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EP4281066A2 - Artemisinin-proteasome inhibitor conjugates and their use in the treatment of disease - Google Patents

Artemisinin-proteasome inhibitor conjugates and their use in the treatment of disease

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Publication number
EP4281066A2
EP4281066A2 EP22743162.4A EP22743162A EP4281066A2 EP 4281066 A2 EP4281066 A2 EP 4281066A2 EP 22743162 A EP22743162 A EP 22743162A EP 4281066 A2 EP4281066 A2 EP 4281066A2
Authority
EP
European Patent Office
Prior art keywords
alkyl
monocyclic
bicyclic
group
heteroaryl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22743162.4A
Other languages
German (de)
French (fr)
Inventor
Gang Lin
Wenhu ZHAN
Hao Zhang
Laura KIRKMAN
Carl Nathan
Daqiang Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cornell University
Original Assignee
Cornell University
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Filing date
Publication date
Application filed by Cornell University filed Critical Cornell University
Publication of EP4281066A2 publication Critical patent/EP4281066A2/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/18—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to Artemi sinin-Proteasome inhibitor conjugates and their use in the treatment of diseases.
  • ART Artemisinin
  • UPS ubiquitin-proteasome degradation system
  • ART resistance is increased tolerance to ART at the early ring stage of the erythrocytic cycle.
  • Multiple mechanisms of resistance are associated with Kelchl3 polymorphisms (Straimer et al., “Drug Resistance. K13- propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015); Mok et al., “Drug Resistance.
  • a first aspect of the present application relates to an Artemisinin-Proteasome inhibitor conjugate including a compound of Formula (I): wherein
  • R a is independently selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle;
  • R b is independently selected from group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle, and wherein R a and R b may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
  • X is O, S, or N
  • Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor
  • Z is O or O-O
  • Proteasome inhibitor is a compound that inhibits either chymotryptic-like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
  • a second aspect of the present application relates to a method of treating infectious diseases in a subject. This method includes administering to the subject in need thereof a compound of the present application.
  • a third aspect of the present application relates to a method of treating cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders in a subject, or for providing immunosuppression for transplanted organs or tissues in a subject.
  • This method includes administering to the subject in need thereof a compound of the present application.
  • a fourth aspect of the present application relates to a pharmaceutical composition including a therapeutically effective amount of the compounds of the present application and a pharmaceutically acceptable carrier.
  • Artemisinin resistance is spreading in Plasmodium falciparum (P ⁇ ) despite combination chemotherapy (ACT), perhaps because differences in pharmacokinetics of each drug result in periodic monotherapy in some anatomical compartments.
  • ACT combination chemotherapy
  • artezomibs single-molecule hybrids of an artemisinin and a proteasome inhibitor are reported.
  • artezomibs create a novel mode of action in which the artemisinin component covalently modifies parasite proteasome substrates and the proteasome is inhibited by the proteasome inhibitor component.
  • artezomibs circumvent the development of both artemisinin resistance conferred by Kelch13 polymorphism and the resistance to the proteasome inhibitor associated with mutations in P ⁇ proteasomes. This mode of action may enable a single molecule to prevent emergence of resistance.
  • proteasome inhibitors not only kill P ⁇ on their own but also make the parasites more susceptible to ART
  • linking a proteasome inhibitor to an ART analog through a tether could yield a hybrid compound with the ability to hijack the parasite ubiquitin proteasome system to produce a host of proteasome inhibitors that overcome resistance to each of the hybrid’s two constituent chemophores.
  • an ART- proteasome inhibitor hybrid would yield ART -modified proteins whose proteasomal degradation products containing a proteasome inhibitor moiety could inhibit the function of P ⁇ 20S by binding to its active proteolytic subunits.
  • the extended peptides of the degradation products could compensate for a loss of binding affinity caused by point mutations near the active sites that would otherwise reduce the efficacy of the proteasome inhibitor.
  • an artezomib can overcome resistance to its individual components and potentially prevent the emergence of resistance to each.
  • Figure 1 shows the synthetic route of Artesunate-based hybrids WZ-13 and WZ- 06 and control WZ-20.
  • Figure 2 is the 1 H nuclear magnetic resonance (NMR) spectrum of
  • Figure 3 is the 13 C NMR spectrum of compound WZ-13.
  • Figure 4 is the liquid chromatography-mass spectrometry (LC-MS) chromatogram of compound WZ-13.
  • Figure 5 is the high-resolution mass spectrometry (HRMS) spectrum of compound WZ-13.
  • Figure 6 is the 1 H NMR spectrum of (3R,5aS, 6R,8aS, 9R, 105, 12R , 12aR)-3, 6,9- Trimethyldecahydro- 12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl 4-(((S)-4-(tert- butylamino)-1,4-dioxo-1-((2-(4-phenylpicolinamido)ethyl)amino)butan-2-yl)amino)-4- oxobutanoate (WZ-06).
  • Figure 7 is the 13 C NMR spectrum of compound WZ-06.
  • Figure 8 is the LC-MS chromatogram of compound WZ-06.
  • Figure 9 is the FIRMS spectrum of compound WZ-06.
  • Figure 10 is the 1 H NMR spectrum of (S)-4-((4-(tert-butylamino)- 1 -((2-(2',4- difluoro-[1,1'-biphenyl]-3 -carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-4- oxobutanoic acid (WZ-20).
  • Figure 11 is the 13 C NMR spectrum of compound WZ-20.
  • Figure 12 is the LC-MS chromatogram of compound WZ-20.
  • Figure 13 is the HRMS spectrum of compound WZ-20.
  • Figure 14 is the 1 H NMR spectrum of 2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-
  • Figure 15 is the 13 C NMR spectrum of compound DeoxoART-AcOH 9.
  • Figure 16 shows the synthetic route of ART-based hybrids ATZ1, ATZ2, ATZ3, and ATZ4.
  • Figure 17 is the 1 H NMR spectrum of 2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-
  • Figure 18 is the 13 C NMR spectrum of compound ART1.
  • Figure 19 is the LC-MS chromatogram of compound ART1.
  • Figure 20 is the HRMS spectrum of compound ART1.
  • Figure 21 is the 1 H NMR spectrum of tert- Butyl (S)-(2-((4-(tert-butyl amino)- 1- ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3-carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-2- oxoethyl)carbamate (WZ-0917).
  • Figure 22 is the 13 C NMR spectrum of compound WZ-0917.
  • Figure 23 is the 1 H NMR spectrum of tert- Butyl (S)-(3 -((4-(tert-butyl amino)- 1 - ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3 -carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-3 - oxopropyl)carbamate (WZ-0918).
  • Figure 24 is the 1 H NMR spectrum of tert- Butyl (S)-(4-((4-(tert-butylamino)-1- ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3 -carboxamido)ethyl)amino)-1,4-dioxobutan-2-yl)amino)-4- oxobutyl)carbamate (PI01).
  • Figure 25 is the 13 C NMR spectrum of compound PI01.
  • Figure 26 is the LC-MS chromatogram of compound PI01.
  • Figure 27 is the HRMS spectrum of compound PI01.
  • Figure 28 is the 1 HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3-carboxamido)ethyl)-2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)succinamide (ATZ1).
  • Figure 29 is the 13 C NMR spectrum of compound ATZ1.
  • Figure 30 is the LC-MS chromatogram of compound ATZ1.
  • Figure 31 is the HRMS spectrum of compound ATZ1.
  • Figure 32 is the 1 HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl )acetami do)acetami do) succinami de (ATZ2) .
  • Figure 33 is the 13 C NMR spectrum of compound ATZ2.
  • Figure 34 is the LC-MS chromatogram of compound ATZ2.
  • Figure 35 is the HRMS spectrum of compound ATZ2.
  • Figure 36 is the 1 HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(3 -(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 ,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)propanamido)succinamide (ATZ3).
  • Figure 37 is the 13 C NMR spectrum of compound ATZ3.
  • Figure 38 is the LC-MS chromatogram of compound ATZ3.
  • Figure 39 is the HRMS spectrum of compound ATZ3.
  • Figure 40 is the 1 HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(4-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 ,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butanamido)succinamide (ATZ4).
  • Figure 41 is the 13 C NMR spectrum of compound ATZ4.
  • Figure 42 is the LC-MS chromatogram of compound ATZ4.
  • Figure 43 is the HRMS spectrum of compound ATZ4.
  • Figure 44 shows proposed synthetic approaches for the formation of diverse Artemisinin-Proteasome inhibitor conjugates.
  • Figures 45A-45H shows the effects of compounds in a ring-stage survival assay.
  • Figure 45A is a flow diagram of the process in which red blood cells infected with highly synchronized ring-stage parasites were treated with DMSO, DHA, ART1, PI01, a 1:1 mixture of PI01 and ART1, ATZ3 or ATZ4 at indicated concentrations. After 6 hours, the compounds were washed off.
  • Figure 45B the parasite cultures were allowed to grow for 66 hours. Viable parasites were analyzed by flow cytometry and their numbers normalized to values for the DMSO control.
  • Figure 45C aliquots of parasites from Figure 45B were cultured for a further 96 hours.
  • Figure 45D shows the inhibition of P ⁇ 20S, P ⁇ 20S( ⁇ 6A117D) and P ⁇ 20S( ⁇ 5A49S) by PI01 or ATZ4 in lysates of Dd2, Dd2( ⁇ 6A117D) and Dd2( ⁇ 5A49S), respectively, was assessed by their ability to block labeling of the parasites’ proteasomes by the activity-based fluorescent probe MV151 with 1 hour preincubation.
  • Figure 45E shows the mode of action of ATZ in parasites was assessed in Dd2, Dd2( ⁇ 6A117D) and Dd2( ⁇ 5A49S) cultures.
  • Figure 46 shows the heme-induced activation of the endoperoxides, yielding reactive radical intermediates of ART1 and ATZ2 capable of two types of covalent modification of ⁇ -casein.
  • Figures 47A-47C show the design of hybrids of ART and proteasome inhibitors and their inhibition of proteasomes and of parasite growth.
  • Figure 47A shows the structures of proteasome inhibitor, ART analog and hybrids.
  • Figure 47B shows the inhibition of P ⁇ 20S, human c-20S and i-20S.
  • Figure 47C shows the growth inhibition of Dd2, Dd2 ⁇ 5A48S and Dd2p6Al 17D by PI01, ART1 and ATZ3.
  • Figures 48A-48D show the mode of action of ATZ in the degradation of ⁇ -casein by 20S.
  • Figure 48A is an illustration of degradation of ⁇ -casein by human i-20S following incubation with ART or ATZ activated by hemin and ascorbate.
  • Figure 48B shows the degradation of ⁇ -casein.
  • ⁇ -casein was treated under indicated conditions (a, b or c).
  • Left panel after dialysis to remove the inhibitors, hemin, and ascorbate, the treated ⁇ -casein was incubated with i-20S and PA28a with bovine serum albumin as an internal control. Aliquots were taken at indicated times and samples run on SDS-page and stained with Coomassie blue.
  • Figure 48C is the MS/MS spectrum of the ATZ2 modified peptide SLVYPFPGP 80 (SEQ ID: 1).
  • the inserted mono-isotope peak at m/z 894.45557 matches the theoretical mass of the aforementioned peptide modified by ATZ2. This peptide was not observed in PI01 treated nor in ART1- treated ⁇ -casein samples through manual check.
  • Figure 48D is the MS/MS spectrum of the ART1 modified peptide F 67 AQTQSLVYPFPGPIPN (SEQ ID:2).
  • the inserted mono-isotope peak at m/z 1101.07361 matches the mass of the aforementioned peptide modified by ART1. This peptide was not observed in PI01 treated nor in ATZ2- treated ⁇ -casein samples through manual check.
  • Figure 49 shows the labelling inhibition of P ⁇ 20S in Dd2 parasites treated with DMSO, PI01, ART1, AZT4, PI01/ART1 (1 : 1) or DHA, assessed by their ability to block labeling of the parasites’ proteasomes by MV151.
  • Parasites were treated with indicated compounds for 6 hours and extracellular compounds were removed prior to hypotonic lysis of red blood cells.
  • Figure 50 is the 3 H NMR spectrum of ((R)-3-methyl-1-(2- ((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)butyl)boronic acid (HZ2082).
  • Figure 51 is the 13 C NMR spectrum of compound HZ2082.
  • Figure 52 is the 3 H NMR spectrum of ((R)-3-methyl-1-(2-(2-)
  • Figure 53 is the 13 C NMR spectrum of compound HZ2083.
  • Figure 54 is the 3 H NMR spectrum of ((R)-3-methyl-1-(3-(2-
  • Figure 55 is the 13 C NMR spectrum of compound HZ2087.
  • Figure 56 is the 3 H NMR spectrum of ((R)-3-methyl-1-(4-(2-
  • Figure 57 is the 13 C NMR spectrum of compound HZ2088.
  • Figure 58 is the 3 H NMR spectrum of N -((1R )-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2- ((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyl decahydro- 12H-3 ,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-3).
  • Figure 59 is the 13 C NMR spectrum of compound DQ-3.
  • Figure 60 is the 1 H NMR spectrum of N-(2-oxo-2-(((1R)-2-phenyl-l-((3aS,4S,6S)-
  • Figure 61 is the 13 C NMR spectrum of compound DQ-4.
  • Figure 62 is the 1 H NMR spectrum of N-((1R)-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-
  • Figure 63 is the 13 C NMR spectrum of compound DQ-7.
  • Figure 64 is the 1 H NMR spectrum of N-((R)-2-(benzofuran-3-yl)-l-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)ethyl)-2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-9).
  • Figure 65 is the 13 C NMR spectrum of compound DQ-9.
  • Figure 66 is the 1 H NMR spectrum of N-((R)-2-(benzofuran-3-yl)-l-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)ethyl)-2-((2R,3R,3aS,3a1R,6R,6aS,9S,10aR )-3,6,9- trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide (DQ-10).
  • Figure 67 is the 13 C NMR spectrum of compound DQ-10.
  • Figure 68 shows that HZ2083 causes apoptosis of multiple myeloma cells.
  • Figure 69 shows apoptotic signal transduction in MM.1S and CAG cell lines following exposure to HZ2083 and control compounds HZ2182 and artesunate.
  • Figure 70 shows that HZ2083 causes activation of caspase 3/7 in MM. IS and cell -based proteasome inhibition.
  • Figure 71 shows HZ3046 labeling profile in MM. 1 S cells
  • a first aspect of the present application relates to an Artemisinin-Proteasome inhibitor conjugate including a compound of Formula (I): wherein
  • R a is independently selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle;
  • R b is independently selected from group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle, and wherein R a and R b may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
  • X is O, S, or N
  • Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor
  • Z is O or 0-0
  • Proteasome inhibitor is a compound that inhibits either chymotryptic-like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
  • alkyl means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 12 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3 -pentyl.
  • alkane refers to aliphatic hydrocarbons of formula C n H 2n+2 , which may be straight or branched having about 1 to about 40 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8) carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkyl chain.
  • Exemplary alkanes include methane, ethane, n-propane, i-propane, n-butane, t-butane, n-pentane, and 3 -pentane.
  • alkylene refers to a divalent group formed from an alkane by removal of two hydrogen atoms.
  • Exemplary' alkylene groups include, but are not limited to, divalent groups derived from the alkanes described above.
  • alkenyl means an aliphatic hydrocarbon group containing a carbon — carbon double bond and which may be straight or branched having about 2 to about 12 carbon atoms in the chain. Particular alkenyl groups have 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkenyl chain. Exemplary alkenyl groups include ethenyl, propenyl, n- butenyl, and i-butenyl. The term “alkenyl” may also refer to a hydrocarbon chain having 2 to 6 carbons containing at least one double bond and at least one triple bond.
  • alkynyl means an aliphatic hydrocarbon group containing a carbon — carbon triple bond and which may be straight or branched having about 2 to about 20 carbon atoms in the chain. Particular alkynyl groups have 2 to about 10 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkynyl chain. Exemplary alkynyl groups include ethynyl, propynyl, n- butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.
  • alkenylene means a group obtained by removal of a hydrogen atom from an alkenyl group.
  • cycloalkyl means a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, preferably of about 3 to about 8 carbon atoms.
  • exemplary monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • cycloalkylalkyl means a cycloalkyl-alkyl-group in which the cycloalkyl and alkyl are as defined herein.
  • exemplary cycloalkylalkyl groups include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclopropyl ethyl, cyclobutyl ethyl, and cyclopentylethyl.
  • the alkyl radical and the cycloalkyl radical may be optionally substituted as defined herein.
  • cycloalkenyl means a non-aromatic mono- or multicyclic ring system containing a carbon — carbon double bond of about 4 to about 12 carbon atoms, preferably of about 5 to about 7 carbon atoms.
  • exemplary monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like.
  • cycloalkenyalkyl means a cycloalkenyl-alkyl-group in which the cycloalkenyl and alkyl are as defined herein.
  • exemplary cycloalkenylalkyl groups include cyclopropenylmethyl, cyclobutenylmethyl, cyclopentenylmethyl, cyclopropenylethyl, cyclobutenylethyl, and cyclopentenyl ethyl.
  • the alkyl radical and the cycloalkenyl radical may be optionally substituted as defined herein.
  • cycloalkynyl means a non-aromatic mono- or multicyclic ring system containing a carbon — carbon triple bond of about 5 to about 12 carbon atoms, preferably of about 5 to about 8 carbon atoms.
  • exemplary monocyclic cycloalkenyls include cyclopentynyl, cyclohexynyl, cycloheptynyl, and the like.
  • cycloalkynyalkyl means a cycloalkynyl-alkyl-group in which the cycloalkynyl and alkyl are as defined herein.
  • exemplary cycloalkynylalkyl groups include cyclopropynylmethyl, cyclobutynylmethyl, cyclopentynylmethyl, cyclopropynylethyl, cyclobutynyl ethyl, and cyclopentynyl ethyl.
  • the alkyl radical and the cycloalkynyl radical may be optionally substituted as defined herein.
  • alkoxy means groups of from 1 to 12 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyl oxy, cyclohexyloxy, and the like. Lower-alkoxy refers to groups containing one to four carbons.
  • alkoxy also includes methylenedi oxy and ethylenedioxy in which each oxygen atom is bonded to the atom, chain, or ring from which the methylenedioxy or ethylenedioxy group is pendant so as to form a ring.
  • phenyl substituted by alkoxy may be, for example,
  • aryl means an aromatic monocyclic or multi cyclic ring system of 6 to about 14 carbon atoms, preferably of 6 to about 10 carbon atoms.
  • Representative aryl groups include phenyl, naphthyl, and anthracenyl.
  • aryloxy means -O-aryl, in which aryl is as defined herein.
  • arylene means a group obtained by removal of a hydrogen atom from an aryl group.
  • Non-limiting examples of arylene include phenylene and naphthylene.
  • arylalkyl or “alkylaryl” means an alkyl substituted with one or more aryl groups, wherein the alkyl and aryl groups are as herein described.
  • arylmethyl or aryl ethyl group in which a single or a double carbon spacer unit is attached to an aryl group, where the carbon spacer and the aryl group can be optionally substituted as described herein.
  • Representative arylalkyl groups include , and
  • aralkoxy or “arylalkoxy” means -O-alkylaryl or -O-arylalkyl, in which arylalkyl and alkylaryl are as defined herein.
  • heteroaryl or “Het” means an aromatic monocyclic or multi cyclic ring system of about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is/are element(s) other than carbon, for example, nitrogen, oxygen, or sulfur. In the case of multicyclic ring system, only one of the rings needs to be aromatic for the ring system to be defined as "Heteroaryl”. Preferred heteroaryls contain about 5 to 6 ring atoms.
  • heteroaryl means that at least a nitrogen, oxygen, or sulfur atom, respectively, is present as a ring atom.
  • a nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N-oxide.
  • heteroaryls include pyridyl, 2-oxo-pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, furanyl, pyrrolyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, indolyl, isoindolyl, benzofuranyl, benzothiophenyl, indolinyl, 2-oxoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, indazolyl, benzimidazolyl, benzooxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, benzotriazolyl,
  • biheteroaryl or “bi-heteroaryl” refers to a heteroaryl group substituted by another heteroaryl group.
  • heterocyclyl or “heterocycle” or “heterocycloalkyl” refers to a stable 3- to 18-membered ring (radical) which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • the heterocycle may be a monocyclic, or a polycyclic ring system, which may include fused, bridged, or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycle may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the ring may be partially or fully saturated.
  • heterocycles include, without limitation, azepinyl, azocanyl, pyranyl dioxanyl, dithianyl, 1,3-dioxolanyl, tetrahydrofuryl, dihydropyrrolidinyl, decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2- oxopyrrolidinyl, 2-oxoazepinyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholiny
  • biheterocyclyl or “bi-heterocyclyl” refers to a heterocyclyl group substituted by another heterocyclyl or heterocycle group.
  • non-aromatic heterocycle means a non-aromatic monocyclic system containing 3 to 10 atoms, preferably 4 to about 7 carbon atoms, in which one or more of the atoms in the ring system is/are element(s) other than carbon, for example, nitrogen, oxygen, or sulfur.
  • Non-aromatic heterocycle groups include pyrrolidinyl, 2-oxopyrrolidinyl, piperidinyl, 2-oxopiperidinyl, azepanyl, 2-oxoazepanyl, 2-oxooxazolidinyl, morpholino, 3- oxomorpholino, thiomorpholino, 1,1 -di oxothiomorpholino, piperazinyl, tetrohydro-2H-oxazinyl, and the like.
  • bicyclic used herein indicates a molecular structure having two ring
  • polycyclic or “multi-cyclic” used herein indicates a molecular structure having two or more rings, including, but not limited to, fused, bridged, or spiro rings.
  • boronic acid complexing agent refers to any compound having at least two functional groups, each of which can form a covalent bond with boron. Nonlimiting examples of suitable functional groups include amino and hydroxyl.
  • moiety derived from a boronic acid complexing agent refers to a moiety formed by removing the hydrogen atoms from two functional groups of a boronic acid complexing agent.
  • a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable.
  • the protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or “deprotection” occurs after the completion of the reaction or reactions in which the functionality would interfere.
  • halo or halogen means fluoro, chloro, bromo, or iodo.
  • benzyl or Bn means -CH 2 -Ph or -CH 2 Ph group.
  • substituted or “substitution” of an atom means that one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded.
  • Up to three H atoms in each residue are replaced with alkyl, halogen, haloalkyl, hydroxy, loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryl oxy.
  • the term “method of treating” means amelioration or relief from the symptoms and/or effects associated with the disorders described herein. As used herein, reference to “treatment” of a patient is intended to include prophylaxis.
  • pharmaceutically acceptable salts means the relatively non-toxic, inorganic, and organic acid addition salts, and base addition salts, of compounds of the present application. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane— sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsul
  • Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed.
  • Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred.
  • Suitable inorganic base addition salts are prepared from metal bases which include, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.
  • Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use, such as ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine,
  • prodrugs as used herein means those prodrugs of the compounds useful according to the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of this application.
  • prodrug means compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. Functional groups which may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this application.
  • alkanoyl such as acetyl, propionyl, butyryl, and the like
  • unsubstituted and substituted aroyl such as benzoyl and substituted benzoyl
  • alkoxycarbonyl such as ethoxycarbonyl
  • trialkylsilyl such as trimethyl- and triethysilyl
  • monoesters formed with dicarboxylic acids such as succinyl
  • the compounds bearing such groups act as pro-drugs.
  • the compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group.
  • prodrugs A thorough discussion of prodrugs is provided in the following: Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309-396 (1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen and H.
  • solvate refers to compounds of the present application in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered.
  • suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate.
  • solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • Compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • Each chiral center may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. This technology is meant to include all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms.
  • Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the basic nitrogen can be quatemized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
  • lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromides and iodides
  • dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates
  • the Artemi sinin-Proteasome inhibitor conjugate includes a compound of Formula (I'): wherein
  • R a is independently selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle;
  • R b is independently selected from group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy, NH 2 , aryl, heteroaryl, and non-aromatic heterocycle, and wherein R a and R b may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
  • X is O, S, or N
  • Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor
  • Proteasome inhibitor is a compound that is known to inhibit either chymotryptic- like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome activity, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
  • One embodiment relates to the Artemi sinin-Proteasome inhibitor conjugate, wherein Linker is selected from the group consisting of
  • the Proteasome inhibitor moiety includes a compound of Formula (II): wherein is the point of attachment to the Linker;
  • R' is H or C 1-6 alkyl
  • R 1 ’ is selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non — aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy
  • R 2’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, — (CH 2 ) m C(O)NHR 6 , — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic
  • R 2 ’ and R y are taken together with the carbon to which they are attached to form a C 3-8 cycloalkyl ring;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, —
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, — CF 3 , C 1-6 alkyl, C 3-8 cycloalkyl, — (CH 2 )kOH, and arylalkyl, wherein C 3-8 cycloalkyl can be optionally substituted with — CF 3 ; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R 12 ; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring;
  • R 10 is H or arylalkyl
  • R 11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF 3 , C 1-6 alkyl, C 3-8 cycloalkyl, aryl, and arylalkyl, wherein C 1-6 alkyl, C 3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R 12 ;
  • R 12 is selected from the group consisting of H, halogen, C 1-6 alkyl, C 3-8 cycloalkyl, and aryl, wherein C 1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
  • R x is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 , — (CH 2 ) m C(O)NR 6 R 7 , and — CH 2 C(O)R 5 ;
  • R y is independently selected at each occurrence thereof from the group consisting of H, D, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy, and the C 1-12 alkyl, C 2-12 alkenyl, and C 2-12 alkynyl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
  • Q is optional and, if present, is C 1-3 alkyl or — C(Y) — ;
  • Q 1 is optional, and, if present, is selected from NH, — (CR 3 ’H) — , — NH- (CR Z H) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle;
  • R z is independently selected at each occurrence thereof from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 3-12 cycloalkyl, C 4-12 cycloalkenyl, C 5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, and (cycloalkynyl)alkyl, wherein C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 3-12 cycloalkyl, C 4-12 cycloalkenyl, C 5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkynyl)alkyl can be optionally substituted 1 time with R z ;
  • R z is independently selected at each occurrence thereof from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;
  • X is a bond, — C(Y)— , — (CH 2 ) q — , — O— , or — (CD 2 ) q — ;
  • Y is O or S
  • Z 1 and Z 2 are each independently OH, C 1-6 alkoxy, aryloxy, or aralkoxy; or Z 1 and Z 2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1, 2, 3, or 4; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2, or 3.
  • Z 1 and Z 2 together with the boron atom to which they are attached form a moiety derived from a boronic acid complexing agent.
  • This moiety derived from a boronic acid complexing agent can be where R 13 can be H or C 1-6 alkyl, R 14 can be H or C 1-6 alkyl, R 15 can be H or C 1-6 alkyl, and R 16 can be H or C 1-6 alkyl.
  • Suitable moi eties derived from a boronic acid complexing agent that can be used according to the present application include
  • the Proteasome inhibitor moiety includes a compound of Formula (II): wherein is the point of attachment to the Linker;
  • R' is H or C 1-6 alkyl
  • R 1 ’ is selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-12 alkyl, C 1-6 alkoxy,
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy; or
  • R 2 ’ and R y are taken together with the carbon to which they are attached to form a C 3-8 cycloalkyl ring;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, —
  • R 5 ’ is selected from the group consisting of H, non-aromatic heterocycle, — NR 6 R 7 , — CR 8 R 9 , C 1-12 alkyl, monocyclic or bicyclic C 3-10 cycloalkyl, C 3-12 cycloalkylalkyl, C 1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C 1-12 alkyl, monocyclic or bicyclic C 3-10 cycloalkyl, C 3-12 cycloalkylalkyl, C 1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with R 11 ;
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, — CF 3 , C 1-6 alkyl, — (CH 2 )kOH, and arylalkyl; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R 12 ; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring;
  • R 10 is H or arylalkyl
  • R 11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF 3 , C 1-6 alkyl, C 3-8 cycloalkyl, aryl, and arylalkyl, wherein C 1-6 alkyl, C 3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R 12 ;
  • R 12 is selected from the group consisting of H, halogen, C 1-6 alkyl, C 3-8 cycloalkyl, and aryl, wherein C 1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
  • R x is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 , — (CH 2 ) m C(O)NR 6 R 7 , and — CH 2 C(O)R 5 ;
  • R y is independently selected at each occurrence thereof from the group consisting of H, D, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy, and the C 1-12 alkyl, C 2-12 alkenyl, and C 2-12 alkynyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
  • Q is optional and, if present, is C 1-3 alkyl or — C(Y) — ;
  • Q 1 is optional, and, if present, is selected from NH, — (CR 3 ’H) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle;
  • X is a bond, — C(Y)— , — (CH 2 ) q — , — O— , or — (CD 2 ) q — ;
  • Y is O or S
  • Z 1 and Z 2 are each independently OH, C 1-6 alkoxy, aryloxy, or aralkoxy; or Z 1 and Z 2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2 or 3.
  • One embodiment relates to the proteasome inhibitor moi eties of Formula (II), where R 1 ’ is selected from the group consisting of , ; and R 11 is selected from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy, wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (II).
  • a further embodiment relates to the proteasome inhibitor moi eties of Formula (II) and wherein is the point of attachment to Q or Q 1 ; and is the point of attachment to halogen, NH 2 , NHCOOC 1-12 alkyl, or C 1-12 alkyl.
  • R 2 is selected from the group consisting of H, Me, — CH 2 (Me) 2 , — CH 2 OMe, wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (II).
  • One embodiment relates to the proteasome inhibitor moieties of Formula (II) where R 3 ’ is selected from the group consisting of H, CH 3 , — CH 2 OMe, — CH 2 C(O)OH, — point of attachment to the corresponding carbon atom of the structure of Formula (II).
  • the Proteasome inhibitor moiety includes a compound of Formula (III): wherein is the point of attachment to the Linker;
  • R 1 ’ is selected from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , C 1-6 alkyl, C 1-6 alk
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, — (CH 2 ) m C(O)NR 6 R 7 , — (CH 2 ) m C(O)0H, and — (CH 2 ) m C(O)OBn, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, wherein C 1-6 alkyl, C 12-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , —
  • R 5 ’ is selected from the group consisting of H, C 1-6 alkyl, C 1-6 alkoxy, non— aromatic heterocycle, — NR 6 R 7 , and — CR 8 R 9 ; and C 3-8 cycloalkyl, wherein C 3-8 cycloalkyl can be optionally substituted with — CF 3 ;
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, C 1-6 alkyl, and — (CH 2 ) k OH; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring;
  • R x is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 , — (CH 2 ) m C(O)NR 6 R 7 , and — CH 2 C(O)R 5 ;
  • R y is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CHiAr, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • X is C(O), — (CH 2 ) q — , — O— , or — (CD 2 ) q — ;
  • Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; s is 0 or 1; and
  • the Proteasome inhibitor moiety includes a compound of Formula (Illa): wherein is the point of attachment to the Linker; R 1 ’ is selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substitute
  • R 2 ’ is H or C 1-6 alkyl
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH 2 ) m C(O)NHR 5 ’, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , — OC 1-6 alkyl, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
  • R 5 ’ is selected from the group consisting of H, C 1-6 alkyl, and C 3-8 cycloalkyl, wherein C 3-8 cycloalkyl can be optionally substituted with — CF 3 ;
  • R 1 ’ is a substituted or unsubstituted biphenyl, or a substituted or unsubstituted hetero aryl;
  • R 2 ’ is H
  • R 3 ’ is — (CH 2 ) m C(O)NHR 5 ’;
  • R 5 ’ is a C 1-6 alkyl
  • Y is O; and n is 1.
  • Exemplary Artemi sinin-Proteasome inhibitor conjugates of the present application with a Proteasome inhibitor moiety of Formula (Illa) include, but are not limited to
  • the Proteasome inhibitor moiety includes a compound of Formula (Illb) : wherein is the point of attachment to the Linker; L is — (CR 3 ’R x ) p — ;
  • M is — (CR 2 ’ y ) r —
  • R 1 ’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy;
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, — (CH 2 ) m C(O)NR 6 R 7 , — (CH 2 ) m C(O)OH, and — (CH 2 ) m C(O)OBn;
  • R 5 ’ is selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, non-aromatic heterocycle, — NR 6 R 7 , and — CR 8 R 9 ;
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, C 1-6 alkyl, and — (CH 2 ) k OH; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring;
  • R x is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 , — (CH 2 ) m C(O)NR 6 R 7 , and — CH 2 C(O)R 5 ;
  • R y is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • X is — (CH 2 ) q — , — O— , or — (CD 2 ) q — ;
  • Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2; and s is 0 or 1.
  • the Proteasome inhibitor moiety includes a compound of Formula (IIIc), Formula (IIId), or Formula (Hie) : wherein is the point of attachment to the Linker;
  • R 1 ’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy;
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, and — (CH 2 )mC(O)NR 6 R 7 ;
  • R 5 ’ is selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, non-aromatic heterocycle, — NR 6 R 7 , and — CR 8 R 9 ;
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, C 1-6 alkyl, and — (CH 2 ) k OH; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring; R x is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 , — (CH 2 ) m C(O)NR 6 R 7 , and — CH 2 C(O)R 5 ;
  • R y is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • X is — (CH 2 )q— , — O— , or — (CD 2 ) q — ;
  • Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; q is 0, 1, or 2; and s is 0 or 1.
  • the Proteasome inhibitor moiety includes a compound of Formula (IIIF): wherein is the point of attachment to the Linker;
  • R 1 ’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy;
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, D, C 1-6 alkyl, — CH 2 OC 1-6 alkyl, — CH 2 Ar, and — CH 2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C 1-6 alkyl, and C 1-6 alkoxy;
  • R 3 ’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH 2 OC 1-6 alkyl, — (CH 2 ) m C(O)NHR 5 ’, and — (CH 2 ) m C(O)NR 6 R 7 ;
  • R 5 ’ is selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, non-aromatic heterocycle, — NR 6 R 7 , and — CR 8 R 9 ;
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of H, D, C 1-6 alkyl, and — (CH 2 ) k OH; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R 8 and R 9 are taken together with the carbon to which they are attached to form an oxetane ring;
  • X is — (CH 2 ) q — , — O— , or — (CD 2 ) q — ;
  • Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; s is 0 or 1; and q is 0, 1, or 2.
