AU2012200798B2 - Compounds for enzyme inhibition - Google Patents

Abstract

Peptide-based compounds including heteroatom-containing, three-membered rings efficiently and selectively inhibit specific activities of N-terminal nucleophile (Ntn) 5 hydrolases associated with the proteasome. The peptide-based compounds include an epoxide or aziridine, and functionalization at the N-terminus. Among other therapeutic utilities, the peptide-based compounds are expected to display anti-inflammatory properties and inhibition of cell proliferation. Oral administration of these peptide-based proteasome inhibitors is possible due to their bioavailability profiles.

Description

AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: Proteolix, Inc. Actual Inventors: Han-jie Zhou and Congcong M. Sun and Kevin D. Shenk and Guy J. Laidig Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: Compounds for enzyme inhibition Details of Original Application No. 2006311584 dated 09 Nov 2006 The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 58531AUP01 COMPOUNDS FOR ENZYME INHIBITION The present application is a divisional application of Australian Application No. 2006311584, which is incorporated in its entirety herein by reference. 5 Background of the Invention In eukaryotes, protein degradation is predominately mediated through the ubiquitin pathway in which proteins targeted for destruction are ligated to the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinated proteins then serve as substrates for the 26S proteasome, a multicatalytic protease, which cleaves proteins into 10 short peptides through the action of its three major proteolytic activities. While having a general function in intracellular protein turnover, proteasome-mediated degradation also plays a key role in many processes such as major histocompatibility complex (MHC) class I presentation, apoptosis, cell division, and NF-KB activation. The 20S proteasome is a 700 kDa cylindrical-shaped multicatalytic protease 15 complex comprised of 28 subunits organized into four rings that plays important roles in cell growth regulation, major histocompatibility complex class I presentation, apoptosis, antigen processing, NF-KB activation, and transduction of pro-inflammatory signals. In yeast and other eukaryotes, 7 different a subunits form the outer rings and 7 different p subunits comprise the inner rings. The a subunits serve as binding sites for the 19S 20 (PA700) and 11 S (PA28) regulatory complexes, as well as a physical barrier for the inner proteolytic chamber formed by the two P subunit rings. Thus, in vivo, the proteasome is believed to exist as a 26S particle ("the 26S proteasome"). In vivo experiments have shown that inhibition of the 20S form of the proteasome can be readily correlated to inhibition of 26S proteasome. Cleavage of amino-terminal prosequences of 25 p subunits during particle formation expose amino-terminal threonine residues, which serve as the catalytic nucleophiles. The subunits responsible for catalytic activity in proteasome thus possess an amino terminal nucleophilic residue, and these subunits belong to the family of N-terminal nucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties). This 30 family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. ]a In addition to the ubiquitously expressed p subunits, higher vertebrates also possess three y-interferon l b inducible 0 subunits (LMP7, LMP2 and MECLI), which replace their normal counterparts, X, Y and Z respectively, thus altering the catalytic activities of the proteasome. Through the use of different peptide substrates, three major proteolytic activities have been defined for the eukaryote 20S proteasome: chymotrypsin-like 5 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. Two additional less characterized activities have also been ascribed to the proteasome: BrAAP activity, which cleaves after branched-chain amino acids; and SNAAP activity, which 10 cleaves after small neutral amino acids. The major proteasome proteolytic activities appear to be contributed by different catalytic sites, since inhibitors, point mutations in s subunits and the exchange of y interferon-inducing P subunits alter these activities to various degrees. In recent years, the proteasome has become an appealing target for 15 therapeutic intervention in cancer, immune and auto-immune disorders, inflammation, ischemic conditions, neurodegenerative disorders and other diseases. To date, the only FDA-approved proteasome inhibitor is bortezomib (VELCADETH), however, several other proteasome inhibitors are currently being evaluated in clinical trials. Thus far, all these therapeutic proteasome inhibitors 20 currently are administered via IV. Clinical application of proteasome inhibitors in the treatment of hematologic malignancies such as myeloma and lymphoma is restricted in part by the necessity of frequent IV administrations and would be improved by oral (PO) administration. However, due to the peptide nature of these molecules, systemic exposure following PO administration of these inhibitors is 25 limited by several factors including gastric pH, gastric and intestinal peptidases, efflux pumps, biliary excretion and intestinal and hepatic metabolic activities. Methods used to overcome the ability of peptides to be enzymatically degraded and to improve absorption into the blood stream from the digestive tract have included making analogs which are less peptide-like in structure and which are 30 reduced in size. Such methods are deemed to be successful when the peptide analog achieves satisfactory blood levels after oral administration, or in the case of -2proteasome inhibitors, when the proteasone activity in blood is satisfactorily reduced. The above mentioned techniques have been applied to preparing analogs of the peptide epoxyketone proteasome inhibitors, thereby rendering them orally 5 bioavailable. Summary of the Invention The invention relates to classes of molecules known as peptide cp' epoxides and peptide a',P'-aziridines. The parent molecules are understood to bind efficiently, irreversibly and selectively to N-terminal nucleophile (Ntn) hydrolases, 10 and can specifically inhibit particular activities of enzymes having multiple catalytic activity. Once thought merely to dispose of denatured and misfolded proteins, the proteasome is now recognized as constituting proteolytic machinery that regulates the levels of diverse intracellular proteins through their degradation in a signal 15 dependent manner. Hence, there is great interest in identifying reagents that can specifically perturb the activities of the proteasome and other Ntn hydrolases and thereby be used as probes to study the role of these enzymes in biological processes. Compounds that target the Ntn hydrolases are herein described, synthesized, and investigated. Peptide epoxides and peptide aziridines that can potently, selectively, 20 and irreversibly inhibit particular proteasome activities are disclosed and claimed. Unlike several other peptide-based inhibitors, the peptide epoxides and peptide aziridines described herein are not expected to substantially inhibit non proteasomal proteases such as trypsin, chymotrypsin, cathepsin B, papain, and calpain at concentrations up to 50 .M. At higher concentrations, inhibition may be 25 observed, but would be expected to be competitive and not irreversible, if the inhibitor merely competes with the substrate. The novel peptide epoxides and peptide aziridines are also expected to inhibit NF-KB activation and to stabilize p53 levels in cell culture. Moreover, these compounds would be expected to have anti inflammatory activity. Thus, these compounds can be unique molecular probes, 30 which have the versatility to explore Ntn enzyme function in normal biological and pathological processes. -3- In one aspect, the invention provides inhibitors comprising a heteroatom containing three-membered ring. These inhibitors can inhibit catalytic activity of N terminal nucleophile hydrolase enzymes (for example, the 20S proteasome, or the 26S proteasome) when said inhibitor is present at concentrations below about 50 5 piM. Regarding the 20S proteasome, particular hydrolase inhibitors inhibit chymotrypsin-like activity of the 20S proteasome when the inhibitor is present at concentrations below about 5 pM, and does not inhibit trypsin-like activity or PGPH activity of the 20S proteasome when present at concentrations below about 5 JAM. The hydrolase inhibitor may be, for example, a peptide a',p'-epoxy ketone or ct',p' 10 aziridine ketone, and the peptide may be a tetrapeptide. The peptide may include branched or unbranched side chains such as hydrogen, C 1 .6alkyl, Ci.

6 hydroxyalkyl, CI.Galkoxyalkyl, aryl, C 1

.

6 aralkyl, C,.ealkylamide, CI-6alkylamine, CI.6carboxylic acid, Ci.6carboxyl ester, Ci- 6 alkylthiol, or Ci-calkylthioether, for example isobutyl, 1-naphthyl, phenylmethyl, and 2-phenylethyl. The W'-carbon of the a',p'-epoxy 15 ketone or a',p'-aziridine ketone may be a chiral carbon atom, such as an (R) or p configured carbon, as these are defined herein. In another aspect, the invention provides pharmaceutical compositions, including a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the hydrolase inhibitor, which ameliorates the effects of 20 neurodegenerative disease (such as Alzheimer's disease), muscle-wasting diseases, cancer, chronic infectious diseases, fever, muscle disuse, denervation, nerve injury, fasting, and immune-related conditions, among others. In another aspect, the invention provides compounds and pharmaceutical compositions that are orally bioavailable. 25 In another aspect, the invention provides anti-inflammatory compositions. In another aspect, the invention provides methods for the following: inhibiting or reducing HIV infection in a subject; affecting the level of viral gene expression in a subject; altering the variety of antigenic peptides produced by the proteasome in an organism; determining whether a cellular, developmental, or 30 physiological process or output in an organism is regulated by the proteolytic activity of a particular Ntn hydrolase; treating Alzheimer's disease in a subject; -4reducing the rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation in a cell; reducing the rate of p53 protein degradation in a cell; inhibiting the growth of p53-related cancers in a subject; inhibiting antigen presentation in a cell; suppressing the immune system of a 5 subject; inhibiting IKB-a degradation in an organism; reducing the content of NF-KB in a cell, muscle, organ or subject; affecting cyclin-dependent eukaryotic cell cycles; treating proliferative disease in a subject; affecting proteasome-dependent regulation of oncoproteins in a cell; treating cancer growth in a subject; treating p53-related apoptosis in a subject; and screening proteins processed by N-terminal nucleophile 10 hydrolases in a cell. Each of these methods involves administering or contacting an effective amount of a composition comprising the hydrolase inhibitors disclosed herein, to a subject, a cell, a tissue, an organ, or an organism. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 15 Detailed Description of the Invention The invention involves compounds useful as enzyme inhibitors. These compounds are generally useful to inhibit enzymes having a nucleophilic group at the N-terminus. For example, activities of enzymes or enzyme subunits having N terminal amino acids with nucleophiles in their side chains, such as threonine, 20 serine, or cysteine can be successfully inhibited by the enzyme inhibitors described herein. Activities of enzymes or enzyme subunits having non-amino acid nucleophilic groups at their N-termini, such as, for example, protecting groups or carbohydrates, can also be successfully inhibited by the enzyme inhibitors described herein. 25 While not bound by any particular theory of operation, it is believed that such N-terminal nucleophiles of Ntn form covalent adducts with the epoxide functional group of the enzyme inhibitors described herein. For example, in the 05/Pre2 subunit of 20S proteasome, the N-terminal threonine is believed to irreversibly form a morpholino or piperazino adduct upon reaction with a peptide 30 epoxide or aziridine such as those described below. Such adduct formation would involve ring-opening cleavage of the epoxide or aziridine. -5- In embodiments including such groups bonded to a' carbons, the stereochemistry of the a'-carbon (that carbon forming a part of the epoxide or aziridine ring) can be (R) or (S). The invention is based, in part, on the structure function information disclosed herein, which suggests the following preferred 5 stereochemical relationships. Note that a preferred compound may have a number of stereocenters having the indicated up-down (or p-a, where P as drawn herein is above the plane of the page) or (R)-(S) relationship (that is, it is not required that every stereocenter in the compound conform to the preferences stated). In some preferred embodiments, the stereochemistry of the a' carbon is (R), that is, the X 10 atom is P, or above the plane of the molecule. Regarding the stereochemistry, the Cahn-Ingold-Prelog rules -for determining absolute stereochemistry are followed. These rules are described, for example, in Organic Chemistry, Fox and Whitesell; Jones and Bartlett Publishers, Boston, MA (1994); Section 5-6, pp 177-178, which section is hereby incorporated by reference. 15 Peptides can have a repeating backbone structure with side chains extending from the backbone units. Generally, each backbone unit has a side chain associated with it, although in some cases, the side chain is a hydrogen atom. In other embodiments, not every backbone unit has an associated side chain. Peptides useful in peptide epoxides or peptide aziridines have two or more backbone units. In some 20 embodiments useful for inhibiting chymotrypsin-like (CT-L) activity of the proteasome, between two and four backbone units are present, and in some preferred embodiments for CT-L inhibition, three backbone units are present. The side chains extending from the backbone units can include natural aliphatic or aromatic amino acid side chains, such as hydrogen (glycine), methyl 25 (alanine), isopropyl (valine), sec-butyl (isoleucine), isobutyl leucinee), phenylmethyl (phenylalanine), and the side chain constituting the amino acid proline. The side chains can also be other branched or unbranched aliphatic or aromatic groups such as ethyl, n-propyl, n-butyl, t-butyl, and aryl substituted derivatives such as 1 phenylethyl, 2-phenylethyl, (I -naphthyl)methyl, (2-naphthyl)methyl, 1 -(1 30 naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1 -naphthyl)ethyl, 2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can be further substituted with branched or unbranched Ci.6alkyl groups, or substituted alkyl groups, acetyl and the like, or -6further aryl groups, or substituted aryl groups, such as benzoyl and the like. Heteroaryl and heterocyclyl groups can also be used as side chain substituents. Heteroaryl groups include nitrogen-, oxygen-, and sulfur-containing aryl groups such as thienyl, benzothienyl, naphthothienyl, thianthrenyl, furyl, pyranyl, 5 isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, indolyl, purinyl, quinolyl, and the like. Heterocyclyl groups include tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. In some embodiments, polar or charged residues can be introduced into the peptide epoxides or peptide aziridines. For example, naturally occurring amino 10 acids such as hydroxy-containing (Thr, Tyr, Ser) or sulfur-containing (Met, Cys) can be introduced, as well as non-essential amino acids, for example, taurine, carnitine, citrulline, cystine, ornithine, norleucine and others. Non-naturally occurring side chain substituents with charged or polar moieties can also be included, such as, for example, Ci.6alkyl chains or Cri 2 aryl groups with one or more hydroxy, short chain 15 alkoxy, sulfide, thio, carboxyl, ester, phospho, amido or amino groups, or such substituents substituted with one or more halogen atoms. In some preferred embodiments, there is at least one aryl group present in a side chain of the peptide moiety. In some embodiments, the backbone units are amide units [-NH-CHR 20 C(=O)-], in which R is the side chain. Such a designation does not exclude the naturally occurring amino acid proline, or other non-naturally occurring cyclic secondary amino acids, which will be recognized by those of skill in the art. In other embodiments, the backbone units are N-alkylated amide units (for example, N-methyl and the like), olefinic analogs (in which one or more amide 25 bonds are replaced by olefinic bonds), tetrazole analogs (in which a tetrazole ring imposes a cis-configuration on the backbone), or combinations of such backbone linkages. In still other embodiments, the amino acid a-carbon is modified by a-alkyl substitution, for example, aminoisobutyric acid. In sorne further embodiments, side chains are locally modified, for example, by AE or Az dehydro modification, in 30 which a double bond is present between the ot and .3 atoms of the side chain, or for example by AE or AZ cyclopropyl modification, in which a cyclopropyl group is -7present between the a and 0 atoms of the side chain. In still further embodiments employing amino acid groups, D-amino acids can be used. Further embodiments can include side chain-to-backbone cyclization, disulfide bond formation, lactam formation, azo linkage, and other modifications discussed in "Peptides and Mimics, 5 Design of Conformationally Constrained" by Hruby and Boteju, in "Molecular Biology and Biotechnology: A Comprehensive Desk Reference", ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664, which is hereby incorporated by reference. One aspect of the invention relates to compounds having a structure of 10 formula (I) or a pharmaceutically acceptable salt thereof: R R 6 0 R 3

