93-12.98(m,1H),10.88-10.96(m,1H),7.94-8.13(m,2H),7.84-7.88(m,1H),7.63-7.68(m,2H),7.28-7.46(m,3H),5.70-5.83(m,2H),5.06-5.12(m,2H),4.90-4.96(m,2H),4.45-4.52(m,4H),3.98-4.06(m,2H),3.86-3.94(m,2H),3.68-3.80(m,2H),3.19-3.27(m,2H),3.05-3.16(m,4H),2.88-3.00(m,2H),2.49(s,6H),2.10(s,3H),1.93-2.03(m,2H),1.28(t,J＝7.04Hz,3H),1.16(t,J＝7.48Hz,3H).
实施例53
Figure PCTCN2020073405-appb-000094
步骤1：(E)-甲基2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-1-(4-(2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)-2-烯-1-基)-7-(甲硫基))-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向51d(139mg，0.193mmol)的DMF(5mL)溶液中加入1-乙基-4-氟-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(49mg，0.232mmol)。反应0.5h后，加入DIPEA(75mg，0.579mmol)和HATU(89mg，0.232mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到53a(108mg，收率62％)。
MS(ESI)m/z＝932.3[M+H]
步骤2：(E)-1-(4-(5-羧基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酸的合成
将53a(108mg，0.12mmol)溶于甲醇、四氢呋喃和水(体积比：1/1/1)的混合溶液中，加入氢氧化锂(49mg，1.16mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物53b(100mg，收率95％)。
MS(ESI)m/z＝904.3[M+H]
步骤3：(E)-1-(4-(5-氨基甲酰基-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-乙基-4-氟-3-甲基-1H-吡唑-5-甲酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酰胺的合成将53b(100mg，0.111mmol)溶于DMF(5mL)中，加入HATU(102mg，0.266mmol)和DIPEA(88mg，0.665mmol)。0.5h后，加入碳酸氢铵(27mg，0.333mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物53(41mg，收率41％)
MS(ESI)m/z＝902.3[M+H]
1H NMR(400M,DMSO-d6)δ12.89-13.08(m,1H),10.47-10.57(m,1H),7.92-8.13(m,2H),7.85-7.89(m,1H),7.61-7.69(m,2H),7.30-7.48(m,3H),5.74-5.79(m,2H),5.09-5.16(m,2H),4.89-4.95(m,2H),4.41-4.49(m,6H),4.03-4.07(m,2H),3.89-3.95(m,2H),3.66-3.75(m,2H),3.21-3.30(m,2H),3.08-3.17(m,2H),2.92-3.02(m,2H),2.45(s,3H),2.10(s,3H),2.08(s,3H),1.91-2.03(m,2H),1.20-1.30(m,6H).
实施例54
Figure PCTCN2020073405-appb-000095
Figure PCTCN2020073405-appb-000096
步骤1：(E)-甲基1-(4-((叔丁氧基羰基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向4c(3.0g，6.27mmol)的DMF(30mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(1.35g，6.90mmol)。反应0.5h后，加入DIPEA(1.62g，12.54mmol)和HATU(2.62g，6.9mmol)，然后升至室温反应3h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到54a(3.21g，收率80％)。
MS(ESI)m/z＝640.3[M+H]
步骤2：(E)-甲基1-(4-氨基丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向54a(3.20g，4.0mmol)的DCM(30mL)中加入TFA(5mL)，然后升至室温反应2h。减压旋干溶剂，加入乙酸乙酯后再减压旋干除去游离的TFA，得到化合物54b(4.2g)，直接用于下一步。
MS(ESI)m/z＝540.3[M+H]