  • the Proteasome inhibitor moiety includes a compound of Formula (Illg): wherein is the point of attachment to the linker;
  • W is CHR 3 ’or NR 3 ’;
  • X 1 is selected from the group consisting of — C(O)-NH — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle;
  • Y 1 is optional and, if present, is — (CH 2 ) m — ;
  • Z 1 is optional and, if present, is aryl or bicyclic heteroaryl, wherein aryl or bicyclic heteroaryl can be optionally substituted 1 or 2 times with H, halogen, NH 2 , NHCOOC 1- 12 alkyl, or C 1-12 alkyl;
  • R' is H or C 1-6 alkyl
  • R 2 ’ is H or C 1-6 alkyl
  • R y is H or C 1-6 alkyl; or R 2 ’ and R y are taken together with the carbon to which they are attached to form a C 3-8 cycloalkyl ring; R 3 is selected from the group consisting of C 1-6 alkyl, and — (CH 2 ) n C(O)NR 6 R 7 , wherein C 1-6 alkyl can be optionally substituted from 1 to
  • R 6 , R 7 are selected from the group consisting of H, C 1-6 alkyl, and arylalkyl; or R 6 and R 7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R 9 ;
  • R 9 is selected from the group consisting of H, halogen, C 1-6 alkyl, C 3-8 cycloalkyl, and aryl, wherein C 1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
  • R 10 is H or arylalkyl; k is 1 or 2; m is 0, 1, or 2; and n is 0, 1, 2, 3, or 4.
  • Another embodiment relates to the proteasome inhibitor moiety of Formula (Illg), where R 2 ’ and R y are taken together with the carbon to which they are attached to form group, and wherein is the point of attachment to NR'; and is the point of attachment to X .
  • R 2 ’ and R y may be taken together with the carbon to which they are attached to form group, and wherein is the point of attachment to NR'; and is the point of attachment to X 1 .
  • One embodiment relates to the Artemi sinin-Proteasome inhibitor conjugate, wherein the proteasome inhibitor is selected from the group consisting of
  • Proteasome inhibitor moieties of Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (IIIf), and Formula (Illg), useful in the present application are disclosed in U.S. Patent Serial No.: 9,988,421 to Lin et al.; and U.S. Patent Application Publication Nos.: 20180221431, 20180282317, and 20200317729 to Lin et al., which are hereby incorporated by reference in their entirety.
  • the Proteasome inhibitor moiety includes a compound of Formula (IV): wherein is the point of attachment to the Linker;
  • Y is wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV);
  • R 1 ’ is a H, branched, cyclic, or linear C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl, wherein the C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl may be optionally substituted from 1 to 3 times with R 3 ’;
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH 2 ) x C(O)NHR 4 ’, wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the
  • R 3 ’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy;
  • R 4 ’ is selected from the group consisting of H, C 1-6 alkyl, and C 3-8 cycloalkyl, wherein C 3-8 cycloalkyl can be optionally substituted with — CF 3 ;
  • the Proteasome inhibitor moiety includes a compound of Formula (IV): wherein is the point of attachment to the Linker; wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV);
  • R 1 ’ is a H, branched, cyclic, or linear C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl, wherein the C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl may be optionally substituted from 1 to 3 times with R 3 ’;
  • R 2 ’ is independently selected at each occurrence thereof from the group consisting of H, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH 2 ) x C(O)NHR 4 ’ , wherein C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO 2 , — CF 3 , — O C 1-6 alkyl, C 1-6 alkyl, C 2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl
  • R 3 ’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF 3 , C 1-6 alkyl, and C 1-6 alkoxy;
  • R 4 ’ is selected from the group consisting of H, C 1-6 alkyl, and C 3-8 cycloalkyl, wherein C 3-8 cycloalkyl can be optionally substituted with — CF 3 ;
  • Z 1 and Z 2 are each independently OH, C 1-6 alkoxy, aryloxy, or aralkoxy; or Z 1 and Z 2 together form a moiety derived from a boronic acid complexing agent; x is 1 or 2; and n is 0, 1, 2, or 3.
  • One embodiment relates to the proteasome inhibitor moieties of Formula (IV), where
  • R 1 ’ is a C4 alkyl
  • Z 1 and Z 2 are OH; and n is 0.
  • Proteasome inhibitor moiety of Formula (IV) include, but are not limited to
  • Exemplary Artemi sinin-Proteasome inhibitor conjugates of the present application with a Proteasome inhibitor moiety of Formula (IV) include, but are not limited to [0156] Further examples of the Proteasome inhibitor moiety of Formula (IV) useful in the present application are disclosed in U.S. Patent Serial No.: 8,871,745; 7,442,830; 7,687,662;
  • DCC dicyclohexyl carbodiimide
  • CDI N,N'- carbonyl diimidazole
  • EEDQ N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline
  • IIDQ N- isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline
  • EDC 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • benzotriazol-1-yl-oxy-tris- pyrrolidinophosphonium hexafluorophosphate commercially available as PyBOP® (Novabiochem, a division of Merck KGaA, Darmstadt, Germany)).
  • the reactions can be conducted in the presence of a base, for example a trialkylamine such as triethylamine or diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, 4-DMAP or 1,8- diazabicycle[5.4.0]undec-7-ene (DBU).
  • a base for example a trialkylamine such as triethylamine or diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, 4-DMAP or 1,8- diazabicycle[5.4.0]undec-7-ene (DBU).
  • the coupling reactions are preferably are conducted in an inert solvent, such as halogenated hydrocarbons, e.g. dichloromethane, chloroform, dipolar aprotic solvents such as acetonitrile, dimethylformamide, dimethylacetamide, DMSO, HMPT, and ethers such as tetrahydrofuran (THF).
  • a second aspect of the present application relates to a method of treating infectious diseases in a subject. This method includes administering to the subject in need thereof a compound of the present application.
  • the infectious disease is caused by bacterial, viral, parasitic, and fungal infectious agents.
  • the infectious disease is caused by a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium-intr acellular e, and Mycobacterium leprosy.
  • the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses, Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.
  • a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses, Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo
  • the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enter obius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.
  • a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium
  • the infectious disease is malaria.
  • another aspect of the present application is a pharmaceutical composition containing a therapeutically effective amount of the compound of Formula (I), Formula (I'), Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (Illf), Formula (Illg), and Formula (IV) or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • the carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof
  • the Artemi sinin-Proteasome inhibitor conjugates can be administered using any method standard in the art.
  • the Artemisinin- Proteasome inhibitor conjugates can be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.
  • the compositions of the present application may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the agents of the present application may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • Agents of the present application may also be administered in a time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage.
  • the agents of the present application may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • compositions and preparations should contain at least 0.1% of the agent, although lower concentrations may be effective and indeed optimal.
  • the percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of an agent of the present application in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • agents of the present application may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the component or components and increase in circulation time in the body examples include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
  • the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, sucrulose, or saccharin.
  • a liquid carrier such as a fatty oil.
  • tablets may be coated with shellac, sugar, or both.
  • a syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
  • the agents of the present application may also be administered parenterally.
  • Solutions or suspensions of the agent can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • agents of the present application When it is desirable to deliver the agents of the present application systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Intraperitoneal or intrathecal administration of the Artemisinin-Proteasome inhibitor conjugates of the present application can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.
  • the agents may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the agents of the present application may also be administered directly to the airways in the form of an aerosol.
  • the agent of the present application in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the agent of the present application also may be administered in a non— pressurized form such as in a nebulizer or atomizer.
  • Effective doses of the compositions of the present application, for the treatment of cancer or pathogen infection vary depending upon many different factors, including type and stage of cancer or the type of pathogen infection, means of administration, target site, physiological state of the patient, other medications or therapies administered, and physical state of the patient relative to other medical complications. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the percentage of active ingredient in the compositions of the present application may be varied such that a suitable dosage is obtained. Obviously, several unit dosage forms may be administered at about the same time. The dose employed will be determined by the physician and depends upon the desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient.
  • the doses are generally from about 0.01 to about 100 mg/kg body weight, preferably about 0.01 to about 10 mg/kg body weight per day by inhalation, from about 0.01 to about 100 mg/kg body weight, preferably 0.1 to 70 mg/kg body weight, more especially 0.1 to 10 mg/kg body weight per day by oral administration, and from about 0.01 to about 50 mg/kg body weight, preferably 0.01 to 10 mg/kg body weight per day by intravenous administration.
  • the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health, and other characteristics which can influence the efficacy of the medicinal product.
  • the Artemi sinin-Proteasome inhibitor conjugates according to the present application may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long— term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the active product may be administered orally 1 to 4 times per day. It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.
  • a third aspect of the present application relates to a method of treating cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders in a subject, or for providing immunosuppression for transplanted organs or tissues in a subject.
  • This method includes administering to the subject in need thereof a compound of the present application.
  • inhibitors could open a new path to the treatment of immunologic, autoimmune, inflammatory, neurodegenerative, and certain neoplastic disorders such as: arthritis, colitis, multiple sclerosis, lupus, Sjogren Syndrome, Systemic Lupus Erythematosus and lupus nephritis, glomerulonephritis, Rheumatoid Arthritis, Inflammatory bowel disease (IBD), ulcerative colitis, Crohn's diseases, Psoriasis, and asthma.
  • immunologic, autoimmune, inflammatory, neurodegenerative, and certain neoplastic disorders such as: arthritis, colitis, multiple sclerosis, lupus, Sjogren Syndrome, Systemic Lupus Erythematosus and lupus nephritis, glomerulonephritis, Rheumatoid Arthritis, Inflammatory bowel disease (IBD), ulcerative colitis, Crohn's diseases, Psoriasis, and asthma.
  • Exemplary inflammatory disorders that may be treated with the Artemisinin- Proteasome inhibitor conjugates, include, but are not limited to, Crohn’s disease, ulcerative colitis, arthritis, or lupus.
  • the Artemisinin-Proteasome inhibitor conjugates may provide immunosuppression useful for transplanted organs or tissues, and can be used to prevent transplant rejection and graft-verse-host disease.
  • cancer refers to a cellular disorder characterized by uncontrolled or disregulated cell proliferation, decreased cellular differentiation, inappropriate ability to invade surrounding tissue, and/or ability to establish new growth at ectopic sites.
  • cancer includes, but is not limited to, solid tumors and bloodborne tumors.
  • cancer encompasses diseases of skin, tissues, organs, bone, cartilage, blood, and vessels.
  • cancer further encompasses primary and metastatic cancers.
  • Non-limiting examples of solid tumors that can be treated with the disclosed proteasome inhibitors include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen— independent prostate cancer; renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma,
  • cancer is treated.
  • the cancer is selected from the group consisting of neoplastic disorders, hematologic malignances, lymphocytic malignancies, multiple myeloma, mantle cell lymphoma, leukemia, Waldenstrom Macroglobulinemia, pancreatic cancer, bladder cancer, colorectal cancer, chordoma cancer, breast cancer, metastatic breast cancer, prostate cancer, androgen-dependent and androgen-independent prostate cancer, renal cancer, metastatic renal cell carcinoma, hepatocellular cancer, lung cancer, non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung, ovarian cancer, progressive epithelial or primary peritoneal cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, squamous cell carcinoma of the head and neck, melanoma, neuroendocrine cancer, metastatic neuroendocrine tumors, brain tumors, gli
  • Another aspect of the present application relates to a pharmaceutical composition including a therapeutically effective amount of the compounds of the present application and a pharmaceutically acceptable carrier.
  • terapéuticaally effective amounts is meant to describe an amount of compound of the present application effective in inhibiting the proteasome or immunoproteasome and thus producing the desired therapeutic effect. Such amounts generally vary according to a number of factors well within the purview of ordinarily skilled artisans given the description provided herein to determine and account for. These include, without limitation: the particular subject, as well as its age, weight, height, general physical condition, and medical history; the particular compound used, as well as the carrier in which it is formulated and the route of administration selected for it; and, the nature and severity of the condition being treated.
  • composition means a composition comprising a compound of the present application and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifingal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
  • pharmaceutically acceptable carriers such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifingal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
  • suspending agents examples include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar — agar and tragacanth, or mixtures of these substances.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin.
  • suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate.
  • disintegrating agents include starch, alginic acids, and certain complex silicates.
  • lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
  • pharmaceutically acceptable means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable dosage forms means dosage forms of the compound of the application, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.
  • Artemisinin a sesquiterpene lactone isolated from the Artemisia annua Chinese herb, and clinical use of its analogues (e.g. artemether, arteether and artesunate) were a major breakthrough in malaria chemotherapy because they produce a very rapid therapeutic response, particularly against ring stage Plasmodium falciparum malaria including multidrug-resistant strains. Despite the rapid clearance of parasites, the short half-lives of these compounds lead to recrudescence of parasitemia after monotherapy. Therefore, artemisinin-based combination therapy (ACT) has now been recommended by the World Health Organization as standard therapy for falciparum malaria.
  • ACT artemisinin-based combination therapy
  • artemisinin and its derivatives have attracted attention as promising anticancer agents because they have potent antineoplastic activity.
  • Antineoplastic activity is postulated to be through a variety of molecular mechanisms in both drug-sensitive and drug-resistant cancer cell lines.
  • Growing experimental evidence demonstrates the great potential of artemisinin compounds for use as a therapeutic alternative to treat highly aggressive cancers and for use as part of anticancer combination therapies without causing drug resistance or added side effects.
  • the proteasome is a large multi -protease complex and is responsible for the controlled degradation of more than 80% of cellular proteins. As such, the proteasome plays a key role in maintaining cellular protein homeostasis and regulates numerous biological processes, such as cell survival, DNA repair, apoptosis, signal transduction, and antigen presentation. To date, the proteasome has been successfully exploited as a therapeutic target to treat human cancers. There are three proteasome inhibitor (PI) drugs in clinical use. Propelled by exemplary academic-industrial partnerships, drug development targeting the proteasome has expanded from cancer to autoimmune diseases and recently to infections.
  • PI proteasome inhibitor
  • Plasmodium proteasome inhibitors are reported to be active at multiple stages of the parasite life cycle and synergize with artemisinins.
  • Hybrid molecules are combinations of two or more drugs that have varied biological activities and mechanisms; these combinations may improve the efficacy of the drugs by enhancing their bioavailability and by avoiding drug resistance.
  • hybridization via the covalent coupling of two biologically active compounds has been considered a useful strategy for drug development.
  • the present application relates to Artemi sinin-proteasome inhibitor hybrid compounds. These compounds are useful for inhibiting the activity of human proteasome and Plasmodium proteasome and may be used in the treatment of human cancers and malaria.
  • the human constitutive proteasome (c-20S, Catalog No.: E-360), human 20S immunoproteasome (i-20S, Catalog No.: E-370), and recombinant human PA28 activator alpha subunit (Catalog NO.: E-381) were purchased from Boston Biochem.
  • the P. falciparum 20S proteasome (Pf20S) was purified as reported (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Sci. USA 115(29):E6863-E6870 (2016), which is hereby incorporated by reference in its entirety).
  • ⁇ -Casein (Catalog No.: C6905), bovine serum albumin (BSA, Catalog No.: 3117057001), hemin (Catalog No.: 51280), sodium ascorbate (Catalog No.: PHR1279), artemisinin (ART, Catalog No.: 361593), and artesunate (ASU, Catalog No.: A3731) were purchased from Sigma-Aldrich. Trypsin (V528A) and chymotrypsin (V106A) were purchased from Promega.
  • Proteasome ⁇ 5 substrate suc-LLVY-AMC, ⁇ 5i substrate Ac-ANW-AMC, 01 and ⁇ 1i substrate Z-LLE-AMC, and 02 and ⁇ 2i substrate Z-VLR-AMC were purchased from Boston Biochem.
  • Activity -based probe MV151 was synthesized as reported (Verdoes et al., “A Fluorescent Broad-Spectrum Proteasome Inhibitor for Labeling Proteasomes in vitro and in vivo,” Chem. Biol. 13(11): 1217-1226 (2006), which is hereby incorporated by reference in its entirety).
  • 02-Specific inhibitor WLW-VS was prepared following the reported method (O'Donoghue et al., “Structure- and Function-based Design of Plasmodium-selective Proteasome Inhibitors,” Nature 530(7589):233-236 (2016), which is hereby incorporated by reference in its entirety).
  • the crude product was purified by prep-HPLC (column: OBD Cl 8 150mm*19mm*5um; mobile phase: [water (0.1%TFA)-ACN]; B%: 5%-95%, 20min) and lyophilisation to afford boronic acid as a white solid.
  • Benzyl N 2 -(tert-Butoxycarbony)-N 4 -(tert-butyl)-L-asparaginate (1) was synthesized by following the general procedure for HATU mediated coupling of Boc-Asp-OBn (3.55g, 11 mmol) and tert-butyl amine (0.73 g, 10 mmol). The isolated off-white product (2.95g, 78%) was used in next step without further purification.
  • N-(2-Aminoethyl)-2',4-difluoro-[ l , l '-biphenyl]-3-carboxamide trifluoroacetate salt (3) was synthesized by two successive steps, one following the general procedure for HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid (141.6 mg, 605 ⁇ mol) and tert- butyl N-(2-aminoethyl)carbamate (88.1 mg, 550 ⁇ mol) and the other following the general procedure for Boc-deprotection of the product in first step.
  • the isolated white product (172.0 mg, two step yield: 80%) was used in next step without further purification.
  • HZ2082, HZ2083, HZ2087, and HZ2088 are HZ2082, HZ2083, HZ2087, and HZ2088.
  • HZ2082 was obtained as a white solid.
  • HZ2087 was obtained as a white solid.
  • HZ2088 was obtained as a white solid.
  • the bulkier catalyst (S, S)-Fe(CF 3 - PDP) could alter the inherent selectivity to favor oxidation at the electron-rich and less sterically hindered C7 position to afford the C7 ketone 13, which converted into 14 by reductive amination.
  • three efficient mutants II-H10, IV-H4 and X-E12 were identified that catalyzed selective hydroxylation of C7(R), C7(S) and C6a of 10 to give 15, 16, and 17, respectively. Esterification of 15, 16, and 17 with succinic anhydride gave 18, 19, 20, respectively.
  • 15 and 17 were converted into primary amine 21 and 25, respectively, in two steps, which further modified into 22 and 26 via late-stage diversifications.
  • 16 and 17 could further functionalized into aryl or heteroaryl ether-based building block 23 and 24, respectively, via Mitsunobu reaction with hydroxy aromatics.
  • IC 50 values of all compounds against Pf20S ⁇ 5, human c-20S ⁇ 5c, ⁇ 2c, ⁇ 1c and i- 20S ⁇ 5i ⁇ 2i, ⁇ 1i were determined in a 96-well format as described (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Set.
  • the fluorogenic substrate suc-LLVY-AMC (SEQ ID:3) was used for Pf20S c-20S and ⁇ 5c at final concentration 25 ⁇ M, and Ac-ANW-AMC was used as substrate of i-20S and ⁇ 5i at final concentration 15 ⁇ M.
  • Activator PA28a at final concentration of 12 nM was used for Pf20S assay in the presence of 0.5 ⁇ M of WLW-VS, whereas 0.02% SDS was used in the assays for c-20S and i-20S, as well as for human ⁇ 5c, ⁇ 2c, ⁇ 1c, ⁇ 5i, ⁇ 2i, and ⁇ 1i.
  • Z-LLE-AMC was used as substrate of ⁇ 1c or ⁇ 1i at final concentration 50 ⁇ M.
  • Z-VLR-AMC was used as substrate of ⁇ 2c or ⁇ 2i at final concentration 50 ⁇ M.
  • Final concentrations of Pf20S, C-20S, and i-20S were 1 nM, 0.2 nM, and 0.4 nM, respectively.
  • the fluorescence of the hydrolyzed AMC at Ex 360nm and Em 460 nm in each well was followed for 1-2 hours. Linear ranges of the time course were used to calculate the velocities in each well, which were fit to a dose-dependent inhibition equation to estimate the IC 50 values (Table 2, Table 3, and Table 4) in PRISM (GraphPad).
  • Table 2 displays the summary of compounds’ enzyme inhibition, parasite growth inhibition, and cytotoxic activity against HepG2 human hepatoma cells for ART1, PI01, ATZ1, ATZ2, ATZ3, and ATZ4.
  • Table 4 displays the growth inhibition of artemisinin-proteasome inhibitor conjugates HZ2082, HZ2083, HZ2087, and HZ2088 against Multiple myeloma MM1S, live cancer HepG2, and P. falciparum 3D7.
  • Ring survival assays were performed as described (Straimer et al., “Drug Resistance. K13-propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015), which is hereby incorporated by reference in its entirety).
  • Parasite cultures, IPC5202 (Cam3.1 R539T ), an artemisinin resistant parasite line from Cambodia, and the genetically engineered artemisinin sensitive revertant Cam3.1 Rev were synchronized several times with 5% sorbitol and then a Percoll-sorbitol gradient was used to obtain tightly synchronized late stage parasites.
  • Isolated late stage parasites were then allowed to reinvade fresh red blood cells for three hours before ring stage parasites were confirmed by microscopy before the cultures were again subjected to 5% sorbitol to obtain 0-3 hour rings.
  • the isolated ring stage cultures were then plated into a 96 well plate at 0.5% parasitemia at the corresponding drug concentrations: DHA 700 nM, PI01 800 nM, ART1 800 nM, ATZ3 700 nM, and ATZ4 700 nM. Plates were incubated at 37 °C in standard gas conditions for six hours before the plates were spun and washed to remove medium with compound and replenished with fresh medium. Plates were then incubated for an additional 66 hours and parasite growth was then assessed using flow cytometry and nucleic acid stains: Hoechst 33342 (HO) and thiazole orange (TO).
  • HO Hoechst 33342
  • TO thiazole orange
  • Pf Dd2, Dd2( ⁇ 6A117D), and Dd2( ⁇ 5A49S) parasites were grown synchronized to a high parasitemia (5-8%).
  • 5 mL of parasite-infected red blood cells were exposed to DMSO, PI01 (800 nM), ART1 (800 nM), ATZ4 (700 nM), and a mixture of ART1 and PI01 in a 1 : 1 ratio both at 800 nM for 6 hours. After centrifugation at 600 rpm for 5 minutes, the supernatant was removed, and red cells were washed with complete medium once and resuspended in 10 mL of fresh medium.
  • Example 8 Inhibition of Pf20S, Pf20S( ⁇ 6A117D0 and Pf20S( ⁇ 5A49S) by PI01 and ATZ4
  • Cell free lysates of P. falciparum Dd2 wild-type and two Dd2-derived resistant (Dd2 ⁇ 5A49S and Dd2 ⁇ 6A117D) were used.
  • 5 - 10 pg of total lysate proteins were incubated with PI01 or ATZ4 at the indicated concentrations for 1 hour at 37 °C prior to addition of MV151 and incubated for a further 1 hour at 37 °C.
  • the samples were then heated with 4X SDS loading buffer at 95 °C for 10 min and run on 12% NovexTM Bis-Tris Protein Gels.
  • the gels were rinsed with double distilled H 2 O and then scanned on the Typhoon Scanner.
  • HepG2 (HB-8065, ATCC) were cultured at 37 °C in a humidified air/5% CO 2 atmosphere in medium supplemented with 10% fetal bovine serum and 100 ug per ml penicillin, 100 pg per ml streptomycin in DMEM medium. HepG2 was used at 5,000 cells per well. Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO 2 . Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
  • ⁇ -Casein dissolved in PBS (10 ⁇ M) was incubated with 100 ⁇ M of PI01, ART1 or ATZ2 in the presence of sodium ascorbate (200 ⁇ M) and hemin (100 ⁇ M) at r.t. for 4 hours.
  • the samples were transferred to Slide-A-Lyzer MINI Dialysis Devices (10K MWCO, Thermo ScientificTM (Waltham, MA) 88401), placed into tubes containing the dialysis buffer (20 mM HEPES and 0.5 mM EDTA, pH7.5), and dialyzed overnight at 4 °C with fresh dialysis buffer changing every 4 hours.
  • MM. IS CRL-2974, ATCC
  • MM. 1S was used at 100,000 cells per well.
  • Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO 2 .
  • Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
  • ⁇ -Casein was treated as in the aforementioned example. After removing the inhibitors, hemin, and ascorbate by dialysis, the treated ⁇ -casein samples were run on SDS-page and stained with Coomassie blue G-250. The gel bands of ⁇ -casein were cut into pieces.
  • Samples were reduced with 5 mM dithiothreitol in 50 mM ammonium bicarbonate buffer for 50 min at 55°C and then dried by acetonitrile. Next, the samples were alkylated with 12.5 mM iodoacetamide in 50 mM ammonium bicarbonate buffer for 45 min in the dark at room temperature and dried by acetonitrile. The samples were then digested by trypsin or chymotrypsin at 37 °C overnight.
  • the fragment peptides were separated by a 120-min gradient elution method at a flow rate of 0.3 ⁇ L/min with a Thermo-Dionex Ultimate 3000 HPLC system that is directly interfaced with a Thermo Orbitrap Fusion Lumos mass spectrometer.
  • the analytical column was a homemade fused silica capillary (75 pm inner- diameter, 150 mm length; Upchurch, Oak Harbor, WA, USA) packed with C-18 resin (pore size 300 A, particle size 5 ⁇ m; Varian, Lexington, MA, USA).
  • Mobile phase A was 0.1% formic acid in water
  • mobile phase B is 100% acetonitrile and 0.1% formic acid.
  • Thermo Orbitrap Fusion Lumos mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 4.0.27.10 software.
  • a single full-scan mass spectrum was done in the Orbitrap (300 -1500 m/z, 120,000 resolution).
  • the spray voltage was 1850 V and the Automatic Gain Control (AGC) target was 200,000.
  • AGC Automatic Gain Control
  • the charge state screening of ions was set at 1-8.
  • the exclusion duration was set at 8 seconds.
  • Mass window for precursor ion selection was set at 2 m/z.
  • the MS/MS resolution was 15,000.
  • the MS/MS maximum injection time was 150 ms and the AGC target was 50,000.
  • WZ-06 and WZ-13 were synthesized ( Figure 1) and their structures confirmed.
  • WZ-20 was synthesized as a proteasome inhibitor control.
  • the compound’s IC 50 values against P ⁇ 20S and human constitutive (c-20S) and immunoproteasomes (i-20S) (Table 3) were then determined.
  • Artesunate itself does not inhibit the ⁇ 5 subunits of P ⁇ 20S, human i-20S, or human c-20S.
  • conjugates WZ-06 and WZ-13 were potent against P ⁇ 20S ⁇ 5 at 6 nM and 2 nM, respectively.
  • the data suggested that ARTs at the P4 position do not interfere with the binding of AsnEDAs to P ⁇ 20S.
  • the semi-ketal ester of the artesunate is not stable in human blood plasma, making it difficult to interpret the activity of ester-based ATZs against P ⁇ parasites in red blood cells.
  • the IC 50 of ATZ1 against P ⁇ 20S increased 10.5-fold to 0.063 ⁇ M compared to PI01.
  • ATZ3 with a propionate linker between ART1 and the proteasome inhibitor, displayed 106-fold and 760-fold selectivity against P ⁇ 20S over i-20S and c-20S, while ATZ4 with a butyrate linker showed 45-fold and 250-fold selectivity, respectively.
  • ATZ3 and ATZ4 showed increased selectivity in enzyme inhibition compared to ATZ1 and ATZ2. The results suggest that the propionate linker best balances potency and selectivity among these compounds.
  • ATZs were more potent than ART1 against P ⁇ Dd2, it was reasoned that the anti-P ⁇ activity of ATZs was not only derived from the ART moiety, but also from the proteasome inhibitor moiety. In agreement with that interpretation, the ATZs were as potent as PI01 in inhibiting the growth of P ⁇ Dd2, and their inhibition activities were only slightly less against the mutant strains: ⁇ 2.9- fold for Dd2 ( ⁇ 6A117D) and ⁇ 3.6- fold for Dd2 ( ⁇ 5A49S), representing ⁇ 5- fold and >100- fold improvement over PI01 against the respective strains. Thus, the ATZs substantially overcame resistance to the proteasome inhibitor moiety alone that were conferred by point mutations in P ⁇ 20S.
  • ⁇ -Casein is intrinsically unstructured and can be degraded by 20S and PA28a without a requirement for ubiquitination.
  • Proteasome inhibitor PI01 and ART analog ART1 served as controls. Degradation of ⁇ -casein treated with ATZ2 was markedly reduced, whereas the degradation of ⁇ -casein treated with PI01 or ART1 alone was almost complete at five hours ( Figure 48B, left). As expected, in a control experiment done without removing small molecules from the reaction mixtures by dialysis, both PI01 and ATZ2 reduced the degradation of ⁇ -casein compared to ART1 ( Figure 48B, right).
  • a proteomic analyses of PI01-, ART1-, and ATZ2- treated ⁇ -casein was conducted in order to identify ART1 and ATZ2 modified ⁇ -casein peptides ( Figure 46 and Table 5).
  • Peptide SLVYPFPGP 80 (SEQ ID: 1) was identified from ATZ2 treated ⁇ -casein in which proline-80 was modified by ATZ2 ( Figure 48C and Table 6), and a peptide F 67 AQTQSLVYPFPGPIPN (SEQ ID:2) from ART1 treated ⁇ -casein, wherein phenylalanine-67 was modified by ART1 ( Figure 48D and Table 7), confirming the covalent modification of ⁇ -casein by activated artemisinin moiety in both ART1 and ATZ2.
  • Bold numbers indicate fragments that were matched with theoretical masses of corresponding fragments; non-bold numbers indicate fragments not detected.
  • ATZs were investigated to determine if their mode of action could circumvent the ART resistance conferred by the Kelch13 polymorphism.
  • a ring-stage survival assay was conducted with strains Cam3.I REV and Cam3.I R539T ; the latter strain has a Kelchl3 polymorphism and is resistant to ART (Straimer et al., “Drug Resistance. K13-propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015), which is hereby incorporated by reference in its entirety).
  • an extended RSA was performed by pulsing parasites as in the standard RSA and then monitoring parasite growth over a further 7 days.
  • parasites pulse- treated with DHA, PI01, or ART1 established normal growth.
  • parasites of both the ART sensitive and resistant lines had significantly lower parasitemia (Figure 45C), indicative of a prolonged growth inhibition profile of ATZs.
  • the ⁇ 6A117D and ⁇ 5A49S mutations prevented PI01 and ATZ4 from inhibiting the labeling 20S( ⁇ 6A117D) and P ⁇ 20S( ⁇ 5A49S) in lysates of Dd2( ⁇ 6A117D) and Dd2( ⁇ 5A49S) parasites (Figure 45D), indicating that the mutations reduced the binding affinity of PI01 and ATZ4 to P ⁇ 20S ⁇ 5.
  • ATZs stable, covalent conjugates of a proteasome inhibitor and an ART analog, termed ATZs, retain both proteasome inhibitory activity and the reactive alkylating activity of ART. These effects are not only synergistic against growth of P ⁇ but can overcome resistance to either moiety.
  • the ability to overcome resistance conferred by point mutations in P ⁇ 20S is associated with ATZ-dependent formation of proteasome-inhibitory activity that is not removed from the parasites by washing procedures that remove ATZ itself. This more robust proteasome-inhibitory activity is ascribed to the demonstrable formation of proteasomal degradation products of ATZ-damaged proteins.
  • the oligopeptides to which the ART-derived radicals are attached appear to stabilize presentation of the proteasome inhibitory moiety of the ATZ at the P ⁇ 20S active site, compensating for the reduced binding affinity conferred by the point mutations.
  • ATZ hybrids hijack the parasite protein degradation machinery to create a pool of proteasome inhibitor-containing oligopeptides. Because the actions of ART and the improved action of the proteasome inhibitor are delivered by a single molecule, a single pharmacokinetic profile will preclude temporary exposure to only one of the components in the combination.
  • the crude product was purified by prep-HPLC (column: OBD C18 150mm*19mm*5um; mobile phase: [water (0.1%TFA)-ACN]; B%: 5%-95%, 20min) and lyophilisation to afford boronic acid as a white solid.
  • HZ2083, HZ2087, HZ2088, HZ3046, and HZ3047 Preparation of ((R)-3-Methyl-l-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimetbyldecabydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butyl)boronic Acid (HZ2082)
  • Hybrid HZ2082 was prepared using artemisinin-derived carboxylic acid (which was synthesized according to Frohlich et al., “Synthesis of Artemi sinin-Estrogen Hybrids Highly Active against HCMV, P. falciparum, and Cervical and Breast Cancer,” ACS Med. Chem. Lett., 9: 1128-1133 (2016), which is hereby incorporated by reference in its entirety) and leucine boronate as starting material by following general procedures I and III and was obtained as a white solid.
  • Hybrid HZ2083 was prepared by following general procedures I, II, I, and III and was obtained as a white solid.
  • Hybrid HZ2087 was prepared by following general procedures I, II, I, and III and was obtained as a white solid.
  • Hybrid HZ2088 was prepared by following general procedures I, II, I, and III and was obtained as a white solid.
  • Probe HZ3046 was prepared by following general procedures I, II, and I and was obtained as a white solid.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.14 7.4 Hz, 1H
  • 6.74 - 6.64 m, 1H
  • 5.37 s, 1H
  • 2.41 - 2.25 m, 3H
  • 2.02 - 1.92 m
  • Inactive probe HZ3047 was prepared by following general procedures I, II, and I and was obtained as a white solid.
  • PCy3.HBF4 tri cyclohexylphosphine tetrafluoroborate
  • SH-SY5Y cells were cultured at 37 °C in a humidified air/5% CO 2 atmosphere in medium supplemented with 10% fetal bovine serum, 100 ug per ml penicillin, and 100 pg per ml streptomycin in DMEM/F-12 medium.
  • SH-SY5Y was used at 10,000 cells per well.
  • Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO 2 .
  • Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit. Cell Viability Assay Applicable for MM. IS, U266, RPMI8226, H929, CAG, and PBMC Cells
  • Cells were cultured at 37 °C in a humidified air/5% CO 2 atmosphere in medium supplemented with 10% fetal bovine serum, 100 ug per ml penicillin, 100 pg per ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 mM Sodium Pyruvate in RPMI 1640 medium.
  • MM.1 S was used at 10,000 cells per well.
  • Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO 2 . Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
  • IC 50 values of all compounds against human c-20S ⁇ 5c, ⁇ 2c, ⁇ 1c and i-20S ⁇ 5i, ⁇ 2i, ⁇ 1i were determined in 96-well plates. Briefly, 1 ⁇ L of compound in a 3-fold series dilution in DMSO at final concentrations from 100 ⁇ M to 0.0017 ⁇ M were spotted to the bottom of a black 96-well plate.
  • reaction buffer (20 mM HEPES, 0.5 mM EDTA, and 0.1 mg/mL BSA, pH 7.4) containing proteasome, substrate, and activator was added to each well and the plate was spun on a desktop plate centrifuge and then placed on an orbital shaker at room temperature for 1 minute. The progress of reactions in each well was followed by the fluorescence of the hydrolyzed AMC at Ex 360nm and Em 460 nm for 1 - 2 hours. Linear ranges of the time course were used to calculate the velocities in each well. The reaction velocity of each well was fit to a dose-dependent inhibition equation using PRISM to determine the IC 50 .
  • the multiple myeloma cell lines MM.1S, CAG, and RPMI8226 were treated with 2 ⁇ M HZ2083, 2 ⁇ M HZ2182, 2 ⁇ M artesunate, or 100 nM BTZ for 15 hours.
  • the cells were lysated in RIPA buffer supplemented with protease cocktail.
  • the protein concentration in the samples were measured with bicinchoninic acid assay (BCA) protein assay.
  • BCA bicinchoninic acid assay
  • MM. IS cells was seeded in 96-well plates at 10,000 cells per well. The cells were treated with various concentrations of test compounds or DMSO for 24 hours at 37 °C in a tissue culture incubator with 5% CO 2 . The effect of compounds on caspase 3/7 activity in MM1.S cells was assessed using the Caspase-Gio 3/7 assay system.
  • MM1S cells were seeded in 96-well plates at 10000 cells per well. The cells were treated with various concentrations of test compounds or DMSO for 5 hours at 37 °C in a tissue culture incubator with 5% CO 2 . The cellular chymotrypsin-like activity was measured using Cell-Based Protesome-Glo Assays kit.
  • MM1S cells were seeded in 6-well plates at 1,000,000 cells per well. The cells were treated with 100 ⁇ M probe HZ3046, 100 ⁇ M inactive-probe HZ3047, or DMSO for 4 hours. The MM. IS cells were collected and lysed in RIPA buffer. The protein concentration of the samples were measured with BCA assay. To each sample (50 pg total protein), Cy3 -azide (10 ⁇ M), TBTA ligand (100 ⁇ M), TCEP (1 mM), and CuSO4 (1 mM) were added. The samples were incubated for 3 hours with shaking at room temperature. The proteins were precipitated and cleaned up using cold acetone. The precipitated proteins were dissolved with IX Laemmli buffer. The samples were resolved with 4-12% bis-tris gel. Finally, gel images were obtained with a Typhoon scanner.
  • HZ2087 with a propionate linker, showed less potency than HZ2083 against ⁇ 5c and ⁇ 5i, while maintaining the selectivity between ⁇ 5c and ⁇ 5i.
  • HZ2088 with a butyrate linker, showed 34-fold less potency against ⁇ 5c than HZ2083.
  • HZ2083 with an acetate linker was more potent than HZ2087 and HZ2088 with a longer linker, and HZ2083 without a linker.
  • HZ2182 showed comparable proteasome inhibition as HZ2083 against all six active subunits.
  • HZ2083 and HZ2182 were then tested against a panel of multiple myeloma cell lines MM. IS, CAG, H929, RPMI8226, and U266. (Table 9). The MM. IS, CAG, and H929 cell lines were more sensitive to hybrid HZ2083 than the RPMI8226 and U266 cell lines. HZ2083 showed higher cytotoxicity against all the five multiple myeloma cell lines than the deoxy compound HZ2182. Bortezomib, the first FDA-approved proteasome inhibitor, was reported as a highly potent Hu-LonP1 protease inhibitor. The off target inhibition might be related to the high toxicity of bortezomib.
  • HZ2083 against Hu-LonP1 was determined.
  • Neuroblastoma cell line SH-SY5Y and peripheral blood mononuclear cell (PBMC) were used as models evaluating the peripheral neuropathy and toxicity of HZ2083, respectively.
  • HZ2083 showed 31 to 33-fold less cytotoxicity against SH- SY5Y and PBMC over MM. IS, demonstrating a large therapeutic window.
  • HZ2083 led to an increase in P21 and Chop protein levels in treated cells ( Figure 68). HZ2083 also induced PARP cleavage and triggered apoptosis in all three cell lines. As shown in Figure 69, both HZ2083 and its deoxy analog HZ2182 increased P21 and CHOP and PARP cleavage in MM.1S and CAG cell line cells; there was no detectable change in protein level of p21, CHOP, and cleaved PARP in artesunate treated CAG cells.
  • HZ2083 and artesunate induced the degradation of ferritin FTH1 in the MM. IS cell line.
  • the inactive analog HZ2182 could not do so.
  • Ferritin is a cytosolic iron storage protein complex capable of chelating as many as 4500 iron atoms. During the process of ferroptosis, lysosomal degradation of ferritin (ferritinophagy) contributes to an increased labile iron pool, leading to elevated lipid peroxidation and oxidation of polyunsaturated fatty acids. HZ2083 might also induce ferroptosis in the MM. IS cell line.
  • Activated caspase-3/-7 are well-recognized markers of apoptosis.
  • Treatment of MM1S cells with HZ2083 for 24 hours induced caspase 3/7 activity which further proved that HZ2083 induced apopotosis (Figure 70).
  • HZ2083 primarily targets chymotrypsin-like activity of the proteasome.
  • the chymotrypsin-like activity inhibition of HZ2083 in the MM.1S cells was measured using a Proteasome-Glo cell based proteasome assay.
  • HZ2083 showed comparable proteasome inhibition in the cellular assay as in biochemical assay using purified proteasome.
  • HZ3046 and HZ3047 were synthesized. Both HZ3046 and HZ3047 showed potent activity against ⁇ 5c and ⁇ 5i.
  • protein targets of HZ3046 in MM.1S cells were visualized by conjugating HZ3046 with a fluorescene dye azide-Cy3 through click chemistry.

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Abstract

The Artemisinin-Proteasome inhibitor conjugate compounds of the present application are represented by the following compounds having Formula (I) where the substituents R1-R5, X, Y, Y', and Z are defined herein. These compounds are used in the treatment of cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders, infectious disease, or for providing immunosuppression for transplanted organs or tissues.

Description

ARTEMISININ-PROTEASOME INHIBITOR CONJUGATES AND THEIR USE IN THE TREATMENT OF DISEASE
[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/139,638, filed January 20, 2021, which is hereby incorporated by reference in its entirety.
[0002] This invention was made with government support under grant numbers AI153485, AI143714, and AI123794 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] The present application relates to Artemi sinin-Proteasome inhibitor conjugates and their use in the treatment of diseases.
BACKGROUND
[0004] Artemisinin (ART) is the backbone of the treatment of malaria, a protozoal infection responsible for 200 million cases and almost half a million deaths each year (WHO, "World Malaria Report 2018," (World Health Organization, Geneva: World Health Organization (2018)). ART is a pro-drug that it is activated by hemoglobin-derived heme within the parasites. Activation converts ART to radicals that cause extensive oxidative damage to lipids and proteins. Oxidized proteins overload the parasites’ ubiquitin-proteasome degradation system (UPS), leading to parasite death (Bridgford et al., “Artemisinin Kills Malaria Parasites by Damaging Proteins and Inhibiting the Proteasome.” Nat. Commun. 9:3801 (2018); Zhou et al., “Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome.” ACS Chem. Bio.l 11 :882-888 (2016); Tilley et al., “Artemisinin Action and Resistance in Plasmodium falciparum,” Trends Par asitol. 32:682-696 (2016); Klonis et al., “Altered Temporal Response of Malaria Parasites Determines Differential Sensitivity to Artemisinin,” Proc. Natl. Acad. Set. USA 110:5157-5162 (2013); Ismail et al., “Artemisinin Activity -based Probes Identify Multiple Molecular Targets Within the Asexual Stage of the Malaria Parasites Plasmodium falciparum 3D7,” Proc. Natl. Acad. Set. USA 113:2080-2085 (2016)). With increasing cases of malaria recrudescence following ART monotherapy, ART-based combination therapy (ACT) was implemented. However, ACT treatment failure is widespread across Southeast Asia and mutations in Kelchl3 associated with ART resistance are appearing in Africa and South America (Mukherjee et al., “Artemisinin Resistance Without Pfkelchl3 Mutations in Plasmodium Falciparum Isolates from Cambodia,” Malar. J. 16: 195 (2017); Ashley et al., “Spread of Artemisinin Resistance in Plasmodium falciparum Malaria,” N Engl. J. Med. 371:411-423 (2014); Muller et al., “Artemisinin Resistance on the Thai-Cambodian Border,” Lancet 374: 1419 (2009); Uwimana et al., “Emergence and Clonal Expansion of in vitro Artemisinin-resistant Plasmodium falciparum Kelchl3 R561H Mutant Parasites in Rwanda,” Nat. Med. 26:602-1608 (2020)). Novel approaches are needed to prevent a potential public health crisis in regions affected by ART resistance. The hallmark of ART resistance is increased tolerance to ART at the early ring stage of the erythrocytic cycle. Multiple mechanisms of resistance are associated with Kelchl3 polymorphisms (Straimer et al., “Drug Resistance. K13- propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015); Mok et al., “Drug Resistance. Population Transcriptomics of Human Malaria Parasites Reveals the Mechanism of Artemisinin Resistance,” Science 347:431- 435 (2015)), including reduced ART activation arising from defects in hemoglobin catabolism that reduce the abundance of free heme (Birnbaum et al., “A Kelch 13 -defined Endocytosis Pathway Mediates Artemisinin Resistance in Malaria Parasites,” Science 367:51-59 (2020)), reduction in proteotoxic stress (Yang et al., “Decreased KI 3 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance,” Cell Rep. 29:2917- 2928 e2915 (2019)), and prolongation of the ring stage of intra-erythrocytic Pƒ development (Hott et al., “Artemisinin-resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes,” Antimicrob. Agents Chemother. 59:3156-3167 (2015)). A challenge for ACT is the divergent pharmacokinetic profiles of the individual drugs; intermittent de-facto monotherapy could forfeit their intended synergistic effect against the emergence of ART resistance (Nguyen et al., “Optimum Population-level Use of Artemisinin Combination Therapies: A Modelling Study,” Lancet Glob. Health 3:e758-766 (2015)).
[0005] Treatment of Pƒ with ART leads to accumulation of polyubiquitinated proteins (Bridgford et al., “Artemisinin Kills Malaria Parasites by Damaging Proteins and Inhibiting the Proteasome.” Nat. Commun. 9:3801 (2018)). Polyubiquitnylation targets proteins for degradation in the proteasome (Pƒ20S). Killing of the malaria parasites by ART occurs when protein damage exceeds the capacity of the protein degradation machinery. The Pƒ20 is thus an appealing target for antimalarials (Zhan et al., “Improvement of Asparagine Ethylenediamines as Anti-malarial Plasmodium-selective Proteasome Inhibitors,” J. Med. Chem. 62:6137-6145 (2019); Stokes et al., “Covalent Plasmodium falciparum-selective Proteasome Inhibitors Exhibit a Low Propensity for Generating Resistance In vitro and Synergize with Multiple Antimalarial Agents,” PLoS Pathog. 15:el007722 (2019); Yoo et al., “Defining the Determinants of Specificity of Plasmodium Proteasome Inhibitors,” J. Am. Chem. Soc. 140: 11424-11437 (2018); Xie et al., “Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome,” J. Med. Chem. 61 : 10053-10066 (2018); LaMonte et al., “Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity,” J. Med. Chem. 60:6721-6732 (2017); Li et al., “Structure- and Function-based Design of Plasmodium-selective Proteasome Inhibitors,” Nature 530:233-236 (2016); Li et al., “Assessing Subunit Dependency of the Plasmodium Proteasome Using Small Molecule Inhibitors and Active Site Probes,” ACS Chem. Biol. 9: 1869-1876 (2014); Li et al., “Identification of Potent and Selective Non-covalent Inhibitors of the Plasmodium falciparum Proteasome,” J. Am. Chem. Soc. 136: 13562-13565 (2014); Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity from Intersubunit Interactions and Fitness Cost of Resistance,” Proc. Natl. Acad. Sci. USA 115:E6863-E6870 (2018)). Pƒ parasites at erythrocytic, liver, gametocyte and gamete activation stages are highly susceptible to proteasome inhibition, suggesting essential functions of Pƒ20S in all lifecycle stages. It has been demonstrated that various classes of proteasome inhibitors with selectivity against the malarial proteasome over human proteasomes showed synergistic anti-malarial effects with ART (Stokes et al., “Covalent Plasmodium falciparum-selective Proteasome Inhibitors Exhibit a Low Propensity for Generating Resistance in vitro and Synergize with Multiple Antimalarial Agents,” PLoS Pathog. 15:el007722 (2019); Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity from Intersubunit Interactions and Fitness Cost of Resistance,” Proc. Natl. Acad. Sci. USA 115:E6863-E6870 (2018)). The synergy may arise in part because treatment of Pƒ with ART leads to accumulation of misfolded proteins with toxic effects and proteasome inhibition prevents the breakdown and removal of damaged proteins. As with other antimalarials, Pƒ parasites can develop resistance to proteasome inhibitors, albeit this was seen in vitro with a higher barrier to resistance than has been seen with other compounds and in some cases with only a minor shift in EC50 (Zhan et al., “Improvement of Asparagine Ethylenediamines as Anti— malarial Plasmodium-selective Proteasome Inhibitors,” J. Med. Chem. 62:6137-6145 (2019); Stokes et al., “Covalent Plasmodium falciparum-selective Proteasome Inhibitors Exhibit a Low Propensity for Generating Resistance In vitro and Synergize With Multiple Antimalarial Agents,” PLoS Pathog. 15:el007722 (2019); Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity from Intersubunit Interactions and Fitness Cost of Resistance,” Proc. Natl. Acad. Sci. USA 115:E6863-E6870 (2018)).
[0006] The present application is directed to overcoming these and other deficiencies in the art. SUMMARY
[0007] A first aspect of the present application relates to an Artemisinin-Proteasome inhibitor conjugate including a compound of Formula (I): wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of H, halogen, -CF3, =CH2, -ORa, -NRaRb, -(CH2)nCOORa, -(CH2)nC(=O)Ra, -(CH2)nCONRaRa, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, aryl, and heteroaryl;
Ra is independently selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle;
Rb is independently selected from group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle, and wherein Ra and Rb may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
X is O, S, or N,
Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor;
Y’ is =O, or — Linker — Proteasome inhibitor, wherein one of Y or Y’ is a — Linker — Proteasome inhibitor;
Z is O or O-O;
Linker is a bond, a branched or unbranched C1-C10 alkylene, a branched or unbranched C2-C10 alkenylene, — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)-(arylene)-C(=O) — , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-C(=O)— , —(CH2)y-NH- C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- (arylene)-C(=O)— , — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y- C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O)— , or — (CH2)y-C(=O) — , wherein, when said Linker is — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)- (arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)—, — (CH2)y-NH-C(=O)-(CH2)y- C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— — (CH2)y-NH-C(=O)-(arylene)-C(=O)— — (CH2)y-NH-(CH2)y-C(=O)—, — (CH2)y-NH-C(=O)- (CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O- (CH2)y)y-C(=O) — , or — (CH2)y-C(=O) — , the carbonyl carbon of the Linker is attached to the Proteasome inhibitor; n is an integer ranging from 0 to 3; y is independently selected at each occurrence from an integer ranging from 0 to 10; and
Proteasome inhibitor is a compound that inhibits either chymotryptic-like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
[0008] A second aspect of the present application relates to a method of treating infectious diseases in a subject. This method includes administering to the subject in need thereof a compound of the present application.
[0009] A third aspect of the present application relates to a method of treating cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders in a subject, or for providing immunosuppression for transplanted organs or tissues in a subject. This method includes administering to the subject in need thereof a compound of the present application.
[0010] A fourth aspect of the present application relates to a pharmaceutical composition including a therapeutically effective amount of the compounds of the present application and a pharmaceutically acceptable carrier.
[0011] Artemisinin resistance is spreading in Plasmodium falciparum (Pƒ) despite combination chemotherapy (ACT), perhaps because differences in pharmacokinetics of each drug result in periodic monotherapy in some anatomical compartments. Here, the construction of artezomibs, single-molecule hybrids of an artemisinin and a proteasome inhibitor are reported. Inside parasites, particularly in plasmodium parasites and in schistosoma parasites, artezomibs create a novel mode of action in which the artemisinin component covalently modifies parasite proteasome substrates and the proteasome is inhibited by the proteasome inhibitor component. In the case of Plasmodium parasites, artezomibs circumvent the development of both artemisinin resistance conferred by Kelch13 polymorphism and the resistance to the proteasome inhibitor associated with mutations in Pƒ proteasomes. This mode of action may enable a single molecule to prevent emergence of resistance.
[0012] Given that proteasome inhibitors not only kill Pƒ on their own but also make the parasites more susceptible to ART, it was hypothesized that linking a proteasome inhibitor to an ART analog through a tether could yield a hybrid compound with the ability to hijack the parasite ubiquitin proteasome system to produce a host of proteasome inhibitors that overcome resistance to each of the hybrid’s two constituent chemophores. It was reasoned that an ART- proteasome inhibitor hybrid would yield ART -modified proteins whose proteasomal degradation products containing a proteasome inhibitor moiety could inhibit the function of Pƒ20S by binding to its active proteolytic subunits. By binding distal to the Pƒ20S active sites, the extended peptides of the degradation products could compensate for a loss of binding affinity caused by point mutations near the active sites that would otherwise reduce the efficacy of the proteasome inhibitor. Here, it is reported that combining the ART and proteasome inhibitor moieties into one small molecule, termed an artezomib (ATZ), can overcome resistance to its individual components and potentially prevent the emergence of resistance to each.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 shows the synthetic route of Artesunate-based hybrids WZ-13 and WZ- 06 and control WZ-20.
[0014] Figure 2 is the 1H nuclear magnetic resonance (NMR) spectrum of
(3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-Trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl 4-(((S)-4-(tert-butyl amino)- 1 -((2-(2',4-difluoro- [1,1 '-biphenyl]-3 -carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-4-oxobutanoate (WZ- 13)
[0015] Figure 3 is the 13C NMR spectrum of compound WZ-13.
[0016] Figure 4 is the liquid chromatography-mass spectrometry (LC-MS) chromatogram of compound WZ-13.
[0017] Figure 5 is the high-resolution mass spectrometry (HRMS) spectrum of compound WZ-13.
[0018] Figure 6 is the 1H NMR spectrum of (3R,5aS, 6R,8aS, 9R, 105, 12R , 12aR)-3, 6,9- Trimethyldecahydro- 12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl 4-(((S)-4-(tert- butylamino)-1,4-dioxo-1-((2-(4-phenylpicolinamido)ethyl)amino)butan-2-yl)amino)-4- oxobutanoate (WZ-06). [0019] Figure 7 is the 13C NMR spectrum of compound WZ-06.
[0020] Figure 8 is the LC-MS chromatogram of compound WZ-06.
[0021] Figure 9 is the FIRMS spectrum of compound WZ-06.
[0022] Figure 10 is the 1H NMR spectrum of (S)-4-((4-(tert-butylamino)- 1 -((2-(2',4- difluoro-[1,1'-biphenyl]-3 -carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-4- oxobutanoic acid (WZ-20).
[0023] Figure 11 is the 13C NMR spectrum of compound WZ-20.
[0024] Figure 12 is the LC-MS chromatogram of compound WZ-20.
[0025] Figure 13 is the HRMS spectrum of compound WZ-20. [0026] Figure 14 is the 1H NMR spectrum of 2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-
3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetic acid (DeoxoART-AcOH 9).
[0027] Figure 15 is the 13C NMR spectrum of compound DeoxoART-AcOH 9.
[0028] Figure 16 shows the synthetic route of ART-based hybrids ATZ1, ATZ2, ATZ3, and ATZ4.
[0029] Figure 17 is the 1H NMR spectrum of 2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-
3,6,9-Trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (ART1).
[0030] Figure 18 is the 13C NMR spectrum of compound ART1. [0031] Figure 19 is the LC-MS chromatogram of compound ART1.
[0032] Figure 20 is the HRMS spectrum of compound ART1.
[0033] Figure 21 is the 1H NMR spectrum of tert- Butyl (S)-(2-((4-(tert-butyl amino)- 1- ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3-carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-2- oxoethyl)carbamate (WZ-0917). [0034] Figure 22 is the 13C NMR spectrum of compound WZ-0917.
[0035] Figure 23 is the 1H NMR spectrum of tert- Butyl (S)-(3 -((4-(tert-butyl amino)- 1 - ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3 -carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-3 - oxopropyl)carbamate (WZ-0918).
[0036] Figure 24 is the 1H NMR spectrum of tert- Butyl (S)-(4-((4-(tert-butylamino)-1- ((2-(2',4-difluoro-[ 1 , 1 '-biphenyl]-3 -carboxamido)ethyl)amino)-1,4-dioxobutan-2-yl)amino)-4- oxobutyl)carbamate (PI01).
[0037] Figure 25 is the 13C NMR spectrum of compound PI01.
[0038] Figure 26 is the LC-MS chromatogram of compound PI01.
[0039] Figure 27 is the HRMS spectrum of compound PI01. [0040] Figure 28 is the 1HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3-carboxamido)ethyl)-2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)succinamide (ATZ1). [0041] Figure 29 is the 13C NMR spectrum of compound ATZ1.
[0042] Figure 30 is the LC-MS chromatogram of compound ATZ1. [0043] Figure 31 is the HRMS spectrum of compound ATZ1. [0044] Figure 32 is the 1HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl )acetami do)acetami do) succinami de (ATZ2) .
[0045] Figure 33 is the 13C NMR spectrum of compound ATZ2. [0046] Figure 34 is the LC-MS chromatogram of compound ATZ2. [0047] Figure 35 is the HRMS spectrum of compound ATZ2. [0048] Figure 36 is the 1HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(3 -(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 ,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)propanamido)succinamide (ATZ3).
[0049] Figure 37 is the 13C NMR spectrum of compound ATZ3. [0050] Figure 38 is the LC-MS chromatogram of compound ATZ3.
[0051] Figure 39 is the HRMS spectrum of compound ATZ3. [0052] Figure 40 is the 1HNMR spectrum of (S)-N4-(tert- Butyl)-N1-(2-(2',4-difluoro- [1,1'-biphenyl]-3 -carboxamido)ethyl)-2-(4-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 ,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butanamido)succinamide (ATZ4).
[0053] Figure 41 is the 13C NMR spectrum of compound ATZ4.
[0054] Figure 42 is the LC-MS chromatogram of compound ATZ4.
[0055] Figure 43 is the HRMS spectrum of compound ATZ4.
[0056] Figure 44 shows proposed synthetic approaches for the formation of diverse Artemisinin-Proteasome inhibitor conjugates.
[0057] Figures 45A-45H shows the effects of compounds in a ring-stage survival assay. Figure 45A is a flow diagram of the process in which red blood cells infected with highly synchronized ring-stage parasites were treated with DMSO, DHA, ART1, PI01, a 1:1 mixture of PI01 and ART1, ATZ3 or ATZ4 at indicated concentrations. After 6 hours, the compounds were washed off. In Figure 45B, the parasite cultures were allowed to grow for 66 hours. Viable parasites were analyzed by flow cytometry and their numbers normalized to values for the DMSO control. In Figure 45C, aliquots of parasites from Figure 45B were cultured for a further 96 hours. Parasitemia was quantified by Giemsa-stained smears. Figure 45D shows the inhibition of Pƒ20S, Pƒ20S(β6A117D) and Pƒ20S(β5A49S) by PI01 or ATZ4 in lysates of Dd2, Dd2(β6A117D) and Dd2(β5A49S), respectively, was assessed by their ability to block labeling of the parasites’ proteasomes by the activity-based fluorescent probe MV151 with 1 hour preincubation. Figure 45E shows the mode of action of ATZ in parasites was assessed in Dd2, Dd2(β6A117D) and Dd2(β5A49S) cultures. Parasites were treated with DMSO, PI01, WZ1840, AZT4, PI01/ART1 (1 : 1) or DHA for 6 hours and compounds were washed off prior to hypotonic lysis of red blood cells. Parasites were then lysed and labelled with MV151. Data in Figures 45B-45C are means of three independent experiments. Images in Figure 45D and in Figure 45E are representative of two and three independent experiments, respectively. Figure 45F displays the raw data (electrophoresis gel) for Figures 45B-45C. Figure 45G displays the raw data (electrophoresis gel) for Figure 45D. Figure 45H displays the raw data (electrophoresis gel) for Figure 45E.
[0058] Figure 46 shows the heme-induced activation of the endoperoxides, yielding reactive radical intermediates of ART1 and ATZ2 capable of two types of covalent modification of β-casein.
[0059] Figures 47A-47C show the design of hybrids of ART and proteasome inhibitors and their inhibition of proteasomes and of parasite growth. Figure 47A shows the structures of proteasome inhibitor, ART analog and hybrids. Figure 47B shows the inhibition of Pƒ20S, human c-20S and i-20S. Figure 47C shows the growth inhibition of Dd2, Dd2β5A48S and Dd2p6Al 17D by PI01, ART1 and ATZ3.
[0060] Figures 48A-48D show the mode of action of ATZ in the degradation of β-casein by 20S. Figure 48A is an illustration of degradation of β-casein by human i-20S following incubation with ART or ATZ activated by hemin and ascorbate. Figure 48B shows the degradation of β-casein. β-casein was treated under indicated conditions (a, b or c). Left panel: after dialysis to remove the inhibitors, hemin, and ascorbate, the treated β-casein was incubated with i-20S and PA28a with bovine serum albumin as an internal control. Aliquots were taken at indicated times and samples run on SDS-page and stained with Coomassie blue. Right panel: without dialysis, aliquots were taken from each reaction at indicated time points and samples run on SDS-page and stained with Coomassie blue. Representative images of three independent experiments. Figure 48C is the MS/MS spectrum of the ATZ2 modified peptide SLVYPFPGP80 (SEQ ID: 1). The inserted mono-isotope peak at m/z 894.45557 matches the theoretical mass of the aforementioned peptide modified by ATZ2. This peptide was not observed in PI01 treated nor in ART1- treated β-casein samples through manual check. Figure 48D is the MS/MS spectrum of the ART1 modified peptide F67AQTQSLVYPFPGPIPN (SEQ ID:2). The inserted mono-isotope peak at m/z 1101.07361 matches the mass of the aforementioned peptide modified by ART1. This peptide was not observed in PI01 treated nor in ATZ2- treated β-casein samples through manual check.
[0061] Figure 49 shows the labelling inhibition of Pƒ20S in Dd2 parasites treated with DMSO, PI01, ART1, AZT4, PI01/ART1 (1 : 1) or DHA, assessed by their ability to block labeling of the parasites’ proteasomes by MV151. Parasites were treated with indicated compounds for 6 hours and extracellular compounds were removed prior to hypotonic lysis of red blood cells.
[0062] Figure 50 is the 3H NMR spectrum of ((R)-3-methyl-1-(2- ((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)butyl)boronic acid (HZ2082). [0063] Figure 51 is the 13C NMR spectrum of compound HZ2082.
[0064] Figure 52 is the 3H NMR spectrum of ((R)-3-methyl-1-(2-(2-
((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)acetamido)butyl)boronic acid (HZ2083).
[0065] Figure 53 is the 13C NMR spectrum of compound HZ2083.
[0066] Figure 54 is the 3H NMR spectrum of ((R)-3-methyl-1-(3-(2-
((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)propanamido)butyl)boronic acid (HZ2087).
[0067] Figure 55 is the 13C NMR spectrum of compound HZ2087.
[0068] Figure 56 is the 3H NMR spectrum of ((R)-3-methyl-1-(4-(2-
((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)butanamido)butyl)boronic acid (HZ2088).
[0069] Figure 57 is the 13C NMR spectrum of compound HZ2088.
[0070] Figure 58 is the 3H NMR spectrum of N -((1R )-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2- ((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyl decahydro- 12H-3 ,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-3).
[0071] Figure 59 is the 13C NMR spectrum of compound DQ-3.
[0072] Figure 60 is the 1H NMR spectrum of N-(2-oxo-2-(((1R)-2-phenyl-l-((3aS,4S,6S)-
3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)-2- ((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 , 6,9-trimethyl decahydro- 12H-3 ,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-4).
[0073] Figure 61 is the 13C NMR spectrum of compound DQ-4.
[0074] Figure 62 is the 1H NMR spectrum of N-((1R)-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-
((2R,3R,3aS,3a1R,6R,6aS,9S,10aR)-3,6,9-trimethyl decahydro-10aH-3a1,9-epoxyoxepino[4,3,2- ij]isochromen-2-yl)acetamide (DQ-7).
[0075] Figure 63 is the 13C NMR spectrum of compound DQ-7.
[0076] Figure 64 is the 1H NMR spectrum of N-((R)-2-(benzofuran-3-yl)-l-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)ethyl)-2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-9).
[0077] Figure 65 is the 13C NMR spectrum of compound DQ-9.
[0078] Figure 66 is the 1H NMR spectrum of N-((R)-2-(benzofuran-3-yl)-l-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)ethyl)-2-((2R,3R,3aS,3a1R,6R,6aS,9S,10aR )-3,6,9- trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide (DQ-10).
[0079] Figure 67 is the 13C NMR spectrum of compound DQ-10.
[0080] Figure 68 shows that HZ2083 causes apoptosis of multiple myeloma cells.
[0081] Figure 69 shows apoptotic signal transduction in MM.1S and CAG cell lines following exposure to HZ2083 and control compounds HZ2182 and artesunate.
[0082] Figure 70 shows that HZ2083 causes activation of caspase 3/7 in MM. IS and cell -based proteasome inhibition.
[0083] Figure 71 shows HZ3046 labeling profile in MM. 1 S cells
DETAILED DESCRIPTION
[0084] A first aspect of the present application relates to an Artemisinin-Proteasome inhibitor conjugate including a compound of Formula (I): wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of H, halogen, -CF3, =CH2, -ORa, -NRaRb, -(CH2)nCOORa, -(CH2)nC(=O)Ra, -(CH2)nCONRaRa, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, aryl, and heteroaryl;
Ra is independently selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle;
Rb is independently selected from group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle, and wherein Ra and Rb may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
X is O, S, or N,
Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor;
Y’ is =O, or — Linker — Proteasome inhibitor, wherein one of Y or Y’ is a — Linker — Proteasome inhibitor;
Z is O or 0-0;
Linker is a bond, a branched or unbranched C1-C10 alkylene, a branched or unbranched C2-C10 alkenylene, — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)-(arylene)-C(=O) — , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH- C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- (arylene)-C(=O)— , — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y- C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O)— , or — (CH2)y-C(=O) — , wherein, when said Linker is — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)- (arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y- C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(arylene)-C(=O)— — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- (CH2)y-(O-(CH2)y)y-C(=O)— — (CH2)y-C(=O)-(CH2)y-C(=O)— — (CH2)y-C(=O) -(CH2)y-(O- (CH2)y)y-C(=O) — , or — (CH2)y-C(=O) — , the carbonyl carbon of the Linker is attached to the Proteasome inhibitor; n is an integer ranging from 0 to 3; y is independently selected at each occurrence from an integer ranging from 0 to 10; and
Proteasome inhibitor is a compound that inhibits either chymotryptic-like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
[0085] As used above, and throughout the description herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this technology belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
[0086] The term "alkyl" means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 12 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3 -pentyl.
[0087] As used herein, the term “alkane” refers to aliphatic hydrocarbons of formula CnH2n+2, which may be straight or branched having about 1 to about 40 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8) carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkyl chain. Exemplary alkanes include methane, ethane, n-propane, i-propane, n-butane, t-butane, n-pentane, and 3 -pentane. The term “alkylene” refers to a divalent group formed from an alkane by removal of two hydrogen atoms.
Exemplary' alkylene groups include, but are not limited to, divalent groups derived from the alkanes described above.
[0088] The term “alkenyl” means an aliphatic hydrocarbon group containing a carbon — carbon double bond and which may be straight or branched having about 2 to about 12 carbon atoms in the chain. Particular alkenyl groups have 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkenyl chain. Exemplary alkenyl groups include ethenyl, propenyl, n- butenyl, and i-butenyl. The term "alkenyl" may also refer to a hydrocarbon chain having 2 to 6 carbons containing at least one double bond and at least one triple bond.
[0089] The term “alkynyl” means an aliphatic hydrocarbon group containing a carbon — carbon triple bond and which may be straight or branched having about 2 to about 20 carbon atoms in the chain. Particular alkynyl groups have 2 to about 10 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkynyl chain. Exemplary alkynyl groups include ethynyl, propynyl, n- butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.
[0090] The term “alkenylene” means a group obtained by removal of a hydrogen atom from an alkenyl group.
[0091] The term "cycloalkyl" means a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, preferably of about 3 to about 8 carbon atoms. Exemplary monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cycloheptyl, and the like.
[0092] The term “cycloalkylalkyl” means a cycloalkyl-alkyl-group in which the cycloalkyl and alkyl are as defined herein. Exemplary cycloalkylalkyl groups include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclopropyl ethyl, cyclobutyl ethyl, and cyclopentylethyl. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined herein.
[0093] The term "cycloalkenyl" means a non-aromatic mono- or multicyclic ring system containing a carbon — carbon double bond of about 4 to about 12 carbon atoms, preferably of about 5 to about 7 carbon atoms. Exemplary monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like.
[0094] The term “cycloalkenyalkyl” means a cycloalkenyl-alkyl-group in which the cycloalkenyl and alkyl are as defined herein. Exemplary cycloalkenylalkyl groups include cyclopropenylmethyl, cyclobutenylmethyl, cyclopentenylmethyl, cyclopropenylethyl, cyclobutenylethyl, and cyclopentenyl ethyl. The alkyl radical and the cycloalkenyl radical may be optionally substituted as defined herein.