R

5 ZL N X I I

R

4 0 R 2

R

7 0 (I) wherein L is selected from C=0, C=S, and SO, preferably C=0; 15 X is selected from 0, S, NH4, and N-CiL-alkyl; Z is absent, C 1 .alkyl, or CI-alkoxy, preferably absent; R1, R 2 , and R 3 are each independently selected from hydrogen, CI-6alkyl, Ci. salkenyl, Ca-6alkynyl, Ci-6hydroxyalkyl, Ci-6alkoxyalkyl, aryl, C,.6aralkyl, heteroaryl, heterocyclyl, CI-6heterocycloalkyl, Cl.heteroaralkyl, carbocyclyl, and 20 CI-6carbocyclolalkyl; R4 is selected from hydrogen, Ci-6aralkyl, and Ci- 6 alkyl; Rs is heteroaryl; and 6 7 R and R 7 are independently selected from hydrogen, Ci-6alkyl, and C 6aralkyl. 25 In certain embodiments, R', R 2 , and R 3 are independently selected from hydrogen, Ci- 6 alkyl, CI.hydroxyalkyl, Ci.alkoxyalkyl, Ci-saralkyl, C,. 6heterocycloalkyl, CI-6heteroaralkyl, and CI.6carbocyclolalkyl. In certain embodiments, any of R', R 2 , and R 3 are independently

C

1 .alkyl selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and isobutyl. In certain -8embodiments, any of R', R2, and R 3 are independently CI6hydroxyalkyl. In certain preferred such embodiments, any of R', R2, and R3 are independently selected from hydroxymethyl and hydroxyothyl, preferably hydroxymethyl. In certain embodiments, any of R', R 2 , and R? are independently Ci6alkoxyalkyl. In certain 5 such embodiments, any of R', R2, and R3 are independently selected from methoxymethyl and methoxyethyl, preferably methoxynethyl. In certain embodiments, any of R', R 2 , and R 3 are independently CI6heteroaralkyl. In certain such embodiments, any of R', R 2 , and R3 are independently selected from imidazolylmethyl, pyrazolylmethyl, and thiazolylmethyl, and pyridylmethyl, 10 preferably imidazol-4-ylmethyl, thiazol-4-ylmethyl, 2-pyridylmethyl, 3 pyridylmethyl, or 4-pyridylmethyl. In certain embodiments, any of R', R 2 , and R2 are independently CI..aralkyl. In certain such embodiments, any of R', R2, and R 3 are independently selected from phenylmethyl (benzyl) and phenylethyl, preferably phenylmethyl. In certain embodiments, any of R', R2, and R3 are independently C.. 15 6carbocycloalkyl. In certain such embodiments R' is cyclohexylmethyl. In certain embodiments R', R 2 , and R3 are all different. In certain embodiments, any two of R', R 2 , and R3 are the same. In certain embodiments,

R

1 , R2, and R 3 are all the same. In certain embodiments, at least one of R' and R 2 is selected from C . 20 6hydroxyalkyl and CI-6alkoxyalkyl. In certain such embodiments, at least one of R' and R2 is alkoxyalkyl. In certain such embodiments, at least one of R' and R2 is selected from methoxymethyl and methoxyethyl. In certain embodiments, R3 is selected from C 1

.

6 alkyl and Ci-saralkyl, preferably CIsalkyl. In certain such embodiments, R3 is selected from methyl, 25 ethyl, isopropyl, sec-butyl, and isobutyl. In certain such embodiments R3 is isobutyl. In certain alternative embodiments,

R

3 is selected from phenylmethyl and phenylethyl, preferably phenylmethyl. In certain embodiments, R4, R 6 , and R 7 are independently selected from hydrogen and methyl, preferably hydrogen. 30 In certain embodiments, Rs is a 5- or 6 -membered heteroaryl. In certain such embodiments, R5 is selected from isoxazole, isothiazole, furan, thiophene, oxazole, thiazole, pyrazole, or imidazole, preferably isoxazole, furan, or thiazole. -9- In certain embodiments,

R

5 is a bicyclic heteroaryl. In certain such embodiments bicyclic heteroaryl is selected from benzisoxazole, benzoxazole, benzothiazole, benzisothiazole. In certain embodiments, L is C=O, Z is absent, and R 5 is an isoxazol-3-yl or 5 isoxazol-5-yl. In certain preferred such embodiments, when the isoxazol-3-yl is substituted, it is substituted at least at the 5-position. In certain preferred embodiments, when the isoxazol-5-yl is substituted, it is substituted at least at the 3 position. In certain embodiments, L is C=O, Z is absent, and RW is an unsubstituted 10 isoxazol-3-yl. In certain embodiments, L is C=O, Z is absent, and R 5 is a substituted isoxazol-3-yl. In certain such embodiments,

R

5 is isoxazol-3-yl substituted with a substituent selected from C1.

6 alkyl, C,- 6 alkoxy, CI-6alkoxyalkyl, CI-6hydroxyalkyl, carboxylic acid, aminocarboxylate, Cisalkylaminocarboxylate, (Ci. 15 6alkyl)2aminocarbdxylate, Ci-6alkylcarboxylate, CI-6heteroaralkyl, Ci-6aralkyl, C. 6heterocycloalkyl, and CI-6carbocycloalkyl. In certain preferred such embodiments

R

5 is isoxazole-3-yl substituted with a substituent selected from methyl, ethyl, isopropyl, and cyclopropylmethyl. In certain embodiments L is C=O, Z is absent, and R' is isoxazol-3-yl 20 substituted with a 4- to 6-membered nitrogen-containing Ci-6heterocycloalkyl. In certain such embodiments,

R

5 is isoxazol-3-yl substituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. In certain alternative such embodiments, Lis C=0, Z is absent, and R 5 is isoxazol-3-yl substituted with , wherein W is 0, NR, or CH 2 , and R is H or C I.6alkyl. In certain such embodiments, W is 0. 25 In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-3-yl substituted with 5-membered nitrogen-containing CI-6heteroaralkyl, such as pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably 1,2, 4 -triazol-5 ylmethyl. In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-3-yl 30 substituted with C,.6alkoxy or C.

6 alkoxyalkyl, preferably methoxy, ethoxy, methoxymethyl, or methoxyethyl. - 10 - In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-3-yl substituted with CI-shydroxyalkyl, preferably hydroxymethyl or hydroxyethyl. In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-3-yl substituted with a carboxylic acid, aminocarboxylate, CI-6alkylaminocarboxylate, 5 (CI.6alkyl)2aminocarboxylate, or Ci-6alkylcarboxylate. In certain such embodiments,

R

5 is substituted with methyl carboxylate or ethyl carboxylate, preferably methyl carboxylate. In certain embodiments, L is C=0, Z is absent, and R 5 is an unsubstituted isoxazol-5-yl. 10 In certain embodiments, L is C=O, Z is absent, and R 5 is a substituted isoxazol-5-yl. In certain such embodiments,

R

5 is isoxazol-5-yl substituted with a substituent selected from CI-6alkyl, CI.6alkoxy, CI.6alkoxyalkyl, Cr-6hydroxyalkyl, carboxylic acid, aminocarboxylate, CI6alkylaminocarboxylate,

(C

1 . 6alkyI)2aminocarboxylate, CI.6alkylcarboxylate, Ci-6heteroaralkyl,

C

1 .6aralkyl, Cj. 15 6heterocycloalkyl, and Ct-6carbocycloalkyl In certain preferred such embodiments R is isoxazole-3-yl substituted with a substituent selected from methyl, ethyl, isopropyl, and cyclopropylmethyl. In certain embodiments L is C=0, Z is absent, and R 5 is isoxazol-3-yl substituted with a 4- to 6 -membered nitrogen-containing CI-6heterocycloalkyl. In 20 certain such embodiments,

R

5 is isoxazol-5-yl substituted with azetidinylmetliyl, preferably azetidin-l-ylmethyl. In certain alternative such embodiments, L is C=0, Z is absent, and R 5 is isoxazol-3-yi substituted with wherein W is 0, NR, or CH 2 , and R is H or CI.alkyl. In certain such embodiments, W is 0. In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-5-yl 25 substituted with 5-membered nitrogen-containing Ci-..heteroaralkyl, such as pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably 1, 2

,

4 -triazol-5 ylmethyl. In certain embodiments, L is C=0, Z is absent, and R 5 is isoxazol-5-yl substituted with CI-6alkoxy or CI- 6 alkoxyalkyl, preferably methoxy, ethoxy, 30 methoxymethyl, or methoxyethyl. - 11 - In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-5-yl substituted with C, shydroxyalkyl, preferably hydroxymethyl or hydroxyethyl. In certain embodiments, L is C=O, Z is absent, and R 5 is isoxazol-3-yl substituted with a carboxylic acid, aminocarboxylate,

C

1 .6alkylaminocarboxylate, 5 (CI-6alkyl)2aminocarboxylate, or C I.6alkylcarboxylate. In certain such embodiments,

R

5 is substituted with methyl carboxylate or ethyl carboxylate, preferably methyl carboxylate. In certain embodiments, a compound of formula I is selected from O~ O--N0 OMe 0 -N O H

N

0 N 0 0 10 e 0 N-O 0 O,: / N 0 N 0--N O O Oj - 12 - OMe HY 0 0 -N

NI

OH 0 0 C 0 0 N1 0 (N .

0 -N 0 0 0 HHe H H0 0 Me 0 -N 0 0 OMe H 0 0 OMe 00 O-N 0 Q-e 0 0 Ome NM eOe H2NOC/, H H 0 0 -0M0 0 H~ N)H- 0 5 0 ThS SY 1 0 O0Me 0 OO 0e~ 0 Th e 0 Me mNIllki~ 0 N 0 0 '.e 00 S 0 N ome 0 Oe N'J Hy'- 14

-

0(H e 0 Me 0 0 0 -N 0 0 O-N 0 OMe 0 0 0 CN OH 00 -- /, i N -- r N O-N0 'O~ 0 0 N. 0 H0 NJ 0 0 N '.OMe 0 / 0 0 H 0 O-N O 0 0 -N O OMe 0 0 OMe 0 H 0 / C

H

2 N -N 0 -. 0 OOMe 5~~ ~ H2C- ~ 00 / 0 HO O-N 0 OMe HO-Me 0 - 16- 0 4H 0 N KJLC Et e:~ H0 ome OMe H 0 N~ N, N 0yH-, H0 O-N 0 00 0 ~ 0 N OMe 0

-

0 0 0 0 ~OMe0 0 0 /- j H -J O-- 17 0 0fH0 0 -N0 0 H 0 NJ N -F 0 2)N 0 '0Me 0 NN H 0 N, 4c N 0~

K

0 00 Q-4 0 NJ 1C 0 0 H~~N E 0 / I 0)H 0 0 - N NHN N0 0 0 N -N 0 C~ H 0 H 0 - N9 H1 0 -N N N N >JN0 N -N H 0.0 M .e H- 0 N'l CD- 0- N 0 H 00 o, 0 N N'YA-N 0 0 -N 0 0 HO 0 OMe 0 00 NN C H - H OMe 0 H 0i 0 NIK 0 -N 0 I10 0 0 O-N 0 -21 - N 0 00 OMe 0- O~ 0 0 IrAH H 0 0 0; N N N 0 *II p -H1j -N 0 0 00N 0 H 0 0 O -0 "10 0 0 H 0 00 HN NH H H 0 N 0 N 0 0 0H 0 0 no-H

H

O - 0 0 0 0 -N 0 -22 - 0 *- OMe 0 HI 0 0 -N 0 0 0 -.- CF 3 O-N0 0 0 0F FF 0 0 0 0 0

N

0 0 0H NI, 0 N ~ 0 / H H ~ H 0 -N 0 -o 0 -0 0H 0 OMe 0- j 0 0 0 H 0 H 0 -23 - 01 H 0 N ON O-N O 0 H 0 and One aspect of the invention relates to a medical device including 5 composition disclosed herein that include an inhibitor having a structure of formula I. In one embodiment, the composition is incorporated within a medical device. In certain embodiments, the medical device is a gel comprising a polymer matrix or ceramic matrix and an inhibitor. Said polymer can be either naturally occurring or synthetic. In another embodiment, said gel serves as a drug depot, an adhesive, a 10 suture, a barrier or a sealant. Another aspect of the invention relates to a medical device comprising a substrate having a surface onto which an inhibitor having a structure of formula I is disposed. In one embodiment, the inhibitor is directly disposed on a medical device. In another embodiment, a coating is so disposed, the coating comprising a polymer 15 matrix or ceramic matrix with an inhibitor having a structure of formula I dispersed or dissolved therein. In one embodiment, the medical device is a coronary, vascular, peripheral, or biliary stent. More particularly, the stent of the present. invention is an expandable stent. When coated with a matrix containing an inhibitor having a structure formula 20 I, the matrix is flexible to accommodate compressed and expanded states of such an expandable stent. In another embodiment of this invention, the stent has at least a portion which is insertable or implantable into the body of a patient, wherein the portion has a surface which is adapted for exposure to body tissue and wherein at -24 least a part of the surface is coated with an inhibitor having a structure of formula I, or a coating comprising a matrix having an inhibitor having a structure of formula I is dispersed or dissolved therein. An example of a suitable stent is disclosed in U.S. Pat. No. 4,733,665, which is incorporated herein by reference in its entirety. 5 In another embodiment, the medical device of the present invention is a surgical implement such as a vascular implant, an intraluminal device, surgical sealant or a vascular support. More particularly, the medical device of the present invention is a catheter, an implantable vascular access port, a central venous catheter, an arterial catheter, a vascular graft, an intraaortic balloon pump, a suture, a 10 ventricular assist pump,a drug-eluting barrier, an adhesive, a vascular wrap, an extra/perivascular support, a blood filter, or a filter adapted for deploymentin a blood vessel, coated with an inhibitor having a structure of formula I either directly or by a matrix containing an inhibitor having a structure of formula I. In certain embodiments, the intraluminal medical device is coated with an 15 inhibitor having a structure of formula I or a coating comprising biologically tolerated matrix and an inhibitor having a structure of formula I dispersed in the polymer, said device having an interior surface and an exterior surface, having the coating applied to at least a part of the interior surface, the exterior surface, or both. In certain embodiments, the medical device may be useful to prevent 20 restenosis after angioplasty. The medical device may also be useful for the treatment of various diseases and conditions by providing localized administration of an inhibitor having a structure of formula I. Such diseases and conditions include restenosis, inflammation, rheumatoid arthritis, tissue injury due to inflammation, hyperproliferative diseases, severe or arthritic psoriasis, muscle-wasting diseases, 25 chronic infectious diseases, abnormal immune response, conditions involving vulnerable plaques, injuries related to ischemic conditions, and viral infection and proliferation, Examples of diseases and conditions that are subject to a treatment including the drug coated medical devices of the present invention include atherosclerosis, acute coronary syndrome, Alzheimer's disease, cancer, fever, 30 muscle disuse (atrophy), denervation, vascular occlusions, stroke, HIV infection, nerve injury, renal failure associated with acidosis, and hepatic failure. See, e.g., Goldberg, United States Patent No. 5,340,736. -25- The term "Cx.yalkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluorornethyl and 2,2,2-trifluoroethyl, etc. Coalkyl indicates a hydrogen where the 5 group is in a terminal position, a bond if internal. The terms "C2.yalkenyl" and "C 2 yalkyny1" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. The term "alkoxy" refers to an alkyl group having an oxygen attached 10 thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxy. The term "Ci.6alkoxyalkyl" refers to a Ci_6alkyl group substituted with an 15 alkoxy group, thereby forming an ether. The term "Ci-aralkyl", as used herein, refers to a Cialkyl group substituted with an aryl group. The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be 20 represented by the general formulae: R9 R 0 --N or --- R1"