步骤3：(E)-甲基2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-1-(4-((4-(甲氧羰基)-2-(甲硫基)-6-硝基苯基)氨基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
向54b(296mg，粗品，0.37mmol)的DMF(10mL)溶液中加入DIPEA(143mg，1.1mmol)和4-氟-3-甲硫基-5-硝基苯甲酸甲酯(91mg，0.37mmol)，室温反应3h。反应完全后混合液倒入水中，过滤、水洗并干燥后得黄色固体54c(120mg，收率42％)
MS(ESI)m/z＝765.3[M+H]
步骤4：(E)-甲基1-(4-(((2-氨基-4-(甲氧羰基)-6-(甲硫基)苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向54c(120mg，0.16mmol)的甲醇(5mL)溶液中加入氨水(1mL)，10分钟后加入连二亚硫酸钠(140mg，0.8mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，再用硅胶柱纯化(洗脱剂：二氯甲烷/甲醇＝10/1，v/v)得到化合物54d(55mg，收率48％)
MS(ESI)m/z＝735.3[M+H]
步骤5：(E)-甲基2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-1-(4-(2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-5-(甲氧羰基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(甲硫基)-1H-苯并[d]咪唑5-羧酸甲酯的合成
在冰浴下，向54d(55mg，0.074mmol)的DMF(5mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(16mg，0.08mmol)。反应0.5h后，加入DIPEA(20mg，0.15mmol)和HATU(34mg，0.09mmol)，然后升至室温反应3.5h。将反应液慢慢倒入水中，有类白色固体析出，过滤并干燥固体得到54e(50mg，收率75％)。
MS(ESI)m/z＝897.0[M+H]
步骤6：(E)-1-(4-(5-羧基-2-(1-乙基-3-甲基-1H-吡唑-5-羧酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-羧酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5-羧酸的合成
将54e(50mg，0.056mmol)溶于甲醇、四氢呋喃和水(体积比：2/1/0.5)的混合溶液(3.5mL)中，加入氢氧化锂(12mg，0.28mmol)。升至75℃搅拌反应过夜。旋蒸除去有机溶剂，冷却至室温，冰浴下以稀盐酸(1M)调至固体不再析出，过滤并干燥固体的到化合物54f(38mg，收率78％)。
MS(ESI)m/z＝867.0[M+H]
步骤7：(E)-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-羧酰胺基)-7-(甲硫基)-1H-苯并[d]咪唑-5–甲酰胺的合成
将54f(38mg，0.043mmol)溶于DMF(5mL)中，加入HATU(49mg，0.129mmol)和DIPEA(30mg，0.215mmol)。0.5h后，加入碳酸氢铵(16mg，0.131mmol)，室温搅拌2h。反应液浓缩后经制备HPLC纯化得到化合物54(18mg，收率47％)，白色固体。
MS(ESI)m/z＝866.3[M+H]
1H NMR(400MHz,DMSO)δ12.98(s,2H),9.79(s,1H),8.06(s,1H),7.95(s,1H),7.87(d,J＝1.3Hz,1H),7.66(s,1H),7.63(d,J＝1.3Hz,1H),7.43(s,1H),7.37(s,1H),7.29(s,1H),6.52(s,1H),6.51(s,1H),5.78–5.61(m,2H),5.12(s,2H), 4.90(s,2H),4.52(p,J＝7.3Hz,4H),4.00(t,J＝5.6Hz,2H),3.94(d,J＝12.4Hz,2H),3.26(d,J＝11.8Hz,3H),3.11(s,3H),2.97(s,3H),2.43(d,J＝8.4Hz,3H),2.12(s,3H),2.10(s,3H),1.96–1.81(m,2H),1.28(td,J＝7.1,4.1Hz,6H).
实施例55
Figure PCTCN2020073405-appb-000097
步骤1:(E)-1-(4-((2-(3-(((叔丁基二甲基甲硅烷基)氧基)丙氧基)-4-氨基甲酰基-6-硝基苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺的合成
化合物1e(3.0g，7.3mmol)、3-(3-((叔丁基二甲基甲硅烷基)氧基)丙氧基)-4-氯-5-硝基苯甲酰胺(2.84g，7.3mmol)和DIPEA(4.7g，36.5mmol)混溶于正丁醇(60mL)中，反应混合液升温至120℃并在此温度下搅拌48h，降温至室温后，加水稀释，乙酸乙酯(30mL×3)萃取，合并的有机相以饱和食盐水洗涤，无水硫酸钠干燥，过滤后旋干溶剂得到的粗品经反相柱层析分离(洗脱剂：乙腈/水＝50/50，v/v)得到化合物55a(4.2g，收率75％)。