[0095] The term "cycloalkynyl" means a non-aromatic mono- or multicyclic ring system containing a carbon — carbon triple bond of about 5 to about 12 carbon atoms, preferably of about 5 to about 8 carbon atoms. Exemplary monocyclic cycloalkenyls include cyclopentynyl, cyclohexynyl, cycloheptynyl, and the like.
[0096] The term “cycloalkynyalkyl” means a cycloalkynyl-alkyl-group in which the cycloalkynyl and alkyl are as defined herein. Exemplary cycloalkynylalkyl groups include cyclopropynylmethyl, cyclobutynylmethyl, cyclopentynylmethyl, cyclopropynylethyl, cyclobutynyl ethyl, and cyclopentynyl ethyl. The alkyl radical and the cycloalkynyl radical may be optionally substituted as defined herein.
[0097] The term “alkoxy” means groups of from 1 to 12 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyl oxy, cyclohexyloxy, and the like. Lower-alkoxy refers to groups containing one to four carbons. For the purposes of the present patent application, alkoxy also includes methylenedi oxy and ethylenedioxy in which each oxygen atom is bonded to the atom, chain, or ring from which the methylenedioxy or ethylenedioxy group is pendant so as to form a ring. Thus, for example, phenyl substituted by alkoxy may be, for example,
[0098] The term "aryl" means an aromatic monocyclic or multi cyclic ring system of 6 to about 14 carbon atoms, preferably of 6 to about 10 carbon atoms. Representative aryl groups include phenyl, naphthyl, and anthracenyl.
[0099] The term “aryloxy” means -O-aryl, in which aryl is as defined herein.
[0100] The term “arylene” means a group obtained by removal of a hydrogen atom from an aryl group. Non-limiting examples of arylene include phenylene and naphthylene.
[0101] The term “arylalkyl” or “alkylaryl” means an alkyl substituted with one or more aryl groups, wherein the alkyl and aryl groups are as herein described. One particular example is an arylmethyl or aryl ethyl group, in which a single or a double carbon spacer unit is attached to an aryl group, where the carbon spacer and the aryl group can be optionally substituted as described herein. Representative arylalkyl groups include , and
[0102] The term “aralkoxy” or “arylalkoxy” means -O-alkylaryl or -O-arylalkyl, in which arylalkyl and alkylaryl are as defined herein.
[0103] As used herein, “biphenyl” or “bi-phenyl” refers to a phenyl group substituted by another phenyl group. [0104] The term "heteroaryl" or “Het” means an aromatic monocyclic or multi cyclic ring system of about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is/are element(s) other than carbon, for example, nitrogen, oxygen, or sulfur. In the case of multicyclic ring system, only one of the rings needs to be aromatic for the ring system to be defined as "Heteroaryl". Preferred heteroaryls contain about 5 to 6 ring atoms. The prefix aza, oxa, thia, or thio before heteroaryl means that at least a nitrogen, oxygen, or sulfur atom, respectively, is present as a ring atom. A nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N-oxide. Representative heteroaryls include pyridyl, 2-oxo-pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, furanyl, pyrrolyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, indolyl, isoindolyl, benzofuranyl, benzothiophenyl, indolinyl, 2-oxoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, indazolyl, benzimidazolyl, benzooxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, benzotriazolyl, benzo [1, 3]dioxolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, pthalazinyl, quinoxalinyl, 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1, 2, 3]triazinyl, benzo[1, 2, 4]triazinyl, 4H-chromenyl, indolizinyl, quinolizinyl, 6aH-thieno[2,3-d]imidazolyl, 1H- pyrrolo[2,3 -b]pyridinyl, imidazof 1 ,2-a]pyridinyl, pyrazolof 1 ,5-a]pyridinyl, [1,2,4]triazolo[4,3 - a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl, thieno[2,3-b]furanyl, thieno[2,3-b]pyridinyl, thieno[3,2-b]pyridinyl, furo[2,3-b]pyridinyl, furo[3,2-b]pyridinyl, thieno[3,2-d]pyrimidinyl, furo[3,2-d]pyrimidinyl, thieno[2,3-b]pyrazinyl, imidazof l,2-a]pyrazinyl, 5, 6,7,8- tetrahydroimidazof 1 ,2-a]pyrazinyl, 6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazinyl, 2-oxo-2,3 - dihydrobenzo[d]oxazolyl, 3,3-dimethyl-2-oxoindolinyl, 2-oxo-2,3-dihydro-lH-pyrrolo[2,3- b]pyridinyl, benzo[c][1,2,5]oxadiazolyl, benzo[c][1,2,5]thiadiazolyl, 3,4-dihydro-2H- benzo[b][1,4]oxazinyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, [1,2,4]triazolo[4,3- a]pyrazinyl, 3-oxo- [1,2,4]triazolo[4,3-a]pyridin-2(3H)-yl, and the like.
[0105] As used herein, “biheteroaryl” or “bi-heteroaryl” refers to a heteroaryl group substituted by another heteroaryl group.
[0106] As used herein, “heterocyclyl” or “heterocycle” or “heterocycloalkyl” refers to a stable 3- to 18-membered ring (radical) which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this application, the heterocycle may be a monocyclic, or a polycyclic ring system, which may include fused, bridged, or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycle may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the ring may be partially or fully saturated. Examples of such heterocycles include, without limitation, azepinyl, azocanyl, pyranyl dioxanyl, dithianyl, 1,3-dioxolanyl, tetrahydrofuryl, dihydropyrrolidinyl, decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2- oxopyrrolidinyl, 2-oxoazepinyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone. Further heterocycles and heteroaryls are described in Katritzky et al., eds., Comprehensive Heterocyclic Chemistry: The Structure, Reactions, Synthesis and Use of Heterocyclic Compounds, Vol. 1-8, Pergamon Press, N.Y. (1984), which is hereby incorporated by reference in its entirety.
[0107] As used herein, “biheterocyclyl” or “bi-heterocyclyl” refers to a heterocyclyl group substituted by another heterocyclyl or heterocycle group.
[0108] The term “non-aromatic heterocycle” means a non-aromatic monocyclic system containing 3 to 10 atoms, preferably 4 to about 7 carbon atoms, in which one or more of the atoms in the ring system is/are element(s) other than carbon, for example, nitrogen, oxygen, or sulfur. Representative non-aromatic heterocycle groups include pyrrolidinyl, 2-oxopyrrolidinyl, piperidinyl, 2-oxopiperidinyl, azepanyl, 2-oxoazepanyl, 2-oxooxazolidinyl, morpholino, 3- oxomorpholino, thiomorpholino, 1,1 -di oxothiomorpholino, piperazinyl, tetrohydro-2H-oxazinyl, and the like.
[0109] The term “monocyclic” used herein indicates a molecular structure having one ring.
[0110] The term “bicyclic” used herein indicates a molecular structure having two ring,
[0111] The term “polycyclic” or “multi-cyclic” used herein indicates a molecular structure having two or more rings, including, but not limited to, fused, bridged, or spiro rings. [0112] The term “boronic acid complexing agent” refers to any compound having at least two functional groups, each of which can form a covalent bond with boron. Nonlimiting examples of suitable functional groups include amino and hydroxyl. The term “moiety derived from a boronic acid complexing agent” refers to a moiety formed by removing the hydrogen atoms from two functional groups of a boronic acid complexing agent.
[0113] Terminology related to “protecting”, “deprotecting,” and “protected” functionalities occurs throughout this application. Such terminology is well understood by persons of skill in the art and is used in the context of processes which involve sequential treatment with a series of reagents. In that context, a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable. The protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or “deprotection” occurs after the completion of the reaction or reactions in which the functionality would interfere. Thus, when a sequence of reagents is specified, as it is in the processes described herein, the person of ordinary skill can readily envision those groups that would be suitable as “protecting groups.” Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1991), which is hereby incorporated by reference in its entirety.
[0114] The term "halo" or "halogen" means fluoro, chloro, bromo, or iodo.
[0115] The term "cyano" means -C=N or -CN group.
[0116] The term "benzyl" or Bn means -CH2-Ph or -CH2Ph group.
[0117] The term "substituted" or "substitution" of an atom means that one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded.
[0118] "Unsubstituted" atoms bear all of the hydrogen atoms dictated by their valency. When a substituent is keto (i.e., =0), then two hydrogens on the atom are replaced. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds; a "stable compound" or "stable structure" is meant to be a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
[0119] The term “optionally substituted” is used to indicate that a group may have a substituent at each substitutable atom of the group (including more than one substituent on a single atom), provided that the designated atom's normal valency is not exceeded and the identity of each substituent is independent of the others. Up to three H atoms in each residue are replaced with alkyl, halogen, haloalkyl, hydroxy, loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryl oxy. “Unsubstituted” atoms bear all of the hydrogen atoms dictated by their valency. When a substituent is keto (i.e., =0), then two hydrogens on the atom are replaced. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds; by “stable compound” or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. [0120] The term “method of treating” means amelioration or relief from the symptoms and/or effects associated with the disorders described herein. As used herein, reference to “treatment” of a patient is intended to include prophylaxis.
[0121] The term "compounds of the invention", and equivalent expressions, are meant to embrace compounds of general Formula (I), Formula (I'), Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (Illf), Formula (Illg), and Formula (IV), as herein described, which expression includes the prodrugs, the pharmaceutically acceptable salts, and the solvates, e.g. hydrates, where the context so permits. Similarly, reference to intermediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits. For the sake of clarity, particular instances when the context so permits are sometimes indicated in the text, but these instances are purely illustrative and it is not intended to exclude other instances when the context so permits. [0122] The term "pharmaceutically acceptable salts" means the relatively non-toxic, inorganic, and organic acid addition salts, and base addition salts, of compounds of the present application. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane— sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66: 1-9 (1977) and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, which are hereby incorporated by reference in their entirety). Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use, such as ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, dicyclohexylamine, and the like.
[0123] The term "pharmaceutically acceptable prodrugs" as used herein means those prodrugs of the compounds useful according to the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of this application. The term "prodrug" means compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. Functional groups which may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this application. They include, but are not limited to, such groups as alkanoyl (such as acetyl, propionyl, butyryl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (such as trimethyl- and triethysilyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like. Because of the ease with which the metabolically cleavable groups of the compounds useful according to this application are cleaved in vivo, the compounds bearing such groups act as pro-drugs. The compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group. A thorough discussion of prodrugs is provided in the following: Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309-396 (1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; "Design and Applications of Prodrugs" p.113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, p.1-38 (1992); J. Pharm. Sci., 77:285 (1988); Nakeya et al, Chem. Pharm. Bull., 32:692 (1984); Higuchi et al., “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press (1987), which are incorporated herein by reference in their entirety. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the invention.
[0124] The term “solvate” refers to compounds of the present application in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
[0125] Compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. This technology is meant to include all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms. Optically active (R)- and (S)-, (-)- and (+)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. [0126] This technology also envisions the “quaternization” of any basic nitrogen— containing groups of the compounds disclosed herein. The basic nitrogen can be quatemized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
[0127] In the characterization of some of the substituents, it is recited that certain substituents may combine to form rings. Unless stated otherwise, it is intended that such rings may exhibit various degrees of unsaturation (from fully saturated to fully unsaturated), may include heteroatoms and may be substituted with lower alkyl or alkoxy.
[0128] In one embodiment, the Artemi sinin-Proteasome inhibitor conjugate includes a compound of Formula (I'): wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of H, halogen, -CF3, =CH2, -ORa, -NRaRb, -(CH2)nCOORa, -(CH2)nC(=O)Ra, -(CH2)nCONRaRa, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl , aryl, and heteroaryl;
Ra is independently selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle;
Rb is independently selected from group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle, and wherein Ra and Rb may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
X is O, S, or N,
Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor;
Y’ is =O, or — Linker — Proteasome inhibitor, wherein one of Y or Y’ is a — Linker — Proteasome inhibitor;
Linker is a bond, a branched or unbranched C1-C10 alkylene, — O-C(=O)-(CH2)y- C(=O)— , — O-C(=O)-(arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y- NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(arylene)-C(=O)— , — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O) — , or — (CH2)y-C(=O) — , wherein, when said Linker is — O-C(=O)-(CH2)y-C(=O)— , — O-C(=O)-(arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y- C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(arylene)-C(=O)— , — (CH2)y-NH- (CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y- C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O)— , or — (CH2)y-C(=O) — , the carbonyl carbon of the Linker is attached to the Proteasome inhibitor; n is an integer ranging from 0 to 3; y is independently selected at each occurrence from an integer ranging from 0 to 10; and
Proteasome inhibitor is a compound that is known to inhibit either chymotryptic- like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome activity, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
[0129] One embodiment relates to the Artemi sinin-Proteasome inhibitor conjugate, wherein Linker is selected from the group consisting of
[0130] In another embodiment of the Artemisinin-Proteasome inhibitors of the present application R1, R2, and R3 are H; R4 and R5 are CH3; X is O; and Linker is — O-C(=O)- (CH2)yC(=O)— , — (CH2)y-C(=O)-(CH2)yC(=O)— , or — (CH2)y-C(=O) — .
[0131] Further examples of the Artemisinin compounds that may be useful in the present application are disclosed in U.S. Patent Serial Nos.: 9,918,972 to Civin et al.; 9,999,621 to Li et al.; and 8,883,765 to Arav-Boger et al., which are hereby incorporated by reference in their entirety.
[0132] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (II): wherein is the point of attachment to the Linker;
R' is H or C1-6 alkyl;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non — aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, NHCOOC1-12alkyl, — B(OR’)2, methylsulfonyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, — (CH2)mC(O)NHR6, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and C1-6 alkoxy; or
R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, —
(CH2)mC(O)NR6R7, — C(O)OR10, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — N3, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein is the point of attachment to the corresponding carbon or nitrogen atom of the structure of Formula (II); R5’ is selected from the group consisting of H, non-aromatic heterocycle, — NR6R7, — CR8R9, C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with R11;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, — CF3, C1-6 alkyl, C3-8 cycloalkyl, — (CH2)kOH, and arylalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R12; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
R10 is H or arylalkyl;
R11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF3, C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl, wherein C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R12;
R12 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy, and the C1-12 alkyl, C2-12 alkenyl, and C2-12 alkynyl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
Q is optional and, if present, is C1-3 alkyl or — C(Y) — ; Q1 is optional, and, if present, is selected from NH, — (CR3’H) — , — NH- (CRZH) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle;
Rz is independently selected at each occurrence thereof from the group consisting of C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-12 cycloalkenyl, C5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, and (cycloalkynyl)alkyl, wherein C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-12 cycloalkenyl, C5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkynyl)alkyl can be optionally substituted 1 time with Rz ;
Rz is independently selected at each occurrence thereof from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;
X is a bond, — C(Y)— , — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1, 2, 3, or 4; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2, or 3.
[0133] In some embodiments, Z1 and Z2 together with the boron atom to which they are attached form a moiety derived from a boronic acid complexing agent. This moiety derived from a boronic acid complexing agent can be where R13 can be H or C1-6 alkyl, R14 can be H or C1-6 alkyl, R15 can be H or C1-6 alkyl, and R16 can be H or C1-6 alkyl. Suitable moi eties derived from a boronic acid complexing agent that can be used according to the present application include
[0134] In yet another embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (II): wherein is the point of attachment to the Linker;
R' is H or C1-6 alkyl;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, NHCOOC1-12 alkyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy; or
R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, —
(CH2)mC(O)NR6R7, — C(O)OR10, — (CH2)mC(O)0H, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C1-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein is the point of attachment to the corresponding carbon or nitrogen atom of the structure of Formula (II);
R5’ is selected from the group consisting of H, non-aromatic heterocycle, — NR6R7, — CR8R9, C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with R11;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, — CF3, C1-6 alkyl, — (CH2)kOH, and arylalkyl; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R12; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
R10 is H or arylalkyl;
R11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF3, C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl, wherein C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R12;
R12 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy, and the C1-12 alkyl, C2-12 alkenyl, and C2-12 alkynyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
Q is optional and, if present, is C1-3 alkyl or — C(Y) — ;
Q1 is optional, and, if present, is selected from NH, — (CR3’H) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle;
X is a bond, — C(Y)— , — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2 or 3.
[0135] One embodiment relates to the proteasome inhibitor moi eties of Formula (II), where R1’ is selected from the group consisting of , ; and R11 is selected from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy, wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (II).
[0136] A further embodiment relates to the proteasome inhibitor moi eties of Formula (II) and wherein is the point of attachment to Q or Q1; and is the point of attachment to halogen, NH2, NHCOOC1-12 alkyl, or C1-12 alkyl.
[0137] Another embodiment relates to the proteasome inhibitor moieties where R2’ is selected from the group consisting of H, Me, — CH2(Me)2, — CH2OMe, wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (II).
[0138] One embodiment relates to the proteasome inhibitor moieties of Formula (II) where R3’ is selected from the group consisting of H, CH3, — CH2OMe, — CH2C(O)OH, — point of attachment to the corresponding carbon atom of the structure of Formula (II).
[0139] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (III): wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-6 alkyl, C1-6 alkoxy, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, — (CH2)mC(O)NR6R7, — (CH2)mC(O)0H, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C12-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R5’ is selected from the group consisting of H, C1-6 alkyl, C1-6 alkoxy, non— aromatic heterocycle, — NR6R7, and — CR8R9; and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CHiAr, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is C(O), — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; s is 0 or 1; and
1 is 0 or 1
[0140] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (Illa): wherein is the point of attachment to the Linker; R1’ is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is H or C1-6 alkyl;
R3’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)mC(O)NHR5’, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R5’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Y is O or S; m is 1 or 2; and n is 1, 2, or 3.
[0141] In a further embodiment of the Proteasome inhibitor moiety of Formula (Illa) R1’ is a substituted or unsubstituted biphenyl, or a substituted or unsubstituted hetero aryl;
R2’ is H;
R3’ is — (CH2)mC(O)NHR5’;
R5’ is a C1-6 alkyl;
Y is O; and n is 1.
[0142] Exemplary Artemi sinin-Proteasome inhibitor conjugates of the present application with a Proteasome inhibitor moiety of Formula (Illa) include, but are not limited to
[0143] In another embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (Illb) : wherein is the point of attachment to the Linker; L is — (CR3’Rx)p— ;
M is — (CR2’ y)r—
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, — (CH2)mC(O)NR6R7, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn;
R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2; and s is 0 or 1.
[0144] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (IIIc), Formula (IIId), or Formula (Hie) : wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, and — (CH2)mC(O)NR6R7 ;
R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring; Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; q is 0, 1, or 2; and s is 0 or 1.
[0145] In another embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (IIIF): wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, and — (CH2)mC(O)NR6R7 ; R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; s is 0 or 1; and q is 0, 1, or 2.
[0146] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (Illg): wherein is the point of attachment to the linker;
W is CHR3’or NR3’;
X1 is selected from the group consisting of — C(O)-NH — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle;
Y1 is optional and, if present, is — (CH2)m — ;
Z1 is optional and, if present, is aryl or bicyclic heteroaryl, wherein aryl or bicyclic heteroaryl can be optionally substituted 1 or 2 times with H, halogen, NH2, NHCOOC1- 12 alkyl, or C1-12 alkyl;
R' is H or C1-6 alkyl;
R2’ is H or C1-6 alkyl;
Ry is H or C1-6 alkyl; or R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring; R3 is selected from the group consisting of C1-6 alkyl, and — (CH2)nC(O)NR6R7, wherein C1-6 alkyl can be optionally substituted from 1 to
3 times with a substituent selected independently at each occurrence thereof from OH or C(O)OR10, wherein A is the point of attachment to the corresponding carbon atom of the structure of Formula (Illg);
R6, R7 are selected from the group consisting of H, C1-6 alkyl, and arylalkyl; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R9;
R9 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
R10 is H or arylalkyl; k is 1 or 2; m is 0, 1, or 2; and n is 0, 1, 2, 3, or 4.
[0147] Another embodiment relates to the proteasome inhibitor moiety of Formula (Illg), where R2’ and Ry are taken together with the carbon to which they are attached to form group, and wherein is the point of attachment to NR'; and is the point of attachment to X .
[0148] Furthermore, in the proteasome inhibitor moiety of Formula (Illg), R2’ and Ry may be taken together with the carbon to which they are attached to form group, and wherein is the point of attachment to NR'; and is the point of attachment to X1. [0149] One embodiment relates to the Artemi sinin-Proteasome inhibitor conjugate, wherein the proteasome inhibitor is selected from the group consisting of
[0150] Further examples of the Proteasome inhibitor moieties of Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (IIIf), and Formula (Illg), useful in the present application are disclosed in U.S. Patent Serial No.: 9,988,421 to Lin et al.; and U.S. Patent Application Publication Nos.: 20180221431, 20180282317, and 20200317729 to Lin et al., which are hereby incorporated by reference in their entirety.
[0151] In another embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (IV): wherein is the point of attachment to the Linker;
Y is wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV);
R1’ is a H, branched, cyclic, or linear C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl, wherein the C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl may be optionally substituted from 1 to 3 times with R3’; R2’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)xC(O)NHR4’, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — N3, — CF3, — O C1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R3’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R4’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; x is 1 or 2; and n is 0, 1, 2, or 3. [0152] In a further embodiment of the Artemi sinin-Proteasome inhibitor conjugates of the present application, the Proteasome inhibitor moiety includes a compound of Formula (IV): wherein is the point of attachment to the Linker; wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV);
R1’ is a H, branched, cyclic, or linear C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl, wherein the C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl may be optionally substituted from 1 to 3 times with R3’;
R2’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)xC(O)NHR4 ’ , wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — O C1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R3’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R4’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; x is 1 or 2; and n is 0, 1, 2, or 3.
[0153] One embodiment relates to the proteasome inhibitor moieties of Formula (IV), where
Linker is — (CH2)-C(=O)— or — CH2-C(=O)-NH-(CH2)y-C(=O)— ;
Y is
R1’ is a C4 alkyl;
Z1 and Z2 are OH; and n is 0.
[0154] Artemisinin-Proteasome inhibitor conjugates of the present application with a
Proteasome inhibitor moiety of Formula (IV) include, but are not limited to
[0155] Exemplary Artemi sinin-Proteasome inhibitor conjugates of the present application with a Proteasome inhibitor moiety of Formula (IV) include, but are not limited to [0156] Further examples of the Proteasome inhibitor moiety of Formula (IV) useful in the present application are disclosed in U.S. Patent Serial No.: 8,871,745; 7,442,830; 7,687,662;
8,003,819; and 8,530,694 to Olhava et al.; 10,604,538 to Elliott et al., 9233115 to Bernardini et al., and 8,703,743 to Fleming et al., which are hereby incorporated by reference in their entirety.
Scheme 1. General synthetic scheme for the formation of exemplary Artemi sinin-Proteasome inhibitor conjugates of the present application. R’, R1-R5, R1’, L, Y, M, Q, Q1, X, and n are as describe supra for the respective moieties of the Artemi sinin-Proteasome inhibitor conjugates.
[0157] One method for the formation of the Artemi sinin-Proteasome inhibitor conjugates of the present application is shown in Scheme 1. The artemisinin moiety can be coupled to the linker- Proteasome inhibitor moiety using (1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) mediated coupling to form an amide bond as shown. Generally, HATU coupling occurs through a two-step process where a carboxylic acid is activated through deprotonation and reaction with HATU forming an active ester. This is followed by nucleophilic addition of an amine to the activated ester to form an amide bond. The formation of the Artemi sinin-Proteasome inhibitor conjugates is not limited to HATU coupling, other common coupling reactions may also be used in the formation of the conjugates of the present application. The direct conversion of carboxylic acids and amines to form amides can be difficult due to the basic amine deprotonating the carboxylic acid thus forming a carboxylate. However, heating the ammonium carboxylate and the removing the water that is formed can push the reaction forward to form the amide (Lundberg et al., “Catalytic Amide Formation from Non-activated Carboxylic Acids and Amines,” Chem. Soc. Rev. 1-29 (2014), which is hereby incorporated by reference in its entirety). More commonly coupling agents are used to activate the carboxylic acid. Some common coupling agent that may be used in the present application include, for example, dicyclohexyl carbodiimide (DCC), N,N'- carbonyl diimidazole (CDI), N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline (EEDQ), N- isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ), 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) or benzotriazol-1-yl-oxy-tris- pyrrolidinophosphonium hexafluorophosphate (commercially available as PyBOP® (Novabiochem, a division of Merck KGaA, Darmstadt, Germany)). In some cases the reactions can be conducted in the presence of a base, for example a trialkylamine such as triethylamine or diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, 4-DMAP or 1,8- diazabicycle[5.4.0]undec-7-ene (DBU). The coupling reactions are preferably are conducted in an inert solvent, such as halogenated hydrocarbons, e.g. dichloromethane, chloroform, dipolar aprotic solvents such as acetonitrile, dimethylformamide, dimethylacetamide, DMSO, HMPT, and ethers such as tetrahydrofuran (THF). Exemplary amide formation reactions that may be used in the formation of the conjugates of the present application are disclosed in U. S. Patent Nos.: 7,705,025 to Finley et al.; 9,309,252 to Brian et al.; 7820821 to Mjalli et al.; 8012939 to Simmen et al.; and 9663519 to Charrier at el., which are hereby incorporated by reference in their entirety.
[0158] A second aspect of the present application relates to a method of treating infectious diseases in a subject. This method includes administering to the subject in need thereof a compound of the present application.
[0159] In one embodiment, the infectious disease is caused by bacterial, viral, parasitic, and fungal infectious agents.
[0160] In one embodiment, the infectious disease is caused by a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium-intr acellular e, and Mycobacterium leprosy.
[0161] In another embodiment, the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses, Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses. [0162] In yet another embodiment, the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enter obius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.
[0163] In one embodiment, the infectious disease is malaria.
[0164] While it may be possible for compounds of Formula (I), Formula (I'), Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (Illf), Formula (Illg), and Formula (IV), to be administered as raw chemicals, it will often be preferable to present them as a part of a pharmaceutical composition. Accordingly, another aspect of the present application is a pharmaceutical composition containing a therapeutically effective amount of the compound of Formula (I), Formula (I'), Formula (II), Formula (III), Formula (Illa), Formula (Illb), Formula (IIIc), Formula (IIId), Formula (Ille), Formula (Illf), Formula (Illg), and Formula (IV) or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. The carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof
[0165] In the methods of the present application, the Artemi sinin-Proteasome inhibitor conjugates can be administered using any method standard in the art. The Artemisinin- Proteasome inhibitor conjugates can be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes. The compositions of the present application may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions. [0166] The agents of the present application may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or they may be incorporated directly with the food of the diet. Agents of the present application may also be administered in a time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage. For oral therapeutic administration, the agents of the present application may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of the agent, although lower concentrations may be effective and indeed optimal. The percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of an agent of the present application in such therapeutically useful compositions is such that a suitable dosage will be obtained.
[0167] Also specifically contemplated are oral dosage forms of the agents of the present application. The agents may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. (Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981), which are hereby incorporated by reference in their entirety). Other polymers that could be used are poly-1, 3-dioxolane and poly-1, 3, 6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.
[0168] The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, sucrulose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to the above types of materials, a liquid carrier such as a fatty oil.
[0169] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
[0170] The agents of the present application may also be administered parenterally. Solutions or suspensions of the agent can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0171] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
[0172] When it is desirable to deliver the agents of the present application systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0173] Intraperitoneal or intrathecal administration of the Artemisinin-Proteasome inhibitor conjugates of the present application can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, CA. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.
[0174] In addition to the formulations described previously, the agents may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [0175] The agents of the present application may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the agent of the present application in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The agent of the present application also may be administered in a non— pressurized form such as in a nebulizer or atomizer.
[0176] Effective doses of the compositions of the present application, for the treatment of cancer or pathogen infection vary depending upon many different factors, including type and stage of cancer or the type of pathogen infection, means of administration, target site, physiological state of the patient, other medications or therapies administered, and physical state of the patient relative to other medical complications. Treatment dosages need to be titrated to optimize safety and efficacy.
[0177] The percentage of active ingredient in the compositions of the present application may be varied such that a suitable dosage is obtained. Obviously, several unit dosage forms may be administered at about the same time. The dose employed will be determined by the physician and depends upon the desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient. In the adult, the doses are generally from about 0.01 to about 100 mg/kg body weight, preferably about 0.01 to about 10 mg/kg body weight per day by inhalation, from about 0.01 to about 100 mg/kg body weight, preferably 0.1 to 70 mg/kg body weight, more especially 0.1 to 10 mg/kg body weight per day by oral administration, and from about 0.01 to about 50 mg/kg body weight, preferably 0.01 to 10 mg/kg body weight per day by intravenous administration. In each particular case, the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state of health, and other characteristics which can influence the efficacy of the medicinal product.
[0178] The Artemi sinin-Proteasome inhibitor conjugates according to the present application may be administered as frequently as necessary in order to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long— term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient. Generally, the active product may be administered orally 1 to 4 times per day. It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.
[0179] A third aspect of the present application relates to a method of treating cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders in a subject, or for providing immunosuppression for transplanted organs or tissues in a subject. This method includes administering to the subject in need thereof a compound of the present application.
[0180] Selective inhibition of the i-20S is believed to impact the immune system but would otherwise be far less toxic than combined inhibition of both constitutive and immuno- proteasomes. Here are presented inhibitors that act both with high selectivity and full reversibility on hu i-20S β5i over hu c-20S. Inhibitors that are selective for the i-20S β5i are expected to be equally if not more efficacious in treating autoimmune disease, with less toxicity. These inhibitors could open a new path to the treatment of immunologic, autoimmune, inflammatory, neurodegenerative, and certain neoplastic disorders such as: arthritis, colitis, multiple sclerosis, lupus, Sjogren Syndrome, Systemic Lupus Erythematosus and lupus nephritis, glomerulonephritis, Rheumatoid Arthritis, Inflammatory bowel disease (IBD), ulcerative colitis, Crohn's diseases, Psoriasis, and asthma.
[0181] Exemplary inflammatory disorders that may be treated with the Artemisinin- Proteasome inhibitor conjugates, include, but are not limited to, Crohn’s disease, ulcerative colitis, arthritis, or lupus. The Artemisinin-Proteasome inhibitor conjugates may provide immunosuppression useful for transplanted organs or tissues, and can be used to prevent transplant rejection and graft-verse-host disease.
[0182] The compounds and pharmaceutical compositions of the present application are particularly useful for the treatment of cancer. As used herein, the term “cancer” refers to a cellular disorder characterized by uncontrolled or disregulated cell proliferation, decreased cellular differentiation, inappropriate ability to invade surrounding tissue, and/or ability to establish new growth at ectopic sites. The term “cancer” includes, but is not limited to, solid tumors and bloodborne tumors. The term “cancer” encompasses diseases of skin, tissues, organs, bone, cartilage, blood, and vessels. The term “cancer” further encompasses primary and metastatic cancers.
[0183] Non-limiting examples of solid tumors that can be treated with the disclosed proteasome inhibitors include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen— independent prostate cancer; renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma.
[0184] In one embodiment, cancer is treated. The cancer is selected from the group consisting of neoplastic disorders, hematologic malignances, lymphocytic malignancies, multiple myeloma, mantle cell lymphoma, leukemia, Waldenstrom Macroglobulinemia, pancreatic cancer, bladder cancer, colorectal cancer, chordoma cancer, breast cancer, metastatic breast cancer, prostate cancer, androgen-dependent and androgen-independent prostate cancer, renal cancer, metastatic renal cell carcinoma, hepatocellular cancer, lung cancer, non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung, ovarian cancer, progressive epithelial or primary peritoneal cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, squamous cell carcinoma of the head and neck, melanoma, neuroendocrine cancer, metastatic neuroendocrine tumors, brain tumors, glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma, bone cancer, and soft tissue sarcoma.
[0185] Another aspect of the present application relates to a pharmaceutical composition including a therapeutically effective amount of the compounds of the present application and a pharmaceutically acceptable carrier.
[0186] The term "therapeutically effective amounts" is meant to describe an amount of compound of the present application effective in inhibiting the proteasome or immunoproteasome and thus producing the desired therapeutic effect. Such amounts generally vary according to a number of factors well within the purview of ordinarily skilled artisans given the description provided herein to determine and account for. These include, without limitation: the particular subject, as well as its age, weight, height, general physical condition, and medical history; the particular compound used, as well as the carrier in which it is formulated and the route of administration selected for it; and, the nature and severity of the condition being treated. [0187] The term "pharmaceutical composition" means a composition comprising a compound of the present application and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifingal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar — agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
[0188] The term "pharmaceutically acceptable" means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
[0189] The term "pharmaceutically acceptable dosage forms" means dosage forms of the compound of the application, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.
[0190] Artemisinin, a sesquiterpene lactone isolated from the Artemisia annua Chinese herb, and clinical use of its analogues (e.g. artemether, arteether and artesunate) were a major breakthrough in malaria chemotherapy because they produce a very rapid therapeutic response, particularly against ring stage Plasmodium falciparum malaria including multidrug-resistant strains. Despite the rapid clearance of parasites, the short half-lives of these compounds lead to recrudescence of parasitemia after monotherapy. Therefore, artemisinin-based combination therapy (ACT) has now been recommended by the World Health Organization as standard therapy for falciparum malaria. Moreover, artemisinin and its derivatives have attracted attention as promising anticancer agents because they have potent antineoplastic activity. Antineoplastic activity is postulated to be through a variety of molecular mechanisms in both drug-sensitive and drug-resistant cancer cell lines. Growing experimental evidence demonstrates the great potential of artemisinin compounds for use as a therapeutic alternative to treat highly aggressive cancers and for use as part of anticancer combination therapies without causing drug resistance or added side effects.
[0191] The proteasome is a large multi -protease complex and is responsible for the controlled degradation of more than 80% of cellular proteins. As such, the proteasome plays a key role in maintaining cellular protein homeostasis and regulates numerous biological processes, such as cell survival, DNA repair, apoptosis, signal transduction, and antigen presentation. To date, the proteasome has been successfully exploited as a therapeutic target to treat human cancers. There are three proteasome inhibitor (PI) drugs in clinical use. Propelled by exemplary academic-industrial partnerships, drug development targeting the proteasome has expanded from cancer to autoimmune diseases and recently to infections. The 20S core proteasomes of Plasmodium falciparum, Trypanosoma and Leishmania have become attractive targets for treatment of malaria, Chagas’ disease and Leishmaniasis, respectively. In particular, Plasmodium proteasome inhibitors are reported to be active at multiple stages of the parasite life cycle and synergize with artemisinins.