R

10 R10' wherein R 9 , R' 0 and R' 0 ' each independently represent a hydrogen, an alkyl, an alkenyl, -(CH 2 )m-R 8 , or R 9 and R' 0 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Ra 25 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or an integer from I to 8. In preferred embodiments, only one of R 9 or R 10 can be a carbonyl, e.g., R 9 , R'", and the nitrogen together do not form an imide. In even more preferred embodiments,

R

9 and R' 0 (and optionally

R'

0 ) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m-R8. In certain -26embodiments, the amino group is basic, meaning the protonated form has a pKa> 7.00. The terms "amide" and "amido" are art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: 0 5 14 wherein

R

9 , R' 0 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable. The term "aryl" as used herein includes 5-, 6-, and 7-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is 10 carbon. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the 15 like. The terms "carbocycle" and "carbocyclyl", as used herein, refer to a non aromatic substituted or unsubstituted ring in which each atom of the ring is carbon. The terms "carbocycle" and "carbocyclyl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two 20 adjoining rings wherein at least one of the rings is carbocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula: 0 0 'R oro 25 .or X Rl' wherein X is a bond or represents an oxygen or a sulfur, and R" represents a hydrogen, an alkyl, an alkenyl, -(CH2)m-R 8 or a pharmaceutically acceptable salt, - 27 - R" represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R 8 , where m and R 8 are as defined above. Where X is an oxygen and R" or R"' is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R" is a hydrogen, the formula represents a. "carboxylic acid". 5 As used herein, "enzyme" can be any partially or wholly proteinaceous molecule which carries out a chemical reaction in a catalytic manner. Such enzymes can be native enzymes, fusion enzymes, proenzymes, apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinated enzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linked enzymes, lipid-linked enzymes, prenylated 10 enzymes, naturally-occurring or artificially-generated mutant enzymes, enzymes with side chain or backbone modifications, enzymes having leader sequences, and enzymes complexed with non-proteinaceous material, such as proteoglycans, proteoliposomes. Enzymes can be made by any means, including natural expression, promoted expression, cloning, various solution-based and solid-based 15 peptide syntheses, and similar methods known to those of skill in the art. The term "C.rheteroaralkyl", as used herein, refers to a C.

6 alkyl group substituted with a heteroaryl group. The terms "heteroaryl" includes substituted or unsubstituted aromatic 5- to 7 membered ring structures, more preferably 5- to 6-membered rings, whose ring 20 structures include one to four heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, 25 for example, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, phosphorus, and sulfur. 30 The terms "heterocyclyl" or "heterocyclic group" refer to substituted or unsubstituted non-aromatic 3- to I 0-membered ring structures, more preferably 3- to - 28 - 7-membered rings, whose ring structures include one to four heteroatoms. The term terms "heterocyclyl" or "heterocyclic group" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic 5 rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. The term "Ci..heterocycloalkyl", as used herein, refers to a CI6alkyl group substituted with a heterocyclyl group. 10 The term "Ci.Thydroxyalkyl" refers to a Ci.

