WO2014018807A1 - Peptide epoxyketone compounds - Google Patents

Peptide epoxyketone compounds Download PDF

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Publication number
WO2014018807A1
WO2014018807A1 PCT/US2013/052143 US2013052143W WO2014018807A1 WO 2014018807 A1 WO2014018807 A1 WO 2014018807A1 US 2013052143 W US2013052143 W US 2013052143W WO 2014018807 A1 WO2014018807 A1 WO 2014018807A1
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compound
proteasome
activity
mmol
formula
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PCT/US2013/052143
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French (fr)
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Xiaoqing Michelle FAN
Jian James CEN
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Centrax International, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Ci_ 4 alkyl -Ci_i 0 alkylene-NRi 2 Ri3, - R12R13, or -OP0 3 MiM 2 ; each of Ri 2 and Ri 3 is independently H, Ci_ioalkyl (e.g. Ci_ 4 alkyl) or substituted Ci_i 0 alkyl (e.g. Ci_ 4 alkyl); each of Mi, and M 2 is independently H, metal (e.g. Na, and K);
  • X is absent or O
  • Z is absent or O; and m is 0, 1, 2, 3, 4 or 5.
  • X is absent or O
  • R 7 is absent, X is O, and Rg is selected from H, metal (e.g. Na, and K), NH 4 , Ci_ioalkyl (e.g. Ci_ 4 alkyl),
  • R 7 is absent, X is absent, and Rg is selected from -NRi 2 Ri 3 , wherein Ri 2 and Ri 3 are defined supra.
  • Each of the chiral carbons in Formula (I) can be independently in
  • a compound of Formula (I) provided herein has a configuration shown in formula(VII): Formula (VII) wherein R is selected from the group consisting of
  • Each of the above compounds of Formula (VIII) is capable of simultaneously inhibiting the CT-L, T-L, and PGPH activities of the 20S proteasome.
  • base salts e.g. aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • Step 2 Reaction of Compound 1002 with isopropenyl bromide affords Compound 1003.
  • the inhibition effect of the compounds can be also tested in vivo.
  • the second active agent that may be administered concomitantly with compounds of the present disclosure for cancer treatment include but not limited to: 5-fluorouracil, doxorubicin, daunorubicin, tamoxifen, leuprolide, goserelin, flutamide, nilutimide, finasteride, dexamethasone, lenalidomide aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, temozolomide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, do
  • fluocinonide fluocorinbutyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halobetasol propionate, halcinonide, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednisolone, dexamethasone, and
  • Example 1 Inhibition of the chymotrypsin-like (CT-L), PGPH, and trypsin-like (T-L) activity of the 20S proteasome in enzymatic assays
  • mice The in vivo proteasome activity was examined in BALB/c nude mice.
  • Mice (5-6 mice/group) were administered intravenously with either vehicle (10-20% (w/v) hydroxypropyl- -cyclodextrin in 10 mM sodium citrate containing 2.5% DMSO (pH 3.0-3.5)), or a compound disclosed herein at a tolerated dose.
  • vehicle 10-20% (w/v) hydroxypropyl- -cyclodextrin in 10 mM sodium citrate containing 2.5% DMSO (pH 3.0-3.5)
  • a compound disclosed herein at a tolerated dose.
  • One hour after dosing whole blood samples were collected by cardiac puncture into tubes containing sodium heparin and centrifuged at 150xg for 5min at 4°C. The resulting pellet was washed using ice-cold phosphate buffered saline (PBS) three times.
  • PBS ice-cold phosphate buffered saline
  • Resin 1033 To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1032 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et 2 0 and allowed to air dry for 24 hours to yield Resin 1033.
  • immunoproteasome by Compound 5 was performed using the enzymatic assay method disclosed in Example 1.
  • the following table presents the IC 50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome.
  • the IC 50 ratio of Compound 5 to Carfilzomib on CT-L activity was determined to be 0.27 and 0.52, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
  • Example 18 Compound 25, Structure, Proteasome Activity
  • immunoproteasome by Compound 25 was performed using the enzymatic assay method disclosed in Example 1.
  • the following table presents the IC 5 o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome.
  • the IC 50 ratio of Compound 25 to Carfilzomib on CT-L activity was determined to be 0.26 and 0.30, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
  • immunoproteasome by Compound 27 was performed using the enzymatic assay method disclosed in Example 1.
  • the following table presents the IC 5 o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome.
  • the IC 50 ratio of Compound 27 to Carfilzomib on CT-L activity was determined to be 0.37 and 0.40, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
  • Example 20 Compound 28, Structure, Proteasome Activity

Abstract

The present disclosure relates to novel compounds and pharmaceutical compositions thereof which are useful as inhibitors of proteasomes. The compounds provided herein are capable of inhibiting all three of CT-L, T-L, and PGPH activities of proteasomes, and are useful in treating various conditions or diseases associated with proteasomes.

Description

PEPTIDE EPOXYKETONE COMPOUNDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority to U.S. Provisional
Patent Application Number 61/675,827 filed on July 26, 2012, and Chinese Patent Application Number 201210352544.5 filed on September 20, 2012, both of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates to compounds that have a backbone of peptide epoxyketone structure. These compounds are useful in, for example, inhibiting proteasomes.
BACKGROUND
[0003] Proteasome is a multicatalytic protease complex that plays a critical role in mediating the regulated degradation of intracellular proteins. In vivo, the proteasome complex is believed to exist as 26S proteasome, which is about 2000 kDa in molecular weight containing one 20S core particle (20S proteasome) and two 19S regulatory particles. The core is hollow and provides an enclosed cavity in which proteins are degraded. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites; it is this structure that recognizes polyubiquitinated proteins and transfers them to the catalytic core. An alternative form of regulatory subunit called the 1 IS particle associates with the core in essentially the same manner as the 19S particle; the 1 IS may play a role in degradation of foreign peptides. The core particle 20S proteasome is about 700kDa in molecular weight and is comprised of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 a subunits form each of the two outer rings and 7 β subunits form each of the two inner rings. The a rings serve as binding sites for the 19S or 1 IS regulatory complex, as well as a physical barrier for the two inner β rings. The two inner β rings contain active proteolytic sites. Degradation of proteins occurs within the central chamber formed by the association of the two β rings. In vivo, inhibition of the 20S proteasome can be directly correlated to inhibition of the 26S proteasome. There are two forms of proteasomes: the constitutive proteasome ubiquitously expressed by the majority of cells in the body, and the immunoproteasome, predominantly expressed in hematopoietic cells and cells that have been exposed to inflammatory cytokines. Proteasome-mediated protein degradation is a highly regulated process that is necessary for a variety of intracellular processes. Through the use of different peptide substrates, three major proteolytic activities have been defined for the eukaryote proteasome: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (T-L), which cleaves after basic residues; and peptidylglutamyl peptide hydrolyzing activity (PGPH), which cleaves after acidic residues. Proteasome has long been recognized as an attractive target for drug development, and was first clinically validated as a therapeutic target in oncology (Orlowski and Kuhn, Clin. Cancer Res. (2008), 14, 1649-1657).
[0004] There are several examples of small molecules which have been used to inhibit proteasome activity, including peptide boronic acids, β-lactones and peptide epoxyketones (Bennett and Kirk, Current Opinion in Drug Discovery & Development (2008), 11, 616-625; Borissenko and Groll, Chem. Rev. (2007) 107, 687-717); however, these compounds generally lack appropriate specificity and/or potency necessary to fully explore and exploit the roles of proteasome at molecular, cellular and in vivo level. For example, peptide boronic acids and β-lactones are non-specific for proteasome, as they also have been found to inhibit other proteases (Borissenko and Groll, Chem. Rev. (2007) 107, 687-717; Myung et al, Medicinal Research Reviews (2001), 21, 245-273). This raises the possibility that these inhibitors could exhibit off-target activities in vivo that are associated with inhibition of non-proteasome targets. On the other hand, the peptide epoxyketones as disclosed in US6831099Bl, WO2005/105827, CN101044157A, and US2007/0105786A1 are highly selective as inhibitors of proteasomes. However, these peptide epoxyketones inhibit only the chymotrypsin-like (CT-L) activity but not the trypsin-like (T-L) activity and/or peptidylglutamyl peptide hydrolyzing (PGPH) activity of proteasomes. It has been reported that simultaneous inhibition of the CT-L, T-L and PGPH activity of proteasome is required to markedly reduce proteolysis (Kisselev et al, J. Biol. Chem. (2006), 281, 8582-8590) and thus induces synergistic effect of inhibiting growth of tumor cells and may lead to improved efficacy in tumor treatment
(Chauchan et al., Blood (2008), 111, 1654-1664). Therefore, there are needs in the art to develop novel proteasome inhibitors that exert simultaneous inhibition of the CT-L, T-L and PGPH activities of proteasomes.
SUMMARY
[0005] Provided herein are compounds that have a peptide ketone epoxy backbone. Also disclosed and claimed herein are compounds capable of
simultaneously inhibiting CT-L, T-L and PGPH activities of the proteasome.
[0006] In certain embodiments, the present disclosure provides compounds having a structure shown in Formula (I), and an enantiomer, diastereomer, tautomer, pharmaceutically acceptable salt or solvate or prodrug thereof:
Figure imgf000004_0001
Figure imgf000005_0001
R2 is -(CH2)mR5; each of R3 is independently H, hydroxyl, Ci_ioalkyl, Ci_ioalkoxyl, Ci_iohydroxyalkyl, C -ioalkylox alkyl, NH2, NHR6, -R7-0(C=0)-R8, -R7-(C=0)X-R8, -R7-OP03MiM2,
Figure imgf000005_0002
R7— XYZ R9—
N
H3C
R5 is phenyl, or Ry;
R7 XYZ R9
Ry is -OH, -OP03MiM2, -Ri0-O(C=O)-Rn,
Figure imgf000005_0003
R6 is Ci_ioalkyl, phenyl, -(C=0)Ci_6alkyl, -(C=0)phenyl; each of R7, R and Rio is independently absent, or Ci_ioalkylene (e.g. -CH2-, -C2H4- -C3H7-, etc.); each of R8 and Rn is independently H, hydroxyl, metal (e.g. Na, and K), Ci_ioalkyl (e.g. Ci_4alkyl), -Ci_i0alkylene-NRi2Ri3, - R12R13, or -OP03MiM2; each of Ri2 and Ri3 is independently H, Ci_ioalkyl (e.g. Ci_4alkyl) or substituted Ci_i0alkyl (e.g. Ci_4alkyl); each of Mi, and M2 is independently H, metal (e.g. Na, and K);
X is absent or O;
Y is absent or -(C=0)-;
Z is absent or O; and m is 0, 1, 2, 3, 4 or 5.
[0007] In certain embodiments, when R5 is phenyl, Ri is not R4. In certain embodiments, when Ri is R4, R5 is not phenyl.
[0008] In certain embodiments, the compounds of Formula (I) comprise a structure selected from Compounds I-l, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, I-ll, 1-12, 1-13, 1-14, 1-15, 1-16.
[0009] In certain embodiments, the compound of Formula (I) as provided herein has a configuration shown in Formula (II):
Figure imgf000006_0001
(II) wherein each of Ri and R2 is as defined supra.
[0010] In certain embodiments, the compounds of Formula (II) comprise a structure selected from Compound 1-5, 7-13, 20, 25, 27 and 28, and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
[0011] In certain embodiments, the compounds of Formula (I) have a structure of Formula (III):
Figure imgf000007_0001
wherein R3 is as defined supra.
[0012] In certain embodiments, the compounds of Formula (I) have a structure of Formulas (IV) or (V):
Figure imgf000007_0002
wherein R is as defined supra.
[0013] In certain embodiments, the compounds of Formula (I) have a structure of Formula (VI):
Figure imgf000008_0001
Formula (VI)
wherein the R4 and Ry group has a definition as provided supra.
[0014] In certain embodiments, the compounds provided herein have a structure of Formula (VII):
Figure imgf000008_0002
wherein R3 is as defined supra.
[0015] In certain embodiments, a compound of Formula (I) provided herein has a configuration shown in formula(VIII):
Figure imgf000008_0003
Formula (VIII), wherein Ri is selected from the group consisting
Figure imgf000009_0001
[0016] In one aspect, the present disclosure provides peptide epoxyketones that can inhibit catalytic activity of the 20S proteasome. In certain embodiments, the peptide epoxyketones provided herein can inhibit catalytic activity of the 20S proteasome at concentration below about 5 μΜ, 2 μΜ, or 1 μΜ. In certain embodiments, the peptide epoxyketones provided herein not only more potently inhibit the CT-L activity of the 20S proteasome, but also potently inhibit the T-L activity and PGPH activity of the 20S proteasome when present at concentrations below about 5 μΜ. In preferred embodiments, the compounds disclosed herein show simultaneous inhibition of the CT-L, T-L and PGPH activities of the 20S proteasome at concentrations below about 5 μΜ, 2 μΜ, or 1 μΜ.
[0017] In another aspect, the compounds disclosed herein simultaneously inhibit the CT-L, T-L and PGPH activities of the 20S proteasome in vivo (for example, in blood collected from mice administered with the compounds disclosed herein at a tolerated dose).