MS(ESI)m/z＝764.3[M+H] +。
步骤2：(E)-1-(4-((2-氨基-6-(3-((叔丁基二甲基甲硅烷基)氧基)丙氧基)-4-氨基甲酰基苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺的合成
在冰浴下，氨水(5.5mL)滴加入化合物55a(4.2g，5.5mmol)的甲醇(40mL)溶液中，反应液在零度下搅拌十分钟后，将连二亚硫酸钠(4.8g，27.5mmol) 水溶液(10mL)缓慢加入。反应混合液缓慢升至室温，继续搅拌3h。过滤除掉无机盐，滤液旋干，所得粗品用反相柱层析(洗脱剂：乙腈/水＝60/40，v/v)分离得到化合物55b(2.8g，收率69％)，白色固体。
MS(ESI)m/z＝734.3[M+H] +。
步骤3：(E)-N-(7-(3-((叔丁基二甲基甲硅烷基)氧基)丙氧基)-5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-1-基)丁-2-烯-1-基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
在冰浴下，将4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(805mg，3.8mmol)加入化合物55b(2.8g，3.8mmol)的DMF(40mL)中，二十分钟后，依次加入HATU(1.59g,4.18mmol)和DIPEA(980mg，7.6mmol)，室温下继续搅拌2h。反应完全后，倒入水中，有固体析出，过滤收集固体并干燥后得到化合物55c(2.5g，收率71％)，
MS(ESI)m/z＝912.4[M+H] +
步骤4：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-7)-甲氧基-1H-苯并[d][咪唑-1-基)丁-2-烯-1-基)-7-(3-羟基丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
向化合物55c(2.5g，2.7mmol)的THF(10mL)溶液中加入四丁基氟化铵(8.1mL,1M四氢呋喃溶液)，室温下搅拌反应4h。旋干溶剂，粗品用反相柱层析(洗脱剂：乙腈/水＝40/60，v/v)分离得到化合物55(1.6g，收率74％)。
MS(ESI)m/z＝798.3[M+H] +
实施例56
Figure PCTCN2020073405-appb-000098
在冰浴下，向化合物55(100mg，0.12mmol)的四氢呋喃(5mL)溶液中 加入三乙胺(36mg，0.36mmol)和甲磺酰氯(14mg，0.13mmol)。升至室温反应2h，LCMS显示反应完全。在向反应液中加入碳酸钾(50mg，0.36mmol)和8-氧杂-3-氮杂双环[3.2.1]辛烷(68mg，0.6mmol)，升至50℃反应2h，旋干溶剂。粗品经制备HPLC纯化得到化合物56(15mg)。
按照化合物56的制备方法，以化合物55和对应的胺为原料，得到化合物57、58、59、60、61、62、63、64。化合物结构和表征如下表5所示：
表5部分实施例化合物结构和表征
Figure PCTCN2020073405-appb-000099
Figure PCTCN2020073405-appb-000100
实施例65
Figure PCTCN2020073405-appb-000101
Figure PCTCN2020073405-appb-000102
步骤1：4-氨基-2-氟-5-甲氧基苯甲酰胺的合成
在室温下，向2-氟-5-甲氧基-4-硝基苯甲酰胺(10g，46.7mmol)的甲醇(100mL)溶液中加入10％钯碳(1.5g)，室温下氢化反应5h，过滤掉钯碳，滤液旋干得到化合物65a(7.7g，收率89％)。
MS(ESI)m/z＝185[M+H] +
步骤2：(E)-(4-((4-氨基甲酰基-5-氟-2-甲氧基苯基)氨基)丁-2-烯-1-基)氨基甲酸叔丁酯的合成
将化合物65a(7.5g，40.7mmol)溶于DMF(150mL)，依次加入碳酸钾(8.4g，61mmol)和(E)-(4-溴丁-2-烯-1-基)氨基甲酸叔丁酯(10.1g，40.7mmol)。升温至60℃反应8h，冷却后过滤除去无机盐，滤液旋干，乙酸乙酯溶解后，分别用水、饱和食盐水洗涤，有机相旋干后经硅胶柱纯化(石油醚/乙酸乙酯＝1/2)得到化合物65b(5.2g，收率36％)。
MS(ESI)m/z＝354[M+H] +
步骤3：(E)-(4-((4-氨基甲酰基-5-氟-2-甲氧基-6-硝基苯基)氨基)丁-2-烯-1-基)氨基甲酸叔丁酯的合成
将化合物65b(5.2g，14.7mmol)溶于乙酸酐(30mL)中，待其在冰浴下降温至0℃，硝酸(1.7g，17.6mmol，65％纯度)滴加到反应液中，维持0℃反应2h。反应液倒入水中，用乙酸乙酯萃取，在用饱和食盐水洗，浓缩有机相后经硅胶柱纯化(石油醚/乙酸乙酯＝1/1)得到化合物65c(2g，收率34％)。
MS(ESI)m/z＝399[M+H] +
步骤4：(E)-(4-((4-氨基甲酰基-5-氟-2-甲氧基-6-胺基苯基)氨基)丁-2-烯-1-基)氨基甲酸叔丁酯的合成