6 alkyl group substituted with a hydroxy group. As used herein, the term "inhibitor" is meant to describe a compound that blocks or reduces an activity of an enzyme (for example, inhibition of proteolytic cleavage of standard fluorogenic peptide substrates such as suc-LLVY-AMC, Box 15 LLR-AMC and Z-LLE-AMC, inhibition of various catalytic activities of the 20S proteasome). An inhibitor can act with competitive, uncompetitive, or noncompetitive inhibition. An inhibitor can bind reversibly or irreversibly, and therefore the term includes compounds that are suicide substrates of an enzyme. An inhibitor can modify one or more sites on or near the active site of the enzyme, or it 20 can cause a conformational change elsewhere on the enzyme. As used herein, the term "orally bioavailable" is meant to describe a compound administered to a mouse at 40 mg/kg or less, 20 mg/kg or less, or even 10 mg/kg or less, wherein one hour after oral administration such a compound shows at least about 50%, at least about 75% or even at least about 90% inhibition of 25 proteasome CT-L activity in the blood. As used herein, the term "peptide" includes not only standard amide linkage with standard a-substituents, but commonly utilized peptidomimetics, other modified linkages, non-naturally occurring side chains, and side chain modifications, as detailed below. 30 The terms "polycyclyl" or "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in -29which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted. The term "preventing" is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a 5 syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable 10 cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount. Prevention of an infection includes, for example, reducing the number of diagnoses of the 15 infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population. Prevention of pain includes, for example, reducing the magnitude of, or alternatively delaying, pain sensations experienced by subjects in a treated population versus an untreated control population. 20 The term "prodrug" encompasses compounds that, under physiological conditions, are converted into therapeutically active agents. A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule, In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. 25 The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, (i.e., it protects the host against developing the unwanted condition), 30 whereas if it is administered after manifestation of the unwanted condition, the -30treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). The term "proteasome" as used herein is meant to include immuno- and constitutive proteasomes. 5 The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that ."substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does 10 not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic 15 compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include, for example, a halogen, a 20 hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an 25 aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. A "therapeutically effective amount" of a compound with respect to the subject method of treatment, refers to an amount of the compound(s) in a 30 preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or -31 slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment. The term "thioether" refers to an alkyl group, as defined above, having a 5 sulfur moiety attached thereto. In preferred embodiments, the "thioether" is represented by -S-alkyl. Representative thioether groups include methylthio, ethylthio, and the like. As used herein, the term "treating" or "treatment" includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a 10 condition in manner to improve or stabilize a subject's condition. Selectivity for 20S Proteasome The enzyme inhibitors disclosed herein are useful in part because they inhibit the action of the 20S proteasome. Additionally, unlike other 20S proteasome inhibitors, the compounds disclosed herein are highly selective toward the 20S 15 proteasome, with respect to other protease enzymes. That is, the instant compounds show selectivities for the 20S proteasome over other proteases such as cathepsins, calpains, papain, chymotrypsin, trypsin, tripeptidyl pepsidase II. The selectivities of the enzyme inhibitors for 20S proteasome are such that at concentrations below about 50 AM, the enzyme inhibitors show inhibition of the catalytic activity of the 20 20S proteasome, while not showing inhibition of the catalytic activity of other proteases such as cathepsins, calpains, papain, chymotrypsin, trypsin, tripeptidyl pepsidase II. In preferred embodiments, the enzyme inhibitors show inhibition of the catalytic activity of the 20S proteasome at concentrations below about 10 AM, while not showing inhibition of the catalytic activity of other proteases at these 25 concentrations. In even more preferred embodiments, the enzyme inhibitors show inhibition of the catalytic activity of the 20S proteasome at concentrations below about I AM, while not showing inhibition of the catalytic activity of other proteases at these concentrations. Enzyme kinetic assays are disclosed in U.S. application serial number 09/569748, Example 2 and Stein et al., Biochem. (1996), 35, 3899 30 3908. -32- Selectivity for Chymotrvpsin-Like Activity Particular embodiments of the enzyme inhibiting compounds described herein are further useful because they can efficiently and selectively inhibit the chymotrypsin-like activity of the 20S proteasome, as compared to the trypsin-like, 5 and PGPH activities. The chymotrypsin-like activity of 20S proteasome is characterized by cleavage of peptides in the immediate vicinity of large hydrophobic residues. In particular, the chymotrypsin-like activity of Ntn hydrolases can be determined by cleavage of a standard substrate. Examples of such substrates are known in the art. For example, a leucylvalinyltyrosine derivative can be used. 10 Enzyme kinetic assays are disclosed in U.S. application serial number 09/569748, Example 2 and Stein et al., Biochen. (1996), 35, 3899-3908. Uses of Enzyme Inhibitors The biological consequences of proteasome inhibition are numerous. Proteasome inhibition has-been suggested as a prevention and/or treatment of a 15 multitude of diseases including, but not limited to, proliferative diseases, neurotoxic/degenerative diseases, Alzheimer's, ischemic conditions, inflammation, immune-related diseases, HIV, cancers, organ graft rejection, septic shock, inhibition of antigen presentation, decreasing viral gene expression, parasitic infections, conditions associated with acidosis, macular degeneration, pulmonary 20 conditions, muscle wasting diseases, fibrotic diseases, bone and hair growth diseases. Therefore, proteasome inhibitor compositions, such as the orally bioavailable peptide epoxy ketone class of molecules as described herein, provide a means of treating patients with these conditions. Proteasome inhibitor compositions may be used to treat conditions mediated 25 directly by the proteolytic function of the proteasome such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome such as NF cB. The proteasome participates in the rapid elimination and post-translational processing of proteins (e.g., enzymes) involved in cellular regulation (e.g., cell cycle, gene transcription, and metabolic pathways), intercellular communication, 30 and the immune response (e.g., antigen presentation). Specific examples discussed -33 below include 0-amyloid protein and regulatory proteins such as cyclins and transcription factor NF-KB. At the cellular level, the accumulation of polyubiquitinated proteins, cell morphological changes, and apoptosis have been reported upon treatment of cells 5 with various proteasoine inhibitors. The proteasome degrades many proteins in maturing reticulocytes and growing fibroblasts. In cells deprived of insulin or serum, the rate of proteolysis nearly doubles. Inhibiting the proteasome reduces proteolysis, thereby reducing both muscle protein loss and the nitrogenous load on kidneys or liver. One aspect of the invention relates to the treatment of cachexia and 10 muscle-wasting diseases. Compounds of the invention may be useful for treating conditions such as cancer, chronic infectious diseases, fever, muscle disuse (atrophy) and denervation, nerve injury, fasting, renal failure associated with acidosis, and hepatic failure. See, e.g., Goldberg, U.S. Patent No. 5,340,736. Certain embodiments of the invention therefore encompass compositions for: reducing the 15 rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation; reducing the rate of degradation of p53 protein in a cell; and inhibiting the growth of p53-related cancers. Each of these methods includes contacting a cell (in vivo or in vitro, e.g., a muscle in a subject) with an effective amount of a pharmaceutical composition comprising a proteasome inhibitor disclosed herein. 20 Intracellular proteolysis generates small peptides for presentation to T lymphocytes to induce MHC class I-mediated immune responses. The immune system screens for autologous cells that are virally infected or have undergone oncogenic transformation. In certain embodiments, the invention relates to a method for inhibiting antigen presentation in a cell, comprising exposing the cell to a 25 compound described herein. Proteasome inhibitors of the invention may be used to treat immune-related conditions such as allergy, asthma, organ/tissue rejection (graft-versus-host disease), and auto-immune diseases, including, but not limited to, lupus, rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). Thus, in certain 30 embodiments, the invention relates to a method for suppressing the immune system of a subject comprising administering to the subject an effective amount of a proteasome inhibitor compound described herein. -34- In certain embodiments, the invention relates to a method for altering the repertoire of antigenic peptides produced by the proteasome or other Ntn with multicatalytic activity. For example, if the PGPH activity of 20S proteasome is selectively inhibited, a different set of antigenic peptides will be produced by the 5 proteasome and presented in MHC molecules on the surfaces of cells than would be produced and presented either without any enzyme inhibition, or with, for example, selective inhibition of chymotrypsin-like activity of the proteasome. Another aspect of the invention relates to the use of proteasome inhibitor compositions disclosed herein for the treatment of neurodegenerative diseases and 10 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 15 Syndrome), HIV/AIDS dementia complex, axonomy, diabetic neuropathy, Parkinson'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 20 atrophy syndromes (such as primary aphasia), metabolic-toxic dementias (such as chronic hypothyroidism or B12 deficiency), and dementias caused by infections (such as syphilis or chronic meningitis). Alzheimer's disease is characterized by extracellular deposits of g-amyloid protein (0-AP) in senile plaques and cerebral vessels. -AP is a peptide fragment of 25 39 to 42 amino acids derived from an amyloid protein precursor (APP). At least three isoforms of APP are known (695, 751, and 770 amino acids). Alternative splicing of mRNA generates the isoforms; normal processing affects a portion of the P-AP sequence, thereby preventing the generation of P-AP. It is believed that abnormal protein processing by the proteasome contributes to the abundance of p 30 AP in the Alzheimer brain.. The APP-processing enzyme in rats contains about ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has an N-terminal - 35 sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the p subunit of human macropain (Kojima, S. et aL., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleaves at the Gln1 5 -- Lys' 6 bond; in the presence of calcium ion, the enzyme also cleaves at the Met-'--Asp' bond, and the 5 Asp'--Ala 2 bonds to release the extracellular domain of P-AP. One aspect of the invention, therefore, relates to a method of treating Alzheimer's disease, comprising administering to a subject an effective amount of a proteasome inhibitor composition disclosed herein. Such treatment includes ~ reducing the rate of P-AP processing, reducing the rate of P-AP plaque formation, 10 reducing the rate of P-AP generation, and reducing the clinical signs of Alzheimer's disease. Fibrosis is the excessive and persistent formation of scar tissue resulting from the hyperproliferative growth of fibroblasts and is associated with activation of the TGF-P signaling pathway. Fibrosis involves extensive deposition of 15 extracellular matrix and can occur within virtually any tissue or across several different tissues. Normally, the level of intracellular signaling protein (Smad) that activate transcription of target genes upon TGF-P stimulation is regulated by proteasome activity (Xu et al., 2000). However, accelerated degradation of the TGF-P signaling components has been observed in cancers and other 20 hyperproliferative conditions. Thus, in certain embodiments the invention relates to a method for treating hyperproliferative conditions such as diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonary fibrosis, collagen vascular disease, 25 sarcoidosis, interstitial lung diseases and extrinsic lung disorders). The treatment of burn victims is often hampered by fibrosis, thus, in certain embodiments, the invention relates to the topical or systemic administration of the inhibitors to treat burns. Wound closure following surgery is often associated with disfiguring scars, which may be prevented by inhibition of fibrosis. Thus, in certain embodiments, the 30 invention relates to a method for the prevention or reduction of scarring. - 36 - Certain proteasome inhibitors block both degradation and processing of ubiquitinated NF-KB in vitro and in vivo. Proteasome inhibitors also block IKB-A degradation and NF-KB activation (Palombella, et a]. Cell (1994) 78:773-785; and Traenckner, et a., EMBO J. (1994) 13:5433-5441). One aspect of the invention 5 relates to a method for inhibiting IxiB-a degradation, comprising contacting the cell with a compound described herein. rn certain embodiments, the invention relates to a method for reducing the cellular content of NF-xB in a cell, muscle, organ, or subject, comprising contacting the cell, muscle, organ, or subject with a proteasome inhibitor compound described herein. 10 NF-KB is a member of the Re! protein family. The Rel family of transcriptional activator proteins can be divided into two groups. The first group requires proteolytic processing, and includes p50 (NF-KB 1, 105 kDa) and p52 (NF 2, 100 kDa). The second group does not require proteolytic processing, and includes p65 (RelA, Rel (c-Rel), and ReIB). Both homo- and heterodimers can be 15 formed by Rel family members; NF-KB, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IKB and p105, the two proteins are degraded and processed, respectively, to produce active NF-KB which translocates from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed by purified proteasomes (Palombella et al., Cell (1994) 78:773-785). Active NF-rB forms a 20 stereospecific enhancer complex with other transcriptional activators and, e.g., HMG I(Y), inducing selective expression of a particular gene. NF-KB regulates genes involved in the immune and inflammatory response, and mitotic events. For example, NF-icB is required for the expression of the immunoglobulin light chain K gene, the IL-2 receptor c-chain gene, the class I major 25 histocompatibility complex gene, and a number of cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony-stimulating factor, and IFN-P (Palombella et al., Cell (1994) 78:773-785). In certain embodiments, the invention relates to methods of affecting the level of expression of IL-2, MHC-I, IL-6, TNFa, IFN-3 or any of the other previously-mentioned proteins, each method comprising 30 administering to a subject an effective amount of a proteasome inhibitor composition disclosed herein. Complexes including p50 are rapid mediators of acute - 37 inflammatory and immune responses (Thanos, D. and Maniatis, T., Cell (1995) 80:529-532). Overproduction of lipopolysaccharide (LPS)-induced cytokines such as TNFa is considered to be central to the processes associated with septic shock. 5 Furthermore, it is generally accepted that the first step in the activation of cells by LPS is the binding of LPS to specific membrane receptors. The a- and P-subunits of the 20S proteasone complex have been identified as LPS-binding proteins, suggesting that the LPS-induced signal transduction may be an important therapeutic target in the treatment or prevention of sepsis (Qureshi, N. et al., J. Inmun. (2003) 10 171: 1515-1525). Therefore, in certain embodiments, the proteasome inhibitor compositions may be used for the inhibition of TNFct to prevent and/or treat septic shock. NF-KB also participates in the expression of the cell adhesion genes that encode E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab. Invest. (1993) 15 68:499-508). In cetain embodiments the invention relates to a method for inhibiting cell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin, ICAM, or VCAM-1), comprising contacting a cell with (or administering to a subject) an effective amount of a pharmaceutical composition comprising a proteasome inhibitor disclosed herein. 20 NF-rB also binds specifically to the HIV-enhancer/promoter. When compared to the Nef of mac239, the HIV regulatory protein Nef of pbj 14 differs by two amino acids in the region which controls protein kinase binding. It is believed that the protein kinase signals the phosphorylation of IxB, triggering 1cB degradation through the ubiquitin-proteasome pathway. After degradation, NF-KB 25 is released into the nucleus, thus enhancing the transcription of HIV (Cohen, J., Science, (1995) 267:960). In certain embodiments, the invention relates to a method for inhibiting or reducing HIV infection in a subject, or a method for decreasing the level of viral gene expression, each method comprising administering to the subject an effective amount of a proteasome inhibitor composition disclosed herein. 30 Viral infections contribute to the pathology of many diseases. Heart conditions such as ongoing myocarditis and dilated cardiomyopathy have been -38linked to the coxsackievirus B3. In a comparative whole-genome microarray analyses of infected mouse hearts, specific proteasome subunits were uniformly up regulated in hearts of mice which developed chronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Some viruses utilize the ubiquitin-proteasome system in 5 the viral entry step where the virus is released from the endosome into the cytosol. The mouse hepatitis virus (MHV) belongs to the Coronaviridae family, which also includes the severe acute respiratory syndrome (SARS) coronvirus. Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment of cells infected with MHV with a proteasome inhibitor resulted in a decrease in viral replication, correlating 10 with reduced viral titer as compared to that of untreated cells. The human hepatitis B virus (HBV), a member of the Hepadnaviridae virus family, likewise requires virally encoded envelop proteins to propagate. Inhibiting the proteasome degradation pathway causes a significant reduction in the amount of secreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005). In addition to 15 HBV, other hepatitis viruses (A, C, D and E) may also utilize the ubiquitin proteasome degradation pathway for secretion, morphogenesis and pathogenesis. Accordingly, in certain embodiments, the invention relates to a method for treating viral infection, such as SARS or hepatitis A, B, C, D and B, comprising contacting a cell with (or administering to a subject) an effective amount of a compound 20 disclosed herein. Ischemia and reperfusion injury results in hypoxia, a condition in which there is a deficiency of oxygen reaching the tissues of the body. This condition causes increased degradation of IK-BaX, thereby resulting in the activation of NF-KB (Koong et al., 1994). It has been demonstrated that the severity of injury resulting in 25 hypoxia can be reduced with the administration of a proteasome inhibitor (Gao et al., 2000; Bao et al., 2001; Pye et al., 2003). Therefore, certain embodiments of the invention relate to a method of treating an ischemic condition or reperfusion injury comprising administering to a subject in need of such treatment an effective amount of a proteasome inhibitor compound disclosed herein. Examples of such conditions 30 or injuries include, but are not limited to, acute coronary syndrome (vulnerable plaques), arterial occlusive disease (cardiac, cerebral, peripheral arterial and vascular - 39 occlusions), atherosclerosis (coronary sclerosis, coronary artery disease), infarctions, heart failure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis. Other eukaryotic transcription factors that require proteolytic processing include the general transcription factor TFIIA, herpes simplex virus VP1 6 accessory 5 protein (host cell factor), virus-inducible IFN regulatory factor 2 protein, and the membrane-bound sterol regulatory element-binding protein L In certain embodiments, the invention relates to methods for affecting cyclin dependent eukaryotic cell cycles, comprising exposing a cell (in vitro or in vivo) to a proteasome inhibitor composition disclosed herein. Cyclins are proteins involved in 10 cell cycle control. The proteasome participates in the degradation of cyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, and cyclin B. Degradation of cyclins enables a cell to exit one cell cycle stage (e.g., mitosis) and enter another (e.g., division). It is believed all cyclins are associated with p 34 cdc2 protein kinase or related kinases. The proteolysis targeting signal is localized to amino acids 42 15 RAALGNISEN-50 (destruction box). There is evidence that cyclin is converted to a form vulnerable to a ubiquitin ligase or that a cyclin-specific ligase is activated during mitosis (Ciechanover, A., Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclin degradation, and therefore inhibits cell proliferation, for example, in cyclin-related cancers (Kumatori et al., Proc. Nat]. Acad. Sci. USA 20 (1990) 87:7071-7075). One aspect of the invention relates to a method for treating a proliferative disease in a subject (e.g., cancer, psoriasis, or restenosis), comprising administering to the subject an effective amount of a proteasome inhibitor composition disclosed herein. The invention also relates to a method for treating cyclin-related inflammation in a subject, comprising adminstering to a subject a 25 therapeutically effective amount of a proteasome inhibitor composition described herein. Additional embodiments of the invention relate to methods for affecting the proteasome-dependent regulation of oncoproteins and methods of treating or inhibiting cancer growth, each method comprising exposing a cell (in vivo, e.g., in a 30 subject, or in vitro) to a proteasome inhibitor composition disclosed herein. HPV-16 and HPV- 1 8-derived E6 proteins stimulate ATP-and ubiquitin-dependent - 40 conjugation and degradation of p53 in crude reticulocyte lysates. The recessive oncogene p53 has been shown to accumulate at the nonpermissive temperature in a cell line with a mutated thermolabile El. Elevated levels of p53 may lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system 5 include c-Mos, c-Fos, and c-Jun. In certain embodiments, the invention relates to a method for treating p53-related apoptosis, comprising administering to a subject an effective. amount of a proteasome inhibitor composition disclosed herein. In certain embodiments, the disclosed compositions may be useful for the treatment of a parasitic infection, such as infections caused by protozoan parasites. 10 The proteasome of these parasites is considered to be involved primarily in cell differentiation and replication activities (Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore, entamoeba species have been shown to lose encystation capacity when exposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res. 1997, 28, Spec No: 139-140). In certain such embodiments, the administrative 15 protocols for the proteasome inhibitor compositions are useful for the treatment of parasitic infections in humans caused by a protozoan parasite selected from Plasmodium sps. (including P. falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria), Trypanosoma sps. (including T. cruzi, which causes Chagas' disease, and T. brucei which causes African sleeping sickness), Leishmania sps. (including 20 L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.), Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS and other immunosuppressed patients), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia. In certain embodiments, the disclosed proteasome inhibitor compositions are useful for the treatment of parasitic infections in animals and 25 livestock caused by a protozoan parasite selected from Plasmodium hermani, Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic diseases are described in WO 98/10779, which is incorporated herein in its entirety. 30 In certain embodiments, the proteasome inhibitor compositions inhibit proteasome activity in a parasite without recovery in red blood cells and white blood cells. In certain such embodiments, the long half-life of blood cells may provide -41prolonged protection with regard to therapy against recurring exposures to parasites. In certain embodiments, the proteasome inhibitor compositions may provide prolonged protection with regard to chemoprophylaxis against future infection. It has also been demonstrated that inhibitors that bind to the 20S proteasome 5 stimulate bone formation in bone organ cultures. Furthermore, when such inhibitors have been administered systemically to mice, certain proteasome inhibitors increased bone volume and bone formation rates over 70% (Garrett, 1. R. et al., J. Clin. Invest. (2003) 111: 1771-1782), therefore suggesting that the ubiquitin proteasome machinery regulates osteoblast differentiation and bone formation. 10 Therefore, the disclosed proteasome inhibitor compositions may be useful in the treatment and/or prevention of diseases associated with bone loss, such as osteroporosis. Proteasome inhibition has already been validated as a therapeutic strategy for the treatment of cancer, particularly multiple myeloma. However, based on both in 15 vitro and in vivo models, one would predict that it could serve as a strategy against other cancers, particularly heme-related malignancies and solid tumors. Therefore, certain embodiments of the invention relate to a method of treating cancers comprising administering to a subject in need of such treatment an effective amount of a proteasome inhibitor compound disclosed herein. 20 Administration Compounds prepared as described herein can be administered in various forms, depending on the disorder to be treated and the age, condition, and body weight of the patient, as is well known in the art. For example, where the compounds are to be administered orally, they may be formulated as tablets, 25 capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories. For application by the ophthalmic mucous membrane route, they may be formulated as eye drops or eye ointments. These formulations can be prepared by conventional means, and if desired, the active ingredient may be 30 mixed with any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying -42 agent, a coating agent, a cyclodextrin, and/or a buffer. Although the dosage will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2000 mg of the 5 compound is recommended for an adult human patient, and this may be administered in a single dose or in divided doses. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. The precise time of administration and/or amount of the composition that 10 will yield the most effective results in terms of efficacy of treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), route of administration, etc. However, the above guidelines 15 can be used as the basis for fine-tuning the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing. The phrase "pharmaceutically acceptable" is employed herein to refer to 20 those ligands, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a 25 pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, 30 glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted 0-cyclodextrin; (3) cellulose, and its derivatives, such as sodium -43 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) 5 polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in 10 pharmaceutical formulations. In certain embodiments, pharmaceutical compositions of the present invention are non-pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient. The term "pharmaceutically acceptable salt" refers to the relatively non toxic, inorganic and organic acid addition salts of the inhibitor(s). These salts can be 15 prepared in situ during the final isolation and purification of the inhibitor(s), or by separately reacting a purified inhibitor(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, 20 maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate salts, and amino acid salts, and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19.) In other cases, the inhibitors useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming 25 pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non toxic inorganic and organic base addition salts of an inhibitor(s). These salts can likewise be prepared in situ during the final isolation and purification of the inhibitor(s), or by separately reacting the purified inhibitor(s) in its free acid form 30 with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or -44 alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylanine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et 5 aL., supra). Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives and antioxidants can also be present in the compositions. 10 Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal 15 chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or 20 non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert matrix, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes, and the like, each containing a predetermined amount of an inhibitor(s) as an active ingredient. A composition may also be administered as a bolus, electuary, or paste. 25 In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, cyclodextrins, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for 30 example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as - 45 agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example,-acetyl-alcohol-and glycelo-mon-osalit C-(8)Talioents uch as 5 kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using 10 such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, 15 preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered inhibitor(s) moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills, and 20 granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, 25 liposomes, and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release 30 the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The -46active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. 5 In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, 10 and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents. 15 Suspensions, in addition to the active inhibitor(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Formulations for rectal or vaginal administration may be presented as a 20 suppository, which may be prepared by mixing one or more inhibitor(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. 25 Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of an inhibitor(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, 30 and inhalants. The active component may be mixed under sterile conditions with a - 47 pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams, and gels may contain, in addition to inhibitor(s), excipients, such as animal and vegetable fats, oils, waxes, paraffins, 5 starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an inhibitor(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain 10 customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. The inhibitor(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the composition. A nonaqueous (e.g., fluorocarbon propellant) 15 suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of 20 the particular composition, but typically include nonionic surfactants (Tweens, Pluronics, sorbitan esters, lecithin, Cremophors), phannaceutically acceptable co solvents such as polyethylene glycol, innocuous proteins like serum albumin, oleic acid, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols generally are prepared from isotonic solutions. 25 Transdermal patches have the added advantage of providing controlled delivery of an inhibitor(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the inhibitor(s) across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing 30 the inhibitor(s) in a polymer matrix or gel. -48- Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more inhibitors(s) in combination with one or more pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile 5 injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, 10 ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. 15 These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include tonicity-adjusting agents, such as sugars, 20 sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow 25 the absorption of the drug from subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of inhibitor(s) in biodegradable polymers such as polylactide-polyglycolide. 30 Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other -49 biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The preparations of agents may be given orally, parenterally, topically, or 5 rectally. They are, of course, given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, infusion; topically by lotion or ointment; and rectally by suppositories. Oral administration is preferred. The phrases "parenteral administration" and "administered parenterally" as 10 used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection, and infusion. 15 The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a ligand, drug, or other material other than directly into the central nervous system, such that it enters the patient's system and thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 20 These inhibitors(s) may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracistemally, and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the inhibitor(s), which 25 may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active 30 ingredient which is effective to achieve the desired therapeutic response for a - 50 particular patient, composition, and mode of administration, without being toxic to the patient. The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the 5 compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration. In general, the compositions of this invention may be provided in an aqueous solution containing about 0.1-10% w/v of a compound disclosed herein, among other substances, for parenteral administration. Typical dose ranges are from about 0.01 to about 50 10 mg/kg of body weight per day, given in 1-4 divided doses. Each divided dose may contain the same or different compounds of the invention. The dosage will be an effective amount depending on several factors including the overall health of a patient, and the formulation and route of administration of the selected compound(s). Another aspect of the invention provides a conjoint therapy wherein one or 15 more other therapeutic agents are administered with the proteasome inhibitor. Such conjoint treatment may be achieved by way of the simultaneous, sequential, or separate dosing of the individual components of the treatment. In certain embodiments, a compound of the invention is conjointly administered with one or more other proteasome inhibitor(s). 20 In certain embodiments, a compound of the invention is conjointly administered with a chemotherapeutic. Suitable chemotherapeutics may include, natural products such as vinca alkaloids (i.e. vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e. etoposide, teniposide), antibiotics (dactinomycin (actinomycin D) daunorubicin, doxorubicin and 25 idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin, enzymes (L-asparaginase which systemically metabolizes