[0018] In further aspect, the present disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a 20S proteasome inhibitor disclosed herein, which are useful, for example, in treatment of human conditions, including but not limited to, cancer, inflammation, neurodegenerative disease (such as Alzheimer's disease),
muscle-wasting disease, chronic infectious diseases, fever, muscle disuse, denervation, nerve injury, and immune -related conditions, among others.
[0019] In certain embodiments, the pharmaceutical compositions comprise about 10~9 g to about 10 g of a compound provided herein. Suitable dosages per subject per day can be from about 0.01 mg to about 5 g.
[0020] In certain embodiments, the compounds or pharmaceutical compositions disclosed herein are formulated into a dosage form which is suitable for delivery to a subject in need thereof through a parenteral route (e.g. subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intra cardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, and/or infusion) and a
non-parenteral route (e.g. oral, enteral, buccal, nasal, intranasal, transmucosal, epidermal, transdermal, dermal, ophthalmic, pulmonary, sublingual, rectal, vaginal or topical).
[0021] Another aspect of the present disclosure relates to methods of treating a condition associated with 20S proteasome, comprising administering a therapeutically effective amount of a compound provided herein. Methods of simultaneously inhibiting the CT-L, T-L and PGPH activities of the 20S proteasome are also provided, comprising administering a therapeutically effective amount of a compound provided herein.
[0022] In a further aspect, the present disclosure provides a method of making a peptide epoxyketone.
[0023] Other features and advantages of the present disclosure will be apparent from the following detailed description, and from the claims.
DETAILED DESCRIPTION
[0024] Compounds and pharmaceutical salts thereof [0025] In one aspect, the present disclosure provides compounds that have a structure of Formula (I), and an enantiomer, diastereomer, tautomer,
harmaceutically acceptable salt or solvate or prodrug thereof:
Figure imgf000011_0001
(Formula I)
wherein Ri is selected from the roup consisting
Figure imgf000011_0002
Figure imgf000011_0003
R2 is -(CH2)mR5; each of R3 is independently H, hydroxyl, Ci_ioalkyl, Ci_ioalkoxyl, Ci_iohydroxyalkyl,
Ci_i0alkyloxyalkyl, NH2, NHR6, -R7-0(C=0)-R8, -R7-(C=0)X-R8, -R7-OP03MiM2,
N ^ R7-XYZ R9— ^\ N^ R7-XYZ— R9—
o H N. 0ΐ
Figure imgf000012_0001
R5 is phenyl, or Ry;
Figure imgf000012_0002
R6 is Ci_ioalkyl, phenyl, -(C=0)Ci_6alkyl, -(C=0)phenyl; each of R7, R and Rio is independently absent, or Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.); each of Rg and Rn is independently H, hydroxyl, metal (e.g. Na, and K), Ci_ioalkyl (e.g. Ci_4alkyl), -Ci_i0alkylene-NRi2Ri3, -NRi2Ri3, or -OP03MiM2; each of R12 and Ri3 is independently H, Ci_ioalkyl (e.g. Ci_4alkyl) or substituted Ci_ioalkyl (e.g. Ci_4alkyl); each of Mi, and M2 is independently H, metal (e.g. Na, and K);
X is absent or O;
Y is absent or -(C=0)-;
Z is absent or O; and m is 0, 1, 2, 3, 4 or 5.
[0026] In certain embodiments, when R5 is phenyl, Ri is not R4. In certain embodiments, when Ri is R4, R5 is not phenyl.
[0027] In certain embodiments, R3 is -R7-0(C=0)-Rg, and R7 and Rg are defined as supra. In certain embodiments, R7 is absent, and Rg is selected from H, Ci_ioalkyl (e.g. Ci_4alkyl), -Ci_ioalkylene-NRi2Ri3, -NRi2Ri3, wherein R12 and Ri3 are defined supra. In certain embodiments, R7 is Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.), and Rg is selected from H, Ci_ioalkyl (e.g. Ci_4alkyl),
-Ci_ioalkylene-NRi2Ri3, -NRi2Ri3, wherein Ri2 and Ri3 are defined supra. [0028] In certain embodiments, R3 is -R7-(C=0)X-R8, and R7 and Rg are defined as supra. In certain embodiments, R7 is absent, X is O, and Rg is selected from H, metal (e.g. Na, and K), NH4, Ci_ioalkyl (e.g. Ci_4alkyl),
-Ci_ioalkylene-NRi2Ri3, and -NRi2Ri3, wherein Ri2 and Ri3 are defined supra. In certain embodiments, R7 is absent, X is absent, and Rg is selected from -NRi2Ri3, wherein Ri2 and Ri3 are defined supra.
[0029] In certain embodiments, R3 is NH2, NHCOMe, NHCOEt ,
NHCOC3H7, or NHBoc,
[
Figure imgf000013_0001
Y, and Z are defined as supra. In certain embodiments, at least one of R7, R9, X, Y, and Z is not absent. In certain embodiments, all of X, Y, Z and R7 are absent, and R is Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.). In certain embodiments, all of R7, R9 and X are absent, Y is -(C=0)- and Z is O. In certain embodiments, both of R7 and X are absent, Rg is Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.), Y is -(C=0)- and Z is O. In certain embodiments, both of R9 and X are absent, R7 is Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.), Y is -(C=0)- and Z is O.
[0031] In certain embodiments, X is absent, both of R7 and R9 are independently Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.), Y is -(C=0)- and Z is O. In certain embodiments, all of R7, R9, X and Z are absent, and Y is -(C=0)-.
[0032] In certain embodiments, Ri is R4, R4 is
X ^^ · or - , and R5 is Ry, Ry is
-OH; -OP03MiM2, wherein Mi and M2 are defined as supra; -Rio-0(C=0)-Rii, wherein Rio is absent, Rn is selected from H, Ci_ioalkyl (e.g. Ci_4alkyl), -Ci_ioalkylene-NRi2Ri3, and -NR12R13, wherein R12 and R13 are defined supra; or
Figure imgf000014_0001
R7 XYZ Ra
H3C , wherein both of R9 and X are absent, Y is (C=0), Z is O, and R7 is optionally absent or Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H-7-, etc.).
[0033] In certain embodiments, each of R3 is independently H, -CH3,
Figure imgf000014_0002
[0034] In certain embodiments, each of R3 is independently -OH, -OCH3,
-OC2H5, -OC3H7, -OP03Na2, -OC(=0)CH3, -OC(=0)C2H5, -OC(=0)C3H7,
-OC(=0)C4¾, -OC(=0)CH2NH2, -OC(=0)CH2N(CH3)2, -OC(=0)CH2N(C2H5)2,
=0)N(C2H5)2,
Figure imgf000014_0003
[0035] In certain embodiments, each of R3 is independently -CH2OH,
-C2H5OH, -C3H7OH, -CH2OC(=0)CH3, -CH2OC(=0)C2H5, -CH2OC(=0)C3H7, -CH2OC(=0)C4¾, -CH2OP03Na2, -CH2OC(=0)CH2NH2, -CH2OC(=0)CH2N(CH3)2,
- Q. -Q
O NH , O
-CH2OC(=0)CH2N(C2H5)2,
-CH2OC(=0)NH2, -CH2OC(=0)N(CH3)2, -CH2OC(=0)N(C2H5)2
° N 0 N N H O N N
[0036] In certain embodiments, each of R3 is independently -(C=0)OH,
-(C=0)ONa, -(C=0)ONH4, -(C=0)OCH3, -(C=0)OC2H5, -(C=0)OC3H7, or -(C=0)OC4¾.
[0037] In certain embodiments, each of R3 is independently -CONH2,
-CON(CH3)2, -CON(C2H5)2,
Figure imgf000015_0001
,
[0038] In certain embodiments, R5 is phenyl or Ry, and Ry is -OH,
-OP03H2, -OP03Na2, -OC(=0)H, -OC(=0)CH3, -OC(=0)C2H5, -OC(=0)C3H7,
- =0)C4¾, -OC(=0)CH2N -OC(=0)CH2N(C2H5> '2, , -OC(=0)N(CH3)2,
Figure imgf000015_0002
[0039] In certain embodiments, ¾ is Ci_ioalkyl(e.g. Ci_4alkyl) or substituted Ci_ioalkyl (e.g. Ci_4alkyl), phenyl, -(C=0)Ci_6alkyl, -(C=0)phenyl.
[0040] Examples of compounds having a structure of Formula (I) include, without limitation: Compounds I-l to Compounds 1-16 as listed below:
Figure imgf000015_0003
Figure imgf000016_0001
[0041] Each of the chiral carbons in Formula (I) can be independently in
R configuration or S configuration.
[0042] In certain embodiments, a compound of Formula (I) has a configuration shown in Formula (II), wherein both Ri and R2 have definitions as provided supra:
Figure imgf000017_0001
[0043] In certain embodiments, the compounds provided herein comprise a structure selected from the group consisting of Compounds 1 to 5, 7 to 13, 20, 25, 27 to 28, and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof:
1)
Figure imgf000017_0002
(Compound 2)
OH
(Compound 3)
Figure imgf000017_0003
Figure imgf000018_0001
(Compound 9)
Figure imgf000018_0002
(Compound 10)
Figure imgf000018_0003
(Compound 11)
Figure imgf000019_0001
Figure imgf000020_0001
(Compound 28)
[0044] In certain embodiments, a compound of Formula (I) has a configuration shown in Formula (III), wherein the R3 group has a definition as provided supra:
Figure imgf000020_0002
[0045] In certain embodiments, the compounds of Formula (III) provided herein comprise a structure selected from the following 53 compounds (i.e. from Compound III-l to Compound 111-53 as shown below) in addition to compound 1, compounds 4, 5, and 7. Also contemplated are enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
Figure imgf000021_0001
Figure imgf000022_0001
21
Figure imgf000023_0001
Figure imgf000024_0001
[0046] In certain embodiments, a compound of Formula (I) has a configuration shown in Formulas (IV) or (V), wherein the R3 group has a definition as provided supra:
Figure imgf000025_0001
Formula (IV)
Figure imgf000025_0002
[0047] In certain embodiments, the compounds in Formula (IV) or
Formula (V) provided herein comprise a structure selected from the following 54 compounds (i.e. from Compound IV-1 to Compound IV-27, and from Compound V-1 to Compound V-27 as shown below), in addition to compound 2, compounds 8-11, compounds 13, and 28. Also contemplated are enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
Figure imgf000026_0001
Figure imgf000027_0001

Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
[0048] In certain embodiments, a compound of Formula (I) has a configuration shown in Formula (VI), wherein R4 and Ry group has a definition as provided supra.
Figure imgf000030_0002
Formula (VI)
[0049] In certain embodiments, the compounds in Formula (VI) provided herein comprise a structure selected from the following 20 compounds (i.e. from Compound VI-1 to Compound VI-20 as shown below), in addition to compound 3 and compound 12. Also contemplated are enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
Figure imgf000031_0001
30
Figure imgf000032_0001
[0050] In certain embodiments, a compound of Formula (I) provided herein has a configuration shown in formula(VII):
Figure imgf000033_0001
Formula (VII) wherein R is selected from the group consisting of
Figure imgf000033_0002
R3 is defined supra.
[0051] In certain embodiments, the compounds in Formula (VII) provided herein comprise a structure selected from the following 43 compounds (i.e. from Compound VII-1 to Compound VII-43 as shown below), in addition to compounds 20, 25, and 27. Also contemplated are enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
Figure imgf000033_0003
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
[0052] In certain embodiments, a compound of Formula (I) provided herein has a configuration shown in formula(VIII):
Figure imgf000036_0002
Formula (VIII),
wherein Ri is selected from the group consisting of:
Figure imgf000037_0001
[0053] In certain embodiments, the compounds in Formula (VIII) provided herein comprise a structure selected from compounds 1-1, 1-2, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, and 1-16. In certain embodiments, the compounds in Formula (VIII) provided herein comprise a structure selected from compounds 1, 2, 4, 5, 7-11, 13, 20, 25, 27, and 28. Also contemplated are enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
[0054] Each of the above compounds of Formula (VIII) is capable of simultaneously inhibiting the CT-L, T-L, and PGPH activities of the 20S proteasome.
In certain embodiments, each of the above compounds of Formula (VIII) inhibits the
CT-L, T-L, and PGPH activities of the 20S proteasome at a concentration below about 5 μΜ, 2 μΜ, or about 1 μΜ. In certain embodiments, these compounds have an IC5o value of below about 5 μΜ, 2 μΜ, or about 1 μΜ on the CT-L, T-L, and
PGPH activities of the 20S proteasome. Such simultaneous inhibition activity of the listed compounds of Formula (VIII) is unexpected, because certain compound having o
NaO P\
a distinct Rl group (for example a ONa group as in Compound 21 shown below) is found to be substantially less potent, if any, in simultaneously inhibiting the CT-L, T-L, and PGPH activities of the 20S proteasome, and therefore are undesirable and unsuitable for simultaneous inhibition of the CT-L, T-L, and PGPH activities of the 20S roteasome:
Figure imgf000038_0001
(Compound 21)
[0055] Pharmaceutically acceptable salts can be any salt or ester that are physiologically acceptable and are suitable for administration to an intended recipient. Examples of pharmaceutically acceptable salts include, acid addition salts (e.g.