[0192] Hybrid molecules are combinations of two or more drugs that have varied biological activities and mechanisms; these combinations may improve the efficacy of the drugs by enhancing their bioavailability and by avoiding drug resistance. Thus, hybridization via the covalent coupling of two biologically active compounds has been considered a useful strategy for drug development.
[0193] The present application relates to Artemi sinin-proteasome inhibitor hybrid compounds. These compounds are useful for inhibiting the activity of human proteasome and Plasmodium proteasome and may be used in the treatment of human cancers and malaria.
[0194] Preferences and options for a given aspect, feature, embodiment, or parameter of the technology described herein should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the technology.
[0195] The following Examples are presented to illustrate various aspects of the present application, but are not intended to limit the scope of the claimed application.
EXAMPLES
Materials and Methods
MATERIALS
[0196] The human constitutive proteasome (c-20S, Catalog No.: E-360), human 20S immunoproteasome (i-20S, Catalog No.: E-370), and recombinant human PA28 activator alpha subunit (Catalog NO.: E-381) were purchased from Boston Biochem. The P. falciparum 20S proteasome (Pf20S) was purified as reported (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Sci. USA 115(29):E6863-E6870 (2018), which is hereby incorporated by reference in its entirety). β-Casein (Catalog No.: C6905), bovine serum albumin (BSA, Catalog No.: 3117057001), hemin (Catalog No.: 51280), sodium ascorbate (Catalog No.: PHR1279), artemisinin (ART, Catalog No.: 361593), and artesunate (ASU, Catalog No.: A3731) were purchased from Sigma-Aldrich. Trypsin (V528A) and chymotrypsin (V106A) were purchased from Promega. Proteasome β5 substrate suc-LLVY-AMC, β5i substrate Ac-ANW-AMC, 01 and β1i substrate Z-LLE-AMC, and 02 and β2i substrate Z-VLR-AMC were purchased from Boston Biochem. Activity -based probe MV151 was synthesized as reported (Verdoes et al., “A Fluorescent Broad-Spectrum Proteasome Inhibitor for Labeling Proteasomes in vitro and in vivo,” Chem. Biol. 13(11): 1217-1226 (2006), which is hereby incorporated by reference in its entirety). 02-Specific inhibitor WLW-VS was prepared following the reported method (O'Donoghue et al., “Structure- and Function-based Design of Plasmodium-selective Proteasome Inhibitors,” Nature 530(7589):233-236 (2016), which is hereby incorporated by reference in its entirety).
[0197] The following parasite strains were obtained through BEI Resources, NIAID, NIH: Plasmodium falciparum,' strain IPC 5202 contributed by Didier Menard and Cam3. lRev, and Dd2 K13R539T contributed by David Fidock.
METHODS
In Vitro Cultivation
[0198] P. falciparum laboratory lines were grown under standard conditions in RPMI
1640 medium with 0.5% Albumax II (Invitrogen), 5% hematocrit, 0.25% sodium bicarbonate, and 0.1 mg/ml gentamicin. Parasites were placed in an incubator under 90% nitrogen, 5% carbon dioxide, and 5% oxygen at 37 °C. Two Dd2-derived resistant strains (Dd2β5A49S and Dd2β6Al 17D) were developed in-house and identified as described (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Sci. USA 115(29):E6863-E6870 (2018); Zhan et al., “Improvement of Asparagine Ethylenediamines as Anti-malarial Plasmodium-Selective Proteasome Inhibitors,” J. Med. Chem. 62(13):6137-6145 (2019), which are hereby incorporated by reference in their entirety).
Chemicals and Spectroscopy
[0199] Unless otherwise stated, all commercially available materials were purchased from Aldrich, P3 BioSystems, Combi-Blocks, or other vendors and were used as received. All reactions in aprotic solvents were performed under argon in oven-dried glassware. Reaction progress was monitored on a Waters Acquity Ultra Performance Liquid Chromatography (UPLC/MS). All HPLC purifications were performed on a Waters Autopure (mass directed purification system) equipped with a Prep C18 5pm OBD (19 X 150 mm) column. 1H- and 13C- NMR spectra were acquired on a Bruker DRX-500 spectrometer. Chemical shift 6 is expressed in parts per million, with the solvent resonance as an internal standard (CDCL, 1H: 7.26; 13C: 77.16 ppm; DMSO-d6, 1H: 2.50 ppm; 13C: 39.52 ppm). NMR data are reported as follows: chemical shift, multiplicity (br = broad, d = doublet, q = quartet, m = multiplet, s = singlet, t = triplet), coupling constant, and integration. High Resolution Mass Spectra (HRMS) of final products were collected on a PE SCIEX API 100.
Example 1 - General Synthetic Procedures for Example 2
General Procedure for HA TU Mediated Amide Bond Formation
[0200] The solution of carboxylic acid (1.1 equivalent) and O-(7-azabenzotriazole-l-yl)- N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU, 1.5 equivalent) in anhydrous DMF was cooled to 0 °C on ice bath prior to addition of amine (1 equivalent) and Hünig base (2 - 3 equiv) sequentially to the reaction mixture at 0 °C. The reaction mixture was stirred at 0 °C and the reaction progress was monitored on an UPLC. The reactions usually completed in 2 - 3 hours. After the completion of reaction, cold water was added to quench the reaction, and the mixture was stirred for 15 minutes. Mixture was extracted twice with ethyl acetate or dichloromethane, the combined organic layer was washed with IN HCl, water, saturated NaHCO3 solution, and saturated brine solution. Organic layer was further dried over anhydrous Na2SO4 and evaporated under vacuum to give product, which was used directly in next step without further purification or purified by flash column chromatography or preparative LCMS.
General Procedure II for Amidation of Carboxylic Acids and Primary Amines [0201] To a solution of carboxylic acid (1.0 mmol, 1.0 eq) and diisopropyl ethyl amine (3.0 mmol, 3.0 eq) in dimethyl formamide (4.0 mL) was added HATU (1.2 mmol, 1.2 eq) at 0°C. The mixture was stirred for 10 min at 0°C. A solution of primary amine (1.1 mmol, 1.1 eq) in dimethyl formamide (1.0 mL) was added to the mixture at 0°C. The mixture was stirred for 2 hours at 25°C. LCMS showed that desired mass was detected. The reaction mixture was poured into water (30 mL) and extracted with dichloromethane (3X). The combined organic phase was washed with saturated sodium bicarbonate aqueous, saturated ammonium chloride, and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel) to afford amide as a colorless oil.
General Procedure for Boc-Deprotection
[0202] The solution of substrate in dichloromethane was cooled to 0 °C prior to addition of trifluoroacetic acid (20% v/v with respect to dichloromethane) dropwise at 0 °C with stirring. The mixture was allowed to warm to room temperature gradually over a period of 1 hour and stirred until the completion of reaction (2 - 3 hours; monitored on an UPLC). Excess trifluoroacetic acid and dichloromethane was evaporated and the residue was dried under vacuum.
General Procedure II for Deprotection of BOC-Protected Amines
[0203] To a solution of Boc-protected compound (0.5 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (1.0 mL) drop-wise. The mixture was stirred at 20°C for 3 hours. LCMS showed that the starting material was consumed. The mixture was concentrated in vacuum to give primary amine as a colorless oil, which was used for the next step without further purification.
General Procedure for Hydrolysis of Boronates to Provide Boronic Acid
[0204] To a solution of boronates (0.2 mmol, 1.0 eq) in MeOH (3.0 ml) was added hexane (3.0 ml), isobutylboronic acid (1.0 mmol, 5.0 equiv), and 1 M HCl (0.6 mL). The resulting mixture was vigorously stirred for 24 hours at 25 °C. The resulting mixture was diluted with MeOH (20 mL) and hexane (20 mL) and was extracted with MeOH (3X). The solvent was removed under reduced pressure and the residue was dissolved in DCM and was washed with 5% NaHCO3, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by prep-HPLC (column: OBD Cl 8 150mm*19mm*5um; mobile phase: [water (0.1%TFA)-ACN]; B%: 5%-95%, 20min) and lyophilisation to afford boronic acid as a white solid.
Example 2 - Synthesis of Representative Compounds
[0205] The synthetic route to Artesunate-based hybrids WZ-13 and WZ-06 and control WZ-20 are shown in Figure 1.
Preparation of Benzyl N2-(tert-Butoxycarbony)-N4-(tert-butyl)-L-asparaginate (1) [0206] Benzyl N2-(tert-butoxycarbonyl)-N4-(tert-butyl)-L-asparaginate (1) was synthesized by following the general procedure for HATU mediated coupling of Boc-Asp-OBn (3.55g, 11 mmol) and tert-butyl amine (0.73 g, 10 mmol). The isolated off-white product (2.95g, 78%) was used in next step without further purification. 1H NMR (500 MHz, CDCL) δ 7.36 - 7.26 (m, 5H), 5.92 - 5.76 (m, 1H), 5.41 (br, 1H), 5.21 (d, J= 12.4 Hz, 1H), 5.14 (d, J= 12.4 Hz, 1H), 4.57 - 4.42 (m, 1H), 2.79 (dd, J= 15.8, 4.9 Hz, 1H), 2.62 (dd, J= 15.8, 4.2 Hz, 1H), 1.42 (s, 9H), 1.29 (s, 9H). ES+ calc, for C20H31N2O5 [M + H]+: 379.2. Found: 379.2. Preparation of N2-(tert-Butoxycarbonyl)-N4-(tert-butyl)-L-asparagine (2)
[0207] 1 (1.89 g, 5 mmol) was dissolved in methanol and Palladium on carbon (10%) was added carefully. Residual air from the flask was removed and flask was flushed with hydrogen. The mixture was stirred at room temperature for 3 hours under hydrogen atmosphere using a hydrogen balloon. After completion of the reaction, the mixture was filtered through celite. The filtrate was evaporated and dried under vacuum to give N2-(tert-butoxycarbonyl)-N4- ftert-butyl)-L-asparagine (2) as a white powder (1.45g, quant.). ES+ calc, for C13H23N2O5 [M - H]-: 287.2. Found: 287.2.
Preparation of N-(2-Aminoetbyl)-2',4-difluoro-[l,l'-biphenylI-3-carboxamide Trifluoroacetate Salt (3)
[0208] N-(2-Aminoethyl)-2',4-difluoro-[ l , l '-biphenyl]-3-carboxamide trifluoroacetate salt (3) was synthesized by two successive steps, one following the general procedure for HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid (141.6 mg, 605 μmol) and tert- butyl N-(2-aminoethyl)carbamate (88.1 mg, 550 μmol) and the other following the general procedure for Boc-deprotection of the product in first step. The isolated white product (172.0 mg, two step yield: 80%) was used in next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 8.61 - 8.50 (m, 1H), 8.04 - 7.76 (m, 4H), 7.76 - 7.67 (m, 1H), 7.61 - 7.51 (m, 1H), 7.49 - 7.39 (m, 2H), 7.38 - 7.30 (m, 2H), 3.59 - 3.46 (m, 2H), 3.07 - 2.93 (m, 2H). ES+ calc, for C15H15F2N2O [M + H]+: 277.2. Found 277.2.
Preparation of tert-Butyl (S)-(4-(tert-butylamino)-l-((2-(2',4-difluoro-[l,l'-biphenyl]- 3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)carbamate (5)
[0209] tert-Butyl (S)-(4-( tert-butylamino)-l-((2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)carbamate (5) was synthesized by following the general procedure for HATU mediated coupling of 2 (63.5 mg, 220 μmol) and 3 (78.0 mg, 200 μmol). After completion of the reaction, the mixture was purified by flash column chromatography to give the product (95.0 mg. 87%) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 8.44 - 8.33 (m, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.83 - 7.75 (m, 1H), 7.74 - 7.67 (m, 1H), 7.60 - 7.52 (m, 1H), 7.48 - 7.42 (m, 1H), 7.42 - 7.29 (m, 4H), 6.74 (d, J = 8.2 Hz, 1H), 4.24 - 4.13 (m, 1H), 3.40 - 3.24 (m, 3H), 3.24 - 3.15 (m, 1H), 2.38 (dd, J = 14.3, 5.4 Hz, 1H), 2.30 (dd, J = 14.3, 8.4 Hz, 1H), 1.34 (s, 9H), 1.19 (s, 9H). HRMS calc, for C28H36F2N4O5Na [M + Na]+: 569.2551. Found: 569.2564. Preparation of (S)-2-Amino-N4-(tert-butyl)-Nl-(2-(2',4-difluoro-[l,l'-biphenyl]-3- carboxamido)ethyl)succinamide Trifluoroacetate Salt (7)
[0210] (S)-2-Amino-N4-(tert-butyl)-N1-(2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)succinamide trifluoroacetate salt (7) was synthesized by following the general procedure for Boc-deprotection of 5 (42.0 mg, 76 μmol). Isolated crude was purified by preparative LCMS to give the product (40.2 mg, 90%) as a colorless gum. 3H NMR (500 MHz, DMSO-d6) δ 8.49 (t, J = 5.2 Hz, 1H), 8.46 - 8.41 (m, 1H), 8.10 (d, J = 4.8 Hz, 3H), 7.82 - 7.78 (m, 2H), 7.73 - 7.69 (m, 1H), 7.59 - 7.54 (m, 1H), 7.49 - 7.39 (m, 2H), 7.36 - 7.30 (m, 2H), 4.00 - 3.94 (m, 1H), 3.43 - 3.29 (m, 3H), 3.29 - 3.19 (m, 1H), 2.65 (dd, J = 16.5, 5.1 Hz, 1H), 2.55 (dd, J = 16.5, 7.8 Hz, 1H), 1.22 (s, 9H). ES+ calc, for C23H29F2N4O3 [M + H]+: 447.2. Found: 447.3.
Preparation of (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-Trimethyldecahydro-12H- 3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl 4-(((S)-4-(tert-butylamino)-l-((2-(2 ',4- difluoro-[1,1'-biphenyl]-3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-4- oxobutanoate (WZ-13)
[0211] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-Trimethyldecahydro-12H-3,12- epoxy[ 1 ,2]dioxepino[4,3-i]isochromen- 10-yl 4-(((S)-4-( tert-butylamino)- 1 -((2-(2',4-difluoro- [1,1'-biphenyl]-3-carboxamido)ethyl)amino)- 1 ,4-dioxobutan-2-yl)amino)-4-oxobutanoate (WZ- 13) was synthesized by following the general procedure for HATU mediated coupling of artesunate (see Scheme 2) (21.0 mg, 55 μmol) and 7 (28.0 mg, 50 μmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (30.5 mg, 75%) as a white powder. 1H NMR (500 MHz, CDCl3) 6 8.14 (d, J= 6.7 Hz, 1H), 7.65 (t, J= 7.1 Hz, 2H), 7.55 (t, J= 5.0 Hz, 1H), 7.42 (t, J= 7.6 Hz, 2H), 7.32 (dd, J= 13.3, 6.8 Hz, 1H), 7.23 - 7.10 (m, 3H), 5.86 (s, 1H), 5.62 (d, J= 9.8 Hz, 1H), 5.39 (d, J= 11.1 Hz, 1H), 4.72 - 4.65 (m, 1H), 3.71 - 3.40 (m, 4H), 3.01 - 2.58 (m, 2H), 2.54 - 2.27 (m, 4H), 2.06 - 1.70 (m, 3H), 1.65 - 1.48 (m, 3H), 1.45 - 1.13 (m, 16H), 0.98 - 0.86 (m, 2H), 0.83 (d, J= 6.2 Hz, 3H), 0.74 (d, J= 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 173.04, 171.59, 171.55, 170.96, 163.77, 163.75, 160.72, 159.15, 158.75, 133.72, 133.67, 132.54, 132.52, 132.21, 130.73, 130.71, 129.71, 129.64, 127.37, 127.26, 124.66, 124.63, 122.00, 121.90, 116.41, 116.36, 116.22, 116.19, 104.52, 104.19, 103.48, 92.80, 91.52, 87.89, 80.23, 51.67, 51.66, 50.59, 45.26, 39.66, 39.51, 37.61, 37.12, 36.33, 34.16, 31.72, 31.18, 29.81, 28.64, 25.92, 24.58, 21.88, 20.19, 12.09. HRMS calc, for C42H55F2N4O10 [M + H]+: 813.3881. Found: 813.3907. Figures 2-5 show the characterization of the product.
Scheme 2. Structures of Artemisinin, Artesunate, AsnEDA-based proteasome inhibitors
PKS21224 and PKS21208 and control compound WZ-20.
Preparation of (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-Trimethyldecahydro-12H- 3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl 4-(((S)-4-(tert-butylamino)-l,4-dioxo-l-((2- (4-phenylpicolinamido)ethyl)amino)butan-2-yl)amino)-4-oxobutanoate (WZ-06)
[0212] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-Trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen- 10-yl 4-(((S)-4-(tert-butylamino)- 1 ,4-dioxo- 1 -((2-(4- phenylpicolinamido)ethyl)amino)butan-2-yl)amino)-4-oxobutanoate (WZ-06) was synthesized by following the general procedure for HATU mediated coupling of artesunate (21.0 mg, 55 μmol) and 8 (26.3 mg, 50 μmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (31.5 mg, 81%) as a white powder. 1 H NMR (500 MHz, CDCl3) δ 8.81 (d, J= 5.5 Hz, 1H), 8.72 (t, J= 5.7 Hz, 1H), 8.59 (s, 1H), 7.99 (d, J= 7.7 Hz, 1H), 7.94 (d, J= 5.3 Hz, 1H), 7.84 - 7.75 (m, 3H), 7.56 (dd, J= 9.9, 5.2 Hz, 3H), 6.75 (s, 1H), 5.65 (d, J= 9.9 Hz, 1H), 5.43 (s, 1H), 4.72 (dd, J= 12.2, 6.6 Hz, 1H), 3.83 (dd, J= 12.8, 5.7 Hz, 1H), 3.64 - 3.47 (m, 2H), 3.39 (dd, J= 12.5, 7.1 Hz, 1H), 2.80 (dd, J= 14.7, 6.6 Hz, 1H), 2.73 - 2.52 (m, 4H), 2.50 - 2.29 (m, 3H), 1.99 (d, J= 14.4 Hz, 1H), 1.86 (dd, J= 8.9, 4.5 Hz, 1H), 1.75 - 1.54 (m, 3H), 1.48 - 1.22 (m, 16H), 1.02 - 0.86 (m, 4H), 0.76 (d, J= 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 173.08, 172.70, 172.15, 171.26, 162.84, 154.12, 147.34, 146.24, 135.98, 131.00, 129.71, 127.75, 124.82, 121.35, 104.83, 93.12, 91.81, 80.32, 52.22, 51.64, 51.31, 45.22, 39.70, 38.97, 37.74, 37.28, 36.31, 34.13, 31.69, 30.51, 29.58, 28.47, 25.80, 24.58, 21.95, 20.26, 12.07. HRMS calc, for C41H56N5O10 [M + H]+: 778.4022. Found: 778.4001. Figures 6-9 show the characterization of the product.
Preparation of (S)-4-((4-(tert-Butylamino)-1-((2-(2',4-difluoro-[1,1'-biphenyl] -3- carboxamido)ethyl)amino)-1,4-dioxobutan-2-yl)amino)-4-oxobutanoic Acid (WZ-20)
[0213] To a stirred solution of 7 (28.0 mg, 50 μmol) and succinic anhydride (5.5 mg, 55 μmol) in dry DMF (1 mL) was added Hünig base (35μL, 200 μmol) at 0 °C. The reaction mixture was allowed to stir at r.t. for 4 hours. After completion of the reaction, the mixture was purified by preparative LCMS to give the product (S)-4-((4-(tert-butylamino)-l-((2-(2',4- difluoro-[1,1'-biphenyl]-3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-4- oxobutanoic acid (WZ-20) See Scheme 2) (24.5 mg, 90%) as a white solid. 1H NMR (500 MHz, CD3OD) 5 8.11 - 7.78 (m, 1H), 7.70 (s, 1H), 7.55 - 7.10 (m, 6H), 4.66 - 4.54 (m, 1H), 3.62 - 3.34 (m, 4H), 2.69 - 2.32 (m, 5H), 1.27 (s, 9H). 13C NMR (125 MHz, CD3OD) 1 δ74.73, 173.82, 171.64, 171.56, 166.85, 161.99, 161.92, 160.03, 159.92, 134.55, 133.67, 132.03, 131.82, 130.95, 130.89, 128.48, 128.38, 125.91, 125.88, 124.37, 124.26, 117.52, 117.34, 117.21, 117.03, 52.13, 52.09, 52.02, 40.51, 40.28, 39.08, 39.03, 28.88. HRMS calc, for C27H33F2N4O6 [M + H]+: 547.2363. Found: 547.2355. Figures 10-13 show the characterization of the product.
Preparation of 2-((3R, 5 aS, 6R, 8aS, 9R,10R, 12R, 12aR)-3, 6, 9- Trimethyldecahydro-12H- 3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetic Acid (DeoxoART-AcOH 9)
Scheme 3. Synthetic route to DeoxoART-AcOH 9.
[0214] Using artemisinin as the starting material, DeoxoART-AcOH 9 was synthesized in four steps according to the literature procedures (see Scheme 3) (Stocks et al., “Evidence for a Common Non-Heme Chelatable-Iron-Dependent Activation Mechanism for Semisynthetic and Synthetic Endoperoxide Antimalarial Drugs,” Angew. Chem. Int. Ed. 46(33):6278-6283 (2007), which is hereby incorporated by reference in its entirety). 1H NMR (500 MHz, CDCl3) 6 5.34 (s, 1H), 4.83 (ddd, J= 9.9, 6.0, 3.7 Hz, 1H), 2.75 - 2.62 (m, 2H), 2.49 (dd, J= 15.6, 3.5 Hz, 1H), 2.31 (td, J= 14.0, 3.7 Hz, 1H), 2.06 - 1.88 (m, 3H), 1.81 - 1.75 (m, 1H), 1.70 - 1.62 (m, 2H), 1.40 (s, 3H), 1.31 - 1.21 (m, 4H), 0.95 (d, J= 5.9 Hz, 3H), 0.86 (d, J= 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 176.45, 103.39, 89.43, 80.95, 71.15, 52.22, 44.03, 37.55, 36.56, 35.94, 34.47, 29.84, 25.93, 24.82, 24.77, 20.21, 12.89. ES+ calc, for C17H26O4 [M - O2]+: 294. Figures 14-15 show the characterization of the product. Preparation of 2-((3R, 5aS, 6R, 8aS, 9R, 1OR, 12R, 12aR)-3,6,9-Trimethyldecahydro-12H- 3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl) acetamide (ART1)
[0215] The general synthetic route for ART-based hybrids ATZ1 to ATZ4 is shown in Figure 16.
[0216] 2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-Trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (ART1) was synthesized by following the general procedure for HATU mediated coupling of Deoxo ART- AcOH 9 (32.6 mg, 100 μmol) and 2 M Ammonia solution in ethanol (1 mL, 2 mmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (31.0 mg, 95%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 6.98 (s, 1H), 5.58 (s, 1H), 5.37 (s, 1H), 4.79 (dd, J= 11.0, 6.1 Hz, 1H), 2.63 - 2.49 (m, 2H), 2.31 (dt, J= 14.0, 7.3 Hz, 2H), 2.04 (d, J= 14.4 Hz, 2H), 1.96 (dd, J = 10.1, 4.4 Hz, 1H), 1.83 - 1.62 (m, 3H), 1.37 (s, 3H), 1.31 - 1.19 (m, 3H), 1.02 - 0.90 (m, 4H), 0.87 (d, J= 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 174.65, 103.06, 90.28, 81.00, 69.79, 51.91, 43.47, 37.64, 37.31, 36.60, 34.34, 30.52, 25.84, 24.93, 24.92, 20.11, 12.11. HRMS calc, for C17H27NNaO5 [M + Na]+: 348.1781. Found: 348.1770. Figures 17-20 show the characterization of the product.
Preparation of Tert-butyl (S)-(2-((4-(tert-butylamino)-l-((2-(2',4-difluoro-[1,l'- biphenyl]-3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-2-oxoethyl)carbamate (WZ-0917)
[0217] tert-Butyl (8)-(2-((4-(tert-butylamino)-l-((2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-2-oxoethyl)carbamate (WZ-0917) was synthesized by following the general procedure for HATU mediated coupling of Boc-Gly-OH (19.3 mg, 110 μmol) and 7 (56.0 mg, 100 μmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (54.2 mg, 90%) as a white solid. 1 H NMR (500 MHz, CDCl3) 6 8.22 - 7.98 (m, 2H), 7.76 (s, 1H), 7.63 - 7.47 (m, 2H), 7.40 (dd, J= 11.1, 4.2 Hz, 1H), 7.33 - 7.27 (m, 1H), 7.20 - 7.08 (m, 3H), 6.30 (s, 1H), 5.74 (s, 1H), 4.69 (d, J = 6.4 Hz, 1H), 3.74 (qd, J= 17.0, 5.5 Hz, 2H), 3.58 (s, 2H), 3.51 - 3.32 (m, 2H), 2.78 (dd, J= 14.6, 3.7 Hz, 1H), 2.44 (dd, J= 14.7, 4.8 Hz, 1H), 1.36 (s, 9H), 1.20 (s, 9H). 13C NMR (125 MHz, CDCl3) 6 174.09, 171.76, 170.41, 170.05, 164.30, 163.23, 160.98, 160.64, 158.99, 158.66, 156.75, 133.75, 133.70, 132.49, 132.47, 131.98, 130.69, 130.67, 129.62, 129.56, 127.24, 127.14, 124.62, 124.59, 121.80, 121.70, 116.36, 116.28, 116.16, 116.10, 80.53, 51.50, 50.62, 44.69, 39.98, 39.91, 37.60, 28.52, 28.30. ES+ calc, for C30H40F2N5O6 [M + H]+: 604.3. Found: 604.3. Figures 21-22 show the characterization of the product. Preparation of tert-Butyl (S)-(3-((4-(tert-butylamino)-l-((2-(2',4-difluoro-[l,l biphenyl]-3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-3-oxopropyl)carbamate (WZ-0918)
[0218] tert-Butyl (S)-(3-((4-(tert-butylamino)- 1 -((2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-3-oxopropyl)carbamate (WZ-0918) was synthesized by following the general procedure for HATU mediated coupling of Boc-P-Ala-OH (20.8 mg, 110 μmol) and 7 (56.0 mg, 100 μmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (50.7 mg, 82%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J= 6.4 Hz, 1H), 7.78 (s, 1H), 7.61 (dd, J= 4.4, 1.6 Hz, 2H), 7.42 (t, J= 7.4 Hz, 2H), 7.31 (dd, J= 12.8, 6.7 Hz, 1H), 7.22 - 7.11 (m, 3H), 6.24 (s, 1H), 5.40 (s, 1H), 4.70 (s, 1H), 3.62 (d, J= 4.3 Hz, 2H), 3.55 - 3.41 (m, 2H), 2.73 (d, J= 13.1 Hz, 1H), 2.56 - 2.37 (m, 4H), 1.37 (s, 9H), 1.24 (s, 9H). ES+ calc, for C31H42F2N5O6 [M + H]+: 618.3. Found: 618.3. Figure 23 shows the characterization of the product.
Preparation of tert-Butyl (S)-(4-((4-(tert-butylamino)-l-((2-(2',4-difluoro-[l,l'- biphenyl]-3-carboxamido)ethyl)amino)-l,4-dioxobutan-2-yl)amino)-4-oxobutyl)carbamate (PI01)
[0219] tert-Butyl (S)-(4-((4-(tert-butylamino)-l-((2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)amino)-1,4-dioxobutan-2-yl)amino)-4-oxobutyl)carbamate (PI01) was synthesized by following the general procedure for HATU mediated coupling of Boc-GABA-OH (22.5 mg, 110 μmol) and 7 (56.0 mg, 100 μmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (56.2 mg, 89%) as a white solid. 1 H NMR (500 MHz, CDCl3) 6 8.14 (d, J= 6.2 Hz, 1H), 8.02 (s, 1H), 7.67 - 7.61 (m, 1H), 7.54 (d, J = 6.1 Hz, 1H), 7.44 (t, J= 13 Hz, 2H), 7.33 (dd, J= 13.6, 6.8 Hz, 1H), 7.18 (ddd, J= 22.0, 17.3, 9.2 Hz, 3H), 6.01 (s, 1H), 4.71 (s, 2H), 3.68 - 3.41 (m, 4H), 3.15 (d, = 4.8 Hz, 1H), 2.98 (s, 1H), 2.79 (d, J= 13.2 Hz, 1H), 2.46 (d, J= 10.0 Hz, 1H), 2.29 (t, J= 6.3 Hz, 2H), 1.89 (s, 1H), 1.63 (s, 1H), 1.41 (s, 9H), 1.25 (s, 9H). 13C NMR (125 MHz, CDCl3) 6 172.69, 172.17, 170.89, 164.02, 162.80, 161.10, 160.75, 159.12, 158.78, 156.78, 133.80, 133.76, 132.64, 132.61, 132.28, 130.80, 130.78, 129.70, 129.64, 127.40, 127.29, 124.69, 124.66, 121.88, 121.78, 116.38, 116.20, 79.67, 51.92, 50.77, 40.55, 39.84, 39.04, 38.14, 32.66, 28.60, 28.52, 26.10. HRMS calc, for C32H43F2N5NaO6 [M + Na]+: 654.3074. Found: 654.3058. Figures 24-27 show the characterization of the product. Preparation of (S)-N4-(tert-Butyl)-Nl-(2-(2',4-difluoro-[l,l'-biphenylJ-3- carboxamido)ethyl)-2- (2- ( (3R,5aS,6R,8aS,9R,10R,12R,12aR )-3,6, 9-trimethyldecahydro-l2H- 3, 12-epoxy [l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)succinamide (ATZ1)
[0220] (S)-N4-( tert-Butyl)-N1 -(2-(2',4-difluoro-[1,1'-biphenyl]-3 -carboxamido)ethyl)-2- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyl decahydro- 12H-3 ,12- epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)succinamide (ATZ1) was synthesized by following the general procedure for HATU mediated coupling of DeoxoART-AcOH 9 (18.0 mg, 55 pmol) and 7 (28.0 mg, 50 pmol). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (29.0 mg, 77%) as a white powder. 1H NMR (500 MHz, CDCh) δ 8.24 (dd, J= 8.3, 6.7 Hz, 1H), 7.73 - 7.59 (m, 2H), 7.53 (d, J= 8.7 Hz,
1H), 7.47 (dd, J= 15.8, 7.8 Hz, 2H), 7.34 (dd, J= 12.8, 6.2 Hz, 1H), 7.24 - 7.13 (m, 3H), 5.65 (s, 1H), 5.15 (s, 1H), 4.86 - 4.78 (m, 2H), 3.73 (dd, J= 18.2, 13.5 Hz, 1H), 3.62 - 3.55 (m, 1H), 3.52 - 3.37 (m, 2H), 3.00 (dd, J= 15.4, 4.4 Hz, 1H), 2.51 - 2.42 (m, 2H), 2.41 - 2.28 (m, 2H), 2.22 (td, J= 14.1, 3.7 Hz, 1H), 1.96 (d, J= 14.3 Hz, 1H), 1.87 - 1.80 (m, 1H), 1.62 - 1.55 (m, 1H), 1.54 - 1.47 (m, 1H), 1.38 - 1.19 (m, 14H), 1.12 (td, J= 11.2, 6.2 Hz, 1H), 1.06 - 0.96 (m,
1H), 0.80 (d, J= 6.2 Hz, 3H), 0.78 - 0.62 (m, 5H). 13C NMR (125 MHz, CDCh) δ 172.04, 171.77, 171.29, 170.11, 163.44, 163.42, 162.52, 161.37, 160.79, 159.37, 158.82, 133.74, 133.70, 133.67, 133.63, 132.56, 132.21, 130.73, 130.71, 129.75, 129.68, 127.25, 127.15, 124.72, 124.69, 122.12, 122.02, 116.48, 116.30, 116.26, 103.00, 90.05, 80.72, 71.69, 51.83, 51.81, 50.13, 43.47, 39.77, 39.62, 39.16, 37.91, 37.21, 36.52, 34.23, 30.24, 28.73, 25.59, 24.74, 24.47, 20.02, 12.46.
HRMS calc, for C40H52F2N4NaO8 [M + Na]+: 777.3645. Found: 777.3657. Figures 28-31 show the characterization of the product.
Preparation of (S)-N4-(tert-Butyl)-Nl-(2-(2',4-difluoro-[l,l'-biphenylJ-3- carboxamido)ethyl)-2- (2- (2- ( (3R, 5aS, 6R, 8aS, 9R, 10R, 12R, 12aR)-3, 6, 9-trimethyldecahydro- 12H-3, 12-epoxy [l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)acetamido)succinamide
(ATZ2)
[0221] (S)-N4-(tert-Butyl)-N1 -(2-(2',4-difluoro-[1,1'-biphenyl]-3 -carboxamido)ethyl)-2-
(2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3, 12- epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)acetamido)succinamide (ATZ2) was synthesized by two successive steps, one following the general procedure for Boc-deprotection of WZ-0917 (30.2 mg, 50 μmol) and the other following the general procedure for HATU mediated coupling of DeoxoART-AcOH 9 (18.0 mg, 55 pmol) with all of the product in first step. After completion of the reaction, the mixture in the second step was purified by preparative LCMS to give the product (33.8 mg, two step yield: 83%) as a white powder. 1H NMR (500 MHz, CDCl3) 6 8.26 (d, J= 7.8 Hz, 1H), 8.11 (d, J= 5.9 Hz, 1H), 7.85 (t, J= 4.7 Hz, 1H), 7.76 (d, J= 6.7 Hz, 1H), 7.65 - 7.56 (m, 2H), 7.44 (t, J= 7.4 Hz, 1H), 7.32 (dd, J= 12.9, 6.4 Hz, 1H), 7.23 - 7.10 (m, 3H), 5.89 (s, 1H), 5.37 (s, 1H), 4.85 (dd, J= 10.8, 6.1 Hz, 1H), 4.64 - 4.57 (m, 1H), 3.81 (d, J= 5.1 Hz, 2H), 3.69 - 3.58 (m, 2H), 3.47 (d, J= 5.1 Hz, 2H), 2.78 (dd, J= 14.7, 3.8 Hz, 1H), 2.60 - 2.50 (m, 2H), 2.39 (dd, J= 14.7, 5.1 Hz, 1H), 2.31 (dd, = 22.6, 9.5 Hz, 2H), 2.06 - 1.99 (m, 1H), 1.94 (d, J= 11.7 Hz, 1H), 1.79 - 1.71 (m, 1H), 1.71 - 1.60 (m, 2H), 1.35 (s, 3H), 1.32 - 1.11 (m, 13H), 1.04 - 0.87 (m, 4H), 0.80 (d, J= 7.4 Hz, 3H). 13C NMR (125
MHz, CDCl3) 6 174.11, 171.41, 170.56, 168.98, 164.08, 164.06, 162.91, 160.99, 160.74, 159.00,
158.77, 133.60, 133.57, 133.53, 133.50, 132.48, 132.46, 132.16, 130.81, 130.79, 129.64, 129.57,
127.40, 127.29, 124.67, 124.64, 122.43, 122.33, 116.35, 116.33, 116.17, 116.13, 103.25, 89.94,
80.93, 70.32, 52.03, 51.68, 50.96, 44.36, 43.64, 40.06, 40.04, 37.57, 37.23, 36.76, 36.55, 34.33, 30.28, 28.55, 25.91, 24.90, 24.85, 20.14, 12.27. HRMS calc, for C42H55F2N5NaO9 [M + Na]+: 834.3860. Found: 834.3876. Figures 32-35 show the characterization of the product.