L

asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, 30 chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, - 51 streptozocin), trazenes - dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2 -chlorodeoxyadenosine); aromatase 5 inhibitors (anastrozole, exemestane, and letrozole); and platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; histone deacetylase (HDAC) inhibitors; hormones (i.e. estrogen) and hormone agonists such as leutinizing hormone releasing hormone (LHRH) agonists (goserelin, leuprolide and triptorelin). Other chemotherapeutic agents may 10 include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analog or derivative variant of the foregoing. In certain embodiments, a compound of the invention is conjointly administered with a cytokine. Cytokines include, but are not limited to, Interferon y, -a, and -0, Interleukins 1-8, 10 and 12, Granulocyte Monocyte Colony 15 Stimulating factor (GM-CSF), TNF-a and -0, and TGF-p. In certain embodiments, a compound of the invention is conjointly administered with a steroid. Suitable steroids may include, but are not limited to, 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, 20 corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, 25 halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25 diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, 30 triamcinolone benetonide, triamcinolone hexacetonide, and salts and/or derivatives thereof. -52- In certain embodiments, a compound of the invention is conjointly administered with an immunotherapeutic agent. Suitable immunotherapeutic agents may include, but are not limited to, MDR modulators (verapamil, valspordar, biricodar, tariquidar, laniquidar), rapamycin, mycophenylate mofetil, 5 cyclophosomide, cyclosporine, thalidomide, and monoclonal antibodies. The monoclonal antibodies can be either naked or conjugated such as rituximab, tositumomab, alemtuzumab, daclizumab, epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib and trastuzumab. Exemplification 10 Example 1 Scheme 1: Synthesis of Compound 010 OMe Ph OMe BOCNH OH + HN O n HOBT, H 'J * Bo N O Pd/C, H H O DIEA. THF. 000 8-: .01C 0 0 Ph Ph 002 003 004 CbrHN TFA, OC HzNF F C BcHN f N N DCM0C H20 DIEA TH R DC Mh o Ph o-N Ph 005 006 007 008 009 010 Compound (003): To a O'C solution of N-Boc serine(methyl ether) (00 1) (2.5 g, 11.4 mmol), 15 L-alanine benzyl ester hydrochloride (002) (3.3 g, 11.4 mmol), HOBT (2.5 g, 18.2 mmol) and HBTU (6.9 g, 18.24 mmol) in tetrahydrofuran (400 mL) was added a solution of N,N-diisopropylethylamine (8.0 mL, 45.6 mmol) in tetrahydrofuran (50 mL) over 10 minutes. The mixture was stirred at room temperature for another 5 hours. Most of the solvents were removed under reduced pressure and the resulting 20 material diluted with ethyl acetate (300 mL). The solution was then washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (100 mL). The organic - 53 layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate), and the desired compound (003) (4.4 g) was isolated and characterized by LC/MS (LCRS (MH) m/z: 457.23). 5 Compound (004): To a 0*C solution of (003) (5.14 g, 11.25 mmol) in tetrahydrofuran (100 mL) was added 10% Pd/C (500 mg). The resulting mixture was allowed to stir under 1 atmosphere of hydrogen for 4 hours. The mixture was filtered through Celite-545 and the filter cake was washed with tetrahydrofuran. The organic filtrate was 10 concentrated under reduced pressure and placed under high vacuum for 2 hours to provide (004) as confirmed by LC/MS (LCRS (MH) m/z: 367.18) which was used without further purification. Compound (006): To a solution of (005) (for a synthesis of (005) see U.S. Patent Application 15 Serial No. 11/131,688) (3.9 g, 13 mmol) in trifluoroacetic acid (50 mL) was added 10 % Pd/C (600 mg). The resulting mixture was allowed to stir under I atmosphere of hydrogen for 6 hours. The mixture was filtered through Celite-545 and the filter cake washed with dichloromethane (200 mL). The filtrate was concentrated under reduced pressure and placed under high vacuum overnight to provide (006) as 20 confirmed by LC/MS (LCRS (MH) m/z: 172.13) and was used in the subsequent transformation without further purification. Compound (007): To a 0*C solution of (004) and (006), HOBT (2.5 g, 18 mmol) and HBTU (6.9 g, 18 mmol) in tetrahydrofuran (400 mL) was added a solution of N,N 25 diethylisopropylamine (8 mL, 46 minol) in tetrahydrofuran (50 mL) over 10 minutes. The mixture was stirred at room temperature for another 5 hours. Most of the solvents were removed under reduced pressure and the remaining material was diluted with ethyl acetate (400 mL). The resulting solution was washed with saturated aqueous sodium bicarbonate (2 x 100 mL) and brine (100 mL). The 30 organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash -54chromatography (hexane and ethyl acetate), to provide (007) (3.5 g) as characterized by LC/MS (LCRS (MH) m/z: 520.29). Compound (008): To a 0*C solution of (007) (320 mg, 0.616 mmol) in dichloromethane (10 5 mL) was added trifluoroacetic acid (10 mL) and the resulting solution was stirred at the same temperature for another hour. The organic layers were concentrated under reduced pressure and then placed under high vacuum for 2 hours to provide (008) as confirmed by LC/MS (LCRS (MH) m/z: 420.24) which was used without further purification. 10 Compound (010): To a 0*C solution of (008), 5-methyl-isoxazole-3-carboxylic acid (009) (94 mg, 0.74 mmol), HOBT (135 mg, 1.0 mmol).and HBTU (350 mg, 1.0 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (2 mL) over 5 minutes. The mixture was stirred at 15 room temperature for another 5 hours and then diluted with ethyl acetate (200 mL). It was then washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL) and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to 20 provide (010) (195 mg) as characterized by LC/MS (LCRS (MH) m/z: 529.26); >90% proteasome CT-L inhibition at 40 mg/kg PO. - 55 - Example 2 Scheme 2: Synthesis of Example 023 0 Ph Ph + Hz 2

N

0 I IF, 0C 0oN nBcHO 011 012 013 014 Ph (006) 0 4 I~iO TAHo~ HOST. HBTU OOCH-Ny MNN Nj( 0CM O DIEA, Tl. f-C B0 0CM 0*C H Ph Ph 015 016 0H 0 OMBS (PhCO) 2 OMeU (aq.) O Br O0n Mo oline CC34U-lO 80V E3Br /N H.rtB, TW-. fl 017 018 019 020 OBn Pdc- H0 OH (016) / HF a (,Noa-.BT U lN TyN DIEA. THF. O'C N O-N o 0 021 022 023 Compound (013): 5 To a 0*C solution of N-Boc L-leucine (011) (2.6 g, 11 mmol),