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate salts), and base salts (e.g. aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts).
[0056] Methods of preparation
Synthesis of the compounds provided herein are illustrated in the below general synthetic schemes. These schemes are illustrative only and are not meant to limit other possible methods that can be used to prepare the compounds provided herein. Additionally, the steps in the Schemes are for better illustration and can be changed as appropriate. The embodiments of the compounds in examples were synthesized overseas for the purposes of research and potentially submission to regulatory agencies.
[0057] Scheme 1
Figure imgf000039_0001
[0058] Step 1 : Reaction of compound 1001 with isobutylchloroformate affords Compound 1002.
[0059] Step 2: Reaction of Compound 1002 with isopropenyl bromide affords Compound 1003.
[0060] Step 3: Reaction of Compound 1003 with DIEA and a solution of
H202 affords epoxy compounds.
[0061] Step 4: Reaction of Compound 1004 with TFA in DC affords
Compound 1005.
[0062] Scheme 2
Figure imgf000039_0002
[0063] Reaction of Compound 1006 with NaCN affords Compoundl007, which is further reacted with KOH and acidified to yield Compound 1008.
[0064] Scheme 3
Figure imgf000040_0001
[0065] Chlorotrityl chloride resin is reacted sequentially with
Fmoc-Phe-OH, Fmoc-Leu-OH, and Fmoc-HomoPhe-OH, to allow synthesis and extension of the peptidyl backbone. The product is subsequently reacted with
Compound 1008, and the resulting compound is released from the resin to afford Compound 1013. Reaction of Compound 1013 with Compound 1005 affords the Compound 1.
[0066] Biological Activity and Selectivity
[0067] The compounds provided herein have biological activity in inhibiting proteasome. Inhibition of the 20S proteasome can be determined by methods and assays known in the art, for example, as disclosed by Stein et al, Biochemistry (1996), 35, 3899-3908; by Lightcap et al, Clinical Chemistry, 2000, 46, 673-683; by Kisselev et al, Journal of Biological Chemistry. (2006), 281, 8582-8590 and in U.S. application number 09/569748. The chymotrypsin-like (CT-L), peptidylglutamyl peptide hydrolyzing (PGPH) and trypsin-like (T-L) activities of the 20S proteasome are measured with a fluorogenic substrate assay method using succinyl-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Glu-AMC and
Boc-Leu-Arg-Arg-AMC , respectively, as the substrates in an assay buffer. The free fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the substrates is quantified using a fluorometer and activity of CT-L, PGPH and T-L of 20S
proteasome is determined.
[0068] The compounds provided herein are useful in part because they inhibit the catalytic activities of proteasome.
[0069] In certain embodiments, the compounds provided herein show inhibition (e.g. at least 50% inhibition) of any of CT-L, T-L and PGPH activity of the 20S proteasome when present at concentrations below 5 μΜ (for example, Compound 1-5, 7-13, 20, 25, 27, and 28). In certain embodiments, the compounds provided herein show inhibition of CT-L activity of the 20S proteasome at below 5 μΜ, 2 μΜ, 1 μΜ (for example, Compound 1-5, 7-13, 20, 25, 27, and 28), or at below 0.01 μΜ (for example, Compound 1-2, 4-5, 7-13, 20, 25, 27, and 28). In certain embodiments, the compounds provided herein show inhibition of T-L activity of the 20 S proteasome at below 5 μΜ (for example, Compound 1-5, 7-13, 20, 25, 27, and 28), below 2 μΜ (for example, Compound 1-5, 8-13, 20, 25, 27, and 28), below 1 μΜ (for example, Compound 1-5, 8-11, 13, 20, 25, 27, and 28), or at below 0.5 μΜ (for example, Compound 1-2, 8-11, 13, 25, and 27). In certain embodiments, the compounds provided herein show inhibition of PGPH activity of the 20S proteasome at below 5 μΜ (for example, Compound 1-5, 7-13, 20, 25, 27, and 28), below 2 μΜ (for example, Compound 1, 2, 4, 5, 7-11, 13, 25, 27, and 28), below 1 μΜ (for example, Compound 1, 2, 4, 5, 8-11, 13, 25, 27, and 28), or below 0.5 μΜ (for example, Compound 2, 5, 8-11, 13, 25, and 27).
[0070] The compounds disclosed herein can simultaneously inhibit CT-L,
T-L and PGPH activity of the 20S proteasome. The term "simultaneously inhibit" or "simultaneous inhibition" as used herein means that a compound substantially inhibits each of the CT-L, T-L and PGPH activity of the 20S proteasome. In preferred embodiments, the compounds disclosed herein simultaneously inhibit CT-L, T-L and PGPH activity of the 20S proteasome at concentrations below about 5 μΜ, 2 μΜ, or about 1 μΜ. In further preferred embodiments, the compounds disclosed herein show simultaneous inhibition of the CT-L, T-L and PGPH activity of the 20S proteasome at concentrations below about 5 μΜ (for example, Compound 1-5, 7-13, 20, 25, 27, and 28), below 2 μΜ (for example, Compound 1, 2, 4, 5, 8-11, 13, 25, 27, and 28), below 1 μΜ (for example, Compound 1, 2, 4, 5, 8-11, 13, 25, 27, and 28), or below 0.5 μΜ (for example, Compound 2, 8-11, 13, 25, and 27).
[0071] In certain embodiments, the compounds provided herein
simultaneously inhibit the CT-L, T-L and PGPH activities of the proteasome when tested in ex vivo or in vivo assays.
[0072] In an exemplary ex vivo assay, blood is collected from a subject
(e.g. a mouse) and is treated with a compound disclosed herein. The compounds disclosed herein can inhibit the CT-L, T-L and PGPH activities of the 20S proteasome prepared in the ex vivo setting. In certain embodiments, the compounds provided herein simultaneously inhibit the CT-L, T-L and PGPH activities of the 20S proteasome in an ex vivo assay (for example Compound 1-5, 7-13, 20, 25, 27, and 28) at concentrations below about 5 μΜ, 2 μΜ , 1 μΜ, and 0.5 μΜ.
[0073] The inhibition effect of the compounds can be also tested in vivo.
For example, the compounds can be administered to subjects (for example, mice) at a tolerated dose, and blood can be collected from the subjects and tested for the CT-L, T-L and PGPH activities of the 20S proteasome in the blood sample. In certain embodiments, the compounds provided herein simultaneously inhibit the CT-L, T-L and PGPH activities of the 20S proteasome in vivo (for example Compound 1-5, 7-13, 20, 25, 27, and 28).
[0074] Uses of the compounds
[0075] Another aspect of the present disclosure relates to methods of simultaneously inhibiting the CT-L, T-L and PGPH activities of the proteasome, comprising administering a therapeutically effective amount of a compound provided herein. "Therapeutically effective amount" as used herein means the amount of a compound that is sufficient to provide intended therapeutic efficacy or activity in the subject receiving the compound. The effective amount may vary depending on various factors such as age, gender, health condition of the subject, dosage form of the compound, severity of the disease in the subject, and etc. A therapeutically effective amount can be determined by a physician or a doctor prescribing the compound, taking account to the aforementioned factors among others.
[0076] Another aspect of the present disclosure relates to methods of treating a condition associated with the proteasome. The method comprises administering an effective amount of a compound provided herein. The compounds provided herein can be used to treat any of the conditions or diseases that are associated with the proteasome, including but not limited to those listed below.
[0077] A variety of conditions or diseases have been known or believed to be mediated by the catalytic functions of the proteasome. Proteasome inhibition has been suggested as a prevention and/or treatment of a multitude of diseases including, but not limited to, cancers, neurotoxic/degenerative diseases, Alzheimer's disease, ischemic conditions, inflammation, immune-related diseases, HIV infection, organ graft rejection, septic shock, inhibition of antigen presentation, parasitic infections, conditions associated with acidosis, macular degeneration, pulmonary conditions, muscle wasting diseases, fibrotic diseases, bone and hair growth diseases. Therefore, proteasome inhibitor compositions such as the peptide epoxyketones disclosed herein provide a means of treating patients with these conditions.
[0078] Proteasome inhibition has been clinically demonstrated as an antitumor therapeutic strategy. Accordingly, the compounds disclosed herein are useful for treating cancers. Exemplary cancers that may be treated include leukemia, lymphoma, myeloma, and carcinomas, such as hepatocellular carcinoma etc. Other cancers that may be treated with the compounds disclosed herein include
adrenocortical carcinoma, AIDS-related carcinomas, astrocytoma, bone carcinoma, osteosarcoma, GBM, malignant fibrous histiocytoma, melanoma, malignant mesothelioma, Pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, neuroblastoma, uterine sarcoma, bile duct cancer, bladder cancer, breast cancers, gastrointestinal cancers, cervical cancers, colon cancers, rectal cancers, esophageal cancers, eye cancers, ovarian cancer, head and neck cancers, kidney cancers, lip and oral cavity cancers, lung cancers, nasal cavity and paranasal sinus cancers, penile cancers, prostate cancers, transitional cell cancers, salivary gland cancers, soft tissue cancers, skin cancers, thyroid, parathyroid cancer, and vaginal cancers.
[0079] Proteasome inhibitors have been associated with NF-κΒ inhibition.
NF-KB is a potent transcription factor which mediates transcription of genes including inflammatory molecules such as TNF, IL-1 , cyclooxygenase, ICAM, and etc.
Accordingly, the compounds provided herein can be used as an immunosuppressant which may be useful in inflammatory disorders or conditions, such as, without limitation, allergy, asthma, rejection of a transplanted organ/tissue, auto-immune diseases lupus, rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel diseases. The compounds provided herein may be administered in an effective amount, optionally in a pharmaceutical composition, to a subject in need thereof, to treat these conditions.
[0080] Proteasome inhibitors have been found to reduce degradation of muscle proteins, therefore useful in inhibiting muscle loss and fiber atrophy.
Accordingly, the compounds provided herein can be used to treat cachexia and muscle-wasting diseases, such as chronic infectious diseases, fever, muscle disuse and denervation, nerve injury, renal failure associated with acidosis, and hepatic failure. In certain embodiments, the compounds provided herein can be administered in an effective amount, optionally in a pharmaceutical composition, to a subject in need thereof, to slow down or reduce muscle protein degradation, intracellular protein degradation or p53 protein degradation in a cell.
[0081] The compounds provided herein can also be used to treat neurodegenerative diseases and conditions, including, but not limited to, stroke, ischemic damage to the nervous system, neural trauma (e.g. percussive brain damage, spinal cord injury, and traumatic damage to the nervous system), multiple sclerosis and other immune-mediated neuropathies (e.g. Guillain-Barre syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy, and Fisher Syndrome), HIV/AIDS dementia complex, axonomy, diabetic neuropathy, Parkinsons's disease, Huntington's disease, multiple sclerosis, bacterial, parasitic, fungal, and viral meningitis, encephalitis, vascular dementia, multi-infarct dementia, Lewy body dementia, frontal lobe dementia such as Pick's disease, subcortical dementias (such as Huntington or progressive supranuclear palsy), focal cortical atrophy syndromes (such as primary aphasia), metabolic-toxic dementias, and dementias caused by infections (such as syphilis or chronic
meningitis).
[0082] The compounds provided herein can further be useful in regulating protein processing that is associated with extracellular deposition of β-amyloid protein (β-ΑΡ), the major cause of Alzheimer's disease. Accordingly, the
compounds provided herein are useful in treating Alzheimer's disease, for example by reducing the rate of β-ΑΡ processing, reducing the rate β-ΑΡ plaque formation, reducing the rate of β-ΑΡ generation, and reducing the clinical symptoms of
Alzheimer's disease.
[0083] Proteasome inhibition has also been further associated with reducing fibrosis. Accordingly, the compounds provided herein can be used for treatment of fibrosis related conditions such as, diabetic nephropathy,
glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis, lung fibrosis, and cardiac fibrosis.
[0084] Pharmaceutical Compositions and Administration
[0085] In another aspect, the present disclosure provides pharmaceutical compositions comprising a compound provided herein and a pharmaceutically acceptable carrier. [0086] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound provided herein from one location, body fluid, tissue, organ (interior or exterior), or portion of the body, to another location, body fluid, tissue, organ, or portion of the body. Pharmaceutically acceptable carriers can be vehicles, diluents, excipients, or other materials that can be used to contact the tissues of an animal without excessive toxicity or adverse effects. Exemplary pharmaceutically acceptable carriers include, sugars, starch, celluloses, malt, tragacanth, gelatin, Ringer's solution, alginic acid, isotonic saline, buffering agents, and etc.
[0087] Each carrier is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients, e.g., a compound provided herein of the formulation and suitable for use in contact with the tissue or organ of a biological subject without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
[0088] Some examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohol, such as ethyl alcohol and propane alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations such as acetone. [0089] The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
[0090] The pharmaceutical composition can be made into any suitable dosage form, such as solid dosage form (e.g. tablets, capsules, powders, granules and etc.) and liquid dosage form (e.g. aqueous solution, emulsion, elixir, syrup, and etc.). Methods of preparing pharmaceutical compositions are well-known in the art, and can be practiced according to routine procedures as described, for example, by Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).