在冰浴下，向65c(2g，5mmol)的甲醇(20mL)溶液中加入氨水(5mL)，10分钟后加入连二亚硫酸钠(4.3g，25mmol)的水溶液，反应2h。过滤除掉无机盐，加乙酸乙酯萃取。有机相用饱和食盐水洗涤，无水硫酸镁干燥后旋干得到粗品，在用硅胶柱纯化(洗脱剂：二氯甲烷/甲醇＝50/1-30/1)得到化合物65d(1.1g，收率64％)
MS(ESI)m/z＝369[M+H] +
步骤5：(E)-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-4-氟-7-甲氧基-1H-苯并[d]咪唑-1-基)-2-烯-1-基)氨基甲酸叔丁酯的合成
在冰浴下，向65d(1.1g，2.9mmol)的DMF(20mL)溶液中加入1-乙基-3-甲基-1H-吡唑-5-羰基异硫氰酸酯(565mg，2.9mmol)。反应0.5h后，加入DIPEA(748mg，5.8mmol)和HATU(1.3g，3.48mmol)，然后升至室温反应12h。将反应液慢慢倒入水中，有黄色固体析出，过滤并干燥固体得到65e(1.1g，收率75％)。
MS(ESI)m/z＝530[M+H] +
步骤6：(E)-1-(4-氨基丁-2-烯-1-基)-2-(4-乙基-2-甲基噻唑-5-甲酰胺基)-7-甲氧基-1H-苯并[d]咪唑-5-羧酸甲酯的合成
在冰浴下，向65e(1.1g，2.1mmol)的DCM(10mL)中加入TFA(5mL)，然后升至室温反应2h。减压旋干溶剂，加入乙酸乙酯后再减压旋干除去游离的TFA，得到化合物65f(1.5g，含三氟乙酸)。
MS(ESI)m/z＝430[M+H] +
步骤7：(E)-1-(4-氨基丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-4-氟-7-甲氧基-1H-苯并[d]咪唑-5-羧酰胺的合成
将65f(1.5g，2.1mmol，57％纯度)分散于正丁醇(10mL)中，再加入4-氯-3-(3-吗啉代丙氧基)-5-硝基苯甲酰胺(720mg，2.1mmol)和DIPEA(1.35g，10.5mmol)。反应液升温至120℃，搅拌18h。减压蒸馏得到的粗品溶于乙酸乙酯，分别用水、饱和食盐水洗涤，无水硫酸钠干燥后旋干，经硅胶柱纯化(洗脱剂：二氯甲烷/甲醇＝30/1)得到65g(770mg，收率49％)。
MS(ESI)m/z＝737.[M+H] +
步骤8：(E)-1-(4-(((2-氨基-4-氨基甲酰基-6-(3-吗啉代丙氧基)苯基)氨基)丁-2-烯-1-基)-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺)-4-氟-7-甲氧基-1H-苯并[d]咪唑-5-甲酰胺的合成
在冰浴下，向65g(500mg，0.68mmol)的甲醇(10mL)溶液中加入氨水(1mL)，10分钟后加入连二亚硫酸钠(591mg，3.4mmol)的水溶液，慢慢升至室温反应2h。过滤除掉无机盐，浓缩滤液得到粗品，再用反相柱色谱(洗脱剂：乙腈/水＝60/40)得到化合物65h(200mg，收率41％)
MS(ESI)m/z＝707[M+H] +
步骤9：(E)-N-(5-氨基甲酰基-1-(4-(5-氨基甲酰基-2-(1-乙基-3-甲基-1H-吡唑-5-甲酰胺基)-4-氟-7-甲氧基-1H)-苯并[d]咪唑-1-基)丁-2-烯-1-基)-7-(3-吗啉代丙氧基)-1H-苯并[d]咪唑-2-基)-4-乙基-2-甲基噻唑-5-甲酰胺的合成
在冰浴下，向65h(100mg，0.14mmol)的DMF(3mL)溶液中加入4-乙基-2-甲基噻唑-5-羰基异硫氰酸酯(30mg，0.14mmol)。反应0.5h后，加入DIPEA(54mg，0.42mmol)和HATU(53mg，0.14mmol)，然后升至室温反应2h。反应液经制备HPLC纯化得到65(20mg，收率16％)。
MS(ESI)m/z＝885[M+H] +
以下用实验例的方式说明本发明的有益效果：
实验例1：本发明化合物与Sting蛋白的结合亲和力测试
(1)实验方法
利用蛋白质热转移实验(TSA)进行化合物同Sting蛋白的结合亲和力测定，在20mM Hepes,150mM NaCl,1mM MgCl 2,1mM DTT,pH＝7.5缓冲液中将100ug/ml Sting蛋白同不同浓度的化合物及5X SYPRO Orange染料混合，在qPCR仪上测定蛋白的溶解曲线，用Protein Thermal Shift Software 1.3软件拟合Tm值，计算加入不同浓度化合物和未加入化合物时蛋白的Tm差值，根据 ΔTm随化合物浓度变化拟合得到解离常数Kd。使用文章(Nature.2018Dec；564(7736):439-443)中报道的compound 3作为阳性参考化合物。试验结果见下表6，其中各化合物的Kd值按照以下说明分类：
“+”表示Kd值大于10μM；
“++”表示Kd值小于10μM大于1μM；
“+++”表示Kd值小于1μM；
Kd越低，表示化合物与Sting蛋白的结合亲和力越强。
(2)实验结果
表6化合物与Sting蛋白的结合亲和力
实施例 Kd
参考化合物 ++
1 ++
2 ++
3 ++
4 ++
5 ++
6 ++
7 ++
8 ++
9 ++
10 ++
11 ++
12 ++
13 ++
14 ++
15 ++
16 ++
17 ++
18 ++
Figure PCTCN2020073405-appb-000103
上述实验结果表明，本发明化合物具有良好的与Sting蛋白结合的能力，与参考化合物的体外亲和力类似，所以本发明化合物可作为一种有效的STING蛋白调节剂。