Preparation of (S)-N4-(tert-Butyl)-Nl-(2-(2',4-difluoro-[1,1'-biphenyl]-3- carboxamido)ethyl)-2-(3-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro- 12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)propanamido)succinamide (ATZ3)
[0222] (S)-N4-(tert-Butyl)-N1-(2-(2',4-difluoro-[1,1'-biphenyl]-3-carboxamido)ethyl)-2- (3-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)propanamido)succinamide (ATZ3) was synthesized by two successive steps, one following the general procedure for Boc-deprotection of WZ-0918 (30.9 mg, 50 μmol) and the other following the general procedure for HATU mediated coupling of DeoxoART-AcOH 9 (18.0 mg, 55 μmol) with all of the product in first step. After completion of the reaction, the mixture in the second step was purified by preparative LCMS to give the product (37.5 mg, two step yield: 91%) as a white powder. 1H NMR (500 MHz, CDCl3) δ 8.16 (d, J= 5.8 Hz, 1H), 7.73 - 7.63 (m, 2H), 7.52 - 7.40 (m, 4H), 7.36 - 7.30 (m, 1H), 7.24 - 7.12 (m, 3H), 5.84 (s, 1H), 5.37 (s, 1H), 4.76 - 4.66 (m, 2H), 3.68 - 3.41 (m, 6H), 2.74 (dd, J= 14.7, 3.7 Hz, 1H), 2.60 - 2.40 (m, 5H), 2.29 (dt, J= 23.4, 8.7 Hz, 2H), 2.01 (dd, J= 11.0, 3.2 Hz, 1H), 1.92 (d, = 12.8 Hz, 1H), 1.76 - 1.72 (m, 1H), 1.65 (dd, J= 15.9, 9.6 Hz, 2H), 1.37 (s, 3H), 1.28 - 1.19 (m, 13H), 0.99 - 0.87 (m, 4H), 0.81 (d, J= 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 172.51, 172.13, 170.80, 164.13, 161.11, 160.76, 159.13, 158.79, 133.96, 133.92, 133.88, 133.85, 132.74, 132.71, 132.30, 130.81, 130.78, 129.74, 129.67, 127.34, 127.23, 124.72, 124.69, 121.77, 121.67, 116.44, 116.41, 116.23, 103.29, 89.80, 81.07, 71.00, 52.10, 51.72, 50.76, 43.83, 40.13, 40.00, 38.37, 37.57, 37.49, 37.22, 36.60, 36.23, 34.41, 30.35, 28.66, 25.97, 24.89, 24.84, 20.17, 12.50. HRMS calc, for C43H57F2N5NaO9 [M + Na]+: 848.4017. Found: 848.4001. Figures 36-39 show the characterization of the product.
Preparation of (S)-N4-(tert-Butyl)-Nl-(2-(2',4-difluoro-[l,l'-biphenyl]-3- carboxamido)ethyl)-2- (4- (2- ( (3R,5aS,6R,8aS,9R,10R,12R,12aR )-3,6, 9-trimethyldecahydro- 12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)butanamido)succinamide (ATZ4)
[0223] (S)-N4-(tert-Butyl)-Nl-(2-(2',4-difluoro-[1,1'-biphenyl]-3-carboxamido)ethyl)-2- (4-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)butanamido)succinamide (ATZ4) was synthesized by two successive steps, one following the general procedure for Boc-deprotection of PI01 (31.5 mg, 50 μmol) and the other following the general procedure for HATU mediated coupling of DeoxoART-AcOH 9 (18.0 mg, 55 μmol) with all of the product in first step. After completion of the reaction, the mixture in the second step was purified by preparative LCMS to give the product (31.5 mg, two step yield: 75%) as a white powder. 1H NMR (500 MHz, CDCl3) δ 8.13 (d, J= 6.3 Hz, 1H), 8.05 (s, 1H), 7.65 (s, 2H), 7.55 - 7.48 (m, 1H), 7.43 (t, J= 7.3 Hz, 1H), 7.40 - 7.30 (m, 2H), 7.24 - 7.11 (m, 3H), 6.25 (s, 1H), 5.44 (s, 1H), 4.74 (s, 1H), 4.64 (dd, J= 11.0, 6.1 Hz, 1H), 3.63 (d, J= 4.4 Hz, 2H), 3.50 (d, J= 4.5 Hz, 2H), 3.33 (dt, = 22.8, 11.6 Hz, 1H), 3.09 (dd, J= 12.4, 5.8 Hz, 1H), 2.71 (dd, J= 14.2, 5.2 Hz, 1H), 2.63 - 2.46 (m, 3H), 2.39 - 2.25 (m, 4H), 2.03 (t, J= 12.5 Hz, 1H), 1.94 (d, J= 10.9 Hz, 2H), 1.79 - 1.60 (m, 4H), 1.36 (s, 3H), 1.30 - 1.11 (m, 13H), 1.00 - 0.87 (m, 4H), 0.81 (d, J= 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) 6 173.75, 173.47, 172.24, 170.85, 164.46, 161.05, 160.73, 159.06, 158.76, 134.02, 133.99, 133.94, 133.91, 132.72, 132.69, 132.14, 130.75, 130.72, 129.78, 129.72, 127.23, 127.13, 124.73, 124.70, 121.65, 121.55, 116.46, 116.40, 116.26, 116.22, 103.50, 89.81, 81.00, 70.87, 52.01, 51.92, 50.95, 43.78, 40.02, 38.38, 37.94, 37.60, 36.89, 36.52, 34.33, 32.23, 30.23, 28.53, 25.86, 25.05, 24.93, 24.80, 20.13, 12.43. HRMS calc, for C44H59F2N5NaO9 [M + Na]+: 862.4173. Found: 862.4147. Figures 40-43 show the characterization of the product.
[0224] The HPLC purity and SMILES for compounds WZ-06, WZ-13, WZ-20, PI01, ART1, ATZ1, ATZ2, ATZ3, and ATZ4 are shown in Table 1. Table 1. HPLC Purity for the Final Compounds
Scheme 4. Synthetic procedures of boronic acid artemisinin-proteasome inhibitor hybrids
HZ2082, HZ2083, HZ2087, and HZ2088.
[0225] The generalized synthetic route for the formation of compounds HZ2082, HZ2083, HZ2087, and HZ2088 is shown in Scheme 4.
Preparation of ((R)-3-Methyl-l-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3,6,9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butyl)boronic Acid (HZ2082) [0226] ((R)-3 -Methyl-1-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butyl)boronic acid (HZ2082) was prepared using compound 9 and leucine boronate as starting material by following the general procedure II for the amidation of carboxylic acids and primary amines and general procedure for Deprotection of BOC-protected amines, as shown in Scheme 4, part b. HZ2082 was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) = δ 5.42 (s, 1H), 4.32 (s, 1H), 2.93 (dd, J = 9.2, 5.6 Hz, 1H), 2.71 - 2.57 (m, 2H), 2.22 - 2.07 (m, 2H), 2.02 (d, J = 14.3 Hz, 1H), 1.90 - 1.80 (m, 1H), 1.72 (d, J = 11.1 Hz, 1H), 1.67 - 1.57 (m, 2H), 1.47 (d, J = 13.5 Hz, 1H), 1.41 - 1.34 (m, 3H), 1.31 (d, J = 7.7 Hz, 1H), 1.28 (s, 3H), 1.21 - 1.13 (m, 1H), 0.92 (d, J = 6.3 Hz, 4H), 0.86 (dd, J = 13.9, 6.5 Hz, 7H), 0.81 ppm (d, J = 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6 + D2O) = 172.7 δ, 103.9, 88.4, 81.4, 73.8, 52.8, 44.8, 40.2, 37.1, 36.7, 35.4, 34.7, 30.0, 26.2, 25.6, 24.9, 24.4, 23.9, 22.5, 20.6, 13.8 ppm. LCMS: retention time = 4.76 min, m/z 462.3 [M+Na]+.
Preparation of ( (R)-3-Methyl-l - (2- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)acetamido)butyl)boronic Acid (HZ2083)
[0227] ((R)-3-Methyl-l-(2-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro-12H7-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)acetamido)butyl)boronic acid (HZ2083) was prepared by following the general procedure II for the amidation of carboxylic acids and primary amines, the general procedure II for the deprotection of BOC-protected amines, and the general procedure for Deprotection of
BOC-protected amines as shown in Scheme 4, part a. HZ2083 was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) = δ 5.42 (s, 1H), 4.38 - 4.28 (m, 1H), 3.83 (d, J = 16.6
Hz, 1H), 3.64 (d, J = 16.6 Hz, 1H), 3.05 (dd, J = 9.3, 5.5 Hz, 1H), 2.73 (t, J = 12.8 Hz, 1H), 2.60
(dd, J = 12.2, 6.0 Hz, 1H), 2.21 - 2.05 (m, 2H), 2.00 (d, J = 14.2 Hz, 1H), 1.89 - 1.77 (m, 1H),
1.71 (d, J = 12.1 Hz, 1H), 1.65 - 1.50 (m, 2H), 1.50 - 1.43 (m, 1H), 1.43 - 1.34 (m, 3H), 1.31 (d,
J = 3.8 Hz, 1H), 1.28 (s, 3H), 1.21 - 1.11 (m, 1H), 0.92 (d, J = 6.2 Hz, 4H), 0.87 - 0.76 ppm (m, 10H). 13C NMR (125 MHz, DMSO-d6 + D2O) = δ 172.6, 169.1, 103.7, 88.2, 81.2, 73.8, 52.7, 44.7, 42.5, 40.1, 36.9, 36.6, 36.0, 34.6, 29.9, 26.2, 25.3, 24.7, 24.3, 23.7, 22.4, 20.5, 13.8 ppm. LCMS: retention time = 4.43 min, m/z 519.2 [M+Na]+ Preparation of ( (R)-3-Methyl-1- (3- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)propanamido)butyl)boronic Acid (HZ2087)
[0228] ((R)-3 -Methyl- 1 -(3 -(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3 ,6, 9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)propanamido)butyl)boronic acid (HZ2087) was prepared by following the general procedure II for the amidation of carboxylic acids and primary amines, the general procedure II for the deprotection of BOC-protected amines, and the general procedure for Deprotection of BOC-protected amines, as shown in Scheme 4, part a. HZ2087 was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) = δ 5.36 (s, 1H), 4.41 - 4.29 (m, 1H), 3.37 (dd, J = 13.3, 6.7 Hz, 1H), 3.23 (dd, J = 13.4, 6.6 Hz, 1H), 2.91 - 2.80 (m, 1H), 2.70 - 2.57 (m, 1H), 2.37 - 2.24 (m, 2H), 2.21 - 2.06 (m, 2H), 2.01 (d, J = 14.3 Hz, 1H), 1.90 - 1.80 (m, 1H), 1.71 (d, J = 12.3 Hz, 1H), 1.64 - 1.53 (m, 2H), 1.46 (d, J = 13.6 Hz, 1H), 1.42 - 1.32 (m, 3H), 1.28 (s, 3H), 1.27 - 1.22 (m, 1H), 1.20 - 1.12 (m, 1H), 0.92 (d, J = 6.3 Hz, 4H), 0.85 (dd, J = 12.3, 6.5 Hz, 7H), 0.80 ppm (d, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6 + D2O) = δ 172.5, 172.3, 103.9, 88.4, 81.4, 73.7, 52.8, 44.8, 40.3, 37.1, 36.7, 36.3, 36.2, 34.9, 34.7, 30.1, 26.2, 25.7, 24.9, 24.5, 23.8, 22.6, 20.7, 13.8 ppm. LCMS: retention time = 4.52 min, m/z 533.0 [M+Na]+.
Preparation of ( (R)-3-Methyl-l -(4- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butanamido)butyl)boronic Acid (HZ2088)
[0229] ((R)-3-Methyl-l-(4-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimethyldecahydro- 12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butanamido)butyl)boronic acid (HZ2088) was prepared by following the general procedure II for the amidation of carboxylic acids and primary amines, the general procedure II for the deprotection of BOC-protected amines, and the general procedure for Deprotection of BOC-protected amines, as shown in Scheme 4, part a. HZ2088 was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) = δ 5.47 (s, 1H), 4.33 (dd, J = 9.2, 6.7 Hz, 1H), 3.25 - 3.13 (m, 1H), 3.05 - 2.95 (m, 1H), 2.80 (dd, J = 9.7, 5.3 Hz, 1H), 2.69 - 2.57 (m, 2H), 2.23 - 2.11 (m, 3H), 2.09 - 1.97 (m, 2H), 1.89 - 1.79 (m, 1H), 1.75 - 1.66 (m, 3H), 1.65 - 1.58 (m, 2H), 1.47 (d, J = 13.6 Hz, 1H), 1.41 - 1.31 (m, 4H), 1.26 (s, 3H), 1.22 - 1.16 (m, 2H), 0.92 (d, J = 6.0 Hz, 4H), 0.86 (d, J = 6.4 Hz, 4H), 0.81 ppm (d, J = 6.6 Hz, 7H). 13C NMR (125 MHz, DMSO-d6 + D2O) 6 = 172.8, 172.0, 103.5, 88.0, 81.0, 74.0, 52.5, 44.5, 39.3, 37.5, 36.8, 36.4, 36.3, 34.4, 31.4, 29.7, 25.8, 25.5, 25.0, 24.6, 24.2, 23.7, 22.0, 20.3, 13.7 ppm. LCMS: retention time = 4.60 min, m/z 547.2 [M+Na]+.
[0230] The proposed syntheses for further exemplary compound of the present application are shown in Figure 44. Over the past decade or so, methods to functionalize sp3 C- H sites in the upper hemisphere of Artemisinin 10, and primarily C6, C6a and C7, have been readily accessed through chemical oxidation via iron-based catalysts and P450-mediated chemoenzymatic synthesis. Shown in Figure 44 is overview of divergent synthetic routes for attachment of the linker to different positions of artemisinin. Selective oxidation of 10 with (S, S)-Fe(PDP) occurred at the most electron-rich and least sterically hindered tertiary aliphatic C-H bond to furnish 11, which then converted into 12 in one step. The bulkier catalyst (S, S)-Fe(CF3- PDP) could alter the inherent selectivity to favor oxidation at the electron-rich and less sterically hindered C7 position to afford the C7 ketone 13, which converted into 14 by reductive amination. Following site-saturation mutagenesis of the P450BM3 FL#62 active site, three efficient mutants (II-H10, IV-H4 and X-E12) were identified that catalyzed selective hydroxylation of C7(R), C7(S) and C6a of 10 to give 15, 16, and 17, respectively. Esterification of 15, 16, and 17 with succinic anhydride gave 18, 19, 20, respectively. 15 and 17 were converted into primary amine 21 and 25, respectively, in two steps, which further modified into 22 and 26 via late-stage diversifications. Besides, 16 and 17 could further functionalized into aryl or heteroaryl ether-based building block 23 and 24, respectively, via Mitsunobu reaction with hydroxy aromatics.
Example 3 - IC50 Determination
[0231] IC50 values of all compounds against Pf20S β5, human c-20S β5c, β2c, β1c and i- 20S β5i β2i, β1i were determined in a 96-well format as described (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Set. USA 115(29):E6863-E6870 (2018); Zhan et al., “Improvement of Asparagine Ethylenediamines as Anti-malarial Plasmodium-Selective Proteasome Inhibitors,” J. Med. Chem. 62(13):6137-6145 (2019); Santos et al., “Structure of Human Immunoproteasome with a Reversible and Noncompetitive Inhibitor That Selectively Inhibits Activated Lymphocytes,” Nat. Commun. 8(1): 1692 (2017), which are hereby incorporated by reference in their entirety). Briefly, 1 μL of compound in a 3-fold series dilution in DMSO at final concentrations from 100 μM to 0.0017 μM was mixed with 99 μL of reaction buffer containing the corresponding proteasome, substrate and activator in a black 96-well plate with a solid bottom. Buffer 50 mM Tris, 5 mM MgCl2, 1 mM dithiothreitol (DTT), pH 7.4 was for Pf20S β5. Buffer 20 mM HEPES, 0.5 mM EDTA and 0.1 mg/mL BSA, pH7.5 for human β5c and β5i . The fluorogenic substrate suc-LLVY-AMC (SEQ ID:3) was used for Pf20S c-20S and β5c at final concentration 25 μM, and Ac-ANW-AMC was used as substrate of i-20S and β5i at final concentration 15 μM. Activator PA28a at final concentration of 12 nM was used for Pf20S assay in the presence of 0.5 μM of WLW-VS, whereas 0.02% SDS was used in the assays for c-20S and i-20S, as well as for human β5c, β2c, β1c, β5i, β2i, and β1i. Z-LLE-AMC was used as substrate of β1c or β1i at final concentration 50 μM. Z-VLR-AMC was used as substrate of β2c or β2i at final concentration 50 μM. Final concentrations of Pf20S, C-20S, and i-20S were 1 nM, 0.2 nM, and 0.4 nM, respectively. The fluorescence of the hydrolyzed AMC at Ex 360nm and Em 460 nm in each well was followed for 1-2 hours. Linear ranges of the time course were used to calculate the velocities in each well, which were fit to a dose-dependent inhibition equation to estimate the IC50 values (Table 2, Table 3, and Table 4) in PRISM (GraphPad). Table 2 displays the summary of compounds’ enzyme inhibition, parasite growth inhibition, and cytotoxic activity against HepG2 human hepatoma cells for ART1, PI01, ATZ1, ATZ2, ATZ3, and ATZ4. Table 4 displays the growth inhibition of artemisinin-proteasome inhibitor conjugates HZ2082, HZ2083, HZ2087, and HZ2088 against Multiple myeloma MM1S, live cancer HepG2, and P. falciparum 3D7.
Table 2. Proteasome Inhibition Activity and Plasmodium Growth Inhibition of Hybrids
All data are means of at least three independent experiments and are presented with standard deviation. Table 3. IC50 Values of Compounds Against β5 of Pf20S, i-20S, and c-20S
Table 4. Growth Inhibition of Artemi sinin-Proteasome Inhibitor Conjugates HZ2082, HZ2083, HZ2087, and HZ2088 Against Multiple Myeloma MM1S, Live Cancer HepG2, and Pfalciparum 3D7 Example 4 - Anti-Malarial Activity in Erythrocytic Stage
[0232] Parasite growth inhibition assays were performed as reported (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity From Intersubunit Interactions and Fitness Cost of Resistance, Proc. Natl. Acad. Sci. USA 115(29):E6863-E6870 (2018), which is hereby incorporated by reference in its entirety). Drug assays were performed on parasites cultured in sterile 96-well plates at a total 200 μL volume per well and a 0.5% initial parasitemia and 2% hematocrit. Plates were placed in an airtight chamber flushed with 5% oxygen, 5% carbon dioxide, and 90% nitrogen for 72 hours. Plates were then placed in the -80 °C freezer to promote cell lysis upon thawing. When thawing was complete, 100 μL of SYBR Green diluted in lysis buffer (0.2 μL SYBR Green per ml lysis buffer) was added to each well and the plates were shaken in the dark at room temperature for 1 hour. Fluorescence was then recorded in a SpectraMax Gemini plate reader using λex=490 nm / λex=530 nm. Data analysis was performed with Graphpad Prism software. Counts were normalized and plotted by non- linear regression to yield EC50 values (Table 2 and Table 3). Example 5 - Ring Survival Assay
[0233] Ring survival assays (RSA) were performed as described (Straimer et al., “Drug Resistance. K13-propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015), which is hereby incorporated by reference in its entirety). Parasite cultures, IPC5202 (Cam3.1R539T), an artemisinin resistant parasite line from Cambodia, and the genetically engineered artemisinin sensitive revertant Cam3.1Rev, were synchronized several times with 5% sorbitol and then a Percoll-sorbitol gradient was used to obtain tightly synchronized late stage parasites. Isolated late stage parasites were then allowed to reinvade fresh red blood cells for three hours before ring stage parasites were confirmed by microscopy before the cultures were again subjected to 5% sorbitol to obtain 0-3 hour rings. The isolated ring stage cultures were then plated into a 96 well plate at 0.5% parasitemia at the corresponding drug concentrations: DHA 700 nM, PI01 800 nM, ART1 800 nM, ATZ3 700 nM, and ATZ4 700 nM. Plates were incubated at 37 °C in standard gas conditions for six hours before the plates were spun and washed to remove medium with compound and replenished with fresh medium. Plates were then incubated for an additional 66 hours and parasite growth was then assessed using flow cytometry and nucleic acid stains: Hoechst 33342 (HO) and thiazole orange (TO).
Example 6 - Parasite Regrowth Assay
[0234] Parasite were synchronized as described for the RSA and treated with the same drug concentrations for six hours in a 96 well plate. After six hours of drug/compound exposure and washing, the 200 μL culture was then transferred to a 3 mL culture to follow for seven days. Parasitemia was checked by smear on day 7.
Example 7 - Intraplasmodial Hybrid Activation and Proteasome Inhibition Assay
[0235] Pf Dd2, Dd2(β6A117D), and Dd2(β5A49S) parasites were grown synchronized to a high parasitemia (5-8%). At the early trophozoite stage, 5 mL of parasite-infected red blood cells were exposed to DMSO, PI01 (800 nM), ART1 (800 nM), ATZ4 (700 nM), and a mixture of ART1 and PI01 in a 1 : 1 ratio both at 800 nM for 6 hours. After centrifugation at 600 rpm for 5 minutes, the supernatant was removed, and red cells were washed with complete medium once and resuspended in 10 mL of fresh medium. The cultures were then placed back in the incubator and shaken for 10 minutes. The procedure was repeated 4 times. After the last wash, the red blood cell pellets were placed on ice and washed with PBS 1 mL once and then lysed with 10% saponin to obtain parasite pellets. Parasite pellets were kept on ice and washed with cold PBS until supernatant was clear (approximately 3 times). Pellets were stored at -80 °C until analysis. The frozen Pƒ pellets were thawed on ice and resuspended in 2 x pellet volume of lysis buffer containing 20 mM Tris-HCl, 5 mM MgCl2, and 1 mM DTT, pH 7.4. The mixtures were kept on ice for 1 hour and vigorously vortexed every 5 min, then centrifuged at 15000 rpm for 20 min at 4 °C. The supernatants were collected and their concentrations were determined by BCA protein assay. Equal amounts of lysates were incubated with MV151 at a final concentration of 2 μM for 1 hour at 37 °C in a 1.5 mL Eppendorf tube wrapped in aluminum foil. The samples were then heated with 4X SDS loading buffer at 95 °C for 10 min and run on a 12% Novex™ Bis-Tris Protein Gel with MOPS SDS running buffer. The gel was rinsed with double distilled H2O and scanned at the TAMRA channel on a Typhoon Scanner (GE Healthcare). In an alternative procedure, wash steps of parasite-infected red blood cells in the above-stated procedure were omitted to examine the permeability of PI01 and ATZ4. However, parasite pellets were thoroughly washed to remove red blood cell constituents to avoid interferences in the MV151 labeling assays.
Example 8 - Inhibition of Pf20S, Pf20S(β6A117D0 and Pf20S(β5A49S) by PI01 and ATZ4 [0236] Cell free lysates of P. falciparum Dd2 wild-type and two Dd2-derived resistant (Dd2β5A49S and Dd2β6A117D) were used. 5 - 10 pg of total lysate proteins were incubated with PI01 or ATZ4 at the indicated concentrations for 1 hour at 37 °C prior to addition of MV151 and incubated for a further 1 hour at 37 °C. The samples were then heated with 4X SDS loading buffer at 95 °C for 10 min and run on 12% Novex™ Bis-Tris Protein Gels. The gels were rinsed with double distilled H2O and then scanned on the Typhoon Scanner.
Example 9 - HepG2 Cell Viability Assay
[0237] HepG2 (HB-8065, ATCC) were cultured at 37 °C in a humidified air/5% CO2 atmosphere in medium supplemented with 10% fetal bovine serum and 100 ug per ml penicillin, 100 pg per ml streptomycin in DMEM medium. HepG2 was used at 5,000 cells per well. Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO2. Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
Example 10 - Degradation of the Modified β-Casein by i-20S
[0238] β-Casein dissolved in PBS (10 μM) was incubated with 100 μM of PI01, ART1 or ATZ2 in the presence of sodium ascorbate (200 μM) and hemin (100 μM) at r.t. for 4 hours. The samples were transferred to Slide-A-Lyzer MINI Dialysis Devices (10K MWCO, Thermo Scientific™ (Waltham, MA) 88401), placed into tubes containing the dialysis buffer (20 mM HEPES and 0.5 mM EDTA, pH7.5), and dialyzed overnight at 4 °C with fresh dialysis buffer changing every 4 hours. After dialysis, the samples were collected and further incubated with i- 20S (50 nM), PA28α (0.5 μM), and bovine serum albumin (10 μM) at 37 °C. Aliquots from the reaction mixtures were removed at designated time intervals, mixed with SDS sample loading buffer, and were run on a SDS-PAGE (4-20%, Tris-Glycine) and stained with Coomassie blue. For the control experiment in Figure 45C, samples were prepared with the same method above, except that dialysis step was skipped.
Example 11 - MM. IS Cell Viability Assay
[0239] MM. IS (CRL-2974, ATCC) were cultured at 37 °C in a humidified air/5% CO2 atmosphere in medium supplemented with 10% fetal bovine serum, 100 ug per ml penicillin, 100 μg per ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 mM Sodium Pyruvate in RPMI 1640 medium. MM. 1S was used at 100,000 cells per well. Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO2. Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
Example 12 - Protein Sample Preparation for Mass Spectrometry Analysis
[0240] β-Casein was treated as in the aforementioned example. After removing the inhibitors, hemin, and ascorbate by dialysis, the treated β-casein samples were run on SDS-page and stained with Coomassie blue G-250. The gel bands of β-casein were cut into pieces.
Samples were reduced with 5 mM dithiothreitol in 50 mM ammonium bicarbonate buffer for 50 min at 55°C and then dried by acetonitrile. Next, the samples were alkylated with 12.5 mM iodoacetamide in 50 mM ammonium bicarbonate buffer for 45 min in the dark at room temperature and dried by acetonitrile. The samples were then digested by trypsin or chymotrypsin at 37 °C overnight. The digestion was stopped with 10% trifluoroacetic acid, after which the digested peptides were extracted twice with 1% formic acid in 50% acetonitrile aqueous solution, and then evaporated to dryness on a Speedvac and resuspended in 20 μL of formic acid/H2O (v:v = 0.1%/99.9%) with sonication.
Example 13 - LC-MS/MS
[0241] For LC-MS/MS analysis, the fragment peptides were separated by a 120-min gradient elution method at a flow rate of 0.3 μL/min with a Thermo-Dionex Ultimate 3000 HPLC system that is directly interfaced with a Thermo Orbitrap Fusion Lumos mass spectrometer. The analytical column was a homemade fused silica capillary (75 pm inner- diameter, 150 mm length; Upchurch, Oak Harbor, WA, USA) packed with C-18 resin (pore size 300 A, particle size 5 μm; Varian, Lexington, MA, USA). Mobile phase A was 0.1% formic acid in water, and mobile phase B is 100% acetonitrile and 0.1% formic acid. The Thermo Orbitrap Fusion Lumos mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 4.0.27.10 software. A single full-scan mass spectrum was done in the Orbitrap (300 -1500 m/z, 120,000 resolution). The spray voltage was 1850 V and the Automatic Gain Control (AGC) target was 200,000. This was followed by 3-second data-dependent MS/MS scans in an ion routing multipole at 30% normalized collision energy (HCD). The charge state screening of ions was set at 1-8. The exclusion duration was set at 8 seconds. Mass window for precursor ion selection was set at 2 m/z. The MS/MS resolution was 15,000. The MS/MS maximum injection time was 150 ms and the AGC target was 50,000.
Example 14 - Mass Data Processing
[0242] Data were searched against the bovine casein database from the Uniprot by using Proteome Discoverer 1.4 software (Thermo Scientific) and peptide sequences were determined by matching protein database with the acquired fragmentation pattern by SEQUEST HT algorithm. The following search parameters were used: the precursor mass tolerance was set to 10 ppm and fragment mass tolerance was 0.02 Da; No-Enzyme (Unspecific); Modification (MOD) A1 (811.39624 Da, Table 5, any amino acids), MOD A2 (751.37511 Da, Table 5, any amino acids), MOD B1 (325.18837 Da, Table 3, any amino acids), MOD B2 (265.16725 Da, Table 5, any amino acids), carbamidomethyl of cysteines (+57.02146 Da), oxidation of methionines (+15.99492 Da) as the variable modifications. Only peptides with the strict target false discovery rate (FDR) below 1% were considered as high-confidence hits.
Table 5. Two Types of Modification of β-Casein by ATZ2 or ART1 (Figure 46).
Figure 46 shows two types of modification of β-casein by ATZ2 or ARTl: (A) For ATZ2, MOD A1 Δmass = 811.39624 and MOD A2 Δmass = 751.37511 (A1-acetate); and (B) For ART1, MOD B1 Δmass =325.18837 and MOD B2 Δmass =265. 16725 (B1-acetate).
Example 15 - Discussion of Examples 1-14
[0243] To establish if an ART could fit into the binding pockets of the Pƒ20S, a commercially available artesunate was conjugated with PKS21224 and PKS21208, two asparagine ethylenediamines in a novel class of proteasome inhibitors (Kirkman et al., “Antimalarial Proteasome Inhibitor Reveals Collateral Sensitivity from Intersubunit Interactions and Fitness Cost of Resistance,” Proc. Natl. Acad. Set. USA 115:E6863-E6870 (2018); Santos et al., “Structure of Human Immunoproteasome with a Reversible and Noncompetitive Inhibitor that Selectively Inhibits Activated Lymphocytes,” Nat. Commun. 8: 1692 (2017), which are hereby incorporated by reference in their entirety), where isoxazoly was replaced with succinate (Scheme 2). The ART moiety was coupled at the P4 position, as the S4 pocket is partially exposed to solvent. It was hypothesized that, at this position, the bulky artemisinin would not interfere with the binding of the rest of the molecule to the active site of the Pƒ20S β5 subunit. WZ-06 and WZ-13 were synthesized (Figure 1) and their structures confirmed. WZ-20 was synthesized as a proteasome inhibitor control. The compound’s IC50 values against Pƒ20S and human constitutive (c-20S) and immunoproteasomes (i-20S) (Table 3) were then determined. Artesunate itself does not inhibit the β5 subunits of Pƒ20S, human i-20S, or human c-20S. In contrast, conjugates WZ-06 and WZ-13 were potent against Pƒ20S β5 at 6 nM and 2 nM, respectively. The data suggested that ARTs at the P4 position do not interfere with the binding of AsnEDAs to Pƒ20S. However, the semi-ketal ester of the artesunate is not stable in human blood plasma, making it difficult to interpret the activity of ester-based ATZs against Pƒparasites in red blood cells.
[0244] Four new ATZs (Figure 47A) were designed with a stable amide instead of an ester tether to improve their stability. In accordance, the control compound artesunate was replaced with ART1, where the semi-ketal ester was changed to acyclic ether with a carboxylic acid, and replaced the proteasome inhibitor control WZ-20 with PI01, a direct proteasome inhibitor moiety of ATZs. Compounds were synthesized as illustrated in Figure 16. The IC50 values against Pƒ20S, c-20S, and i-20S were then determined (Figure 47B and Table 2). ART1 showed weak inhibitory activity against Pƒ20S at 9.23 μM. The IC50 of ATZ1 against Pƒ20S increased 10.5-fold to 0.063 μM compared to PI01. ATZ3, with a propionate linker between ART1 and the proteasome inhibitor, displayed 106-fold and 760-fold selectivity against Pƒ20S over i-20S and c-20S, while ATZ4 with a butyrate linker showed 45-fold and 250-fold selectivity, respectively. ATZ3 and ATZ4 showed increased selectivity in enzyme inhibition compared to ATZ1 and ATZ2. The results suggest that the propionate linker best balances potency and selectivity among these compounds.
[0245] These compounds were then tested against Pƒ20S and two proteasome inhibitor- resistant strains derived from Dd2, Dd2(β6A117D), and Dd2(β5A49S), which harbor a mutation in the β6 subunit (A117D) or in the β5 subunit (A49S) of Pƒ20S, respectively (Figure 47C and Table 2). As expected, the growth inhibitory activity of ART analog ART1 was similar for Dd2 and the proteasome inhibitor mutants. Also as expected, Dd2(β6A117D) and Dd2(β5A49S) were 16- and >370- fold more resistant to PI01, respectively, than the parental strain Dd2. Since ATZs were more potent than ART1 against PƒDd2, it was reasoned that the anti-Pƒ activity of ATZs was not only derived from the ART moiety, but also from the proteasome inhibitor moiety. In agreement with that interpretation, the ATZs were as potent as PI01 in inhibiting the growth of PƒDd2, and their inhibition activities were only slightly less against the mutant strains: < 2.9- fold for Dd2 (β6A117D) and ≤ 3.6- fold for Dd2 (β5A49S), representing ≥ 5- fold and >100- fold improvement over PI01 against the respective strains. Thus, the ATZs substantially overcame resistance to the proteasome inhibitor moiety alone that were conferred by point mutations in Pƒ20S.