[0091] In certain embodiments, the pharmaceutical compositions comprise about 10"9 g to about 10 g of a compound provided herein (e.g. about 0.01 mg to about 10 g, about 0.1 mg to about 10 g, about 1 mg to about 10 g, about 5 mg to about 10 g, about 10 mg to about 10 g, about 20 mg to about 10 g, about 30 mg to about 10 g, about 40 mg to about 10 g, about 50 mg to about 10 g, about 80 mg to about 10 g, about 100 mg to about 10 g, about 150 mg to about 10 g, about 200 mg to about 10 g, about 300 mg to about 10 g, about 400 mg to about 10 g, about 500 mg to about 10 g, about 600 mg to about 10 g, about 700 mg to about 10 g, about 800 mg to about 10 g, about 900 mg to about 10 g, about 1 g to about 10 g, about 10 mg to about 5 g, about 10 mg to about 3 g, about 10 mg to about lg, about 10 mg to about 900 mg, about 10 mg to about 700 mg, about 10 mg to about 500 mg, or about 10 mg to about 300 mg). Suitable dosages per subject per day can be from about 0.01 mg to about 5 g.
[0092] In certain embodiments, the compounds or pharmaceutical compositions disclosed herein are formulated into a dosage form which is suitable for delivery to a subject in need thereof through a parenteral route (e.g. subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intra cardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, and/or infusion) and a
non-parenteral route (e.g. oral, enteral, buccal, nasal, intranasal, transmucosal, epidermal, transdermal, dermal, ophthalmic, pulmonary, sublingual, rectal, vaginal or topical).
[0093] The suitable dosage forms include, without limitation, formulations for parenteral use such as injectable emulsions, solutions, and suspensions, formulations for oral use such as tablets, capsules, pills, dragees, powders, and granules, formulations for topical or transdermal use such as sprays, omintments, pastes, creams, lotions, gels, solutions, patches and inhalants, and formulations for intravaginal or intrarectal use such as suppositories. These formulations can be prepared by associating the compound with the suitable excipients at suitable conditions, in accordance with methods known in the art such as described by Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).
[0094] The pharmaceutical compositions can be administered to a subject in any suitable route of administration, such as via oral, intravenous, intranasal, topical, intramuscular, intradermal, transdermal, or subcutaneous routes.
[0095] In certain embodiments, the compounds or pharmaceutical compositions provided herein can be administered concomitantly with the second active agent, such that combinatorial or even synergetic pharmaceutical effects can be achieved in the subject in need. For example, the compounds provided herein and the second active agent may be administered in a single pharmaceutical composition, or simultaneously in separate compositions, or sequentially in separate compositions. The second active agent that may be administered concomitantly with compounds of the present disclosure for cancer treatment include but not limited to: 5-fluorouracil, doxorubicin, daunorubicin, tamoxifen, leuprolide, goserelin, flutamide, nilutimide, finasteride, dexamethasone, lenalidomide aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, temozolomide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin adriamycine, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, f uorouracil, f uoxymesterone, f utamide, gemcitabine, genistein, goserelin, tamoxifen, teniposide, testosterone, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.
[0096] In certain embodiments, a compound of the present disclosure may be administered concomitantly with non-chemical methods of cancer treatment. In certain embodiments, a compound of the present disclosure may be administered concomitantly with radiation therapy. In certain embodiments, a compound of the present disclosure may be conjointly administered with surgery, with thermoablation, with focused ultrasound therapy, with cryotherapy, or with any combination of these.
[0097] In certain embodiments, a compound of the present disclosure is administered concomitantly with a steroid, including but are not limited to,
amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, sortiazol, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, flumethasone, flunisolide, flucloronide, fluocinolone acetonide,
fluocinonide, fluocorinbutyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halobetasol propionate, halcinonide, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednisolone, dexamethasone, and
25-diethylaminoacetate.
[0098] In certain embodiments, a compound of the present disclosure is administered concomitantly with an immunotherapeutic agent, including MDR modulators such as verapamil, rapamycin, mycophenylate mofetil, thalidomide, cyclophosomide, cyclosporine, and monoclonal antibodies.
[0099] EXAMPLES
[00100] Example 1 : Inhibition of the chymotrypsin-like (CT-L), PGPH, and trypsin-like (T-L) activity of the 20S proteasome in enzymatic assays
[00101 ] The CT-L, PGPH, and T-L activities of the 20S proteasome were determined using succinyl-Leu-Leu-Val-Tyr-AMC (10 μιηοΙ/L),
Z-Leu-Leu-Glu-AMC (10 μπιοΙ/L) and Boc-Leu-Arg-Arg-AMC (50 μιηοΙ/L), respectively, as the substrates with purified human 20S proteasome at 2, 4 and 8 nmol/L, respectively, in the assay buffer containing 20 mM Tris (pH 8.0), 0.5 mM EDTA, 0.001% sodium dodecyl sulfate (SDS) and 0.05% NP-40. Stock solutions of the 20S proteasome inhibitors disclosed herein were prepared in dimethyl sulfoxide (DMSO) and the final DMSO concentration in the assay mixture was 1%. Reaction was conducted at 27°C. Proteasome activity was measured based on detection of the fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the substrates with a plate-based spectrofluorometer. IC50 (half maximal inhibitory concentration) values were determined based on the reaction velocity measured between 60 and 75 min. IC50 is a quantitative measure indicating the concentration of an inhibitor at which the catalytic activity of the 20S proteasome is inhibited 50%.
[00102] Example 2: Inhibition of CT-L, T-L and PGPH activity of the 20S proteasome in blood
[00103] Eight hundred micro liter of fresh whole blood samples were collected from CD-I mice into tubes containing sodium heparin by cardiac puncture and centrifuged at 150xg for 5min at 4°C. The resulting pellet was washed using ice-cold phosphate buffered saline (PBS) three times. Every time, the pellet is re-suspended in 1 mL cold PBS and centrifuged at 6000xg for 10 min at 4°C. After the last wash, the pelleted cells were lysed by the addition of 200 of the lysis buffer (PBS containing 5 mM EDTA, pH 8.0) for 1 hr and then centrifuged at 6000xg for 10 min at 4°C. The blood lysate's protein concentration is measured by BCA method. 100 μg of proteins were taken into HEPES buffer containing 0.5 mM EDTA, 0.05% NP-40, and 0.002% SDS (pH 7.5). A compound disclosed herein was spiked at 0.2, 1 and 5 μΜ to the above mixture. Succinyl-Leu-Leu-Val-Tyr-AMC (25 μιηοΙ/L), Z-Leu-Leu-Glu-AMC (10 μπιοΙ/L) or Boc-Leu-Arg-Arg-AMC (10 μπιοΙ/L) was then added as the substrate, respectively, for CT-L, PGPH or T-L. The final volume of the reaction mixture was 50 μΐ^. The mixture was then incubated for 30 minutes at 37°C. The free AMC fluorescence was quantified using a 360/460 nm filter set in a fluorometer to measure the CT-L, PGPH, or T-L activities of the 20S proteasome. Percent inhibition was calculated by comparing to control samples.
[00104] Example 3 : Inhibition of CT-L, T-L and PGPH activity of the 20S proteasome in vivo
[00105] The in vivo proteasome activity was examined in BALB/c nude mice. Mice (5-6 mice/group) were administered intravenously with either vehicle (10-20% (w/v) hydroxypropyl- -cyclodextrin in 10 mM sodium citrate containing 2.5% DMSO (pH 3.0-3.5)), or a compound disclosed herein at a tolerated dose. One hour after dosing, whole blood samples were collected by cardiac puncture into tubes containing sodium heparin and centrifuged at 150xg for 5min at 4°C. The resulting pellet was washed using ice-cold phosphate buffered saline (PBS) three times. Every time, the pellet was re-suspended in 1 mL cold PBS and centrifuged at 6000xg for 10 min at 4°C. After the last wash, the pelleted cells were lysed by the addition of 200 μΐ^ of the lysis buffer (PBS containing 5 mM EDTA, pH 8.0) for 1 hr and then centrifuged at 6000xg for 10 min at 4°C. The protein concentrations of the blood lysates were measured by BCA method. About 100 μg of proteins were taken into HEPES buffer containing 0.5 mM EDTA, 0.05% NP-40, and 0.002% SDS (pH 7.5). Succinyl-Leu-Leu-Val-Tyr-AMC (25 μηιοΙ/L), Z-Leu-Leu-Glu-AMC (10 μηιοΙ/L), or Boc-Leu-Arg-Arg-AMC (10 μηιοΙ/L) was then added as the substrate, respectively, for CT-L, PGPH or T-L activity assay. The mixture was then incubated for 30 minutes at 37°C. The free AMC fluorescence was quantified using a 360/460 nm filter set in a fluorometer to measure the CT-L, PGPH or T-L activities of the 20S proteasome. Percent inhibition was calculated by comparing to samples collected from the vehicle treated animals.
[00106] Example 4: Compound 1, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00107] Shown below is the structure of Compound 1 :
Figure imgf000052_0001
Compound 1
[00108] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 1 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 1 to Carfilzomib (IC50 Compound 1/ IC50 Carfilzomib) on CT-L activity was determined to be 0.18 and 0.26, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000052_0002
[00109] The inhibitory effect of Compound 1 on proteasome activity in mouse blood lysates was performed using the assay method disclosed in Example 2. At concentration of 0.2 μΜ of Compound 1, CT-L activity was inhibited by 96.7%, T-L activity was inhibited by 60.5%, and PGPH activity was inhibited by 40.2%. At concentration of 1 μΜ of Compound 1, CT-L activity was inhibited by 99.2%, T-L activity was inhibited by 82.5%, and PGPH activity was inhibited by 61.5%. And at concentration of 5 μΜ of Compound 1, CT-L activity was inhibited by 98.9%>, T-L activity was inhibited by 87.3%>, and PGPH activity was inhibited by 74.3%.
[00110] In vivo proteasome activity inhibition of Compound 1 is evaluated using the method disclosed in Example 3. Compound 1 simultaneously inhibits mouse blood CT-L, T-L, and PGPH activities by >40% at a dose of 2 mg/kg.
[00111 ] Synthesis of Compound 1
[00112] Scheme 1
Figure imgf000053_0001
[00113] Compound 1002. A free base dimethyl hydroxylamine solution was prepared: To a suspension of N, O-dimethylhydroxyamine HCI (8.2g, 84 mmol) in DCM (100 mL), was added a solution of TEA (11.7 mL, 84 mmol) in DCM (50 mL) over 20 min. To a solution of compound 1001 (18.5g, 80 mmol) in DCM (200 mL) was added isobutylchloroformate 10.9g, 80 mmol) and the resulting mixture was cooled down -10°C and NMM (8.1 g, 80 mmol) was added. Above prepared dimethyl hydroxylamine solution was slowly added. Then the resulting solution was stirred at room temperature overnight and diluted with water. The layers were separated, and the aqueous layer was extracted by DCM. The combined organic layers were washed with brine, dried over MgS04, and filtrated. The solvent was removed under reduced pressure to give compound 1002 (17.5g).
[00114] Compound 1003. Under argon atmosphere, to a -78°C solution of compound 1002 (8.2 g, 30 mmol) in THF (100 mL) was added a solution of t-BuLi (46 mL of 1.3M in Hexane, 60 mmol). After the solution was stirred at -78°C, Isopropenyl bromide (3.63g, 30 mmol) was added. The reaction was stirred at -78°C for 1 hour, after which it was poured into a saturated aqueous ammonium chloride solution (100 mL). The mixture was extracted with EtOAc. The combined organic layers were then washed with brine. The organic layer was dried with Na2S04, filtered and concentrated under reduced pressure. Purification by column chromatography gave compound 1003.
[00115] Compound 1004. To a solution of compound 1003 (5.1 g, 20 mmol) in MeOH (50 mL) was added DIEA and a solution of H202 (4.6 mL 30%, 80mmol). After the solution was stirred for 2 hours, it was poured into an aqueous NaHS03 solution (100 mL). The mixture was extracted with EtOAc. The combined organic layers were then washed with brine. The organic layer was dried with Na2S04, filtered and concentrated under reduced pressure. Purification by column chromatography gave desired stereoisomer compound 1004.
[00116] Compound 1005. To a solution of compound 1004 (2.7 g, 10 mmol) in DCM was added TFA. After the solution was stirred, it was concentrated under reduced pressure to give compound 1005.
[00117] Scheme 2
Figure imgf000054_0001
[00118] Compound 1007. To a solution of compound 1006 (2.6 g, 20 mmol) in DMSO (10 mL) was added NaCN (2.0 g, 40 mmol). After the solution was stirred overnight at room temperature, it was diluted with water, and extracted with AcOEt. The combined organic layer was washed with brine. It was dried with Na2S04, filtered and concentrated under reduced pressure. Purification by recrystallized gave compound 1007.
[00119] Compound 1008. To a solution of compound 1007 (1.2 g, 10 mmol) in dioxane (20 mL) was added a solution of KOH (1 mL, 3M). After the solution was stirred overnight, it was poured slowly into water. It was filtrated and the filter was recrystallized gave compound 1008.