实验例2、本发明化合物对Sting蛋白的激动功能测试
(1)实验方法
本实验通过检测化合物刺激人外周血单核细胞系THP1细胞(上海细胞库)产生的IFN-β和CXCL10(IP10)细胞因子变化来评价sting激动剂的功能。实验第一天按IFN-β(R&D，#DY814-05)和IP10(BD,#550926)ELISA检测试剂盒说明书包被ELISA板。取化合物DMSO溶解成储液，并用培养基稀释成2X工作浓度，加入96孔板，每孔100μL；取对数生长期的THP1细胞计数，并稀释成2*10 6/mL浓度，加入上述含化合物的96孔板中，每孔100μL，混匀，于37℃，5％CO 2培养箱中培养18小时。第二天取上述细胞培养上清，每孔100μL，分别按IFN-β和IP10 ELISA检测试剂盒进行检测，读取OD450值，根据标准曲线换算成IFN-β和IP10浓度，并用GraphPad 5.0拟合剂效曲线计算EC 50值。EC 50为半最大效应浓度(concentration for 50％of maximal effect,EC50)，是指能引起50％个体有效的药物浓度。
使用文章(Nature.2018 Dec；564(7736):439-443)中报道的compound 3作为阳性参考化合物。
(2)实验结果
实验结果见下表7，其中各化合物的EC 50值按照以下说明分类：
“+”表示EC 50值大于1μM；
“++”表示EC 50值小于1μM大于100nM；
“+++”表示EC 50值小于100nM大于10nM；
“++++”表示EC 50值小于10nM。
表7化合物对IFN-β和CXCL10(IP10)细胞因子的影响
实施例 IFN-βEC 50 IP10 EC 50
参考化合物 ++ +++
1 ++ +++
2 +++ +++
3 ++ +++
4 +++ ++++
5 +++ ++++
6 ++ ++
7 ++ ++
8 ++ +++
9 ++ +++
10 ++ +++
11 ++ +++
12 ND ++
13 ++ +++
14 ++ ++
15 +++ +++
16 ND ++
17 ++ +++
18 ++ +++
19 ++++ ++++
20 ++ +++
21 ++ +++
22 ++ +++
23 +++ +++
29 ++ +++
30 ++ +++
35 +++ +++
40 +++ ND
41 ++ +++
47 + +++
48 ++ +++
49 +++ ++++
50 +++ +++
51 +++ ++++
52 +++ ++++
53 +++ ++++
表7中，“ND”表示还未进行检测。
上述实验数据表明，本发明化合物具有良好的刺激THP1细胞产生的IFN-β和CXCL10(IP10)细胞因子的活性，具有较好的STING蛋白激动功能。特别是化合物2、4、5、15、19、23、35、40、49、50、51、52、53等，相比于参考化合物，本发明化合物具有相当的、甚至更高的刺激免疫细胞产生细胞因子的活性。
实验例3、小鼠体内IFN-β诱导实验
(1)实验方法
Balb/C小鼠背部皮下接种5×10 5CT26细胞。接种后挑选瘤体积在200-300mm3范围内的荷瘤小鼠用于实验。依据瘤体积大小区段随机法进行分组，分为21组，每组3只小鼠。分别通过尾静脉注射给以不同剂量的药物(本发明化合物)，对照组给以对照溶媒(5％DMSO，40％PEG400配制于生理盐水)。给药后3小时，通过小鼠眼眶静脉取血，EDTA·2K作为抗凝剂。将收集的小鼠全血8000g离心5分钟后取上层血浆待测。给药后3小时，取小鼠肿瘤组织，称重并加入9倍体积PBS匀浆。将匀浆液10000rpm离心15分钟后，收集上清待测。分别按IFN-β检测试剂盒(R&D，#DY814-05)进行检测，读取OD450值，根据标准曲线换算成IFN-β浓度。
(2)实验结果
实验结果显示小鼠尾静脉给药能够在血液和肿瘤中诱导STING通路下游的IFN-β释放。本发明化合物给药后肿瘤内IFN-β水平是血浆中IFN-β水平的3倍以上，表明细胞因子在肿瘤组织中相对富集，血液中细胞因子较低，提示有较高的治疗窗口。
实施例4、本发明化合物的肿瘤抑制效果(CT26肿瘤模型)
(1)实验方法
Balb/c小鼠静养一周后，背部皮下接种CT26细胞，每只小鼠接种100uL含有5×10 5细胞的细胞悬液。平均肿瘤体积长到120mm3左右时，依据瘤体积大小随机分组，每组5只。分别在分组后的第1，4，8天为各组小鼠尾静脉静脉给药，对照组给以对照溶媒(5％DMSO，40％PEG400配制于生理盐水)。分组当天、首次给药后每周3次、安乐死前，利用游标卡尺测量并记录肿瘤长、短径，计算肿瘤体积，并根据肿瘤体积绘制肿瘤生长曲线。按照以下公式计算肿瘤体积：V＝1/2×长径×短径×短径。
(2)实验结果
实验结果如图1所示，给药20天后，本发明实施例制得的化合物均可有效抑制肿瘤生长，并呈现剂量依赖的肿瘤生长抑制效果。
实施例5、本发明化合物的肿瘤抑制效果(H22肿瘤模型)
(1)实验方法
昆明鼠静养一周后，背部皮下接种H22细胞，每只小鼠接种100uL含有 5×10 6细胞的细胞悬液。平均肿瘤体积长到200mm3左右时，依据瘤体积大小随机分组，每组7只。分别在分组后的第1，4，8天为高中低各剂量组小鼠尾静脉静脉给药(本发明化合物)，或第1，8，15天为高剂量组小鼠尾静脉静脉给药，对照组给以对照溶媒(5％DMSO，40％PEG400配制于生理盐水)。分组当天、首次给药后每周3次、安乐死前，利用游标卡尺测量并记录肿瘤长、短径，计算肿瘤体积，并根据肿瘤体积绘制肿瘤生长曲线。按照以下公式计算肿瘤体积：V＝1/2×长径×短径×短径。