L

phenylalanine benzyl ester hydrochloride (012) (2.9 g, 10 mmol), HOBT (1.7 g, 1 mmol) and HBTU (3.9 g, 11 mmol) in tetrahydrofuran (200 mL) was added a solution of N,N-diisopropylethylamine (4.9 mL, 30 mmol) in tetrahydrofuran (10 mL) over 5 minutes. The mixture was stirred at room temperature for another 5 10 hours and became homogenous. It was then diluted with ethyl acetate (300 mL) and washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (100 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (013) (4.4 g) as 15 characterized by LC/MS (LCRS (MH) m/z: 469.26). Compound (014): To a 0*C solution of (013) (4.32 g, 9.24 mmol) in tetrahydrofuran (100 mL) was added 10% Pd/C (500 mg). The resulting mixture was allowed to stir under I -56atmosphere of hydrogen for 4 hours. The mixture was filtered through Celite-545 and the filter cake was washed with tetrahydrofuran. The organic filtrate was concentrated under reduced pressure and placed under high vacuum to provide (014) (3.5 g) as confirmed by LC/MS (LCRS (MH) m/z: 378.22) which was used without 5 further purification. Compound (015): To a 0*C solution of (014) (3.5 g, 9.24 mmol) and (006) (2.4 g, 11 mmol), HOBT (1.7 g, 11 mmol) and HBTU (3.9 g, 11 mmol) in tetrahydrofuran (200 mL) was added a solution of N,N-diisopropylethylamine (4.9 mL, 30 mmol) in 10 tetrahydrofuran (10 mL) over 5 minutes. The mixture was stirred at room temperature for another 5 hours and became homogenous. It was then diluted with ethyl acetate (400 mL), and washed with saturated aqueous sodium bicarbonate (2 x 100 mL) and brine (100 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and 15 the residue was purified by flash chromatography (hexane and ethyl acetate), and the desired compound (015) (5.0 g) was isolated and characterized by LC/MS (LCRS (MH) m/z: 532.33). . Compound (016): To a 0*C solution of (015) (5.0 g, 9.40 mmol) in dichloromethane (50 mL) 20 was added trifluoroacetic acid (20 mL) over 5 minutes, and the resulting solution was stirred at the same temperature for another hour. The organic layers were concentrated under reduced pressure and placed under high vacuum to provide (016) as confirmed by LC/MS (LCRS (MH) m/z: 432.33) which was used without further purification. 25 Compound (018): To a solution of methyl 5-methyl-3-isoxazolecarboxylate (017) (14.lg, 100 mmol) in carbon tetrachloride (500 mL) was added N-bromosuccunimide (23 g, 130 mmol) and benzoyl peroxide (2.5 g, 10 mmol) at room temperature. The resulting mixture was stirred at 80 0 C under an atmosphere of argon ovemight. The reaction 30 was the cooled and diluted with 500 mL of dichloromethane and washed with saturated aqueous sodium bicarbonate (3 x 100 mL). The aqueous phase was -57extracted with 200 mL of dichloromethane, and the combined organic layers were washed with brine and dried over MgSO 4 . The solvents were removed and the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (018) (7.9 g) which was characterized by LC/MS (LCRS (MH) m/z: 219.95). 5 Compound (019): To a 0*C solution of (018) (12 g, 55 mmol) in tetrahydrofuran (20 mL) was added aqueous lithium hydroxide (35 mL, 4N). The resulting mixture was stirred at room temperature overnight. It was then acidified with hydrochloric acid (2N) to pH =1 and extracted with tetrahydrofuran (3 x 200 mL). The combine organic layers 10 were washed with brine (10 mL), dried over sodium sulfate, and filtered. The solvents were removed and the residue was lyophilized to yield (019) (8.2 g), which was confirmed by LC/MS (LCRS (MH) m/z: 205.95) and used without further purification. Compound (020): 15 A solution of (019) (6.0 g, 30 mmol), benzyl alcohol (3.5 mL), and p toluenesulfonyl acid (1.lg, 6 mmol) in toluene (100 mL) was stirred at 100*C overnight. It was then allowed to cool, diluted with 300 mL of ethyl acetate and washed with saturated aqueous sodium bicarbonate. The aqueous phase was then extracted with 200 mL of ethyl acetate. The combine organic layers were washed 20 with brine, dried over sodium sulfate, and filtered. The solvents were removed and the residue was purified by flash chromatography (hexane and ethyl acetate), to provide (020) (5.8 g) which was characterized by LC/MS (LCRS (MH) m/z: 295.98). Compound (021): 25 A solution of (020) (2.0 g, 6.8 mmol) and morpholine (3.0 mL) in tetrahydrofuran (50 mL) was stirred at room temperature for two hours. The solvents were then removed and the residue was purified by flash chromatography (hexane and ethyl acetate/methanol) to provide (021) (820 mg) which was characterized by LC/MS (LCRS (MH) m/z: 303.13). 30 Compound (022): - 58 - To a solution of (021) (400 mg, 1.32 mmol) in tetrahydrofuran (40 nL) was added 10 %Pd/C (100 mg) and the mixture was stirred at room temperature under one atmosphere of hydrogen for 2 hours. It was then filtered through Celite and concentrated to give (022), which was confirmed by LC/MS (LCRS (MH) m/z: 5 213.08) and was used without further purification. Compound (023): To a 0*C solution of (016) (130 mg, 0.3 mmol) and (022) (70 mg, 0.4 mmol), HOBT (70 mg, 0.5 mmol) and HBTU (170 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in 10 tetrahydrofuran (5 mL). The mixture was stirred at room temperature for another 5 hours and became homogenous. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by 15 HPLC (aqueous ammonium acetate and acetonitrile) to provide compound (023) (125 mg) which was characterized by LC/MS (LCRS (MH) m/z: 626.35); >80% proteasome CT-L inhibition at 40 mg/kg PO. - 59 - Example 3 Scheme 3: Synthesis of Example 029 HMPB rei raH 0 1. 2%/ Piperldine Wer OHFoHN)~ 2. Fmnoc-Ser(oMe,.CH rogq N FmocHNSO H B 2F H(M) FmocHN Z O DFHBTU. DMF 0 024 025 026 O0M e e 1. 20% Piperldine 0A Me 0 ML OH DIA, H DC C OH HBTU, OMF 0-N 0 z- 0;, (009) 027 0 027 028 Me (006), HOBT, HBTU H DIEA, THF, 0*C H 029 Compound (025): 5 To a 0*C solution of Fmoc-Val-OH (024) (348 mg, 1.6 mmol) in dichloromethane (4 mL) were added MSNT (474 mg, 1.6 mmol) and N-methyl imidazole (0.13 mL, 1.6 mmol). HMPB resin (400 mg, 0.32 mmol) was added once the mixture became homogenous, The resulting reaction mixture was allowed to shake for two hours at room temperature. The resin was filtered off, washed with 10 N,N-dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL), and was allowed to air dry to yield (025). Compound (026): Resin (025) was placed in a solution of 20% piperidine in N,N dimethylfornamide (20m]) and the resulting mixture was shaken at room 15 temperature for I hour. The resin was filtered off and washed with N,N dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL). To a 0*C solution of Fmoc-Ser(OMe)-OH (546 mg, 1.6 mmol) in N,N dimethylformamide (4 mL) were added HOBT (245 mg, 1.6 mmol), HBTU (606 - 60 mg, 1.6 mmol,) and N,N-diisopropylethylamine (0.6 mL, 3.2 mmol). The resin was added once the reaction mixture became homogenous. The resulting mixture was allowed to shake overnight. The resin was then filtered off and washed with N,N dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL), and allowed to 5 air dry to yield (026). Compound (027): Resin (026) was placed in a solution of 20% piperidine in N,N dimethylformamide (20mL) and the resulting mixture was shaken at room temperature for 1 hour. The resin was filtered off and washed with N,N 10 dimethylformamide (3 x 10 nL) and dichloromethane (3 x 10 mL). To a 0*C solution of 5-methyl-isoxazole-3-carboxylic acid (009) (162 mg, 1.6 mmol) in N,N-dimethylformamide (4 mL) were added HOBT (245 mg, 1.6 mmol), HBTU (606 mg, 1.6 mmol) and N,N-diisopropylethylamine (0.6 mL, 3.2 mmol). Once the resulting mixture became homogenous, the resin was added and 15 the resulting reaction mixture was allowed to shake overnight. The resin was then filtered off and washed with N,N-dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 nL), and the resin was allowed to air dry to yield (027). Compound (028): To resin (027) was added a solution of 50% trifluoroacetic acid in 20 dichloromethane (10 mL), and the resulting mixture was allowed to shake for 30 minutes. The resin was then filtered off and washed with dichloromethane (3 x 10 mL). The volatiles were removed under reduced pressure to provide (028) which was characterized by LC/MS (LCRS (NH) m/z: 328.14) and used without further purification. 25 Compound (029): To a 0*C solution of (029) and (006) (117 mg, 0.4 mmol), HOBT (70 mg, 0.5 mmol) and HBTU (170 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (5 mL). The mixture was stirred at room temperature for another 5 hours and became 30 homogenous. It was then diluted with ethyl acetate (200 nL) and washed with - 61 saturated aqueous sodium bicarbonate (2 x 10 m.L) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (029) (125 mg) which was 5 characterized by LC/MS (LCRS (MH) m/z: 481.26);>70% proteasome CT-L inhibition at 20 mg/kg PO. Example 4 Scheme 4: Synthesis of Example 035 N=-\ s 0 1. 20% Piperine FmocHN H H r FmocN2. Fmoc-Leu-OH FmocHN O FmocH DIE T OEA, HOBT HBTU D3, I HBTU. DMF 0 030 031032 1. 20/o PiperidIne/ N Hj O TFA O1- OH 2. DIEA. H-OBT I C~M, 0-N HBTU, DMF DM-N 0*C (C22 033 N-N 034 s -(006), HOT, HBTU DIEA, -HF, o*C o-N 0 0 035 Q 10 Compound (031): To a 0*C solution of Fmoc-L-4-thiazolylalanine (030) (2.0 g, 2.5 mmol) in dichloromethane (4 mL) were added N-methyl-imidazole (150 uL, 1.9 mmol), MSNT (755 mg, 2.55 mmol) and HMPB resin ( 8 0 0mg, 0.5 immol) was added once the mixture became homogenous. The resulting reaction mixture was allowed to 15 shake for two hours at room temperature. The resin was then filtered off and washed with NN-dimethylfornamide (3 x 20 mL) and dichloromethane (3 x 20 mL), and allowed to air dry to yield (031). - 62 - Compound (032) Resin (031) (360 mg, 0.23 mmol) was placed in a solution of 20% piperidine in N,N-dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 1 hour. The resin was then filtered off and washed with NN 5 dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL). To a 0*C solution of Fmoc-L-Leucine (204 mg, 0.58 mmol) in N,N dimethylformamide (4 mL) were added HOBT (124 mg, 0.92 mmol), HBTU (349 mg 0.92 mmol) and N,N-diisopropylethylamine (402 uL, 2.3 mmol). The resin was then added once the reaction mixture became homogenous. The resulting mixture 10 was allowed to shake at 5*C for five 5 hours. The resin was then filtered off and washed with N,N-dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL), and the resulting resin was allowed to air dry to yield (032). Compound (033): Resin (032) (0.23 mmol) was placed in a solution of 20% piperidine in N,N 15 dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 1 hour. The resin was then filtered off and washed with N,N dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL). To a 0*C solution of (022) (123 mg 0.58 mmol) in N,N-dimethylformamide (4 mL) were added HOBT (124 mg, 0.92 mmol), HBTU (349 mg 0.92 mmol) and 20 N,N-diisopropylethylamine (402 uL, 2.3 mmol). Once the resulting mixture became homogenous, the resin was added and the resulting reaction mixture was allowed to shake at room temperature ovemight. The resin was then filtered off, washed with N,N-dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL), and the resulting resin was allowed to air dry to yicld (033). 25 Compound (034): To resin (033) was added a solution of 50% of trifluoroacetic acid in dichlorometbane (10 mL), and the resulting mixture was allowed to shake for 30 minutes. It was then filtered off and the resin washed with dichloromethane (3 x 10 mL). The volatiles were removed under reduced pressure to provide (34) as - 63 characterized by LC/MS (LCRS (MH) m/z: 480.18) which was used without further purification. Compound (035): To a O'C solution of (034) and (006) (70 mg, 0.23 mmol), H4OBT (50 mg, 0.37 5 mmol) and HBTU (140 mg, 0.37 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (5 mL). The mixture was stirred at room temperature for 5 hours and then diluted with ethyl acetate (200 mL). It was then washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over 10 sodium sulfate and filtered through Celite-545 and the solvents were removed under reduced pressure. The resulting residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (035) (15 mg) which was characterized by LC/MS (LCRS (MH) m/z: 633.3); >90% proteasome CT-L inhibition at 40 mg/kg PO. 15 Example 5 Scheme 5: Synthesis of Example 039 W~e 0O0 e FncNN 1. 20% PipeFidmine 1. 20% Piperdine - 2. F oc-LejOH- 2. 0-EA; , 005 B. HOT -N 0T, N 032Ys 036 (009) 037 s W~e 00 j o 0u TFA c OH (006), HOBT. HBTU N N DCM. 0 0 C 0 -N 0 Z ~ e o-c / N DEA, THF, O-C o-N 038 ' 039 Compound (036): Resin (031) (800 mg, 0.23 mmol) was placed in a solution of 20% piperidine 20 in N,N-dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 1 hour. The resin was filtered off and washed with N,N dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL). To a 0*C solution of Fmoc-L-Ser(OMe)-OH (435 mg, 1.3 mmol) in N,N dimethylformamide (10 mL) were added HOBT (276 nig, 2.0 mmol), HBTU (710 - 64 mg, 2.0 mmol) and N,N-diisopropylethylamine (0.9 mL, 5.1 mmol). The resin was then added once the reaction mixture became homogenous. The resulting mixture was allowed to shake at 5*C for 5 hours. The resin was then filtered off and washed with N,N-dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL) and 5 allowed to air dry to yield (036). Compound (037): Resin (036) was placed in a solution of 20% piperidine in N,N dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 1 hour. The.resin was filtered off and washed with N,N 10 dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL). To a O*C solution of (009) (162 mg, 1.3 mmol) in N,N-dimethylformamide (4 mL) were added HOBT (276 mg, 2.0 mmol), HBTU (710 mg, 2.0 mmol) and N,N-diisopropylethylamine (0.9 mL, 5.1 mmol). Once the resulting mixture became homogenous, the resin was added and the resulting reaction mixture was allowed to 15 shake at room temperature overnight. The resin was then filtered off, washed with N,N-dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL), and allowed to air dry to yield (037). Compound (038): To (037) was added a solution of 50% of trifluoroacetic acid in 20 dichloromethane (10 mL), and the resulting mixture was allowed to shake for 30 minutes. The resin was then filtered off and washed with dichloromethane (3 x 10 mL). The volatiles were removed under reduced pressure to provide (38) which was characterized by LC/MS (LCRS (MH) m/z: 383.09) and used without further purification. 25 Compound (039): To a 0*C solution of (038) and (006) (156 mg, 0.51 mmol), HOBT (111 Img, 0.82 mmol) and HBTU (311 mg, 0.82 mmol) in tetrahydrofuran (50 mL) was added a solution of NN-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrahydrofuran (5 mL). The mixture was stirred at room temperature for 5 hours and became 30 homogenous. It was then diluted with ethyl acetate (200 mL) and washed with - 65 saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (039) (22 mg) which was 5 characterized by LC/MS (LCRS (MH) m/z: 536.21); >75% proteasome CT-L inhibition at 20 mg/kg PO. Example 6 Scheme 6: Synthesis of Example 045 (approach A) HMPB resin 0 i. 20% Piperidine OMe FmocHN OOH BTH , EmocHN. I2. F SeOMe)-O FmocHNJO 0 HBTU, DMF C 040 041 042 1. 20% Piperdine 2. IDEA, HCOBT DM0C HBTU, DMF --N O DcM, 0*C H (009) 043 044 (00), HOBT, HBTU / 0 DIEA THF, 0"C O-N - 045 10 Compound (041): To a O'C solution of Fmoc-L-alanine (040) (1.0 g, 3.2 mmol) in dichloromethane (30 rnL) were added N-methyl-imidazole (190 pL, 12.4 mmol), MSNT (950 mg, 3.2 mmol), and HMPB resin (1.0 g, 0.64 mmol) which was then added once mixture became homogenous. The resulting reaction mixture was allowed to shake for two 15 hours at room temperature. The resin was then filtered off and washed with N,N dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL) to yield (041). Compound (042); Resin (041) was placed in a solution of 20% piperidine in N,N dimethylformamide (20 mL) and the resulting mixture was shaken at room - 66 temperature for 1 hour. The resin was filtered off and washed with N,N dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL). To a 0*C solution of Fmoc-Ser(OMe)-OH (546 mg, 1.6 mmol) in NN dimetbylformamide (10 mL) were added HOBT (346 mg, 2.6 mmol), HBTU (970 5 mg 2.6 mmol) and N,N-diisopropylethylamine (1.1 mL, 6.4 mmol). The resin was then added once the reaction mixture became homogenous. The resulting mixture was allowed to shake at 5C for 5 hours. The resin was then filtered off and washed with N,N-dimethylfornamide (3 x 20 mL) and dichloromethane (3 x 20 mL), and allowed to air dry to yield (042). 10 Compound (043): Resin (042) (0.23 mmol) was placed in a solution of 20% piperidine in N,N dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 1 hour. The resin was filtered off and washed with N,N dimethylformamide (3 x 10 mL) and dichloromethane (3 x 10 mL). 15 To a 0*C solution of (009) (203 mg, 1.6 mmol) in N,N-dimethylformamide (10 mL) were added HOBT (346 mg, 2.6 mmol), HBTU (970 mg, 2.6 mmol) and N,N-diisopropylethylamine (1.1 mL, 6.4 mmol). Once the resulting mixture became homogenous, the resin was added and the resulting reaction mixture was allowed to shake at room temperature overnight. The resin was then filtered off, washed with 20 N,N-dimethyformamide (3 x 20 mL) and dichloromethane (3 x 20 mL), and allowed to air dry to yield (043). Compound (044): To (043) was added a solution of 50% of trifluoroacetic acid in dichloromethane (10 mL), and the resulting mixture was allowed to shake for 30 25 minutes. The resin was then filtered off and washed with dichloromethane (3 x 10 mL). The volatiles were removed under reduced pressure to provide (044) which was characterized by LC/MS (LCRS (MH) m/z: 300.11) and used without further purification. Compound (045): - 67 - To a 0*C solution of aforementioned intermediates (044) and (006) (195 nig, 0.64 mmol), HOBT (137 mg, 1.0 mmol) and HBTU (357 mg, 1.0 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL, 2.5 mmol) in tetrhydrofuran (5 mL). The mixture was stirred at room 5 temperature for 4 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (045) (84 mg) which 10 was characterized by LC/MS (LCRS (MH) m/z: 453.23); >80% proteasome CT-L inhibition at 20.mg/kg PO. Example 7 Scheme 7: Synthesis of Example 045 (approach B) OMe OMe BoNH OH +OBn HOST, HBTU o TFA o O DIEA. THF. O 0 C O O DCM, 0 C 001 046 0 04 OMe H+ CO2H HOBT. HBTU 0 OM 0 o Me DIEA, THF. O"C / N-><N>-lOBn 048 009 Cj 049 045 0 OMe e Pd&C, H12 Qfj1~ HOWF, O He 11-F, ODC o OH HIEA, HTF0 MJ N--A 0 -N 0o 044 045 15 Compound (047): To a 0*C solution of N-Boc-serine(methyl ether) (001) (6.57 g, 33 mmol), L alanine benzyl ester hydrochloride (046) (6.45 g, 30 mmol), HOBT (5.05 g, 33 mmol) and HBTU (11.8 g, 33 mmol) in tetrahydrofuran (400 mL) was added a solution of N,N-diisopropylethylamine (9.0 g, 70 mmol) in tetrahydrofuran (50 mL) 20 over 10 minutes. The mixture became homogenous and was stirred at room -68 temperature for another 5 hours. Most of the solvent Was then removed under reduced pressure and the resulting material diluted with ethyl acetate (500 mL). It was washed with saturated aqueous sodium bicarbonate (2 x 150 mL) and brine (200 mL) and the organic layers were dried over sodium sulfate and filtered through 5 Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (047) (11.8 g) which was characterized by LC/MS (LCRS (MH) m/z: 381.19). Compound (048): To a 0*C solution of (047) (11.8 g, 31.0 mmol) in dichloromethane (100 mL) 10 was added trifluoroacetic acid (50 mL) over 10 minutes, and the resulting mixture was stirred at the same temperature for another 3 hours. The solvents were then removed under reduced pressure and the residue was placed under high vacuum overnight to provide the TFA salt of (048), which was characterized by LC/MS (LCRS (MH) m/z: 281.15) and was used without further purification. 15 Compound (049): To a 0*C solution of (048), 5-methyl-isoxazole-3-carboxylic acid (009) (3.93 g, 31 mmol), HOBT (4.7 g, 35 mmol) and HBTU (12.5 g, 35 mmol) in tetrahydrofuran (400 mL) was added a solution of N,N-diisopropylethylamine (20 mL) in tetrahydrofuran (100 mL) over 10 minutes, and the pH of the resulting 20 mixture was -8. The mixture was stirred at room temperature for another 5 hours. Most of the solvent was then removed under reduced pressure and diluted with ethyl acetate (1.0 L). It was then washed with saturated aqueous sodium bicarbonate (2 x 100 mL) and brine (100 mL) and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure 25 and residue was purified by flash chromatography (hexane and ethyl acetate) to provide (049) (10.8 g) which was characterized by LC/MS (LCRS (MH) m/z: 390.16). Compound (044): To a 0"C solution of (049) (3.28 g, 8.4 mmol) in tetrahydrofuran (100 mL) 30 was added 10 % Pd/C (500 mg). The resulting mixture was allowed to stir under I atmosphere of hydrogen for 4 hours. The mixture was then filtered through Celite -69- 545 and the filter cake was washed with tetrahydrofuran. The organic filtrate was concentrated under reduced pressure and placed under high vacuum for 2 hrs to yield (044), which was characterized by LC/MS (LCRS (MH) m/z: 281.15) and was used without further purification. 5 Compound (045): To a 0*C solution of (044) and (006) (1.9 g, 8.5 mmol), HOBT (2.0 g, 13 mmol) and HBTU (5.4 g, 14 mmol) in tetrahydrofuran (200 mL) was added a solution of N,N-diisopropylethylamine (5.4 g, 42 mmol) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature for another 5 hours. Most of the solvent 10 was then removed under reduced pressure and the resulting material diluted with ethyl acetate (400 mL). It was then washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (50 mL) and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and residue was purified by HPLC (aqueous ammonium acetate 15 and acetonitrile) to provide (045) (1.35 g) which was characterized by LC/MS (LCRS (MH) m/z: 453.23). Example 8 Scheme 8: Synthesis of Example 055 PhI 08On -OBTHTJ P&CL, 142 OH +O .n F cNH On BocNH N \ OH 0 0+_ Ph "Ph 050 002 051 052 'N N C HOB T, H BT TFA +DIEA TH. 0 0 C DNEA, P coc Ph (006) 053 054 N '\ + ~N HOBT, HBTU 0 H N DIEA THF. O'c eV~ (009) 0 -N 0 50 Ph O 055 - 70 - Compound (051): To a 0*C solution of N-Boc-L-2-pyridylalanine (050) (1.0 g, 3.76 mmol),