[00120] Scheme 3
Figure imgf000055_0001
[00121] Resin 1009. Chlorotrityl chloride resin (3.2 mmol) and
Fmoc-Phe-OH (6.4 mmol) in DCE (30 mL) were stirred. A solution of DIEA (1.2 mL, 6.6 mmol) was added and the system was stirred for 1 hour. The resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry. To the resin in DMF (40 mL) was added 20% piperidine in DMF and heterogeneous mixture was allowed to shake The solution was filtered and the resin was washed by DMF, MeOH and DCM and allowed to air dry to yield Resin 1009.
[00122] Resin 1010. To the resin 1009 (3.2 mmol) was added DCE (40 mL), and Fmoc-Leu-OH (6.2 mmol), DIEA (13.2 mmol), HOBT (6.4mmol), and BOP (6.4 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol, and Et20 and allowed to air dry. To the resin in DMF (40 mL) was added 20% piperidine in DMF, and heterogeneous mixture was allowed to shake. The solution was filtered and the resin was washed by DMF and DCM, and allowed to air dry to yield Resin 1010.
[00123] Resin 1011. To the resin 1010 (3.2 mmol) was added DCE (40 mL), and Fmoc-HFE-OH (2.56g, 6.4 mmol), DIEA (2.3 mL, 13.2 mmol), HOBT (0.86g, 6.4mmol), and BOP (1.78g, 6.4 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol, and Et20 and allowed to air dry. To the resin in DMF (40 mL) was added 20% piperidine in DMF and heterogeneous mixture was allowed to shake. The solution was filtered and the resin was washed by DMF, MeOH, and DCM and allowed to air dry to yield Resin 1011.
[00124] Resin 1012. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1008 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1012.
[00125] Compound 1013. To the resin 1012 (1.6 mmol) was added 10 mL of a mixture solution of TFA/DCM=1/4(V/V). The mixture was stirred at room temperature. The resin was separated by filtration. The volatiles were removed under reduced pressure to get compound 1013.
[00126] Compound 1. To a stirred solution of compound 1005 (0.33 mmol) in MeCN was added compound 1013 (0.3 mmol), DIEA (1.5mmol), HOBT
(0.6mmol), and BOP (0.64mmol) and the mixture was stirred at room temperature. The reaction mixture was diluted with water and extracted with EtOAc. The organic layers were washed with water and dried over Na2S04. The Na2S04 was removed by filtration and the volatiles were removed under reduced pressure. The crude was purified by flash chromatography to yield compound 1. 1HNMR (400 Hz,
Methanol-^) δ 7.32-7.28 (m, 2H), 7.29-7.19 (m, 3H), 7.14-7.10 (m, 5H), 6.20 (s, 1H), 4.61-4.53 (m, 2H), 4.31-4.27 (m, 2H), 3.70 (dd, 2H), 3.23-3.14 (m, 2H), 2.93-2.82 (m, 2H), 2.74-2.66 (m, 2H), 2.44 (s, 3H), 2.12-2.02 (m,lH), 2.01-1.89 (m,lH),
1.75-1.38 (m, 9H), 0.96-0.87 (m, 12H). LRMS (M+H+) C4oH53N507 m/z: calculated: 715.4, found: 716.3.
[00127] Example 5 : Compound 2, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00128] Shown below is the structure of Compound 2:
Figure imgf000057_0001
[00129] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 2 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 2 to Carfilzomib on CT-L activity was determined to be 0.05 and 0.21, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000057_0002
[00130] The inhibitory effect of Compound 2 on proteasome activity in mouse blood lysates was performed using the assay method disclosed in Example 2. At concentration of 0.2 μΜ of Compound 2, CT-L activity was inhibited by 98.0%, T-L activity was inhibited by 70.1%, and PGPH activity was inhibited by 44.7%. At concentration of 1 μΜ of Compound 2, CT-L activity was inhibited by 98.1%, T-L activity was inhibited by 81.8%, and PGPH activity was inhibited by 68.4%. And at concentration of 5 μΜ of Compound 2, CT-L activity was inhibited by 93.0%, T-L activity was inhibited by 89.3%>, and PGPH activity was inhibited by 68.5%>.
[00131 ] In vivo proteasome activity inhibition of Compound 2 was evaluated using the method disclosed in Example 3. Compound 2 simultaneously inhibited 97.1%, 57.8% and 45.7% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00132] Synthesis of Compound 2
[00133] Scheme 4
Figure imgf000058_0001
[00134] Resin 1015. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1014 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1015.
[00135] Compound 1016. The same procedure was used in the preparation of compound 1013 to get compound 1016.
[00136] Compound 2. To a solution of compound 1016 (0.17 mmol) in
DCM was added DEPBT (0.21 mmol) and DIEA (0.34 mol). The mixture was stirred for 0.5 hour, then compound 1005 (0.20 mmol) was added, the mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 2. 1HNMR(400 MHz, DMSO-d6): δ 0.76-0.87 (m, 12H), 1.26-1.39 (m, 7H), 1.45-1.63 (m, 2H), 1.73-1.87 (m, 2H), 2.44-2.54 (t, 2H), 2.57 (s, 3H), 2.76-2.90 (m, 1H), 2.93-2.98 (m, 2H), 3.08-3.10 (d, J= 5.2 Hz, 1H), 3.65-3.76 (m, 2H), 4.20-4.4 (m, 3H), 4.51-4.55 (t, 1H), 7.06-7.19 (m, 8H), 7.25-7.29 (t, 2H), 7.38 (s, 1H), 7.82-7.84 (d, J= 7.6 Hz, 1H), 7.99-8.01 (d, J= 8.4 Hz, 1H), 8.20-8.22 (d, J= 7.2 Hz, 1H), 8.39-8.41 (d, J= 7.6 Hz, 1H); LRMS (M+H+) C4oH53N506S m/z: calculated: 731.4, found: 732.5.
[00137] Example 6: Compound 3, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00138] Shown here is the structure of Compound 3 :
Figure imgf000059_0001
Compound 3
[00139] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 3 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 3 to Carfilzomib on CT-L activity was determined to be 2.40 and 4.08, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000059_0002
[00140] The inhibitory effect of Compound 3 on the proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 5 μΜ of Compound 3, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 50%, and PGPH activity is inhibited > 40%.
[00141 ] In vivo proteasome activity inhibition of Compound 3 is evaluated using the method disclosed in Example 3. Compound 3 simultaneously inhibits >30%> of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00142] Synthesis of Compound 3 [00143] Scheme 5
Figure imgf000060_0001
[00144] Resin 1017. Chlorotrityl chloride resin (3.2 mmol) and
Fmoc-Ser(tBu)-OH (6.4 mmol) in DCE (30 mL) were stirred. A solution of DIEA (6.6 mmol) was added and the system was stirred for 1 hour. The resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry. To the resin in DMF (40 mL) was added 20%> piperidine in DMF and heterogeneous mixture was allowed to shake The solution was filtered and the resin was washed by DMF, MeOH and DCM and allowed to air dry to yield Resin 1017.
[00145] Resin 1018. The same procedure was used in the preparation of
Resin 1010 to yield Resin 1018. [00146] Resin 1019. The same procedure was used in the preparation of
Resin 1011 to yield Resin 1019.
[00147] Resin 1021. To the resin 1019 (1.6 mmol) was added DCE, and
Compound 1020 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, and Et20 and allowed to air dry to yield Resin 1021.
[00148] Compound 1022. The same procedure was used in the preparation of compound 1013 to get compound 1022.
[00149] Compound 3. To a solution of compound 1022 (0.2 mmol) in DCM was added DEPBT (0.26 mmol) and DIEA (0.4 mol). The mixture was stirred for 0.5 hour, then compound 1005 (0.24 mmol) was added, the mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give pure desired compound 3. 1HNMR(400 MHz, Methanol-^): δ 0.92-1.00 (m, 12H), 1.30-1.42 (m, 2H), 1.46-1.55 (m, 4H), 1.63-1.75 (m, 4H), 1.99-2.20 (m, 1H), 2.00-2.20 (m, 1H), 2.65-2.79 (m, 6H), 2.92-2.94 (d, J= 4.8 Hz, 1H), 3.23-3.25 (d, J= 5.2 Hz, 1H), 3.77-3.80 (m, 6H), 4.40-4.48 (m, 3H), 4.56-4.60 (m, 1H), 4.62-4.80 (br, 1H), 7.19-7.32 (m, 5H), LRMS (M+H+) C34H53N508 m/z: calculated: 659.4, found: 660.3.
[00150] Example 7: Compound 4, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00151 ] Shown below is the structure of Compound 4:
Figure imgf000061_0001
Compound 4
[00152] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 4 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 4 to Carfilzomib on CT-L activity was determined to be 0.66 and 0.93, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000062_0002
[00153] The inhibitory effect of Compound 4 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 4, CT-L activity is inhibited >90.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00154] In vivo proteasome activity inhibition of Compound 4 is evaluated using the method disclosed in Example 3. Compound 4 simultaneously inhibits >30%> of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00155] Synthesis of Compound 4
[00156] Scheme 6
Figure imgf000062_0001
1030 1031 1032
[00157] Compound 1024. A mixture of compound 1023 (0.6 mol), hydroxyl amine hydrochloride (0.718 mol) in methanol was stirred at room
temperature overnight. After evaporation of the solvent, water was added and the product was extracted with dichloromethane. The organic phase was washed with brine, dried over sodium sulfate, and concentrated to give crude compound 1024.
[00158] Compound 1026. To compound 1024 (0.479 mol) in DMF was added NCS (0.5 mol) and the reaction was heated to 60 °C and stirred for one hour to get compound 1025. After cooling the reaction to 0 °C, prop-2-yn-l-ol (0.5 mol) was added. Et3N (0.5 mol) was added to the reaction. The reaction was stirred for another 10 min. Water and ethyl acetate was added. The aqueous was further extracted with ethyl acetate. The organic layers were washed with brine, dried over sodium sulfate, and concentrate in vacuo. The residue was purified by column chromatography to give compound 1026.
[00159] Compound 1027. To a mixture of compound 1026 (40 mmol) and imidazole (123 mmol) in dichloromethane was added TBDPSC1 (45 mmol). The mixture was stirred at room temperature for 2 hours. Water was added and extracted with dichloromethane. The organic phase was washed with brine, dried over Na2S04 and filtered. The filtrate was concentrated to give compound 1027.
[00160] Compound 1028. To a mixture of compound 1027 (37 mmol) in
THF was added DIBAL-H (15 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was quenched with water and extracted with dichloromethane. The organic phase was washed with sodium bicarbonate, dried over Na2S04, and concentrated to give compound 1028.
[00161 ] Compound 1029. A mixture of compound 1028 (40.87 mmol), carbon tetrabromide (44.96 mmol) and triphenylphosphine (44.96 mmol) in
dichloromethane was stirred at 0 °C for 1 hour. The mixture was concentrated and the residue was purified by column chromatography to give compound 1029.
[00162] Compound 1030. A mixture of compound 1029 (22.8 mmol) and sodium cyanide (27.3 mmol) in DMSO was stirred at room temperature for 3 hours. The mixture was added to water and extracted with ethyl acetate. The organic layer was dried over Na2S04, and concentrated to give compound 1030.
[00163] Compound 1031. A mixture of compound 1030 (16 mmol) in
HCl/MeOH was stirred overnight. Water was added to the mixture and stirred for 1 hour. The solvent was removed in vacuo. The residue was purified by column chromatography to give compound 1031.
[00164] Compound 1032. A mixture of compound 1031(4.68 mmol) and
NaOH (9.35 mmol) in methanol was stirred at room temperature overnight. The methanol was removed in vacuum. The pH was adjusted to 5 to give compound 1032.
[00165] Scheme 7
Figure imgf000064_0001
[00166] Resin 1033. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1032 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1033.
[00167] Compound 1034. The same procedure was used in the preparation of compound 1013 to get compound 1034.
[00168] Compound 4. To a solution of compound 1034 (0.24 mmol) in
DCM was added DEPBT (0.31 mmol) and 0.72 mol). The mixture was stirred for 2 hours at 0 °C, then compound 1005 (0.31 mmol) was added, the mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 4. 1HNMR(400 MHz, Methanol-^): δ 0.84-0.94 (m, 12H), 1.36-1.68 (m, 9H), 1.91-2.10 (m, 2H), 2.57-2.68 (m, 2H), 2.78-2.89 (m, 2H), 3.10-3.19 (m, 2H), 3.70 (q, 2H), 4.24-4.30 (m, 2H), 4.49-4.64 (m, 5H), 6.37 (s, 1H), 7.06-7.28 (m, 10H); LRMS (M+H+) C4oH53N508 m/z: calculated: 731.4, found: 732.3.
[00169] Example 8: Compound 5, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00170] Shown below is the structure of Compound 5:
Figure imgf000065_0001
[00171 ] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 5 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 5 to Carfilzomib on CT-L activity was determined to be 0.27 and 0.52, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000065_0002
[00172] The inhibitory effect of Compound 5 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 5, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00173] In vivo proteasome activity inhibition of Compound 5 is evaluated using the method disclosed in Example 3. Compound 5 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00174] Synthesis of Compound 5
[00175] Scheme 8
Figure imgf000066_0001
[00176] Compound 1036. A mixture of compound 1035 (7 mmol) and
CDI (7.7 mmol) in DCM was stirred for 4 hours. The mixture was concentrated to give the compound 1036 which was used for the next step directly.