(2)实验结果
给药30天后，本发明化合物均可有效抑制肿瘤生长，，并呈现剂量依赖的肿瘤生长抑制效果。
实施例6、本发明化合物的肿瘤抑制效果(肿瘤再激发模型)
(1)实验方法
将上述实验例4中CT26抗肿瘤药效模型中肿瘤消退的各组小鼠继续观察，末次给药结束30天后重新在背部皮下接种CT26细胞，每只小鼠接种100uL含有5×10 5细胞的细胞悬液；未经过药物治疗的小鼠作为对照组以相同方式接种CT26细胞。观察肿瘤记录肿瘤生长情况。
(2)实验结果
实验结果如图2所示，对照组CT26肿瘤在接种后能够生长，但经过本发明化合物治疗肿瘤完全消失的小鼠接种后肿瘤不能够重新生长，说明这些化合物激活了小鼠的免疫记忆机制，使小鼠对再次接种的肿瘤细胞有免疫作用。
综上，本发明公开了式I所示化合物，该类化合物能够与STING有效结合，具有良好的STING蛋白激动功能，对多种肿瘤表现出良好的抑制效果，而且还能激活小鼠的免疫记忆机制，抑制肿瘤再激发。所以，本发明的化合物可作为STING激动剂并用于治疗各种相关病症。本发明化合物在制备治疗与STING活性相关疾病的药物(特别是治疗炎性疾病、变应性疾病、自身免疫性疾病、感染性疾病、癌症或癌前期综合征的药物)以及在制备免疫佐剂中具有非常好的应用前景，为临床上筛选和/或制备与STING活性相关的疾病的药物提供了一种新的选择。

Claims (19)

  1. 式I所示的化合物、或其立体异构体、或其药学上可接受的盐：
    Figure PCTCN2020073405-appb-100001
    其中，
    L选自C 2～C 6亚烷基、C 2～C 6亚烯基、C 2～C 6亚炔基；
    R 1、R 3、R 1’、R 3’分别独立选自氢、卤素、C 1～C 6烷基、氰基、硝基、羟基、氨基、C 1～C 6烷氧基、C 1～C 6烷胺基、卤素取代的C 1～C 6烷基；
    R 2、R 2’分别独立选自氢、-C(O)NR aR b、-NR aC(O)R b、-C(O)R a、-C(O)OR a、卤素、C 1～C 6烷基、氰基、硝基、羟基、氨基、C 1～C 6烷氧基、C 1～C 6烷胺基、卤素取代的C 1～C 6烷基；
    R 4、R 4’分别独立选自氢、C 1～C 6烷基；
    X、X’分别独立选自-CH 2-、-NH-、-O-、-S-；
    A环、A’环分别独立选自被0～4个R c任选取代的5～6元芳杂环；
    且X、X’选自-CH 2-、-NH-、-O-时，A环、A’环至少有一个选自被0～4个R c任选取代的含硫的5～6元芳杂环或含氧的5～6元芳杂环；
    R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
    R a、R b分别独立选自氢、C 1～C 6烷基；
    R c分别独立选自卤素、-CN、-OR a、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
    R d分别独立选自卤素、-OR a、-NR aR b、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基、被0～4个R e任选取代的7～10元环烷基、被0～4个R e任选取代的7～10元杂环烷基、被0～4个R e任选取代的7～10元螺环烷基、被0～4个R e任选取代的7～10元螺杂环烷基、被0～4个R e任选取代的5～10元桥环烷基、被0～4个R e任选取代的5～10元桥杂环烷基、被0～4个R e任选取代的7～10元稠环烷基、被0～4个R e任选取代的7～10元稠杂环烷基；
    R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
  2. 根据权利要求1所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物的结构如式I所示：
    Figure PCTCN2020073405-appb-100002
    其中，
    L选自C 2～C 6亚烷基、C 2～C 6亚烯基、C 2～C 6亚炔基；
    R 1、R 3、R 1’、R 3’分别独立选自氢、卤素、C 1～C 6烷基；
    R 2、R 2’分别独立选自氢、-C(O)NR aR b；
    R 4、R 4’分别独立选自氢、C 1～C 6烷基；
    X、X’分别独立选自-CH 2-、-NH-、-O-、-S-；
    A环、A’环分别独立选自被0～4个R c任选取代的5～6元芳杂环；
    且X、X’选自-CH 2-、-NH-、-O-时，A环、A’环至少有一个选自被0～4个R c任选取代的含硫的5～6元芳杂环或含氧的5～6元芳杂环；
    R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
    R a、R b分别独立选自氢、C 1～C 6烷基；
    R c分别独立选自卤素、-CN、-OR a、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
    R d分别独立选自卤素、-OR a、-NR aR b、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基；