L

phenylalanine benzyl ester hydrochloride (002) (1.3 g, 3.76 mmol), HOBT (0.68 g, 5.0 mmol) and HBT"U (1.8 g, 5.0 mmol) in tetrahydrofuran (100 mL) was added a 5 solution of N,N-diisopropylethylamine (1.6 mL) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature for another 3 hours and then diluted with ethyl acetate (200 mL), washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (100 mL) and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvent was removed under reduced pressure and 10 the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (051) (1.45 g) which was characterized by LC/MS (LCRS (MH) m/z: 504.24). Compound (052): To a 00C solution of (051) in tetrahydrofuran (100 mL) was added 10 % Pd/C 15 (100 mg) and the resulting mixture was allowed to stir under 1 atmosphere of hydrogen for 4 hours. The mixture was then filtered through Celite-545 and the filter cake was washed with tetrahydrofuran. The organic filtrate was then concentrated under reduced pressure and placed under high vacuum to provide (052) by LC/MS (LCRS (MH) m/z: 414.2) which was used without further purification. 20 Compound (053):. To a 0*C solution of (052) and (006) (0.85 g, 3.9 mmol), HOBT (0.70 g, 5.3 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (3 mL) in tetrahydrofuran (10 mL) and the mixture was stirred at room temperature overnight. It was then diluted with ethyl 25 acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545, and the solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate) and HPLC (aqueous ammonium acetate and acetonitrile) to provide (053) (1.51 g) which 30 was characterized by LC/MS (LCRS (MH) m/z: 567.21). Compound (054): -71- To a 0*C solution of (053) (200 mg, 0.352 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL), and the resulting solution was stirred at the same temperature for another hour. The solution was concentrated under reduced pressure and placed under high vacuum to provide (054) as confirmed by LC/MS 5 (LCRS (MH) m/z: 467.26) which was used without further purification. Compound (055): To a 0*C solution of (054) and 5-methyl-isoxazole-3-carboxylic acid (009) (127 mg, 1.0 mmol), HOBT (135 mg, 1.0 mmol) and HBTU (350 mg, 1.0 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (0.5 10 mL) in tetrahydrofuran (2 mL). The mixture was stirred at room temperature for 5 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545, the solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous 15 ammonium acetate and acetonitrile) to provide (055) (40 mg) which was characterized by LC/MS (LCRS (MH) m/z: 576.27); >80% proteasome CT-L inhibition at 20 mg/kg PO. Example 9 Scheme 9: Synthesis of Example 061 BocN OH + 2N OBn HOBT HTc BocNH O BocNH OH DIEA, THF. OOC BocNOO 000 Ph 0 Ph 056 002 057 058

H

2 N D BocHNT OJ( C0Ck

.

H2N M059 060 + %- IA B U § o HOST , 8 c H +-N 0 Co 20 022 . O-N 0 Ph O -72- Compound (057): To a 0"C solution of N-Boc-L-n-valine (056) (1.0 g, 4.6 mmol), L phenylalanine benzyl ester hydrochloride (002) (1.4 g, 4.6 mmol), HOBT (1.0 g, 7.4 mmol) and HBTU (2.8 g, 7.4 mmol) in tetrahydrofuran (100 mL) was added a 5 solution of N,N-diisopropylethylamine (3.2 mL, 18.4 mmol) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature for 3 hours and then diluted with ethyl acetate (200 mL), washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (100 mL), and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvent was removed under reduced pressure and 10 the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (057) which was characterized by LC/MS (LCRS (MH) m/z: 455.25). Compound (058): To a 0"C solution of (057) (1.30g, 2.875 mmol) in tetrahydrofuran (100 mL) was added 10% Pd/C (100 mg). The resulting mixture was allowed to stir under I 15 atmosphere of hydrogen for 4 hours. The mixture was filtered through Celite-545 and the filter cake was washed with tetrahydrofuran. The filtrate was then concentrated under reduced pressure and placed under high vacuum to provide (058) as confirmed by LC/MS (LCRS (MH) m/z: 365.2) which was used without further purification. 20 Compound (059): To a 0*C solution of (058) and (006) (0.99 g, 4.6 mmol), HOBT (0.62 g, 4.6 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (2.4 mL) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature overnight and was then diluted with ethyl 25 acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were then removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (059) (1.21 g) which was characterized by LC/MS (LCRS (MH) m/z: 30 518.32). -73- Compound (060): To a 0*C solution of (059) (250 mg, 0.48 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL), and the resulting solution was stirred at the same temperature for another hour. The organic layers were concentrated under 5 reduced pressure and placed under high vacuum to provide (060) as confirmed by LC/MS (LCRS (MH) m/z: 418.26) which was used without further purification. Compound (061): To a 0*C solution of (060) and (022) (122 mg, 0.58 mmol), HOBT (104 mg, 0.77 mmol) and HBTU (292 mg, 0.72 mmol) in tetrahydrofuran (100 mL) was 10 added a solution of N,N-diisopropylethylamine (0.35 mL) in tetrahydrofuran (2 mL) and the mixture was stirred at room temperature for another 4 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under 15 reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (061) (88.4 mg) which was characterized by LC/MS (LCRS (MH) m/z: 612.33); >80% proteasome CT-L inhibition at 40 mg/kg PO. -74- Example 10 Scheme 10: Synthesis of Example 068 MOO MeO MeO BaCH H O~n HOBT HM ~ J1 dC. H 2 0 SoN OH C HBocNH ogn BocNH OH O 0 8I hF o THF,0 0 M 062 002 063 064 MMeO 4 N 0 HOBr. HBT!U 0 NJ o TFA ,4 H BOlHN tEZ. Nw Ii O ~IEA THF, 0QC " cocHN NAJI

CO

2 H +. / N -OT. o IEA THF. 0*C 067 -N 068 Ph Compound (063): 5 To a 0*C solution of N-Boc-HoSer(OMe)--OH (062) (1.0 g, 4.3 mmol),

L

phenylalanine benzyl ester hydrochloride (002) (1.3 g, 4.3 mmol), HOBT (0.88 g, 6.5 nmol) and HBTU (2.3 g, 6.5 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropyletliylamine (2.0 mL) in tetrahydrofuran (5 mL). The mixture was stirred at room temperature for another 3 hours and then diluted with 10 ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x i0 mL) and brine (100 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (063) (1.81 g) which was characterized by LC/MS (LCRS (MH) m/z: 15 471.24). Compound (064): To a 0*C solution of (063) (1.35 g, 2.875 mmol) in tetrahydrofuran (100 mL) was added 10 % Pd/C (100 mg). The resulting mixture was allowed to stir under 1 atmosphere of hydrogen for 4 hours. The mixture was filtered through Celite-545 - 75 and the filter cake was washed with tetrahydrofuran. The organic filtrate was concentrated under reduced pressure and placed under high vacuum to provide (064) as confirmed by LC/MS (LCRS (MH) m/z: 381.19) which was used without further purification. 5 Compound (065): To a 0*C solution of (065) and (006) (0.99 g, 4.6 mmol), HOBT (0.62 g, 4.6 mmol) and HBTU (1.70 g, 4.9 mmol) in tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (2.4 mL) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature overnight and then diluted with ethyl 10 acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (065) (1.11 g) which was characterized by LC/MS (LCRS (MH) m/z: 15 534.31). Compound (066): To a 0*C solution of (065) (230 mg, 0.43 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (10 mL), and the resulting solution was stirred at the same temperature for another one hour. The reaction mixture was then concentrated 20 under reduced pressure and placed under high vacuum to provide (066) as confirmed by LC/MS (LCRS (MH) m/z: 434.26) which was used without further purification. Compound (068): To a 0*C solution of (066) and 5-isopropylisoxazole-3-carboxylic acid (067) (81 mg, 0.52 mmol), HOBT (93 mg, 0.69 mmol) and HBTU (262 mg, 0.69 mmol) in 25 tetrahydrofuran (100 mL) was added a solution of N,N-diisopropylethylamine (0.30 mL) in tetrahydrofuran (2 mL) and the mixture was stirred at room temperature for another 4 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 m.L) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents 30 were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (068) (75.7 mg) which was - 76 characterized by LC/MS (LCRS (MH) m/z: 571.3 1); >70% proteasome CT-L inhibition at 40 mg/kg PO. Example 11 Scheme 11: Synthesis of Example 075 (approach A) oo De ______resin_ 0 1.200M Pi eid*n OH Pmoc COCI OH HMPB resi _________ H 2 N HFmocHN o1N1 NiO.DOan HBTLJ. OMF

--

I-BTU, DMP 069 070 071 e (070) OMe OH Ome FmocHNcoH 20% n DcM. OOC IN 2. OIEA, H01T O HBTU, OMF O-N o 0 OMe OMe 072 009 073 H OH + H 2 Di F N M O 02 uIEiA THF, H ~ 5 074 006 075 Compound (070): To a solution of L-serine (methyl ether) hydrochloride (069) (1.0 g, 6 .4mmol) in water/dioxane (1:1, 80ml) was added sodium hydroxide

(

7 68mg, 1 9

.

2 mmol). After the mixture was stirred at room temperature for 30 minutes, it was 10 cooled to 0 *C, and a solution of 9 -fluorenylmethyl chloroformate (1.65 g, 6.4mmol) in dioxane (16 mL) was added dropwisely. The reaction mixture was allowed to stir at room temperature for another 4 hours. The solvents were then removed, the residue was diluted with water and the pTJ was adjusted to -1 with IN NCl, and the aqueous layer was extracted with ethyl acetate (4 x 100 mL). The organic layers 15 were concentrated under reduced pressure and placed under high vacuum to provide (070) (1.8 g) as confirmed by LC/MS (LCRS (MH) m/z: 342.13) which was used without further purification. - 77 - Compound (071): The resin HMPB-BHA (500 mg, 0.32 mmol ) was washed with dichloromethane. In a dry flask, Fmoc-Ser(Me)-OH (070) (546 mg, 1.6 mmol) was dissolved in dichloromethane and to the solution was added I -methylimidazole (95 5 .L, 1.2 mmol) followed by MSNT (474 mg, 1.6 mmol). Once the resulting mixture had become homogenous (10 minutes) it was added to the HMPB-BHA resin as a suspension in dichloromethane (5 mL). The resulting reaction mixture was allowed to shake overnight. The resin was then filtered off and washed with DMF (3 x 20 mL), MeOH (3 x 20 mL), DCM (3 x 20 mL), and allowed to air dry to yield (071). 10 Compound (072): Resin (071) (300 mg, 0.192 mmol) was placed in a solution of 20% piperidine in N,N-dimethylformamide (20 mL) and the resulting mixture was shaken at room temperature for 30 minutes. The resin was filtered off and washed with N,N-dimethylforrnamide (3 x 20 mL) and dichloromethane (3 x 20 mL) twice. 15 To a 00C solution of Fmoc-Ser(Me)-OH (070) (0.48 mmol, 163 mg) in N,N dimethylformamide (10 mL) was added HOBT (104 mg, 0.77 mmol), HBTU (291 mg, 0.77 mmol) and diisopropylethylamine (0.34 mL, 1.92 mmol). Once the resulting mixture became homogenous, the resin (0.13 mmol, 200 mg) was added and the resulting reaction mixture was allowed to shake overnight. The resin was 20 then filtered off and washed with DMF (10 mL), DCM (10 mL), MeOH (10 mL),