[0246] Next, a model system was devised to explore the mechanism of action of the hybrids and test the hypothesis that degradation products of a protein covalently attached to an ATZ can lead to inhibition of 20S (Figure 48A). Because of the scarcity of purified Pƒ20S, human i-20S was used for a proof of concept. The highly potent i-20S inhibitor ATZ2 was incubated with β-casein in the presence of PA28a to activate 20S and ascorbate and hemin to activate the ART moiety, as described (Zhou et al., “Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome.” ACS Chem. Bio.l 11 :882-888 (2016), which is hereby incorporated by reference in its entirety). β-Casein is intrinsically unstructured and can be degraded by 20S and PA28a without a requirement for ubiquitination. Proteasome inhibitor PI01 and ART analog ART1 served as controls. Degradation of β-casein treated with ATZ2 was markedly reduced, whereas the degradation of β-casein treated with PI01 or ART1 alone was almost complete at five hours (Figure 48B, left). As expected, in a control experiment done without removing small molecules from the reaction mixtures by dialysis, both PI01 and ATZ2 reduced the degradation of β-casein compared to ART1 (Figure 48B, right). A proteomic analyses of PI01-, ART1-, and ATZ2- treated β-casein was conducted in order to identify ART1 and ATZ2 modified β-casein peptides (Figure 46 and Table 5). Peptide SLVYPFPGP80 (SEQ ID: 1) was identified from ATZ2 treated β-casein in which proline-80 was modified by ATZ2 (Figure 48C and Table 6), and a peptide F67AQTQSLVYPFPGPIPN (SEQ ID:2) from ART1 treated β-casein, wherein phenylalanine-67 was modified by ART1 (Figure 48D and Table 7), confirming the covalent modification of β-casein by activated artemisinin moiety in both ART1 and ATZ2. The result suggests that degradation of ART-damaged protein by the proteasome was not affected by the covalent modification by ART, yet the proteasome inhibitor-coupled ART, ATZ, inhibited degradation either directly or via the degradation products of ATZ- containing oligopeptides (Figure 48A).
Table 6. Fragment Ions of Peptide (SLVYPFPGP80 (SEQ ID: 1)) Modified by ATZ2
Bold numbers indicate fragments that were matched with theoretical masses of corresponding fragments; non-bold numbers indicate fragments not detected.
Table 7. Fragment Ions of Peptide (F67AQTQSLVYPFPGPIPN (SEQ ID:2)) Modified by ART1 Bold numbers indicate fragments that were matched with theoretical masses of corresponding fragments; non-bold numbers indicate fragments not detected.
[0247] Next, ATZs were investigated to determine if their mode of action could circumvent the ART resistance conferred by the Kelch13 polymorphism. A ring-stage survival assay was conducted with strains Cam3.IREV and Cam3.IR539T; the latter strain has a Kelchl3 polymorphism and is resistant to ART (Straimer et al., “Drug Resistance. K13-propeller Mutations Confer Artemisinin Resistance in Plasmodium falciparum Clinical Isolates,” Science 347:428-431 (2015), which is hereby incorporated by reference in its entirety). Highly synchronized 0-3 hour ring stage parasites were treated with DMSO, dihydroartemisinin (DHA), ART1, PI01, ATZ3, or ATZ4 at indicated concentrations for 6 hours (Figure 45A). Then, the compounds were washed off and the parasite cultured at 37 °C for a further 66 hours. Live parasites in each condition were analyzed by flow cytometry and survival expressed relative to the DMSO control. As expected, Cam3.IR539T was highly resistant to DHA and slightly resistant to ART1 (Figure 45B), whereas Cam3.IR539T was as susceptible as Cam3.IRev to ATZ3 and ATZ4, respectively. Interestingly, Cam3.IR539T was more sensitive than Cam3.IRev to the proteasome inhibitor PI01. These data suggest that ATZs can circumvent ART resistance associated with Kelchl3 mutation at the early ring stages. In the ring survival assay (RSA), even DHA-sensitive parasites pulsed with DHA will recover normal growth. More extended RSAs have been proposed to better reflect clinical efficacy (Davis et al., “The Extended Recovery Ring-stage Survival Assay Provides a Superior Association with Patient Clearance Half-life and Increases Throughput,” Malar. J. 19:54 (2020), which is hereby incorporated by reference in its entirety). Accordingly, an extended RSA was performed by pulsing parasites as in the standard RSA and then monitoring parasite growth over a further 7 days. As expected, parasites pulse- treated with DHA, PI01, or ART1 established normal growth. In contrast, parasites of both the ART sensitive and resistant lines had significantly lower parasitemia (Figure 45C), indicative of a prolonged growth inhibition profile of ATZs.
[0248] To relate growth inhibition to pharmacodynamic effect, a covalently reactive, irreversible probe compound, MV151 was used to label Pƒ20S (Verdoes et al., “A Fluorescent Broad-spectrum Proteasome Inhibitor for Labeling Proteasomes in vitro and in vivo,” Chem. Biol. 13: 1217-1226 (2006), which is hereby incorporated by reference in its entirety). First it was established that a 1-hour incubation of either PI01 or ATZ4 with Dd2 Pƒ20S prior to labeling with MV151 dose-dependently blocked labeling of the Pƒ208β5 of Dd2 wild type by the probe (Figure 45D). Under the same conditions, the β6A117D and β5A49S mutations prevented PI01 and ATZ4 from inhibiting the labeling 20S(β6A117D) and Pƒ20S(β5A49S) in lysates of Dd2(β6A117D) and Dd2(β5A49S) parasites (Figure 45D), indicating that the mutations reduced the binding affinity of PI01 and ATZ4 to Pƒ20S β5. The seemingly contradictory results between the growth inhibition potency of ATZ3 and ATZ4 against the Dd2 and two Pƒ20S inhibitor resistant strains (Table 2) and the enzyme labeling (Figure 45D) could be explained by ATZs being transformed to intracellularly retained moieties with proteasome inhibiting potential that arose inside the parasites following activation of ATZs and that were active not only against wild type Pƒ20S but also against Pƒ20S with the β6A117D and β5A49S mutations.
[0249] To test this hypothesis, late stage Dd2 wild type, Dd2(β6A117D), and Dd2(β5A49S) parasites were pulse-treated with DMSO as a vehicle control, DHA (700 nM), PI01 (800 nM), ART1 (800 nM), ATZ4 (700 nM), or a 1 : 1 combination of PI01 and ART1 for six hours. After thoroughly washing off the compounds and recovering the parasites from the red blood cells, they were lysed and Pƒ20S was labeled with MV151 (Figure 45E). The labeling of Pƒ20S in lysates from parasites treated with DHA, PI01, ART1, and the combination of PI01 with ART1 (1 : 1) was not inhibited compared to the labeling of Pƒ20S β5 by MV 151 in DMSO treated samples. This demonstrated the effectiveness of the washing procedure at lowering the intracellular concentration of compounds below a functionally detectable level, because when the washing steps were omitted, PI01 and ATZ4 did block labelling of Pƒ20S β5 by MV151 (Figure 49), confirming that both of the compounds could enter red bloods cells and the parasites within them. Despite the extensive washing, Pƒ20S in the lysate from the parasites treated with ATZ4 showed substantial inhibition of labeling of Pƒ20S β5 by MV151 (Figure 45E).
Moreover, this activity was similarly effective against intracellular Pƒ20S β5 with the β6A117D and β5A49S mutations (Figure 45E), in contrast to the minimal inhibition of Pƒ20S β6A117D and β6A49S labeling by ATZ4 itself (Figure 45D). These results are consistent with transformation of ATZ4 in the parasites to a persistently-retained intracellular moiety capable of sustained inhibition of Pƒ20S β5.
[0250] In summary, stable, covalent conjugates of a proteasome inhibitor and an ART analog, termed ATZs, retain both proteasome inhibitory activity and the reactive alkylating activity of ART. These effects are not only synergistic against growth of Pƒ but can overcome resistance to either moiety. The ability to overcome resistance conferred by point mutations in Pƒ20S is associated with ATZ-dependent formation of proteasome-inhibitory activity that is not removed from the parasites by washing procedures that remove ATZ itself. This more robust proteasome-inhibitory activity is ascribed to the demonstrable formation of proteasomal degradation products of ATZ-damaged proteins. The oligopeptides to which the ART-derived radicals are attached appear to stabilize presentation of the proteasome inhibitory moiety of the ATZ at the Pƒ20S active site, compensating for the reduced binding affinity conferred by the point mutations. In short, withPƒ's dependence on the proteasome to remove ART-damaged proteins, ATZ hybrids hijack the parasite protein degradation machinery to create a pool of proteasome inhibitor-containing oligopeptides. Because the actions of ART and the improved action of the proteasome inhibitor are delivered by a single molecule, a single pharmacokinetic profile will preclude temporary exposure to only one of the components in the combination.
Example 16 - Materials and Methods for Examples 17-20
General Procedure I: Amidation of Carboxylic Acids and Primary Amines
[0251] To a solution of carboxylic acid (1.0 mmol, 1.0 eq) and diisopropyl ethyl amine (3.0 mmol, 3.0 eq) in dimethyl formamide (4.0 mL) was added HATU (1.2 mmol, 1.2 eq) at 0°C. The mixture was stirred for 10 min at 0°C. A solution of primary amine (1.1 mmol, 1.1 eq) in dimethyl formamide (1.0 mL) was added to the mixture at 0°C. The mixture was stirred for 2 hours at 25°C. LCMS showed desired mass was detected. The reaction mixture was poured into water (30 mL) and extracted with dichloromethane (3X). The combined organic phase was washed with saturated sodium bicarbonate aqueous, saturated ammonium chloride, and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel) to afford amide as a colorless oil.
General Procedure II: Deprotection of BOC-Protected Amines
[0252] To a solution of Boc-protected compound (0.5 mmol) in dichloromethane (3.0 mL) was added trifluoroacetic acid (1.0 mL) drop-wise. The mixture was stirred at 20°C for 3 hours. LCMS showed the starting material was consumed. The mixture was concentrated in vacuum to give primary amine as a colorless oil, which was used for the next step without further purification.
General Procedure III: Hydrolysis of Boronates to Provide Boronic Acid
[0253] To a solution of boronates (0.2 mmol, 1.0 eq) in MeOH (3.0 ml) was added hexane (3.0 ml), isobutylboronic acid (1.0 mmol, 5.0 equiv), and 1 M HCl (0.6 mL). The resulting mixture was vigorously stirred for 24 hours at 25 °C. The resulting mixture was diluted with MeOH (20 mL) and hexane (20 mL) and was extracted with MeOH (3X). The solvent was removed under reduced pressure and the residue was dissolved in DCM and was washed with 5% NaHCO3, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by prep-HPLC (column: OBD C18 150mm*19mm*5um; mobile phase: [water (0.1%TFA)-ACN]; B%: 5%-95%, 20min) and lyophilisation to afford boronic acid as a white solid.
Scheme 4. Synthesis of Boronic Acid Artemi sinin-Proteasome Inhibitor Hybrids HZ2082,
HZ2083, HZ2087, HZ2088, HZ3046, and HZ3047 Preparation of ((R)-3-Methyl-l-(2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimetbyldecabydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butyl)boronic Acid (HZ2082)
[0254] Hybrid HZ2082 was prepared using artemisinin-derived carboxylic acid (which was synthesized according to Frohlich et al., “Synthesis of Artemi sinin-Estrogen Hybrids Highly Active Against HCMV, P. falciparum, and Cervical and Breast Cancer,” ACS Med. Chem. Lett., 9: 1128-1133 (2018), which is hereby incorporated by reference in its entirety) and leucine boronate as starting material by following general procedures I and III and was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) = δ 5.42 (s, 1H), 4.32 (s, 1H), 2.93 (dd, J = 9.2, 5.6 Hz, 1H), 2.71 - 2.57 (m, 2H), 2.22 - 2.07 (m, 2H), 2.02 (d, J = 14.3 Hz, 1H), 1.90 - 1.80
(m, 1H), 1.72 (d, J = 11.1 Hz, 1H), 1.67 - 1.57 (m, 2H), 1.47 (d, J = 13.5 Hz, 1H), 1.41 - 1.34 (m, 3H), 1.31 (d, J = 7.7 Hz, 1H), 1.28 (s, 3H), 1.21 - 1.13 (m, 1H), 0.92 (d, J = 6.3 Hz, 4H), 0.86 (dd, J = 13.9, 6.5 Hz, 7H), 0.81 ppm (d, J = 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6 + D2O) δ = 172.7, 103.9, 88.4, 81.4, 73.8, 52.8, 44.8, 40.2, 37.1, 36.7, 35.4, 34.7, 30.0, 26.2, 25.6, 24.9, 24.4, 23.9, 22.5, 20.6, 13.8 ppm. LCMS: retention time = 4.76 min, m/z 462.3 [M+Na]+. Figures 50-51 show the characterization of the product.
Preparation of ( (R)-3-Methyl-l - (2- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)acetamido)butyl)boronic Acid (HZ2083)
[0255] Hybrid HZ2083 was prepared by following general procedures I, II, I, and III and was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) δ = 5.42 (s, 1H), 4.38 - 4.28 (m, 1H), 3.83 (d, J = 16.6 Hz, 1H), 3.64 (d, J = 16.6 Hz, 1H), 3.05 (dd, J = 9.3, 5.5 Hz, 1H), 2.73 (t, J = 12.8 Hz, 1H), 2.60 (dd, J = 12.2, 6.0 Hz, 1H), 2.21 - 2.05 (m, 2H), 2.00 (d, J = 14.2 Hz, 1H), 1.89 - 1.77 (m, 1H), 1.71 (d, J = 12.1 Hz, 1H), 1.65 - 1.50 (m, 2H), 1.50 - 1.43 (m, 1H), 1.43 - 1.34 (m, 3H), 1.31 (d, J = 3.8 Hz, 1H), 1.28 (s, 3H), 1.21 - 1.11 (m, 1H), 0.92 (d, J = 6.2 Hz, 4H), 0.87 - 0.76 ppm (m, 10H). 13C NMR (125 MHz, DMSO-d6 + D2O) = δ 172.6, 169.1, 103.7, 88.2, 81.2, 73.8, 52.7, 44.7, 42.5, 40.1, 36.9, 36.6, 36.0, 34.6, 29.9, 26.2, 25.3, 24.7, 24.3, 23.7, 22.4, 20.5, 13.8 ppm. LCMS: retention time = 4.43 min, m/z 519.2 [M+Na]+. Figures 52-53 show the characterization of the product.
Preparation of ( (R)-3-Methyl-l - (3- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)propanamido)butyl)boronic Acid (HZ2087)
[0256] Hybrid HZ2087 was prepared by following general procedures I, II, I, and III and was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) δ = 5.36 (s, 1H), 4.41 - 4.29 (m, 1H), 3.37 (dd, J = 13.3, 6.7 Hz, 1H), 3.23 (dd, J = 13.4, 6.6 Hz, 1H), 2.91 - 2.80 (m, 1H), 2.70 - 2.57 (m, 1H), 2.37 - 2.24 (m, 2H), 2.21 - 2.06 (m, 2H), 2.01 (d, J = 14.3 Hz, 1H), 1.90 - 1.80 (m, 1H), 1.71 (d, J = 12.3 Hz, 1H), 1.64 - 1.53 (m, 2H), 1.46 (d, J = 13.6 Hz, 1H), 1.42 - 1.32 (m, 3H), 1.28 (s, 3H), 1.27 - 1.22 (m, 1H), 1.20 - 1.12 (m, 1H), 0.92 (d, J = 6.3 Hz, 4H), 0.85 (dd, J = 12.3, 6.5 Hz, 7H), 0.80 ppm (d, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6 + D2O) δ = 172.5, 172.3, 103.9, 88.4, 81.4, 73.7, 52.8, 44.8, 40.3, 37.1, 36.7, 36.3, 36.2, 34.9, 34.7, 30.1, 26.2, 25.7, 24.9, 24.5, 23.8, 22.6, 20.7, 13.8 ppm. LCMS: retention time = 4.52 min, m/z 533.0 [M+Na]+. Figures 54-55 show the characterization of the product.
Preparation of ( (R)-3-Methyl-l -(4- (2-((3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9- trimethyldecahydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10- yl)acetamido)butanamido)butyl)boronic Acid (HZ2088)
[0257] Hybrid HZ2088 was prepared by following general procedures I, II, I, and III and was obtained as a white solid. 1H NMR (500 MHz, DMSO-d6 + D2O) 6 = 5.47 (s, 1H), 4.33 (dd, J = 9.2, 6.7 Hz, 1H), 3.25 - 3.13 (m, 1H), 3.05 - 2.95 (m, 1H), 2.80 (dd, J = 9.7, 5.3 Hz, 1H), 2.69 - 2.57 (m, 2H), 2.23 - 2.11 (m, 3H), 2.09 - 1.97 (m, 2H), 1.89 - 1.79 (m, 1H), 1.75 - 1.66 (m, 3H), 1.65 - 1.58 (m, 2H), 1.47 (d, J = 13.6 Hz, 1H), 1.41 - 1.31 (m, 4H), 1.26 (s, 3H), 1.22 - 1.16 (m, 2H), 0.92 (d, J = 6.0 Hz, 4H), 0.86 (d, J = 6.4 Hz, 4H), 0.81 ppm (d, J = 6.6 Hz, 7H). 13C NMR (125 MHz, DMSO-d6 + D2O) δ = 172.8, 172.0, 103.5, 88.0, 81.0, 74.0, 52.5, 44.5, 39.3, 37.5, 36.8, 36.4, 36.3, 34.4, 31.4, 29.7, 25.8, 25.5, 25.0, 24.6, 24.2, 23.7, 22.0, 20.3, 13.7 ppm. LCMS: retention time = 4.60 min, m/z 547.2 [M+Na]+. Figures 56-57 show the characterization of the product.
Preparation of (S)-5-Azido-N-((R)-3-metbyl-l-((3aS,4S,6S,7aR)-3a,5,5- trimetbylhexabydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)butyl)-2-(2- ( (3R,5aS,6R,8aS,9R,10R,12R,12aR )-3, 6, 9-trimethyldecahydro-l2H-3,12- epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamido)pentanamide (HZ3046)
[0258] Probe HZ3046 was prepared by following general procedures I, II, and I and was obtained as a white solid. 1H NMR (500 MHz, Chloroform-d) δ 7.14 (d, J = 7.4 Hz, 1H), 6.74 - 6.64 (m, 1H), 5.37 (s, 1H), 4.77 (ddd, J= 11.5, 6.3, 2.1 Hz, 1H), 4.43 (td, J= 7.7, 5.8 Hz, 1H), 4.27 (dd, J= 8.8, 2.1 Hz, 1H), 3.29 (q, J= 6.7 Hz, 2H), 3.13 (td, J= 7.6, 5.1 Hz, 1H), 2.63 - 2.49 (m, 2H), 2.41 - 2.25 (m, 3H), 2.15 (dtd, J= 10.7, 6.1, 2.2 Hz, 1H), 2.05 (ddd, J= 14.6, 4.7, 3.0 Hz, 1H), 2.02 - 1.92 (m, 3H), 1.91 - 1.83 (m, 2H), 1.82 - 1.63 (m, 9H), 1.45 (d, J= 7.7 Hz, 1H), 1.42 (s, 3H), 1.37 (s, 3H), 1.26 (d, J= 4.3 Hz, 6H), 0.97 (d, J= 5.5 Hz, 3H), 0.89 (dd, J= 6.6, 2.2 Hz, 6H), 0.87 (d, J= 7.6 Hz, 3H), 0.83 (s, 3H). 13C NMR (126 MHz, CDCl3) 6 171.92, 171.83, 103.14, 90.17, 85.40, 80.66, 77.58, 69.12, 51.82, 51.76, 51.46, 51.09, 43.46, 40.11, 39.62, 38.15, 37.48, 37.35, 36.55, 35.64, 34.18, 30.17, 28.61, 28.01, 27.13, 26.29, 25.89, 25.52, 25.11, 24.74, 24.05, 23.00, 22.19, 19.97, 12.19.
Preparation of (S)-5-Azido-N-((R)-3-methyl-l-((3aS,4S,6S,7aR)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)butyl)-2-(2- ((2R,3R,3aS,3a1R,6R,6aS,9S,10aS)-3,6,9-trimethyldecahydro-10aH-3a1,9- epoxyoxepino[4,3,2-ij]isochromen-2-yl) acetamido)pentanamide (HZ3047)
[0259] Inactive probe HZ3047 was prepared by following general procedures I, II, and I and was obtained as a white solid. 1H NMR (500 MHz, Chloroform-d ) 6 6.73 (t, J= 6.0 Hz, 2H), 5.28 (s, 1H), 4.52 (ddt, J= 8.5, 6.3, 3.0 Hz, 2H), 4.28 (dd, J= 8.8, 2.2 Hz, 1H), 3.29 (td, J= 6.8, 2.6 Hz, 2H), 3.02 (td, J= 7.8, 4.5 Hz, 1H), 2.46 - 2.21 (m, 4H), 2.15 (dtd, J= 10.6, 6.1, 2.1 Hz, 1H), 2.03 - 1.93 (m, 3H), 1.91 - 1.86 (m, 2H), 1.85 - 1.76 (m, 2H), 1.74 - 1.58 (m, 9H), 1.54 (s, 3H), 1.52 - 1.46 (m, 1H), 1.38 (s, 4H), 1.27 (s, 3H), 1.23 - 1.19 (m, 2H), 0.91 - 0.86 (m, 12H), 0.84 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 172.34, 171.79, 107.85, 97.08, 85.00, 84.99, 82.50, 65.37, 51.63, 51.27, 51.25, 51.08, 45.15, 40.23, 39.87, 39.73, 38.47, 38.15, 35.81, 35.56, 34.39, 34.37, 29.53, 28.71, 28.52, 27.17, 26.32, 25.47, 25.18, 24.96, 24.09, 23.79, 22.72, 22.63, 22.08, 18.71, 11.92. Preparation of (R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)ethan-l-amine Hydrochloride a. Preparation of 2-(Benzofuran-3-yl)acetaldehyde
[0260] To a solution of 2-(benzofuran-3-yl)ethan-1-ol (350 mg, 2.2 mmol) in 12 mL dry DCM and 4 mL dry dimethylsulfoxide (DMSO) at 0°C was added dry trimethylamine (1.5 ml, 10.8 mmol) and followed by pyridine sulfur trioxide (1.03 g, 6.47 mmol) in three potions within 10 min. The mixture was allowed to warm slowly to room temperature, and stirred at room temperature for 45 min. It was then poured into 20 mL water and extracted with DCM (2x20 mL). The combined organic fractions were dried over anhydrous Na2SO4 and the solvent was removed by reduced pressure. The residue was purified by flash column chromatography (hexane/EtOAc, 19: 1) to give 2-(benzofuran-3-yl)acetaldehyde (206 mg, 60%) as a light yellow oil. 1H NMR (500 MHz, CDCl3) δ 9.82 (t, J= 2.0 Hz, 1H), 7.66 (s, 1H), 7.53 - 7.47 (m, 2H), 7.37 - 7.31 (m, 1H), 7.30 - 7.24 (m, 1H), 3.78 (dd, J= 2.0, 1.0 Hz, 2H) ppm. b. Preparation of (R,E)-N-(2-(Benzofuran-3-yl)ethylidene)-2-methylpropane-2- sulfinamide
[0261] To a solution of(R)-tert-butanesulfinamide (78 mg, 0.64 mmol) in dry methylene chloride (5 mL) was added pyridinium p-toluenesulfonate (PPTS, 8.0 mg, 0.032 mmol), anhydrous MgSO4 (387 mg, 3.22 mmol), and 2-(benzofuran-3-yl)acetaldehyde (103 mg, 0.64 mmol). The reaction was stirred at room temperature overnight, filtered through a pad of celite, and washed again with methylene chloride. After solvent evaporation the residue was purified by flash column chromatography (hexane/EtOAc, 7:3) to obtain (R,E)-N-(2-(benzofuran-3- yl)ethylidene)-2-methylpropane-2-sulfinamide (136 mg, 80%) as a colorless oil. 1H NMR (500 MHz, CDCl3) 6 8.19 (t, J= 4.8 Hz, 1H), 7.59 (s, 1H), 7.54 (d, J= 7.7 Hz, 1H), 7.49 (d, J= 8.2 Hz, 1H), 7.35 - 7.29 (m, 1H), 7.27 - 7.22 (m, 1H), 3.91 (dt, J= 4.8, 1.2 Hz, 2H), 1.18 (s, 9H) ppm. ES+ calc, for C10H8O2 [M+H]+: 264.1 Found: 264.3. c. Preparation of (R)-N-((R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)ethyl)-2-methylpropane-2-sulfinamide
[0262] In a 10 mL round-bottom flask were added tri cyclohexylphosphine tetrafluoroborate (PCy3.HBF4, 2.0 mg, 0.006 mmol), toluene (0.1 mL), a 30 mM aqueous solution of CuSO4 (0.2 mL, 0.006 mmol), and benzylamine (2.64 μL, 0.024 mmol). After stirring vigorously for 10 minutes, toluene (0.9 mL), the corresponding (R,E)-N-(2-(benzofuran- 3-yl)ethylidene)-2-methylpropane-2-sulfinamide (130 mg, 0.49 mmol), and bis(pinacolato)diboron (B2pin2, 251 mg, 0.99 mmol) were added to the catalyst mixture and stirred at room temperature overnight. After diluting with ethyl acetate, the precipitate was filtered through a short pad of deactivated silica gel (SiO2/H2O 100:35, m/m) and washed with ethyl acetate. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (hexane/EtOAc, 7:3) using deactivated silica gel (SiO2/ftO 100:35, m/m) and cyclohexane/ethyl acetate mixtures to obtain (R)-N-((R)-2-(benzofuran-3-yl)-l- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)ethyl)-2-methylpropane-2-sulfinamide (145 mg, 75%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.67 (dd, J= 7.6, 0.7 Hz, 1H), 7.59 (s, 1H), 7.44 (d, J= 8.2 Hz, 1H), 7.30 - 7.25 (m, 1H), 7.23 (td, J= 7.5, 1.0 Hz, 1H), 3.46 - 3.36 (m, 2H), 3.17 - 3.06 (m, 2H), 1.20 (s, 9H), 1.13 (s, 6H), 1.11 (s, 6H) ppm. ES+ calc, for C10H8O2 [M+H]+: 392.2 Found: 392.3. d. Preparation of (R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2- dioxahorolan-2-yl) ethan-1 -amine Hydrochloride
[0263] A solution of (R)-N-((R)-2-(benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)ethyl)-2-methylpropane-2-sulfinamide (135 mg, 0.51 mmol) in 4N HCl- dioxane (2.2 mL) and methanol (0.2 mL) was stirred at room temperature for 2 hours. The volatiles were removed under vacuo. The (R)-2-(benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)ethan-l-amine hydrochloride was obtained as a light yellow solid and was used for next step without further purification. ES+ calc, for C10H8O2 [M+H]+: 288.2 Found: 288.2.
Preparation of tert-Butyl (2-oxo-2-(((lR)-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)carbamate
[0264] A mixture of (tert-butoxycarbonyl)glycine (26 mg, 0.15 mmol), (1R)-2-phenyl-l-
((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethan-l- amine hydrochloride (55 mg, 0.17 mmol), HATU (85 mg, 0.23 mmol), and N,N- diisopropylethylamine (DIPEA, 50 μL, 0.30 mmol) in DMF (0.9 mL) was stirred at room temperature for 1 hour. The mixture was diluted with EtOAc (10 mL) and H2O (10 mL). The organic layer was separated and washed with saturated NaHCO3 (10 mL x 2) and brine (10 mL x 1), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography to provide tert-butyl (2-oxo-2-(((1R)-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)carbamate (40 mg, 58%) as a white solid. ES+ calc, for C10H8O2 [M+Na]+: 479.3 Found: 479.4.
Preparation of 2-A mino-N-((lR)-2-phenyl-l - ( (3 aS, 4S, 6S)-3a, 5, 5-trimethylhexahydro- 4, 6-methanobenzo[d][l, 3, 2]dioxaborol-2-yl)ethyl)acetamide Hydrochloride
[0265] A solution of tert-butyl (2-oxo-2-(((1R)-2-phenyl-l-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)carbamate (25 mg, 0.041 mmol) in 4N HCl-dioxane (0.3 mL) and DCM (0.3 mL) was stirred at room temperature for 30 min. The volatiles were removed under vacuo. The 2-amino-N-((1R)-2- phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2- yl)ethyl)acetamide hydrochloride was obtained as a white solid and was used for next step without further purification. ES+ calc, for C10H8O2 [M+H]+: 357.2 Found: 357.3. Preparation ofN-((1R)-2-Phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][l,3,2]dioxaborol-2-yl)ethyl)-2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9- trimetbyldecabydro-12H-3,12-epoxy[l,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-3)
[0266] N-((1R)-2-Phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((3A,5a5,6A,8a5,9A,10A,12A,12aR)-3,6,9- trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-3) was synthesized by following the general procedure for the HATU mediated coupling of DeoxoART -AcOH 9 (10.0 mg, 30 μM) and (R)-BoroPhe-(+)-Pinanediol-HCl (11.0 mg, 33 μM). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (6.5 mg, 36%) as a white powder. 3H NMR (500 MHz, CDCl3) δ 7.67 (s, 1H), 7.30 - 7.23 (m, 4H), 7.19 - 7.13 (m, 1H), 5.23 (s, 1H), 4.62 (ddd, J= 11.3, 6.3, 2.4 Hz, 1H), 4.20 (dd, J = 8.6, 2.1 Hz, 1H), 3.05 - 2.93 (m, 2H), 2.77 (td, J= 12.4, 4.0 Hz, 1H), 2.69 - 2.57 (m, 2H), 2.51 (dd, J= 17.0, 2.4 Hz, 1H), 2.33 - 2.22 (m, 2H), 2.06 - 1.88 (m, 4H), 1.85 - 1.73 (m, 3H), 1.69 - 1.61 (m, 2H), 1.39 - 1.31 (m, 4H), 1.29 - 1.20 (m, 9H), 1.15 (qd, J= 13.4, 3.2 Hz, 1H), 0.99 - 0.90 (m, 4H), 0.87 - 0.82 (m, 6H) ppm. 13C NMR (125 MHz, CDCl3) 6 175.99, 140.77, 128.97, 128.63, 126.15, 103.39, 89.49, 83.37, 80.72, 76.59, 69.75, 52.47, 52.03, 43.84, 40.23, 38.29, 37.64, 37.61, 36.66, 36.52, 34.36, 32.83, 30.02, 29.34, 27.54, 26.50, 25.75, 24.84, 24.83, 24.40, 20.18, 12.67 ppm. LCMS: retention time = 4.52 min, m/z 608.6 [M+H]+. Figures 58-59 show the characterization of the product.
Preparation ofN-(2-Oxo-2-(((lR)-2-phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro- 4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)-2-
((3R, 5aS, 6R, 8aS, 9R, 10R, 12R, 12aR)-3,6, 9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl) acetamide (DQ-4)
[0267] N-(2-Oxo-2-(((1R)-2-phenyl-1-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)amino)ethyl)-2-
((3R,5aS,6R,8aS,9A,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-4) was synthesized by following the general procedure for the HATU mediated coupling of DeoxoART -AcOH 9 (12.8 mg, 39 μM) and 2-amino-N-((1R)-2-phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][1, 3, 2]dioxaborol-2-yl)ethyl)acetamide hydrochloride (16.1 mg, 41 μM). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (7.2 mg, 28%) as a white powder. 1H NMR (500 MHz, CDCl3) δ 7.30 (dd, J= 12.5, 6.7 Hz, 3H), 7.22 - 7.15 (m, 3H), 6.58 (s, 1H), 5.32 (s, 1H), 4.66 (ddd, J= 11.3, 6.1, 1.8 Hz, 1H), 4.28 (dd, J= 8.7, 1.9 Hz, 1H), 4.04 (dd, J= 16.5, 6.4 Hz, 1H), 3.85 (dd, J= 16.5, 5.4 Hz, 1H), 3.15 (dt, J= 9.1, 4.4 Hz, 1H), 2.96 (dd, J= 14.1, 5.1 Hz, 1H), 2.80 (dd, J= 14.0, 9.9 Hz, 1H), 2.59 - 2.50 (m, 1H), 2.44 (dd, J= 16.3, 11.4 Hz, 1H), 2.36 - 2.24 (m, 3H), 2.16 - 2.09 (m, 1H), 2.06 - 1.94 (m, 3H), 1.89 - 1.83 (m, 2H), 1.83 - 1.76 (m, 2H), 1.75 - 1.66 (m, 2H), 1.37 (s, 3H), 1.34 (s, 3H), 1.29 - 1.14 (m, 7H), 1.01 - 0.93 (m, 4H), 0.88 - 0.82 (m, 6H) ppm. 13C NMR (125 MHz, CDCl3) 6 172.50, 171.32, 139.86, 129.21, 128.65, 126.42, 103.09, 90.18, 85.27, 80.88, 77.60, 69.68, 51.92, 51.81, 43.48, 42.08, 39.85, 38.30, 37.65, 37.33, 37.19, 36.59, 35.90, 34.33, 30.41, 28.80, 27.33, 26.45, 25.93, 24.94, 24.91, 24.24, 20.12, 12.18 ppm. LCMS: retention time = 4.33 min, m/z 665.5 [M+H]+. Figures 60-61 show the characterization of the product.