[00177] Compound 1037. To a mixture of compound 1036 (7 mmol) and
Et3N (21 mmol) in DCM was added dimethylamine hydrochloride (14 mmol) at 0 °C. The mixture was stirred at room temperature for 3 hours. The mixture was
concentrated and the residue was purified to give pure compound 1037.
[00178] Compound 1038. A mixture of compound 1037 (2.07 mmol) and
Lithium hydroxide (4.14 mmol) in methanol was stirred at room temperature for 4 hours. Methanol was removed in vacuo. The residue was added to water. The pH of the mixture was adjusted to 6 with acetic acid. The mixture was extracted with ethyl acetate, and the organic phase was washed with brine, dried over Na2S04, and concentrate to give compound 1038.
[00179] Scheme 9
Figure imgf000067_0001
[00180] Resin 1039. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1038 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1039.
[00181 ] Compound 1040. The same procedure was used in the preparation of compound 1013 to get compound 1040.
[00182] Compound 5. To a solution of compound 1040(0.20 mmol) in DCM was added DEPBT (0.26 mmol) and DIEA (0.60 mmol). The mixture was stirred for 2 hours at 0 °C, then compound 1005 (0.26 mmol) was added, the mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 5. 1HNMR(400 MHz, CDC13): δ 0.84-0.94 (m, 12H), 1.32-1.72 (m, 9H), 1.91-2.10 (m, 2H), 2.50-2.64 (m, 2H), 2.80-2.93 (m, 7H), 3.10-3.17(m, 1H), 3.21-3.30 (m,lH), 3.50-3.70 (m, 2H), 4.49-4.90 (m, 5H), 5.17 (s, 1H), 6.30-6.45 (d, J = 13.2 Hz, 1H), 7.00-7.26 (m, 1 1H); LRMS (M+H+) C43H58N609 m/z: calculated: 802.4, found: 803.4.
[00183] Example 9: Compound 7, Structure, Proteasome Activity Inhibitory
Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00184] Shown below is the structure of Compound 7:
Figure imgf000068_0001
Compound 7
[00185] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 7 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 7 to Carfilzomib on CT-L activity was determined to be 1.61 and 2.00, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000068_0003
[00186] The inhibitory effect of Compound 7 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 5 μΜ of Compound 7, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00187] In vivo proteasome activity inhibition of Compound 7 is evaluated using the method disclosed in Example 3. Compound 7 simultaneously inhibits >40%> of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00188] Synthesis of Compound 7
[00189] Scheme 10
Figure imgf000068_0002
[00190] Compound 1042. To a solution of compound 1041 (57 mmol) in methanol, a solution of NH2OH HC1 (149 mmol) in H20 was added at room temperature. The mixture was stirred at room temperature overnight. Methanol was removed, the residue was treated with ethyl acetate, the filtered solution was dried over Na2S04, and the solvent was removed to give compound 1042.
[00191] Compound 1043. A mixture of compound 1042 (53 mmol) and 100 mL of 1.5 N NaOH solution in methanol was stirred for 3 hours. Methanol was removed; the resulting solution was acidified, and then extracted with ethyl acetate. The organic phases were dried over Na2S04, and concentrated to give compound 1043 as red oil. 00192] Scheme 11
Figure imgf000069_0001
[00193] Resin 1044. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1043 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1044.
[00194] Compound 1045. The same procedure was used in the preparation of compound 1013 to get compound 1045.
[00195] Compound 7. To a solution of compound 1045 (0.137 mmol) in
DCM was added DEPBT (0.179 mmol) and DIEA (0.411 mmol). The mixture was stirred for 2 hours at 0 °C, then compound 1005 (0.179 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 7. 1HNMR(400 MHz, Methanol-^): δ 0.83-0.91 (m, 12H), 1.25-1.61 (m, 9H), 1.85-2.02 (m, 2H), 2.62-2.68 (m, 2H), 2.85-2.88 (m, 2H),
3.07-3.19 (m, 2H), 3.48-3.60 (m, 2H), 4.23-4.31 (m, 2H), 4.47-4.60 (m, 2H), 7.06-7.28 (m, 11H); LRMS (M+H+) C39H5iN508 m/z: calculated: 717.4, found: 718.4.
[00196] Example 10: Compound 8, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00197] Shown below is the structure of Compound 8:
Figure imgf000070_0001
Compound 8
[00198] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 8 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 8 to Carfilzomib on CT-L activity was determined to be 0.28 and 0.55, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000070_0002
[00199] The inhibitory effect of Compound 8 on proteasome activity in mouse blood lysates was performed using the assay method disclosed in Example 2. At concentration of 0.2 μΜ of Compound 8, CT-L activity was inhibited by 98.0%, T-L activity was inhibited by 65.2%, and PGPH activity was inhibited by 50.1%. At concentration of 1 μΜ of Compound 8, CT-L activity was inhibited by 100%, T-L activity was inhibited by 79.5%, and PGPH activity was inhibited by 75.1%. And at concentration of 5 μΜ of Compound 8, CT-L activity was inhibited by 98.5%, T-L activity was inhibited by 90.2%, and PGPH activity was inhibited by 89.4%.
[00200] In vivo proteasome activity inhibition of Compound 8 is evaluated using the method disclosed in Example 3. Compound 8 simultaneously inhibits 92.5%, 50.5% and 41.0% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 5 mg/kg.
[00201 ] Synthesis of Compound 8
[00202] Scheme 12
Figure imgf000071_0001
[00203] Compound 1047. A solution of compound 1046 (0.120 mol) and
KCN (0.156 mol) in DMF was stirred overnight at room temperature. After completed, H20 and ethyl acetate was added to the reaction to extract in turn. Then the organic layers were concentrated. The residue was purified by chromatography to give compound 1047.
[00204] Compound 1048. A solution of compound 1047 (25.2 mmol) in
HCl/MeOH (50 mL, 4N) was stirred for 3 hours at room temperature. The reaction mixture was evaporated. The residue was purified to give compound 1048.
[00205] Compound 1049. A mixture of compound 1048 (15.7 mmol) in
NaOMe/MeOH (3N, 30 mL) was stirring at 40 °C for 3 hours. The mixture was cooled to room temperature and H20 was added to the reaction mixture. Then the reaction mixture was stirring for 3 hours at room temperature. The reaction was extracted with DCM. The organic layers were concentrated and purified to give compound 1049. [00206] Scheme 13
Figure imgf000072_0001
[00207] Resin 1050. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1049 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1050.
[00208] Compound 1051. The same procedure was used in the preparation of compound 1013 to get compound 1051.
[00209] Compound 8. To a solution of compound 1051 (0.16 mmol) in
DCM was added DEPBT (0.21 mmol) and DIEA (0.48 mmol). The mixture was stirred for 2 hours at 0 °C, then compound 1005 (0.213 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 8. 1HNMR(400 MHz, Methanol-^): δ 0.74-0.91 (m, 12H), 1.32-1.58 (m, 9H), 1.90-2.02 (m, 2H), 2.52-2.65 (m, 2H), 2.85-2.90 (m, 2H),
3.06-3.16 (m, 2H), 3.45 (s, 2H), 4.26-4.33 (m, 2H), 4.49-4.60 (m, 2H), 6.59 (s, 1H), 7.06-7.26 (m, 10H); LRMS (M+H+) C39H5iN507S m/z: calculated: 733.4, found: 734.3.
[00210] Example 11 : Compound 9, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00211 ] Shown below is the structure of Compound 9: Compound 9
[00212] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 9 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 9 to Carfilzomib on CT-L activity was determined to be 0.35 and 0.54, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000073_0001
[00213] The inhibitory effect of Compound 9 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 9, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00214] In vivo proteasome activity inhibition of Compound 9 is evaluated using the method disclosed in Example 3. Compound 9 simultaneously inhibits >40%> of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00215] Synthesis of Compound 9
[00216] Scheme 14
Figure imgf000074_0001
1055 1056
[00217] Compound 1054. A mixture of compound 1052 (40 mmol) and compound 1053 (20 mmol) in DMF was heated to reflux for 12 hours. The mixture was added to water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2S04, and concentrated. The residue was purified by column chromatography to give compound 1054.
[00218] Compound 1055. A mixture of compound 1054 (10 mmol) and con. H2S04 (5 mL) in EtOH was heated to reflux for 6 hours. The mixture was concentrated in vacuo. The residue was added to water and extracted with EtOAc. Then the pH of water phase was adjusted to 8 with Na2C03. The mixture was extracted with EtOAc. The organic phase was washed with brine, dried over Na2S04, and concentrated to give compound 1055.
[00219] Compound 1056. A mixture of compound 1055 (10 mmol) and sodium hydroxide (8 mmol) in ethanol was stirred at room temperature overnight. The ethanol was removed in vacuo. The pH was adjusted to 5. The mixture was filtered and the filtrate was concentrated to give compound 1056.
[00220] Scheme 15
Figure imgf000075_0001
[00221] Resin 1057. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1056 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1057.
[00222] Compound 1058. The same procedure was used in the preparation of compound 1013 to get compound 1058.
[00223] Compound 9. To a solution of compound 1058 (0.50 mmol) in
DCM was added DEPBT (0.65 mmol) and DIEA (1.5 mmol). The mixture was stirred for 2 hours, then compound 1005 (0.65 mmol) was added. The mixture was stirred overnight. The mixture was concentrated and purified to give compound 9.
1HNMR(400 MHz, Methanol-^): δ 0.83-0.90 (m, 12H), 1.32-1.60 (m, 9H), 1.90-2.04 (m, 2H), 2.59-2.63 (m, 2H), 2.81-2.87(m, 2H), 3.05-3.16 (m, 2H), 3.73 (s, 2H), 4.26-4.32 (m, 2H), 4.48-4.60 (m, 2H), 4.79 (s, 2H), 7.06-7.30 (m, 11H); LRMS (M+H+) C4oH53N507S m/z: calculated: 747.4, found: 748.3.
[00224] Example 12: Compound 10, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00225] Shown below is the structure of Compound 10: Compound 10
[00226] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 10 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 10 to Carfilzomib on CT-L activity was determined to be 0.27 and 0.35, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000076_0001
[00227] The inhibitory effect of Compound 10 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 10, CT-L activity is inhibited >80.0%, T-L activity is inhibited >60%, and PGPH activity is inhibited >40%.
[00228] In vivo proteasome activity inhibition of Compound 10 is evaluated using the method disclosed in Example 3. Compound 10 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00229] Synthesis of compound 10
[00230] Scheme 16
Figure imgf000077_0001
[00231] Compound 10. To a solution of compound 9 (0.087 mmol) in 2 mL Pyridine was added 1 mL of Ac20. The mixture was stirred for overnight at room temperature. The mixture was concentrated and purified to give compound 10.
1HNMR(400 MHz, Methanol-^): δ 0.83-0.90 (m, 12H), 1.32-1.57 (m, 9H), 1.91-2.07 (m, 2H), 2.06 (s, 3H), 2.60-2.63 (m, 2H), 2.81-2.87(m, 2H), 3.05-3.16 (m, 2H), 3.76 (d, J = 5.2 Hz, 2H), 4.26-4.32 (m, 2H), 4.48-4.51 (m, 1H), 4.56-4.57 (m,lH), 5.31 (s, 2H), 7.06-7.27 (m, 10H), 7.40 (s, 1H); LRMS (M+H+) C42H55N508S m/z: calculated: 789.4, found: 790.3.
[00232] Example 13 : Compound 11, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo
Proteasome Activity Inhibitory Property, and Synthesis
[00233] Shown below is the structure of Compound 11 :
Figure imgf000077_0002
[00234] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 11 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 11 to Carfilzomib on CT-L activity was determined to be 0.36 and 0.56, respectively, for 20S constitutive proteasome and 20S immunoproteasome. IC50 (μΜ)
Compound
20S Constitutive Proteasome 20S Immunoproteasome Number
CT-L T-L PGPH CT-L T-L PGPH
11 <0.01 0.36 0.25 <0.01 0.46 0.49
[00235] The inhibitory effect of Compound 11 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 11, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00236] In vivo proteasome activity inhibition of Compound 11 is evaluated using the method disclosed in Example 3. Compound 11 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00237] Synthesis of Compound 11 00238] Scheme 17
Figure imgf000078_0001
[00239] Compound 11. To a solution of compound 9 (0.1 mmol) in DCM were added N,N-dimethyl-glycine (0.1 mmol), EDCI (0.1 mmol), HOBt (0.05 mmol) and DIE A (0.3 mmol). The mixture was stirred at room temperature overnight. The mixture was concentrated and purified to give compound 11. 1HNMR(400 MHz, Methanol-d4): δ 0.83-0.90 (m, 12H), 1.32-1.60 (m, 9H), 1.91-2.06 (m, 2H), 2.30 (s, 6H), 2.61-2.66 (m, 2H), 2.80-2.87 (m, 2H), 3.04-3.06 (m, 1H), 3.15 (d, J = 4.8 Ηζ,ΙΗ), 3.24 (s, 2H), 3.76 (d, J =7.2 Hz, 2H), 4.27-4.33 (m, 2H), 4.47-4.51 (m, 1H), 4.56-4.57 (m,lH), 5.38 (s, 2H), 7.06-7.27 (m, 10H), 7.41 (s, 1H); LRMS (M+H+) C44H6oN608S m/z: calculated: 832.4, found: 833.4.