    R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
  3. 根据权利要求2所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    R 1、R 3、R 1’、R 3’分别独立选自氢；
    R 2、R 2’分别独立选自-C(O)NR aR b；
    R 4、R 4’分别独立选自氢；
    X、X’分别独立选自-O-、-S-；
    A环、A’环分别独立选自被0～3个R c任选取代的5元芳杂环；
    且X、X’选自-O-时，A环、A’环至少有一个选自被0～3个R c任选取代的含硫的5元芳杂环；
    R 5、R 5’分别独立选自被0～1个R d任选取代的C 1～C 6烷基；
    R c分别独立选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
    R d分别独立选自被0～2个R e任选取代的3～6元环烷基、被0～2个R e任选取 代的3～6元杂环烷基；
    R e分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
  4. 根据权利要求3所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    X、X’至少有一个选自-S-，A环、A’环分别独立选自被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100003
    R 5、R 5’分别独立选自C 1～C 6烷基、
    Figure PCTCN2020073405-appb-100004
    Figure PCTCN2020073405-appb-100005
    R c’选自C 1～C 6烷基、卤素取代的C 1～C 6烷基；R c选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
    R e’分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基，R a选自氢、C 1～C 6烷基。
  5. 根据权利要求3所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：
    X、X’选自-O-，A环、A’环至少有一个选自被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100006
    R 5、R 5’分别独立选自C 1～C 6烷基、
    Figure PCTCN2020073405-appb-100007
    R c选自卤素、C 1～C 6烷基、卤素取代的C 1～C 6烷基；
    R e’分别独立选自C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基，R a选自氢、C 1～C 6烷基。
  6. 根据权利要求3所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物的结构如式II所示：
    Figure PCTCN2020073405-appb-100008
    其中，X、X’分别独立选自-O-、-S-；
    A环、A’环分别独立选自被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100009
    且当X、X’均为-O-时，A环、A’环至少有一个选自被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100010
    R c’选自C 1～C 2烷基、卤素取代的C 1～C 2烷基；
    R c选自卤素、C 1～C 2烷基、卤素取代的C 1～C 2烷基；所述卤素优选为F；
    R 5、R 5’分别独立选自C 1～C 3烷基、
    Figure PCTCN2020073405-appb-100011
    Figure PCTCN2020073405-appb-100012
    R e’选自C 1～C 3烷基、3～6元环烷基、-C(O)R f；
    R f选自-OR a’、C 1～C 3烷基、3～4元环烷基，R a’选自C 1～C 3烷基。
  7. 根据权利要求6所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：X选自-O-、-S-，X’选自-O-；
    R 5选自C 1～C 3烷基；
    R 5’选自
    Figure PCTCN2020073405-appb-100013
    R e’选自C 1～C 3烷基、3～6元环烷基、-C(O)R f；
    R f选自-OR a’、C 1～C 3烷基、3～4元环烷基，R a’选自C 1～C 3烷基。
  8. 根据权利要求6所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100014
    为
    Figure PCTCN2020073405-appb-100015
    所述被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100016
    为