Preparation ofN-((lR)-2-Phenyl-l-((3aS,4S,6S)-3a,5,5-trimetbylhexabydro-4,6- methanobenzo[d][l,3,2]dioxaborol-2-yl)ethyl)-2-((2R,3R,3aS,3a1R,6R,6aS,9S,10aR)-3,6,9- trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide (DQ- 7)
[0268] N-((1R)-2-Phenyl-l-((3aS,4S,6S)-3a,5,5-trimethylhexahydro-4,6- methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-((2A,3A,3a5,3a1A,6A,6a5,95,10aR)-3,6,9- trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide (DQ-7) was synthesized by following the general procedure for the HATU mediated coupling of 2- ((2R,3R,3aS,3a1R,6R,6aS,9S,10aS)-3,6,9-trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2- ij]isochromen-2-yl)acetic acid (9.3 mg, 30 μM)-and (1R)-2-phenyl-1-((3aS,4S,6S)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d] [1,3,2]dioxaborol-2-yl)ethan-1-amine hydrochloride (11 mg, 33 μM). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (6.9 mg, 39%) as a white powder. 1H NMR (500 MHz, CDCl3) δ 7.58 (s, 1H), 7.30 - 7.27 (m, 3H), 7.26 - 7.23 (m, 1H), 7.20 - 7.14 (m, 1H), 5.16 (s, 1H), 4.41 (td, J= 7.8, 5.2 Hz, 1H), 4.19 (dd, J= 8.6, 2.1 Hz, 1H), 3.01 - 2.93 (m, 2H), 2.79 - 2.71 (m, 1H), 2.52 - 2.46 (m, 2H), 2.34 - 2.20 (m, 2H), 2.06 - 1.99 (m, 1H), 1.98 - 1.89 (m, 2H), 1.88 - 1.79 (m, 3H), 1.78 - 1.60 (m, 4H), 1.54 (ddd, J= 11.5, 9.7, 6.0 Hz, 1H), 1.37 (s, 3H), 1.31 (s, 3H), 1.25 (s, 3H), 1.21 - 1.08 (m, 4H), 0.90 - 0.82 (m, 10H) ppm. 13C NMR (125 MHz, CDCl3) 6 176.42, 140.94, 129.06, 128.53, 126.03, 107.75, 96.96, 83.14, 82.41, 76.48, 64.32, 52.54, 45.21, 40.27, 40.07, 38.28, 37.66, 36.79, 35.65, 34.46, 34.44, 33.76, 29.41, 29.40, 27.56, 26.57, 25.14, 24.41, 23.54, 22.25, 18.82, 12.14 ppm. LCMS: retention time = 4.74 min, m/z 592.6 [M+H]+. Figures 62-63 show the characterization of the product.
Preparation of N-((R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl) ethyl)-2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12- epoxy[l,2]dioxepino[4,3-i]isochromen-l 0-yl) acetamide (DQ-9)
[0269] N-((R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)ethyl)-2-((3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro- 12H -3, 12- epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)acetamide (DQ-9) was synthesized by following the general procedure for the HATU mediated coupling of DeoxoART-AcOH 9 (10 mg, 30 μM) and (R)-2-(benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)ethan-l -amine hydrochloride (10.7 mg, 33 μM). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (5.5 mg, 31%) as a white powder. 1H NMR (500 MHz, CDCl3) δ 8.04 (s, 1H), 7.59 - 7.56 (m, 1H), 7.53 (s, 1H), 7.43 (d, J= 8.1 Hz, 1H), 7.29 - 7.27 (m, 1H), 7.24 - 7.20 (m, 1H), 5.23 (s, 1H), 4.62 (ddd, J= 11.4, 6.4, 2.5 Hz, 1H), 3.04 - 2.96 (m, 2H), 2.75 (dd, J= 16.7, 11.9 Hz, 1H), 2.67 - 2.52 (m, 3H), 2.27 - 2.18 (m, 1H), 1.96 - 1.87 (m, 2H), 1.80 - 1.73 (m, 1H), 1.70 - 1.62 (m, 3H), 1.36 - 1.29 (m, 1H), 1.26 - 1.19 (m, 14H), 1.17 - 1.11 (m, 1H), 1.03 (s, 3H), 0.95 (d, J= 5.8 Hz, 3H), 0.85 (d, J= 7.6 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3) 6 177.30, 155.57, 141.47, 128.34, 124.39, 122.46, 119.95, 119.64, 111.42, 103.33, 89.72, 80.67, 80.48, 69.04, 51.89, 43.67, 37.64, 36.44, 34.30, 32.25, 30.01, 25.48, 25.41, 25.36, 25.25, 24.83, 24.81, 20.12, 12.49 ppm. LCMS: retention time = 4.10 min, m/z 596.5 [M+H]+. Figures 64-65 show the characterization of the product. Preparation ofN-((R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2-dioxahorolan- 2-yl)ethyl)-2-((2R,3R,3aS,3a1R,6R,6aS,9S,10aR)-3,6,9-trimethyldecahydro-10aH-3a1,9- epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide
[0270] N-((R)-2-(Benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)ethyl)-2-((2R,3 R,3aS,3a1R,6R,6aS,9S,10aR)-3,6,9-trimethyldecahydro-10aH-3a1,9- epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetamide (DQ-10) was synthesized by following the general procedure for the HATU mediated coupling of 2-((2R,3R,3aS,3a1R,6R,6aS,9S,10aS)- 3,6,9-trimethyldecahydro-10aH-3a1,9-epoxyoxepino[4,3,2-ij]isochromen-2-yl)acetic acid (9.3 mg, 30 μM) and (R)-2-(benzofuran-3-yl)-l-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)ethan-l- amine hydrochloride (10.7 mg, 33 μM). After completion of the reaction, the mixture was purified by preparative LCMS to give the product (5.8 mg, 33%) as a white powder. 1 H NMR (500 MHz, CDCl3) δ 7.89 (s, 1H), 7.58 (dd, J= 7.6, 0.5 Hz, 1H), 7.55 (s, 1H), 7.44 (d, J= 8.1 Hz, 1H), 7.28 (dd, J= 7.2, 1.2 Hz, 1H), 7.24 - 7.19 (m, 1H), 5.12 (s, 1H), 4.40 - 4.32 (m, 1H), 3.05 - 2.94 (m, 2H), 2.80 - 2.70 (m, 1H), 2.52 - 2.47 (m, 2H), 2.20 (dd, J= 15.5, 7.6 Hz, 1H), 1.94 - 1.87 (m, 1H), 1.85 - 1.80 (m, 1H), 1.76 - 1.69 (m, 1H), 1.68 - 1.62 (m, 1H), 1.58 - 1.52 (m, 1H), 1.50 - 1.42 (m, 1H), 1.23 (d, J= 3.1 Hz, 12H), 1.15 - 1.07 (m, 7H), 0.87 (d, J = 5.9 Hz, 3H), 0.83 (d, J= 7.6 Hz, 4H) ppm. 13C NMR (125 MHz, CDCl3) 6 177.59, 155.62, 141.81, 128.43, 124.28, 122.41, 120.01, 119.42, 111.34, 107.81, 96.94, 82.36, 80.36, 64.12, 45.16, 39.95, 35.61, 34.42, 34.27, 32.99, 29.34, 25.43, 25.38, 25.21, 25.16, 23.15, 22.14, 18.79, 12.07 ppm. LCMS: retention time = 4.28 min, m/z 580.5 [M+H]+. Figures 66-67 show the characterization of the product.
SH-SY5Y Cell Viability Assay
[0271] SH-SY5Y cells were cultured at 37 °C in a humidified air/5% CO2 atmosphere in medium supplemented with 10% fetal bovine serum, 100 ug per ml penicillin, and 100 pg per ml streptomycin in DMEM/F-12 medium. SH-SY5Y was used at 10,000 cells per well. Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO2. Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit. Cell Viability Assay Applicable for MM. IS, U266, RPMI8226, H929, CAG, and PBMC Cells
[0272] Cells were cultured at 37 °C in a humidified air/5% CO2 atmosphere in medium supplemented with 10% fetal bovine serum, 100 ug per ml penicillin, 100 pg per ml streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 mM Sodium Pyruvate in RPMI 1640 medium. MM.1 S was used at 10,000 cells per well. Cells plated in a 96-well plate were treated with various concentrations of test compounds or DMSO for 72 hours at 37 °C in a tissue culture incubator with 5% CO2. Cell viability was measured using CellTiter-Glo® Assay (Promega, Madison, WI) kit.
IC50 Determination
[0273] IC50 values of all compounds against human c-20S β5c, β2c, β1c and i-20S β5i, β2i, β1i were determined in 96-well plates. Briefly, 1 μL of compound in a 3-fold series dilution in DMSO at final concentrations from 100 μM to 0.0017 μM were spotted to the bottom of a black 96-well plate. 100 μL of reaction buffer (20 mM HEPES, 0.5 mM EDTA, and 0.1 mg/mL BSA, pH 7.4) containing proteasome, substrate, and activator was added to each well and the plate was spun on a desktop plate centrifuge and then placed on an orbital shaker at room temperature for 1 minute. The progress of reactions in each well was followed by the fluorescence of the hydrolyzed AMC at Ex 360nm and Em 460 nm for 1 - 2 hours. Linear ranges of the time course were used to calculate the velocities in each well. The reaction velocity of each well was fit to a dose-dependent inhibition equation using PRISM to determine the IC50. Final concentrations of human c-20S and i-20S were 0.2 nM and 0.4 nM, respectively. Suc-LLVY-AMC was used for human β5c at final concentration 25μM. Ac-ANW-AMC was used as substrate of β5i at final concentration 15 μM. Z-LLE-AMC was used as substrate of β1c or β1i at final concentration 50 μM. Z-VLR-AMC was used as substrate of β2c or β2i at final concentration 50 μM. 0.02% SDS was used as activator in the assays for human β5c, β2c, β1c, β5i, β2i, and β1i.
Western Blot
[0274] The multiple myeloma cell lines MM.1S, CAG, and RPMI8226 were treated with 2 μM HZ2083, 2 μM HZ2182, 2 μM artesunate, or 100 nM BTZ for 15 hours. The cells were lysated in RIPA buffer supplemented with protease cocktail. The protein concentration in the samples were measured with bicinchoninic acid assay (BCA) protein assay. The proteins were separated on 4%-20% tris-glycine gel. Primary antibodies against p21 (#2947), CHOP (#5554), PARP (#9532), cleaved PARP (#5625), Bcl-2 (#4223), Mcl-1 (#94296), FTH1 (#3998), and 0- actin (#58169) were from Cell Signaling technology. Caspase 3/7 Activation Assay
[0275] MM. IS cells was seeded in 96-well plates at 10,000 cells per well. The cells were treated with various concentrations of test compounds or DMSO for 24 hours at 37 °C in a tissue culture incubator with 5% CO2. The effect of compounds on caspase 3/7 activity in MM1.S cells was assessed using the Caspase-Gio 3/7 assay system.
Cell Based Proteasome Inhibition Assay
[0276] MM1S cells were seeded in 96-well plates at 10000 cells per well. The cells were treated with various concentrations of test compounds or DMSO for 5 hours at 37 °C in a tissue culture incubator with 5% CO2. The cellular chymotrypsin-like activity was measured using Cell-Based Protesome-Glo Assays kit.
In Gel Labeling Assay
[0277] MM1S cells were seeded in 6-well plates at 1,000,000 cells per well. The cells were treated with 100 μM probe HZ3046, 100 μM inactive-probe HZ3047, or DMSO for 4 hours. The MM. IS cells were collected and lysed in RIPA buffer. The protein concentration of the samples were measured with BCA assay. To each sample (50 pg total protein), Cy3 -azide (10 μM), TBTA ligand (100 μM), TCEP (1 mM), and CuSO4 (1 mM) were added. The samples were incubated for 3 hours with shaking at room temperature. The proteins were precipitated and cleaned up using cold acetone. The precipitated proteins were dissolved with IX Laemmli buffer. The samples were resolved with 4-12% bis-tris gel. Finally, gel images were obtained with a Typhoon scanner.
Example 17 - Discussion of Example 16
[0278] A series of artemisinin boronates hybrids were synthesized as illustrated in scheme 4. HZ2083, with an acetate linker between ART and lucine boronate, was potent against β5c (IC50 = 21 nM) and showed 25-fold selectivity over β5i (Table 8). HZ2087, with a propionate linker, showed less potency than HZ2083 against β5c and β5i, while maintaining the selectivity between β5c and β5i. HZ2088, with a butyrate linker, showed 34-fold less potency against β5c than HZ2083. HZ2082, without a linker between ART and lucine boronate, showed moderate selectivity against β5i (IC50 = 1.8 μM) over β5c (IC50 = 6.0 μM) and showed more potency against pic (IC50 = 0.49 μM) and β1i (IC50 = 0.39 μM) than β5c and β5i. HZ2083 with an acetate linker was more potent than HZ2087 and HZ2088 with a longer linker, and HZ2083 without a linker. HZ2182, the deoxy analog of HZ2083, was then synthesized and used as the artemisinin inactive control compound. HZ2182 showed comparable proteasome inhibition as HZ2083 against all six active subunits. [0279] DQ-9, a benzofuran moiety in the P1 position, displayed a superior inhibitory activity against β5i (IC50 = 2.3 nM) and 153-fold selectivity of β5i over β5c (IC50 = 353 nM). By contrast, the deoxy compound DQ-10 showed better inhibitory activities against β5i (IC50 = 1.4 nM) and reduced selectivity of β5i over β5c (IC50 = 95 nM). Replacement of benzofuran with a phenyl moiety in the P1 position resulted in DQ-3 and DQ-7 which showed dramatically decreased potency against β5i (IC50 = 100 nM and 98, respectively) in comparison with DQ-9 and DQ-10. The incorporation of glycine in the P2 position resulted in the dipeptide compound DQ-4 with highly potent inhibitory activities against β5i (IC50 = 12 nM) and β5c (IC50 = 30 nM). All the compounds DQ-3, DQ-4, DQ-7, DQ-9, and DQ-10 represented superior selectivity for β5i over other subunits (β1i/β1c/β2i/β2c).
Table 8. Enzyme Inhibition IC50 Values
[0280] HZ2083 and HZ2182 were then tested against a panel of multiple myeloma cell lines MM. IS, CAG, H929, RPMI8226, and U266. (Table 9). The MM. IS, CAG, and H929 cell lines were more sensitive to hybrid HZ2083 than the RPMI8226 and U266 cell lines. HZ2083 showed higher cytotoxicity against all the five multiple myeloma cell lines than the deoxy compound HZ2182. Bortezomib, the first FDA-approved proteasome inhibitor, was reported as a highly potent Hu-LonP1 protease inhibitor. The off target inhibition might be related to the high toxicity of bortezomib. The IC50 value for HZ2083 against Hu-LonP1 was determined. HZ2083 showed negligible inhibition against Hu-LonP1 (IC50 = 50 μM) and displayed 2385- fold selectivity against β5c over Hu-LonP1. Neuroblastoma cell line SH-SY5Y and peripheral blood mononuclear cell (PBMC) were used as models evaluating the peripheral neuropathy and toxicity of HZ2083, respectively. HZ2083 showed 31 to 33-fold less cytotoxicity against SH- SY5Y and PBMC over MM. IS, demonstrating a large therapeutic window.
[0281] Although the artemisinin hybrid compounds DQ-3, and DQ-9 had reduced potency against β5i, and β5c in comparison with deoxy compounds DQ-4, and DQ-10, DQ-3, and DQ-9 demonstrated nearly 5-fold increased antiproliferative activity against the MM. IS cell line. This trend was also observed for the comparison between DQ-9 and DQ-10 in other multiple myeloma cells (CAG, H929, and RPMI8226), which may result from the synergistic effects of artemisinin and proteasome inhibition. The dipeptide compound DQ-4 with both inhibitory activities against β5 i and β5c showed a better cytotoxicity against MM.1 S (ECso = 13.5 nM).
Table 9. Cytotoxicities of Hybrid Compounds [0282] Myeloma cell lines MM1S, CAG, and RPMI8226 were treated with 2 μM HZ2083 for 15hours. HZ2083 led to an increase in P21 and Chop protein levels in treated cells (Figure 68). HZ2083 also induced PARP cleavage and triggered apoptosis in all three cell lines. As shown in Figure 69, both HZ2083 and its deoxy analog HZ2182 increased P21 and CHOP and PARP cleavage in MM.1S and CAG cell line cells; there was no detectable change in protein level of p21, CHOP, and cleaved PARP in artesunate treated CAG cells. Both HZ2083 and artesunate induced the degradation of ferritin FTH1 in the MM. IS cell line. However, the inactive analog HZ2182 could not do so. Ferritin is a cytosolic iron storage protein complex capable of chelating as many as 4500 iron atoms. During the process of ferroptosis, lysosomal degradation of ferritin (ferritinophagy) contributes to an increased labile iron pool, leading to elevated lipid peroxidation and oxidation of polyunsaturated fatty acids. HZ2083 might also induce ferroptosis in the MM. IS cell line.
[0283] Activated caspase-3/-7 are well-recognized markers of apoptosis. Treatment of MM1S cells with HZ2083 for 24 hours induced caspase 3/7 activity which further proved that HZ2083 induced apopotosis (Figure 70). HZ2083 primarily targets chymotrypsin-like activity of the proteasome. The chymotrypsin-like activity inhibition of HZ2083 in the MM.1S cells was measured using a Proteasome-Glo cell based proteasome assay. HZ2083 showed comparable proteasome inhibition in the cellular assay as in biochemical assay using purified proteasome. [0284] To identify the direct binding targets of hybrid HZ2083 and its MOA in MM.1S cells, an alkyne-tagged probe HZ3046 and its deoxy analog HZ3047 were synthesized. Both HZ3046 and HZ3047 showed potent activity against β5c and β5i. As shown in Figure 71, protein targets of HZ3046 in MM.1S cells were visualized by conjugating HZ3046 with a fluorescene dye azide-Cy3 through click chemistry.
[0285] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:
1. An Artemi sinin-Proteasome inhibitor conjugate comprising a compound of
Formula (I): wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of H, halogen, -CF3, =CH2, -ORa, -NRaRb, -(CH2)nCOORa, -(CH2)nC(=O)Ra, -(CH2)nCONRaRa, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, aryl, and heteroaryl;
Ra is independently selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle;
Rb is independently selected from group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle, and wherein Ra and Rb may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
X is O, S, or N,
Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor;
Y’ is =O, or — Linker — Proteasome inhibitor, wherein one of Y or Y’ is a — Linker — Proteasome inhibitor;
Z is O or O-O;
Linker is a bond, a branched or unbranched C1-C10 alkylene, a branched or unbranched C2-C10 alkenylene, — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)-(arylene)-C(=O) — , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH- C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- (arylene)-C(=O)— , — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y- C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O)— , or — (CH2)y-C(=O) — , wherein, when said Linker is — O-C(=O)-(CH2)y-C(=O) — , — O-C(=O)- (arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)—, — (CH2)y-NH-C(=O)-(CH2)y- C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— — (CH2)y-NH-C(=O)-(arylene)-C(=O)— — (CH2)y-NH-(CH2)y-C(=O)—, — (CH2)y-NH-C(=O)- (CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O- (CH2)y)y-C(=O) — , or — (CH2)y-C(=O) — , the carbonyl carbon of the Linker is attached to the Proteasome inhibitor; n is an integer ranging from 0 to 3; y is independently selected at each occurrence from an integer ranging from 0 to 10; and
Proteasome inhibitor is a compound that inhibits either chymotryptic-like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
2. The Artemi sinin-Proteasome inhibitor conjugate of claim 1, comprising a compound of Formula (I'): wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of H, halogen, -CF3, =CH2, -ORa, -NRaRb, -(CH2)nCOORa, -(CH2)nC(=O)Ra, -(CH2)nCONRaRa, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 cycloalkyl, C3-7 heterocycloalkyl, aryl, and heteroaryl;
Ra is independently selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle;
Rb is independently selected from group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl, heteroaryl, and aralkyl, wherein said C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl is optionally substituted with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, aryl, heteroaryl, and non-aromatic heterocycle, and wherein Ra and Rb may be taken together with the adjacent nitrogen atom forming a heterocyclic group;
X is O, S, or N,
Y is, at each occurrence thereof, either H or — Linker — Proteasome inhibitor;
Y’ is =O, or — Linker — Proteasome inhibitor, wherein one of Y or Y’ is a — Linker — Proteasome inhibitor;
Linker is a bond, a branched or unbranched C1-C10 alkylene, — O-C(=O)-(CH2)y- C(=O)— , — O-C(=O)-(arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y- NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)- O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(arylene)-C(=O)— , — (CH2)y-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y-C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O) — , or — (CH2)y-C(=O) — , wherein, when said Linker is — O-C(=O)-(CH2)y-C(=O)— , — O-C(=O)-(arylene)-C(=O)— , — (CH2)y-C(=O)-NH-(CH2)y- C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-NH-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-O-(CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(arylene)-C(=O)— , — (CH2)y-NH- (CH2)y-C(=O)— , — (CH2)y-NH-C(=O)-(CH2)y-(O-(CH2)y)y-C(=O)— , — (CH2)y-C(=O)-(CH2)y- C(=O)— , — (CH2)y-C(=O) -(CH2)y-(O-(CH2)y)y-C(=O)— , or — (CH2)y-C(=O) — , the carbonyl carbon of the Linker is attached to the Proteasome inhibitor; n is an integer ranging from 0 to 3; y is independently selected at each occurrence from an integer ranging from 0 to 10; and
Proteasome inhibitor is a compound that is known to inhibit either chymotryptic- like beta5, tryptic-like beta2, or caspase-like betal activity of proteasome activity, or an oxide thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.
3. The Artemi sinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein
R1, R2, and R3 are H;
R4 and R5 are CH3;
X is O; and
Linker is — O-C(=O)-(CH2)yC(=O)— , — (CH2)y-C(=O)-(CH2)yC(=O)— , or — (CH2)y-C(=O) — .
4. The Artemi sinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the
Proteasome inhibitor moiety comprises a compound of Formula (II): wherein is the point of attachment to the Linker;
R' is H or C1-6 alkyl;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, NHCOOC1-12 alkyl, — B(OR’)2, methylsulfonyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, — (CH2)mC(O)NHR6, — CH2OC1-6 alkyl, — CH2Ar, and — CH2 heteroaryl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and C1-6 alkoxy; or
R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring; R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, —
(CH2)mC(O)NR6R7, — C(O)OR10, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — N3, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein is the point of attachment to the corresponding carbon or nitrogen atom of the structure of Formula (II);
R5’ is selected from the group consisting of H, non-aromatic heterocycle, — NR6R7, — CR8R9, C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3- 12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkyl alkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with R11;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, — CF3, C1-6 alkyl, C3-8 cycloalkyl, — (CH2)kOH, and arylalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R12; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
R10 is H or arylalkyl;
R11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF3, C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl, wherein C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R12; R12 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy, and the C1-12 alkyl, C2-12 alkenyl and C2-12 alkynyl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
Q is optional and, if present, is C1-3 alkyl or — C(Y) — ;
Q1 is optional, and, if present, is selected from NH, — (CR3’H) — , — NH- (CRzH) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle;
Rz is independently selected at each occurrence thereof from the group consisting of C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-12 cycloalkenyl, C5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, and (cycloalkynyl)alkyl, wherein C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-12 cycloalkenyl, C5-12 cycloalkynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkynyl)alkyl can be optionally substituted 1 time with Rz ;
Rz is independently selected at each occurrence thereof from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;
X is a bond, — C(Y)— , — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1, 2, 3, or 4; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2, or 3.
5. The Artemi sinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (II): wherein
L is — (CR3’Rx)p— , or NR3’;
M is — (CR2’Ry)r; is the point of attachment to the Linker;
R' is H or C1-6 alkyl;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-12 alkyl, C1-6 alkoxy, NH2, NHCOOC1-12 alkyl, — B(OR’)2, methylsulfonyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy; or
R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, —
(CH2)mC(O)NR6R7, — C(O)OR10, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl, wherein is the point of attachment to the corresponding carbon or nitrogen atom of the structure of Formula (II);
R5’ is selected from the group consisting of H, non-aromatic heterocycle, — NR6R7, — CR8R9, C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-12 alkyl, monocyclic or bicyclic C3-10 cycloalkyl, C3-12 cycloalkylalkyl, C1-12 alkoxy, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with R11;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, — CF3, C1-6 alkyl, — (CH2)kOH, and arylalkyl; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or a morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R12; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
R10 is H or arylalkyl;
R11 is selected independently at each occurrence thereof from the group consisting of halogen, — CF3, C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl, wherein C1-6 alkyl, C3-8 cycloalkyl, aryl, and arylalkyl can be optionally substituted 1 to 3 times with R12;
R12 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy, and the C1-12 alkyl, C2-12 alkenyl and C2-12 alkynyl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of a substituted or unsubstituted aryl or heteroaryl group;
Q is optional and, if present, is C1-3 alkyl or — C(Y) — ;
Q1 is optional, and, if present, is selected from NH, — (CR3’H) — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non — aromatic heterocycle;
X is a bond, — C(Y)— , — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; and s is 0, 1, 2, or 3.
6. The Artemi sinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the
Proteasome inhibitor moiety comprises a compound of Formula (III): wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non— aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, C1-6 alkyl, C1-6 alkoxy, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, — (CH2)mC(O)NR6R7, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, wherein C1-6 alkyl, C1-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R5’ is selected from the group consisting of H, C1-6 alkyl, C1-6 alkoxy, non- aromatic heterocycle, — NR6R7, and — CR8R9; and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is C(O), — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2, 3, or 4; s is 0 or 1; and
1 is 0 or 1.
7. The Artemi sinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (Illa): wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, aryl, heteroaryl, non-aromatic heterocycle, and non-aromatic heterocycle substituted with =O;
R2’ is H or C1-6 alkyl;
R3’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)mC(O)NHR5’, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — OC1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; R5’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Y is O or S; m is 1 or 2; and n is 1, 2, or 3.
8. The Artemi sinin-Proteasome inhibitor conjugate of claim 7, wherein
R1’ is a substituted or unsubstituted biphenyl, or a substituted or unsubstituted hetero aryl; R2’ is H;
R3’ is — (CH2)mC(O)NHR5’;
R5’ is a C1-6 alkyl;
Y is O; and n is 1.
9. The Artemi sinin-Proteasome inhibitor conjugate of claim 8, wherein the conjugate is selected from the group consisting of:
10. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the
Proteasome inhibitor moiety comprises a compound of Formula (Illb): wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2 heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy; R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, — (CH2)mC(O)NR6R7, — (CH2)mC(O)OH, and — (CH2)mC(O)OBn;
R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or a morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ;
Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; p is 1 or 2; q is 0, 1, or 2; r is 1 or 2; and s is 0 or 1.
11. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (IIIc), Formula (IIId), or Formula (IIIe) : wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, and — (CH2)mC(O)NR6R7 ;
R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
Rx is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5, — (CH2)mC(O)NR6R7, and — CH2C(O)R5 ; Ry is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; q is 0, 1, or 2; and s is 0 or 1.
12. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (Illf) : wherein is the point of attachment to the Linker;
R1’ is selected from the group consisting of monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle, wherein monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl and bi- heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocycle can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R2’ is independently selected at each occurrence thereof from the group consisting of H, D, C1-6 alkyl, — CH2OC1-6 alkyl, — CH2Ar, and — CH2heteroaryl, wherein aryl (Ar) can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, C1-6 alkyl, and C1-6 alkoxy;
R3’ is independently selected at each occurrence thereof from the group consisting of H, D, — CH2OC1-6 alkyl, — (CH2)mC(O)NHR5’, and — (CH2)mC(O)NR6R7 ; R5’ is selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, non-aromatic heterocycle, — NR6R7, and — CR8R9;
R6, R7, R8, and R9 are each independently selected from the group consisting of H, D, C1-6 alkyl, and — (CH2)kOH; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, or morpholine ring; or R8 and R9 are taken together with the carbon to which they are attached to form an oxetane ring;
X is — (CH2)q— , — O— , or — (CD2)q— ;
Y is O or S; k is 1, 2, or 3; m is 0, 1, 2, 3, 4, or 5; s is 0 or 1; and q is 0, 1, or 2.
13. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (Illg): wherein is the point of attachment to the linker;
W is CHR3’, or NR3’;
X1 is selected from the group consisting of — C(O)-NH — , monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and monocyclic and bicyclic non- aromatic heterocycle;
Y1 is optional and, if present, is — (CH2)m — ;
Z1 is optional and, if present, is aryl or bicyclic heteroaryl, wherein aryl or bicyclic heteroaryl can be optionally substituted 1 or 2 times with H, halogen, NH2, NHCOOC1- 12 alkyl, or C1-12 alkyl;
R' is H or C1-6 alkyl;
R2 is H or C1-6 alkyl;
Ry is H or C1-6 alkyl; or R2’ and Ry are taken together with the carbon to which they are attached to form a C3-8 cycloalkyl ring;
R3 is selected from the group consisting of C1-6 alkyl, , and — (CH2)nC(O)NR6R7, wherein C1-6 alkyl can be optionally substituted from 1 to
3 times with a substituent selected independently at each occurrence thereof from OH or C(O)OR10, wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (Illg);
R6, R7 are selected from the group consisting of H, C1-6 alkyl, and arylalkyl; or R6 and R7 are taken together with the nitrogen to which they are attached to form a piperidine, pyrrolidine, azepane, or morpholine ring, wherein piperidine, pyrrolidine, azepane, or morpholine ring can be optionally substituted 1 to 3 times with R9;
R9 is selected from the group consisting of H, halogen, C1-6 alkyl, C3-8 cycloalkyl, and aryl, wherein C1-6 alkyl can be optionally substituted 1 to 3 times with halogen;
R10 is H or arylalkyl; k is 1 or 2; m is 0, 1, or 2; and n is 0, 1, 2, 3, or 4.
14. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (IV): wherein is the point of attachment to the Linker; wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV); R1’ is a H, branched, cyclic, or linear C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl, wherein the C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl may be optionally substituted from 1 to 3 times with R3’;
R2’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)xC(O)NHR4’, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — N3, — CF3, — O C1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R3’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R4’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; x is 1 or 2; and n is 0, 1, 2, or 3.
15. The Artemisinin-Proteasome inhibitor conjugate of claims 1 or 2, wherein the Proteasome inhibitor moiety comprises a compound of Formula (IV): wherein is the point of attachment to the Linker;
Y is wherein is the point of attachment to the corresponding carbon atom of the structure of Formula (IV); R1’ is a H, branched, cyclic, or linear C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl, wherein the C1-12 alkyl, C2-12 alkenyl, or C2-12 alkynyl may be optionally substituted from 1 to 3 times with R3’;
R2’ is independently selected at each occurrence thereof from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl, and — (CH2)xC(O)NHR4’, wherein C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic and bicyclic heterocyclyl can be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — OH, — NO2, — CF3, — O C1-6 alkyl, C1-6 alkyl, C2-6 alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;
R3’ is an aryl or heteroaryl, wherein the aryl or heteroaryl may be optionally substituted from 1 to 3 times with a substituent selected independently at each occurrence thereof from the group consisting of halogen, cyano, — CF3, C1-6 alkyl, and C1-6 alkoxy;
R4’ is selected from the group consisting of H, C1-6 alkyl, and C3-8 cycloalkyl, wherein C3-8 cycloalkyl can be optionally substituted with — CF3;
Z1 and Z2 are each independently OH, C1-6 alkoxy, aryloxy, or aralkoxy; or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent; x is 1 or 2; and n is 0, 1, 2, or 3.
16. The Artemisinin-Proteasome inhibitor conjugate of claims 14 or 15, wherein
Linker is — (CH2)-C(=O)— or — CH2-C(=O)-NH-(CH2)y-C(=O)— ;
Y is
R1’ is a C4 alkyl;
Z1 and Z2 are OH; and n is 0.
17. The Artemisinin-Proteasome inhibitor conjugate of claims 14 or 15, wherein the conjugate is selected from the group consisting of:
18. The Artemisinin-Proteasome inhibitor conjugate of claim 15, wherein the conjugate is selected from the group consisting of:
19. A method of treating an infectious disease in a subject, said method comprising: administering to the subject in need thereof a compound of any one of claims 1- 18.
20. The method according to claim 19, wherein the said administering is carried out orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.
21. The method according to claim 19, wherein the infectious disease is caused by bacterial, viral, parasitic, and fungal infectious agents.
22. The method according to claim 21, wherein the infectious disease is caused by a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium-intracellular e, and Mycobacterium leprosy.
23. The method according to claim 21, wherein the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses, Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.
24. The method according to claim 21, wherein the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.
25. The method according to claim 21, wherein the infectious disease is malaria.
26. A method of treating cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders in a subject or for providing immunosuppression for transplanted organs or tissues in a subject, said method comprising: administering to the subject in need thereof a compound of any one of claims 1- 18.
27. The method of claim 26, wherein an autoimmune disorder is treated, said autoimmune disorder being selected from the group consisting of arthritis, colitis, multiple sclerosis, lupus, Sjogren Syndrome, Systemic Lupus Erythematosus and lupus nephritis, glomerulonephritis, Rheumatoid Arthritis, Inflammatory bowel disease (IBD), ulcerative colitis, Crohn's diseases, Psoriasis, and asthma.
28. The method of claim 26, wherein immunosuppression is provided for transplanted organs or tissues, said immunosuppression being used to prevent transplant rejection and graft-verse-host disease.
29. The method of claim 26, wherein an inflammatory disorder is treated, said inflammatory disorder being Crohn’s disease, ulcerative colitis, arthritis, or lupus.
30. The method of claim 26, wherein cancer is treated, said cancer being selected from the group consisting of neoplastic disorders, hematologic malignances, lymphocytic malignancies, multiple myeloma, mantle cell lymphoma, leukemia, Waldenstrom Macroglobulinemia, pancreatic cancer, bladder cancer, colorectal cancer, chordoma cancer, breast cancer, metastatic breast cancer, prostate cancer, androgen-dependent and androgen— independent prostate cancer, renal cancer, metastatic renal cell carcinoma, hepatocellular cancer, lung cancer, non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung, ovarian cancer, progressive epithelial or primary peritoneal cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, squamous cell carcinoma of the head and neck, melanoma, neuroendocrine cancer, metastatic neuroendocrine tumors, brain tumors, glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma, bone cancer, and soft tissue sarcoma.
31. The method according to claim 26, wherein the said administering is carried out orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.
32. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1-18 and a pharmaceutically acceptable carrier.
EP22743162.4A 2021-01-20 2022-01-20 Artemisinin-proteasome inhibitor conjugates and their use in the treatment of disease Pending EP4281066A2 (en)

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