[00240] Example 14: Compound 12: Structure, Proteasome Activity Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00241 ] Shown below is the structure of Compound 12:
Figure imgf000079_0001
Compound 12
[00242] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 12 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 12 to Carfilzomib on CT-L activity was determined to be 1.16 and 1.71, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000079_0002
[00243] The inhibitory effect of Compound 12 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 5 μΜ of Compound 12, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 40%, and PGPH activity is inhibited > 30%.
[00244] In vivo proteasome activity inhibition of Compound 12 is evaluated using the method disclosed in Example 3. Compound 12 simultaneously inhibits >30% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00245] Synthesis of Compound 12 [00246] Scheme 18
Figure imgf000080_0001
12
[00247] Compound 12. Compound 3 (0.11 mmol) was dissolved in THF, and 7 mg of sodium hydride was added. The mixture was stirred at room temperature for 30 minutes. 31.6 mg of dimethylcarbamic chloride was added, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was concentrated and the residue was purified to give compound 12. 1HNMR(400 MHz, CDC13): δ 0.87-0.95 (m, 12H), 1.26-1.35 (m, 3H), 1.50-1.72 (m, 7H), 1.96-2.06 (m, 1H), 2.15-2.25 (m, 1H), 2.53 (d, J = 2.4 Hz, 4H), 2.63-2.70 (m, 2H), 2.82-2.90 (m, 6H), 3.04 (s, 2H), 3.28-3.30 (m, 1H), 3.73 (d, J = 2.4 Hz, 4H), 4.27-4.35 (m, 2H), 4.40-4.50 (m, 2H), 4.55-4.63 (m, 2H), 7.16-7.32 (m, 5H), 6.71(d, J = 6.0 Hz, 1H), 6.91(d, J = 8.0 Hz, 1H), 7.54-5.78 (m, 2H), LRMS (M+H+) C37H58N609 m/z: calculated: 730.4, found: 731.4.
[00248] Example 15: Compound 13, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00249] Shown below is the structure of Compound 13:
Figure imgf000080_0002
[00250] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 13 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 13 to Carfilzomib on CT-L activity was determined to be 0.24 and 0.34, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000081_0002
[00251 ] The inhibitory effect of Compound 13 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 13, CT-L activity is inhibited >80.0%, T-L activity is inhibited by 60%, and PGPH activity is inhibited by 40%.
[00252] In vivo proteasome activity inhibition of Compound 13 is evaluated using the method disclosed in Example 3. Compound 13 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00253] Synthesis of Compound 13 00254] Scheme 19
Figure imgf000081_0001
1055 1059 1060
[00255] Compound 1059. A mixture of compound 1055 (6 mmol) and CDI
(6.6 mmol) in DCM was stirred at room temperature for 2 hours. The mixture was concentrated to give the crude compound 1059 without purification.
[00256] Compound 1060. To a mixture of compound 1059 (6 mmol) and
Et3N (18 mmol) in DCM was added dimethylamine hydrochloride (12 mmol) at 0 °C. The mixture was stirred at room temperature for 3 hours. The mixture was concentrated. The residue was purified to give compound 1060. [00257] Scheme 20
Figure imgf000082_0001
[00258] Resin 1061. To the resin 1011 (1.6 mmol) was added DCE (30 mL), and Compound 1060 (2.8 mmol), DIEA (6.3 mmol), HOBT (3.2 mmol), and BOP (3.2 mmol) and the reaction mixture was allowed to shake. The reaction mixture was filtered and the resin was washed DCE, DMF, 2-Propanol and Et20 and allowed to air dry for 24 hours to yield Resin 1061.
[00259] Compound 1062. The same procedure was used in the preparation of compound 1013 to get compound 1062.
[00260] Compound 13. To a solution of compound 1062 (0.20 mmol) in
DCM was added DEPBT (0.26 mmol) and DIEA (0.60 mmol). The mixture was stirred for 2 hours at 0 °C, then compound 1005 (0.26 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was concentrated and purified to give compound 13. 1HNMR(400 MHz, CDC13): δ 0.76-0.89 (m, 12H), 1.22-1.60 (m, 9H), 1.85-1.97 (m, 1H), 2.00-2.10 (m, 1H), 2.56-2.62 (m, 2H), 2.83 (d, J = 4.8 Hz, 1H), 2.91(d, J = 6.8 Hz, 6H), 2.98-3.07 (m, 1H), 3.09-3.13 (m, 1H), 3.27 (d, J = 4.8 Hz, 1H), 3.70 (s, 2H), 4.15-4.27 (m, 2H), 4.45-4.52 (m, 1H), 4.58-4.68 (m, 1H), 5.33 (s, 2H), 6.46 (d, J = 6.4 Hz, 1H), 6.51 (d, J = 7.6 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 7.15-7.30 (m, 11H); LRMS (M+H+) C43H58N608S m/z: calculated: 818.4, found: 819.3.
[00261] Example 16: Compound 20, Structure, Proteasome Activity Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00262] Shown below is the structure of Compound 20:
Figure imgf000083_0001
[00263] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 20 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 20 to Carfilzomib on CT-L activity was determined to be 0.17 and 0.18, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000083_0002
[00264] The inhibitory effect of Compound 20 on proteasome in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 5 μΜ of Compound 20, CT-L activity is inhibited >80.0%, T-L activity is inhibited by 50%, and PGPH activity is inhibited by 40%.
[00265] In vivo proteasome activity inhibition of Compound 20 is evaluated using the method disclosed in Example 3. Compound 20 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00266] Synthesis of Compound 20 [00267] Scheme 21
Figure imgf000084_0001
[00268] Compound 1064. To a solution of compound 1001 (70.1 mmol), compound 1063 (69.8 mmol), HATU (69.8 mmol) in DMF and DIEA (24 mL) was added, then stirred at RT overnight. The reaction mixture was concentrated and the residue was diluted with water and extracted with ethyl acetate. The organic phase was dried over MgS04, concentrated and the residue was purified by chromatography to give compound 1064.
[00269] Compound 1065. To a solution of compound 1064 (40.8 mmol) in
DCM and TFA (40 mL) in DCM was added, then stirred for 3 h. The reaction mixture was diluted with water and extracted with DCM; the combined DCM was washed with water, dried over Na2S04, then concentrated and dried to give compound 1065 as white solid.
[00270] Compound 1066. To a solution of compound 1065 (25.7 mmol),
Boc-HFE-OH (25.7 mmol), HATU (9.8 g, 25.7 mmol) in DMF and DIEA (9.5 mL) was added, then stirred at RT overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic phase was dried over MgS04, concentrated and the residue was purified by flash chromatography to give compound 1066 as white solid.
[00271] Compound 1067. To a solution of compound 1066 (3.6 mmol) in
MeOH/ THF(10: lOmL) cooled to 0 °C was added LiOH H20 (10.8 mmol), then stirred at room temperature for 3 h. The reaction mixture was quenched and acidified to pH < 6, then extracted with ethyl acetate. The combined organic phase was washed with water and dried over Na2S04, then concentrated to give compound 1067 as colorless oil.
[00272] Compound 1068. To a solution of compound 1067 (3.1 mmol), compound 1005 (3.5 mmol), HATU (3.2 mmol) in DMF and DIEA (0.5 mL) was added, then stirred at RT overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with water and dried over Na2S04, concentrated and the residue was purified to give compound 1068 as white solid.
[00273] Compound 1069. To a solution of compound 1068 (4.3 mmol) in
DCM and TFA (10 mL) was added, then stirred at room temperature for 4 h. The reaction mixture was concentrated to give compound 1069 (500 mg) as brown oil without further purification.
[00274] Scheme 22
Figure imgf000085_0001
[00275] Compound 20. To a solution of compound 1069 (0.15 mmol), compound 1070 (0.2 mmol), HATU (0.16 mmol) in DMF and DIEA (0.1 mL) was added, then stirred at RT overnight. The reaction mixture was diluted with water and extracted with DCM. The organic phase was washed with water and dried over Na2S04, concentrated and the residue was purified to give compound 20 as white solid. 1HNMR (400 MHz, DMSO-d6): δ 0.79-0.88 (m, 12H), 1.28-1.39 (m, 7H), 1.51-1.53 (m, 2H), 1.99-2.03 (t, 2H), 2.50-2.58 (m, 2H), 2.71-2.77 (m, 1H), 2.93-2.97 (m, 2H), 3.10-3.12 (d, J= 5.2 Hz, 1H), 4.32-4.42 (m, 2H), 4.52-4.59 (m, 2H), 7.04-7.26 (m, 10H), 8.01-8.03 (d, J= 8 Hz, 1H), 8.22-8.24 (d, J= 7.6 Hz, 2H), 8.76-8.80 (m, 2H), 8.90-8.91 (d, J= 2.4 Hz, 1H), 9.19-9.20 (d, J= 1.6 Hz, 1H); LRMS (M+H)+ Cs HsoNeOe calculated: 698.4, found: 699.4.
[00276] Example 17: Comparison study: Compound 21: Structure,
Proteasome Activity Inhibitory Property and Synthesis
[00277] Shown below is the structure of Compound 21 :
Figure imgf000086_0001
[00278] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 21 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against
CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 21 to Carfilzomib on CT-L activity was determined to be 23 and 39, respectively, for 20S constitutive proteasome and
20S immunoproteasome. The IC50 of Compound 21 on the T-L activity was as high as 181 μΜ and 39.7 μΜ, respectively, for the 20S constitutive proteasome and 20S immunoproteasome. The IC50 of Compound 21 on the PGPH activity was also high
(4.41 μΜ and 12.3 μΜ, respectively, for the 20S constitutive proteasome and 20S immunoproteasome). The inhibitory potency of Compound 21 on proteasome is o
NaO
O ^
undesirable. When Rl group was changed to ONa , the inhibition potency of the compound on the CT-L, T-L and PGPH activity of the 20S proteasome decreased substantially; the compound could not simultaneously inhibiting all of CT-L, T-L, PGPH activities of the 20S proteasome, which makes it undesirable for the intended therapeutic effects. This indicates the Rl group provided herein renders the compounds unpredictable with respect to simultaneous inhibition of the CT-L, T-L, and PGPH activities.
Figure imgf000087_0002
[00279] Synthesis of Compound 21
[00280] Scheme 23
Figure imgf000087_0001
[00281] Compound 1073. To a solution of compound 1071 (27.7 mmol) in
THF, TEA (27.7 mmol) was added at 0°C. Compound 1072(27.7 mmol) was added and stirred for 5 h at RT. The reaction mixture was filtered, concentrated and purified by flash chromatography to give compound 1073 as yellow oil.
[00282] Compound 1074. To a solution of compound 1073 (0.38mmol) in
THF was added LiOH H20 (0.58 mmol), then stirred at room temperature overnight. The reaction mixture was concentrated and the residue was quenched and acidified to pH < 6, then extracted with DCM. The organic phase was washed with water and dried over Na2S04, then concentrated and purified by flash chromatography to give compound 1074 as colorless oil.
[00283] Compound 1075. To a solution of compound 1074(0.50 mmol), compound 1069 (0.50 mmol), HATU (0.55 mmol) in DMF and DIEA (0.2 mL) was added, then stirred at RT overnight. The reaction mixture was concentrated and the residue was diluted with water. The aqueous solution was extracted with DCM. The organic phase was dried over MgS04, concentrated and the residue was purified to give compound 1075 as white solid.
[00284] Compound 21. To a solution of compound 1075 (0.08mmol) in
THF was added H20 and 10% pd/C. The reaction mixture was stirred under H2 at RT for 2 hour. The reaction mixture was filtered and the filtrate was treated with Na2C03. The solids were collected by filtration to give the compound 21 as white solid.
1HNMR(400 MHz, DMSO-d6): δ 0.72-0.86 (m, 12H), 1.06-1.11 (m, 1H), 1.21-1.49 (m, 7H), 1.66-1.89 (t, 1H), 1.90-1.97 (m, 2H), 2.49-2.67 (m, 2H), 2.90-2.94 (m, 3H), 2.98-3.19 (m, 1H), 4.10-4.46 (m, 6H), 7.06-7.35 (m, 10H), 8.10-8.11 (d, 1H), 8.46 (s, 1H), 8.78-8.79 (t, 1H), 9.04-9.05 (t, 1H); LRMS (M+H)+ C36H49N4Na2Oi0P calculated: 774.4, found: 775.3.
[00285] Example 18: Compound 25, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00286] Shown below is the structure of Compound 25:
Figure imgf000088_0001
[00287] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 25 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 25 to Carfilzomib on CT-L activity was determined to be 0.26 and 0.30, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000089_0002
[00288] The inhibitory effect of Compound 25 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 1 μΜ of Compound 25, CT-L activity is inhibited >80.0%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00289] In vivo proteasome activity inhibition of Compound 25 is evaluated using the method disclosed in Example 3. Compound 25 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 2 mg/kg.