    Figure PCTCN2020073405-appb-100017
  9. 根据权利要求1～8任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物为：
    Figure PCTCN2020073405-appb-100018
    Figure PCTCN2020073405-appb-100019
    Figure PCTCN2020073405-appb-100020
    Figure PCTCN2020073405-appb-100021
    Figure PCTCN2020073405-appb-100022
    Figure PCTCN2020073405-appb-100023
    Figure PCTCN2020073405-appb-100024
    Figure PCTCN2020073405-appb-100025
  10. 根据权利要求1所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物中，R 5、R 5’分别独立选自被0～2个R d任选取代的C 1～C 6烷基；
    R d分别独立选自卤素、-OH、被0～4个R e任选取代的3～6元环烷基、被0～4个R e任选取代的3～6元杂环烷基、被0～4个R e任选取代的7～10元环烷基、被0～4个R e任选取代的7～10元杂环烷基、被0～4个R e任选取代的7～10元螺环烷基、被0～4个R e任选取代的7～10元螺杂环烷基、被0～4个R e任选取代的5～10元桥环烷基、被0～4个R e任选取代的5～10元桥杂环烷基；
    R e分别独立选自卤素、C 1～C 6烷基、3～6元环烷基、-C(O)R f；
    R f独立选自-OR a、C 1～C 6烷基、3～6元环烷基。
  11. 根据权利要求10所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物如式III所示：
    Figure PCTCN2020073405-appb-100026
    其中，R 1、R 3、R 1’、R 3’分别独立选自氢、卤素；
    A环、A’环分别独立选自被0～2个R c任选取代的
    Figure PCTCN2020073405-appb-100027
    R c’选自C 1～C 2烷基、卤素取代的C 1～C 2烷基；
    R c选自卤素、C 1～C 2烷基、卤素取代的C 1～C 2烷基；
    R 5’选自被0～2个R d任选取代的C 1～C 3烷基；
    R d分别独立选自-OH、被0～2个R e任选取代的3～6元环烷基、被0～2个R e任选取代的3～6元杂环烷基、被0～2个R e任选取代的7～10元环烷基、被0～2个R e任选取代的7～10元杂环烷基、被0～2个R e任选取代的7～10元螺环烷基、被0～2个R e任选取代的7～10元螺杂环烷基、被0～2个R e任选取代的5～10元桥环烷基、被0～2个R e任选取代的5～10元桥杂环烷基、被0～2个R e任选取代的7～10元稠环烷基、被0～2个R e任选取代的7～10元稠杂环烷基；R e分别独立选自卤素、C 1～C 3烷基。
  12. 根据权利要求11所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：A环、A’环分别独立选自
    Figure PCTCN2020073405-appb-100028
    R 5’选自被0～2个R d任选取代的C 1～C 3烷基；
    R d分别独立选自-OH、
    Figure PCTCN2020073405-appb-100029
    Figure PCTCN2020073405-appb-100030
  13. 根据权利要求10～12任一项所述的化合物、或其立体异构体、或其药学上可接受的盐，其特征在于：所述化合物为：
    Figure PCTCN2020073405-appb-100031
    Figure PCTCN2020073405-appb-100032
  14. 权利要求1～13任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备激活STING类药物中的用途。
  15. 权利要求1～13任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备治疗与STING活性相关的疾病的药物中的用途。
  16. 根据权利要求15所述的用途，其特征在于：所述与STING活性相关的疾病是与炎性、自身免疫性疾病、感染性疾病、癌症、癌前期综合征相关的疾病中的一种或几种。
  17. 权利要求1～13任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备治疗炎性、自身免疫性疾病、感染性疾病、癌症或癌前期综合征的药物中的用途。
  18. 权利要求1～13任一项所述的化合物、或其立体异构体、或其药学上可接受的盐在制备免疫佐剂中的用途。
  19. 一种药物，其特征在于：它是以权利要求1～13任一项所述的化合物、或其立体异构体、或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
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