H

2 0 (10 mL), DMF (10 mL), MeOlH (10 mL), and DCM (10 mL), and allowed to air dry to yield (072). Compound (073): To (072) (300 mg, 0.19 mmol) was added a solution of 20% piperidine in 25 NN-dimethylforrnamide (20 mL) and the resulting mixture was shaken at room temperature for 30 minutes. The resin was filtered off and washed with N,N dimethylformamide (3 x 20 mL) and dichloromethane (3 x 20 mL) twice. To a 00C solution of (009) (61 ng, 0.48 mmol) in NN-dimethylformamide (2 mL) was added HOBT (104 mg, 0.77 mmol), HBTU (291 mg 0.77 mmol) and 30 N,N-diisopropylethylamine (0.34 mL, 1.92 mmol). Once the resulting mixture - 78 became homogenous, the resin (300 mg, 0.192 mmol) was added and the resulting reaction mixture was allowed to shake at room temperature overnight. The resin was then filtered off, washed with DMF (10 mL), DCM (10 mL), MeOH (10 nL), H20 (10 mL), DMF (10 mL), MeOH (10 mL), and DCM (10 mL), and allowed to air dry 5 to yield (073). Compound (074): To (073) was added a solution of 50% of trifluoroacetic acid in dichloromethane (10 nL), and the resulting mixture was allowed to shake for 30 minutes. The resin was then filtered off and washed with dichloromethane (3 x 10 10 mL). The volatiles were removed under reduced pressure, and the desired compound (074) was characterized by LC/MS (LCRS (MH) m/z: 330.12) and used without further purification. Compound (075): To a 0*C solution of (074) and (006) (78 mg, 0.38 mmol), HOBT (41 mg, 15 0.30 mmol) and HBTU ( 1 1 6 mg, 0.30 mmol) in acetonitrile (50 mL) was added a solution of N,N-diisopropylethylamine (0.1 mL, 0.6 mmol). The mixture was stirred at 0 - 4*C overnight and then diluted with ethyl acetate (200 mL). It was then washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL) and the organic layers were dried over sodium sulfate and filtered through Celite 20 545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (075) (29 mg) which was characterized by LC/MS (LCRS (MH) m/z: 483.24); > 8 0% proteasome CT-L inhibition at 20 mg/kg PO. - 79 - Example 12 Scheme 12: SynthesIs of Example 075'(approach B) OMe OMe OMe OMe OH BnCO 2 CI, DMAP . TFA (OMe1 BocNH TEA, 0cM,0*C BocNH O DCM.C H 2 N HOBT,HBTU BocNH On ODIEA, THF, 0"c OMS 001 076 077 078 WPe OMB Pd/C, H 2 N HOBT.HBTLJM BocNH OH + H2N o BocHN 0HF -- 1 om DIEA, THF,.0 0 C 079 006 080 TFA H9 0 2 H o u OGM,0WC IH2 / N HO/T 1BT C 0 H c DIEA. THF 00 / I NOMe 0 O-N \~ 081 009 075 5 Compound (076): To a 0*C solution of N-Boc serine(methyl ether)-OH (43.8 g, 200 mmol), triethylamine (26.5 g, 260 mmol) and 4-(dimethylamino)pyridine in dichloromethane (1.2 L) was added a solution of benzyl chloroformate (41 g, 240 mmol) in dichloromethane (250 mL) over 30 minutes and the resulting mixture was 10 stirred at the same temperature for another 3 hours. Saturated aqueous sodium bicarbonate (200 nL) was then added and the organic layer was washed with saturated aqueous sodium bicarbonate (200 mL) and brine (200 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and residue was purified by flash 15 chromatography (hexane and ethyl acetate) to provide (076) (54 g) which was characterized by LC/MS (LCRS (MH) m/z: 310.16). Compound (077): To a 0*C solution of (076) (54 g, 174.6 mmol) in dichloromethane (200 mL) was added trifluoroacetic acid (200 mL) over 10 minutes, and the resulting mixture 20 was stirred at the same temperature for another 3 hours. The solvents were then removed under reduced pressure and the residue was placed under high vacuum - 80 overnight giving the TFA salt of (077), confirmed by LC/MS (LCRS (MH) m/z: 210.11), and was used without further purification, Compound (078): To a 00C solution of (077) (43.8 g, 200 mmol), N-Boe serine(methyl ether) 5 OH (36.7 g, 167 mmol), HOBT (27 g, 200 mmol) and HBTU (71.4 g, 200 mmol) in tetrahydrofuran (1.2 L) was added a solution of N,N-diisopropylethylamine (75 g, 600 mmol) in tetrahydrofuran (250 mL) over 10 minutes, and pH of the resulting mixture was -8. The mixture was stirred at room temperature for another 5 hours. Most of the solvent was then removed under reduced pressure and the resulting 10 material diluted with ethyl acetate (1.0 L). It was then washed with saturated aqueous sodium bicarbonate (2 x 150 mL) and brine (200 mL) and the organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and residue was purified by flash chromatography (hexane and ethyl acetate) to provide (078) (65 g) which was 15 characterized by LC/MS (LCRS (MH) m/z: 411.21). Compound (079): To a 0"C solution of (079) (13.4 g, 32.7 mmol) in tetrahydrofuran (300 mL) was added 10 % Pd/C (2.7 g) and the resulting mixture was allowed to stir under 1 atmosphere of hydrogen for 4 hours. The mixture was filtered through Celite-545 20 and the filter cake was washed with tetrahydrofuran. The organic layers were concentrated under reduced pressure and placed under high vacuum to provide (079) as confirmed by LC/MS (LCRS (MH) m/z: 321.16) which was used without further purification. Compound (080): 25 To a 0*C solution of (079) and (006) (5.6 g, 26 mmol), HOBT (6.0 g, 41.4 mmol) and HBTU (14.8 g, 41.4 mmol) in tetrahydrofuran (400 mL) was added a solution of NN-diisopropylethylamine (23 mL) in tetrahydrofuran (40 mL) and the mixture was stirred at room temperature overnight. Most of the solvent was then removed under reduced pressure and the resulting material diluted with ethyl acetate 30 (500 mL) and washed with saturated aqueous sodium bicarbonate (2 x 100 mL) and brine (100 nL). The organic layers were dried over sodium sulfate and filtered - 81 through Celite-545. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (hexane and ethyl acetate) to provide (080) (9.2 g) which was characterized by LCIMS (LCRS (MH) m/z: 474.27). Compound (081): 5 To a 0*C solution of (080) (200 mg, 0.43 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL), and the resulting solution was stirred at the same temperature for another hour. The organic layers were concentrated under reduced pressure and placed under high vacuum to provide (081) as confirmed by LC/MS (LCRS (MH) m/z: 374.22) which was used without further purification. 10 Compound (07S). To a 0*C solution of (081) and 5-methyl-isoxazole-3-carboxylic acid (009) (65 mg, 0.5 mmol), HOBT (65 mg, 0.5 mmol) and HIBTU (175 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N-diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL) and the mixture was stirred at room temperature for 15 another 5 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (075) (85 mg) which was 20 characterized by LC/MS (LCRS (MH) m/z: 483.24). Example 13 Scheme 13: Synthesis of Example 083 OMe 0 0H0 OMe0 HO0H HOBT, HBTU 0k

H

2 N + DEATH ;/FC N 0 Me O-N N 081 082 083 Compound (083): 25 To a 0*C solution of (081) (160 mg, 0.43 mmol) and isoxazole-3-carboxylic acid (082) (60 mg, 0.5 mmol), HOBT (65 mg, 0.5 mmol) and HBTU (175 mg, 0.5 - 82 mmol) in tetrahydrofuran (50 mL) was added a solution of NN diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL) and the mixture was stirred at room temperature for another 5 hours. It was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and 5 brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (083) (74 mg) which was characterized by LC/MS (LCRS (MH) m/z: 469.22); >80% proteasome CT-L inhibition at 20 mg/kg PO. 10 Example 14 Scheme 14: Synthesis of Example 085 081O~ O-e N- O _Oe 081 085 Compound (08):~ To a 0*C solution of (081) ( 16 0 mg, 0.43 mmol) and isoxazole-3-carboxylic 15 acid (084) (65 mg, 0.5 mmol), HOBT (65 mg, 0.5 mmol) and HBTU (175 mg, 0.5 mmol) in tetrahydrofuran (50 mL) was added a solution of N,N diisopropylethylamine (0.5 mL) in tetrahydrofuran (2 mL) and the mixture was stirred at room temperature for another 5 hours. The reaction was then diluted with ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 20 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through Celite-545. The solvents were removed under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (085) (71 mg) which was characterized by LC/MS (LCRS (MH) m/z: 483.24); >50% proteasome CT-L inhibition at 20 mg/kg PO. - 83 - Scheme 15: Synthesis of Example 088 CbzNH O Pd/C, H 2 086 rFA, 0*C OoMe N 0'LO + O H- 0o, H2 HOT H "c O-MO D EA, THF, OOC -N -YJ2 07 I(0MM 004 088 Compound (087): 5 To a solution of (086) (prepared using the same procedure as (005) except that Cbz-phenylalanine was substituted for Cbz-Leucine) (0.100 g, .0295 mmol) in trifluoroacetic acid (10 mL) was added 10 % Pd/C (20 mg). The resulting mixture was allowed to stir under 1 atmosphere ofhydrogen for 6 hours. The mixture was then filtered through Celite-545 and the filter cake washed with dichloromethane (50 10 mL). The filtrate was concentrated under reduced pressure and placed under high vacuum ovemight to provide (087) as confirmed by LCIMS (LCRS (MH) Mz: 206.1) which was used in the subsequent transformation without further purification. Compound (088): To a 0*C solution of (087) and (074) (166 mg, 0.354 mrnol), HOBT (54 mg, 15 0.354 mmol) and HBTU (134 mg, 0.354 mmol) in tetrahydrofuran (20 mL) was added N,N-diisopropylethylamine (0.2 mL, 1.18 mmol). The mixture was stirred 0*C overnight and became homogenous. It was then diluted with ethyl acetate (20 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine (10 mL). The organic layers were dried over sodium sulfate and filtered through 20 Celite-545 and concentrated under reduced pressure and the residue was purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (088) (10 mg) as characterized by LC/MS (LCRS (MH) m/z: 517.69); >80% proteasome CT-L inhibition at 20 mg/kg PO. - 84- Scheme 16: Synthesis of Example 091 F CbzNH Pd/c, H 2 089 TFA. 0FC FF OMMB 0 0 0 O- OM e HNJ DIEA, THF, 0 0 C / 074 090 091 Me Compound (090): 5 To a solution of (089) (prepared using the same procedure as for (005) except that Cbz- 4 -fluorophenylalanine was substituted for Cbz-leucine) (0.100 g, 0.28 mmol) in trifluoroacetic acid (10 mL) was added 10 % Pd/C (20 mg). The resulting mixture was allowed to stir under I atmosphere of hydrogen for 6 hours. The mixture was filtered through Celite-545 and the filter cake washed with 10 dichloromethane (50 mL). The filtrate was concentrated under reduced pressure and placed under high vacuum overnight to provide (090) as confirmed by LC[MS (LCRS (MH) m/z: 224.1) which was used in the subsequent transformation without further purification. Compound (091): 15 To a 0*C solution of (090) and (074) (110 mg, 0.336 mmol), HOBT (51 mg, 0.336 mmol) and HBTU (127 mg, 0.336 mmol) in tetrahydrofuran (20 mL) was added N,N-diisopropylethylamine (0.2 mL, 1.18 mmol). The mixture was stirred 0*C overnight and became homogenous. It was then diluted with ethyl acetate (20 mL) and washed with saturated aqueous sodium bicarbonate (2 x 10 mL) and brine 20 (10 mL). The organic layers were dried over sodium sulfate, filtered through Celite 545, and concentrated under reduced pressure. The residue was then purified by HPLC (aqueous ammonium acetate and acetonitrile) to provide (091) (60 mg) which was characterized by LC/MS (LCRS (MH) m/z: 535.69); >80% proteasome CT-L inhibition at 20 mg/kg PO. - 85 - Biological Activity Compounds were formulated in 10% PS80/NaCitrate (pH 3) vehicle and administered to mice orally (PO) (3 animals/cohort). One hour post-dosing, the 5 animals were sacrificed and the following tissues harvested: blood, brain, adrenal gland, heart and liver. Whole blood (-200 pl) was washed twice with PBS and lysed byhypotonic shock (300 pl 50 mM Tris pH 8, 5 mM EDTA). Blood lysates were stored at -80*C until assayed. Blood lysates were clarified by centrifugation in a microcentrifuge. The CT-L specific activity of proteasome in each lysate was 10 evaluated by determining: a) the protein concentration by modified Bradford assay with bovine gamma globulin as a standard; and b) the rate of cleavage of the fluorogenic proteasome substrate LLVY-AMC. The percent proteasorne activity for the analog-treated animals was calculated by dividing of the average specific activity for each analog-dosed cohort by the average specific activity of the vehicle-dosed 15 cohort. Percent proteasome inhibition was calculated by subtracting percent proteasome activity from 100. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods 20 of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. All of the above-cited references and publications are hereby incorporated by reference. -86-

Claims (27)

1. A compound having a structure of formula (I) or a pharmaceutically acceptable salt thereof: R 1 R' 0 R 3 RSZL N X R 4 0 R 2 R 7 0 () 5 wherein L is C=O; X is 0; Z is absent; R', R 2 and R 3 are each independently selected from C 1 6 alkyl, C 1 - 6 alkoxyalkyl, 10 C 1 - 6 aralkyl and Ci 6 heteroaralkyl; R 4 is hydrogen; R' is heteroaryl; and R6 and R are hydrogen.

2. A compound of claim 1, wherein R , R 2 and R 3 are independently selected from 15 C 1 -6alkoxyalkyl and Cl. aralkyl.

3. A compound of claim 2, wherein any one of R1, R2 or R 3 is independently C 1 . 6 hydroxyalkyl.

4. A compound of claim 3, wherein any one of R', R 2 or R 3 is independently selected from hydroxymethyl and hydroxyethyl. 87

5. A compound of claim 1, wherein any one of R', R2 or R3 is independently C 1 . 6 alkoxyalkyl.

6. A compound of claim 5, wherein any one of R', R 2 or R 3 is independently selected from methoxymethyl and methoxyethyl. 5

7. A compound of claim 1, wherein any one of R1, R 2 or R 3 is independently C 1 . 6 heteroaralkyl.

8. A compound of claim 7, wherein any one of R', R2 or R3 is independently selected from imidazolylmethyl, pyrazolylmethyl, thiazolylmethyl and pyridylmethyl.

9. A compound of any one of claims 1 to 8, wherein R', R 2 and R 3 are all different.

10 10. A compound of claim 1, wherein at least one of R' and R2 is C 1 -6 alkoxyalkyl.

11. A compound of claim 10, wherein at least one of R' and R 2 is selected from methoxymethyl and methoxyethyl.

12. A compound of any one of claims 1, 10 or 11 wherein R 3 is selected from Ci- 6 alkyl and C 1 - 6 aralkyl. 15

13. A compound of claim 12, wherein R 3 is C 1 . 6 alkyl.

14. A compound of claim 13, wherein R 3 is selected from methyl, ethyl, isopropyl, sec-butyl and isobutyl.

15. A compound of claim 14, wherein R 3 is isobutyl.

16. A compound of claim 12, wherein R 3 is C 1 -aralkyl. 20

17. A compound of claim 16, wherein R 3 is phenylmethyl.

18. A compound ofany one of claims I to 17, wherein R 5 is a 5- or 6-membered heteroaryl.

19. A compound of claim 1, wherein R 5 is isoxazol-3-yl or isoxazol-5-yl. 88

20. A compound of claim 19, wherein R 5 is isoxazol-3-yl that has a substituent at the 5-position.

21. A compound of claim 19, wherein R 5 is isoxazol-5-yl that has a substituent at the 3-position. 5

22. A compound of claim 20 or 21, wherein the substituent is selected from C 1 . 6 alkyl and C1- 6 heteroaralkyl.

23. A compound of claim 22, wherein the substituent is selected from methyl and isopropyl.

24. A compound of claim 20 or 21, wherein the substituent is selected from 10 CI- 6 heteroaralkyl and CI- 6 heterocycloalkyl.

25. A pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of: inflammation, neurodegenerative diseases, viral infections, ischemia, repurfusion injury, parasitic infections, muscle-wasting diseases, cancer, chronic infectious diseases, a hyperproliferative condition, muscle disuse, 15 diseases associated with bone loss and immune-related conditions, the composition comprising a compound of any one of the preceding claims or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.

26. A pharmaceutical composition of claim 25, wherein said composition is formulated such that said compound is orally bioavailable. 20

27. A compound according to claim 1; or a pharmaceutical composition according to claim 25, substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. 89