[00290] Synthesis of Compound 25
[00291] Scheme 24
Figure imgf000089_0001
[00292] Compound 25. To a solution of compound 1069 (0.33 mmol), compound 1076 (0.35 mmol), BOP (0.35 mmol) in DMF and TEA (0.2 mL) was added, then stirred at RT overnight. The reaction mixture was concentrated and the residue was diluted with water. The aqueous solution was extracted with DCM. The organic phase was dried over MgS04, concentrated and the residue was purified to give compound 25 as white solid. 1HNMR 400 MHz, DMSO-d6): δ 0.71-0.87 (m, 12H), 1.23-1.52 (m, 7H), 1.58-1.63 (m, 2H), 1.85-1.95 (m, 2H), 2.44-2.60 (m, 2H), 2.63-2.76 (m, 1H), 2.95-2.98 (t, 2H), 3.08-3.11 (t, 1H), 4.22-4.35 (m, 3H), 4.52-4.53 (d, J= 4 Hz, 1H), 7.08-7.30 (m, 10H), 7.46-7.49 (d, J= 12.4 Hz, 2H), 7.77 (s, 1H), 7.85-7.96 (m, 2H), 8.18-8.22 (t, 2H); LRMS (M+H])+ C38H50N6O6S calculated: 718.4, found: 719.4.
[00293] Example 19: Compound 27, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00294] Shown below is the structure of Compound 27:
Figure imgf000090_0001
Compound 27
[00295] Inhibition of 20S constitutive proteasome and 20S
immunoproteasome by Compound 27 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC5o values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 27 to Carfilzomib on CT-L activity was determined to be 0.37 and 0.40, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000090_0002
[00296] The inhibitory effect of Compound 27 on proteasome activity in mouse blood lysates is performed using the assay method disclosed in Example 2. At concentration of 5 μΜ of Compound 27, CT-L activity is inhibited >80%, T-L activity is inhibited > 60%, and PGPH activity is inhibited > 40%.
[00297] In vivo proteasome activity inhibition of Compound 27 is evaluated using the method disclosed in Example 3. Compound 27 simultaneously inhibits >40% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00298] Synthesis of Compound 27 00299] Scheme 25
Figure imgf000091_0001
[00300] Compound 1078. Compound 1077 (8.54 mmol) was added to a solution of sodium formate (7.05 mmol) dissolved in formic acid (2.15 mL) and the mixture was stirred at RT for 4 h and filtered. Acetic anhydride (2.92 mL) was added to the filtrate and the mixture was stirred at RT for 4 h. The solvent was evaporated and the residue was distilled under high vacuum to give ethyl N-formyl sarcosine ester. Potassium-t-butoxide (6.14 mmol) was suspended in diethylether and cooled to 0° C. A solution of N-formyl sarcosine ether ester (6.2 mmol) and methyl formate (4.32 mmol) in diethylether (5 mL) was added over 30 minutes and the mixture was stirred for 1 hour. The solid product was filtered and was dissolved in water and concentrated HC1 (1.8 mL) was added. The mixture was heated on a steam bath for 30 minutes, cooled and the pH was adjusted to 5. Cyanamide (5.95 mmol) was added and the mixture was heated at 100° C. for 1 hour. The mixture was allowed to cool; the solid was filtered to give compound 1078.
[00301] Compound 1079. Compound 1078 (0.52 mmol) was dissolved in
THF. (Boc)20(1.29 mmol) and DMAP(1.03 mmol) were added and the mixture was stirred overnight at room temperature. The reaction mixture was extracted with ethyl acetate .The organic layer was evaporated and purification to get compound 1079. [00302] Compound 1080. Compound 1079 (0.43 mmol) was dissolved in a mixture (10 mL THF: H20 2: 1), then LiOH.H20 (4.3 mmol) was added. The mixture was stirred for 4 hours. The mixture was evaporated and adjusted to pH 5. The mixture was extracted with ethyl acetate. The residue was dried and evaporated to get product 1080.
[00303] Compound 1081. To a solution of compound 1080 (0.33 mmol), compound 1069 (0.34 mmol), HATU (0.37 mmol) in DMF and DIEA (0.2 mL) was added, then stirred at RT overnight. The reaction mixture was concentrated and the residue was diluted with water. The aqueous solution was extracted with DCM. The organic phase was dried over MgS04, concentrated and the residue was purified to give compound 1081 as white solid
[00304] Compound 27. To a solution of compound 1081 (0.1 mmol) in
DCM and TFA (0.2 mL) was added, and then stirred at room temperature for 3 h. The reaction mixture was concentrated and the residue was purified to give compound 27 as white solid. 1HNMR(400 MHz, DMSO-d6): δ 0.77-0.87 (m, 12H), 1.23-1.39 (m, 7H), 1.46-1.68 (m, 2H), 1.88-1.94 (m, 2H), 2.49-2.50 (t, 1H), 2.72-2.76 (t, 2H), 2.94-2.99 (m, 2H), 3.10-3.11 (d, J= 5.2 Hz, 1H), 3.5 (s, 3H), 4.23-4.36 (m, 3H), 4.52-4.53 (d, J= 4.8 Hz, 1H), 5.82 (s, 2H), 7.08-7.33 (m, 11H), 7.82-7.89 (m, 3H), 8.17-8.19 (d, J= 8 Hz, 1H); LRMS (M+H+) Cs HssNvOe calculated: 715.4, found: 716.5.
[00305] Example 20: Compound 28, Structure, Proteasome Activity
Inhibitory Property, Ex Vivo Proteasome Activity Inhibitory Property, In Vivo Proteasome Activity Inhibitory Property, and Synthesis
[00306] Shown below is the structure of Compound 28:
Figure imgf000092_0001
28 [00307] Inhibition of 20S constitutive proteasome and 20S immunoproteasome by Compound 28 was performed using the enzymatic assay method disclosed in Example 1. The following table presents the IC50 values against CT-L, T-L, and PGPH activities of both 20S constitutive proteasome and 20S immunoproteasome. The IC50 ratio of Compound 28 to Carfilzomib on CT-L activity was determined to be 0.16 and 0.15, respectively, for 20S constitutive proteasome and 20S immunoproteasome.
Figure imgf000093_0001
[00308] The inhibitory effect of Compound 28 on proteasome activity in mouse blood lysates was performed using the assay method disclosed in Example 2. At concentration of 0.2 μΜ of Compound 28, CT-L activity was inhibited by 96.1%, T-L activity was inhibited by 57.8%, and PGPH activity was inhibited by 36.0%. At concentration of 1 μΜ of Compound 28, CT-L activity was inhibited by 98.4%, T-L activity was inhibited by 73.5%, and PGPH activity was inhibited by 48.2%. And at concentration of 5 μΜ of Compound 28, CT-L activity was inhibited by 100%, T-L activity was inhibited by 83.2%, and PGPH activity was inhibited by 64.9%.
[00309] In vivo proteasome activity inhibition of Compound 28 was evaluated using the method disclosed in Example 3. Compound 28 simultaneously inhibited 97.5%, 72.1% and 68.4% of the CT-L, T-L and PGPH activities of the blood proteasome at the dose of 4 mg/kg.
[00310] Synthesis of Compound 28
[00311] Scheme 26
Figure imgf000094_0001
[00312] Compound 28. To a solution of compound 1082 (0.19 mmol), compound 1069 (0.17 mmol), HATU (0.18 mmol) in DMF and DIEA (0.1 mL) was added, then stirred at RT overnight. The reaction mixture was concentrated and the residue was diluted with water. The aqueous solution was extracted with DCM. The organic phase was dried over MgS04, concentrated and the residue was purified to give compound 28 as white solid. !NMR (400 MHz, DMSO-d6): δ 0.73-0.87 (m, 12H), 1.19-1.40 (m, 7H), 1.39-1.50 (m, 1H), 1.52-1.63 (m, 1H), 1.73-1.76 (m, 1H), 1.84-1.88 (m, 1H), 2.33-2.44 (m, 2H), 2.56-2.76 (m, 1H), 2.93-3.22 (m, 5H),
4.22-4.36 (m, 3H), 4.51-4.53 (t, 1H), 6.25 (s, 1H), 6.88 (s, 2H), 7.06-7.29 (m, 10H), 7.88-7.90 (d, J= 8.4 Hz, 1H), 7.97-7.99 (d, J= 8 Hz, 1H), 8.13-8.15 (d, J = 8 Hz, 1H), 8.20-8.22 (d, J= 7.2 Hz, 1H); LRMS (M+H+) Cs^NeOeS calculated: 732.4, found: 733.4.

Claims

Claims
1. A compound having a structure shown in Formula (I), and an enantiomer,
diastereomer, tautomer, pharmaceutically acceptable salt or solvate or prodrug thereof:
Figure imgf000095_0001
R2 is -(CH2)mR5; each of R3 is independently H, hydroxyl, Ci_ioalkyl, Ci_ioalkoxyl, Ci_iohydroxyalkyl, Ci-ioalkyloxyalkyl, NH2, NHR6, -R7-0(C=0)-R8, -R7-(C=0)X-R8, -R7-OP03MiM2,
Figure imgf000096_0001
R is phenyl, or Ry. Ry is -OH, -OPO3M1M2, -Ri0-O(C=O)-Rn,
Figure imgf000096_0002
R6 is Ci_ioalkyl, phenyl, -(C=0)Ci_6alkyl, -(C=0)phenyl; each of R7, R and Rio is independently absent, or Ci_ioalkylene (e.g. -CH2-, -C2H4-, -C3H7-, etc.); each of Rg and Rn is independently H, hydroxyl, metal (e.g. Na, and K), Ci_ioalkyl (e.g. Ci_4alkyl), -Ci_i0alkylene-NRi2Ri3, -NRi2Ri3, or -OP03MiM2; each of Ri2 and Ri3 is independently H, Ci_ioalkyl (e.g. Ci_4alkyl) or substituted Ci_ioalkyl (e.g. Ci_4alkyl); each of Mi, and M2 is independently H, metal (e.g. Na, and K);
X is absent or O;
Y is absent or -(C=0)-;
Z is absent or O; and m is 0, 1, 2, 3, 4 or 5.
2. The compound of claim 1, comprising a structure selected from Compounds I-l, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, I-ll, 1-12, 1-13, 1-14, 1-15, 1-16, and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
3. The compound of claim 1 or 2, wherein the compound of Formula (I) has a configuration shown in Formula (II):
Figure imgf000097_0001
wherein each of Ri and R2 is as defined in claim 1.
4. The compound of claim 3, comprising a structure selected from Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7,
Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 20, Compound 25, Compound 27 and Compound 28, and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts or solvates or prodrugs thereof.
5. The compound of claim 1 or 3, wherein the compound of Formula (I) has a configuration shown in Formula (III):
Figure imgf000097_0002
wherein R3 is as defined in claim 1.
6. The compound of claim 1 or 3, wherein the compound of Formula (I) has a configuration shown in Formula (V):
Figure imgf000098_0001
wherein R3 is as defined in claim 1
7. The compound of claim 1 or 3, wherein the compound of Formula (I) has a confi uration shown in Formula (V).
Figure imgf000098_0002
wherein R3 is as defined in claim 1.
8. The compound of claim 1 or 3, wherein the compound of Formula (I) has a configuration shown in Formula (VI):
Figure imgf000098_0003
wherein the R4 and Ry is defined as claim 1.
9. The compound of claim 1 or 3, wherein the compound of Formula (I) has a configuration shown in Formula (VII):
Figure imgf000099_0001
wherein R is selected from the group consisting of
Figure imgf000099_0002
wherein the R3 group is as defined in claim 1.
The compound of claim 1 or 3, wherein the compound of Formula (I) has confi uration shown in Formula(VIII):
Figure imgf000099_0003
Formula (VIII), wherein Ri is selected from the group consisting of:
Figure imgf000099_0004
11. A pharmaceutical composition comprising a compound of any of claims 1-10, and a pharmaceutically acceptable carrier.
12. A method of specifically inhibiting catalytic activity of 20S proteasome,
comprising administering a therapeutically effective amount of a compound of any of claims 1-10.
13. The method of claim 12, wherein the CT-L activity, T-L activity and PGPH activity of the 20S proteasome are simultaneously inhibited.
14. A method of treating a proteasome-related disease or condition, comprising administering a therapeutically effective amount of a compound of any of claims 1-10.
15. Use of a compound of any of claims 1-10 in the manufacture of a medicament for treating a 20S proteasome-related disease or condition.
16. The method of claim 14 or the use of claim 15, wherein the 20S
proteasome-related disease or condition is selected from the group consisting of cancer, neurotoxic/degenerative disease, Alzheimer's disease, ischemic condition, inflammation, immune -related disease, HIV infection, organ graft rejection, septic shock, inhibition of antigen presentation, decreasing viral gene expression, parasitic infection, condition associated with acidosis, macular degeneration, pulmonary condition, muscle wasting disease, fibrotic disease, and bone and hair growth diseases.
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