EP1021407A1 - Synthesis of clasto-lactacystin beta-lactone and analogs thereof - Google Patents

Abstract

The present invention is directed to an improved synthesis of clasto-lactacystin-β-lactone, and analogs thereof, that proceeds in fewer steps and in much greater overall yield than syntheses described in the prior art. The synthetic pathway relies upon a novel stereospecific synthesis of an oxazoline intermediate and a unique stereoselective addition of a formyl amide to the oxazoline. Also described are novel clasto-lactacystin-β-lactones, and analogs thereof and their use as proteosome inhibitors.

Description

Synthesis of Clasto-Lactacystin β-Lactone and Analogs

Thereof

Background of the Invention

Field of the Invention

The invention relates generally to methods for preparing lactacystin and related compounds, to novel analogs of lactacystin and c/αsto-lactacystin β- lactone, and their uses as proteasome inhibitors.

Description of Related Art

The Streptomyces metabolite lactacystin (1) inhibits cell cycle progression and induces neurite outgrowth in cultured neuroblastoma cells (Omura et al, J. Antibiotics 44:\ \1 (1991); Omura et al, J. Antibiotics 44:113 (1991); Fenteany et al, Proc. Natl Acad. Sci. (USA) 91:335% (1994)). The cellular target mediating these effects is the 20S proteasome, the proteolytic core of the 26S proteasome, which is the central component of the ubiquitin-proteasome pathway for intracellular protein degradation. Mechanistic studies have established that lactacystin inhibits the proteasome through the intermediacy of the active species, c/αsto-lactacystin β-lactone (2), which specifically acylates the N-terminal threonine residue of the proteasome X/MBl subunit (Fenteany, et al, Science 268:726 (1995); Dick et al, J. Biol. Chem. 271:7273 (1996)). Lactacystin analogs are disclosed by Fenteany et al (WO 96/32105).

The ubiquitin-proteasome pathway is involved in a variety of important physiological processes (Goldberg et al, Chemistry & Biology 2:503 (1995); Ciechanover Cell 79:13 (1994); Deshaies, Trends Cell Biol.5:43 \ (1995)). In fact, the bulk of cellular proteins are hydrolyzed by this pathway. Protein substrates are first marked for degradation by covalent conjugation to multiple molecules of a small protein, ubiquitin. The resultant polyubiquitinated protein is then recognized and degraded by the 26S proteasome. Long recognized for its role in degradation of damaged or mutated intracellular proteins, this pathway is now also known to be responsible for selective degradation of various regulatory proteins. For example, orderly cell cycle progression requires the programmed ubiquitination and degradation of cyclins. The ubiquitin-proteasome pathway also mediates degradation of a number of other cell cycle regulatory proteins and tumor suppressor proteins (e.g., p21, p27, p53). Activation of the transcription factor NF-κB , which plays a central role in the regulation of genes involved in the immune and inflammatory responses, is dependent upon ubiquitination and degradation of an inhibitory protein, IκB-α (Palombella et al , ^"WO 95/25533). In addition, the continual turnover of cellular proteins by the ubiquitin-proteasome pathway is essential to the processing of antigenic peptides for presentation on MHC class I molecules (Goldberg and Rock, WO 94/17816).

The interesting biological activities of lactacystin and c/αsto-lactacy stin β- lactone and the scarcity of the natural materials, as well as the challenging chemical structures of the molecules, have stimulated synthetic efforts directed toward lactacystin and related analogs. Corey and Reichard J. Am. Chem. Soc. 114:10677 (1992); Tetrahedron Lett. 34:6977 (1993)) achieved the first total synthesis of lactacystin, which proceeded in 15 steps and 10% overall yield. The key feature of the synthesis is a stereoselective aldol reaction of a cw-oxazolidine aldehyde derived from N-benzylserine to construct the C(6)-C(7) bond. In the synthesis reported by (Uno et al, J. Am. Chem. Soc. 116:2139 (1994)), stereoselective Mukaiyama-aldol reaction of a bicyclic oxazolidine silyl enol ether intermediate derived from D-pyroglutamic acid is employed in C(5)-C(9) bond construction. This synthesis proceeds in 19 steps and 5% overall yield. Aldol reactions under basic conditions of a similar bicyclic oxazolidine intermediate form the basis of model studies reported by (Dikshit et al, Tetrahedron Lett. 36:6131 (1995)).

Aldol reactions of oxazoline-derived enolates feature prominently in the synthesis of lactacystin reported by Smith and coworkers (Suazuka et al, J. Am.

Chem. Soc. 115:5302 (1993); Νagamitsu et al , J. Am Chem. Soc. 775:3584 (1996)) and in the synthesis of (<57?)-lactacystin reported by (Corey and Choi Tetrahedron Lett. 34:6969 (1993)); Choi Ph.D., Thesis, Harvard University, 44 (1995). In the former synthesis, which proceeds in 20 steps and 9% overall yield, the enolate is condensed with formaldehyde to install a single carbon atom, which must then be elaborated in a number of additional steps. In the Corey and Choi synthesis, the aldol reaction selectively provides the product of undesired stereochemistry, resulting in the eventual preparation of the C(6) epimer of lactacystin, which is devoid of biological activity. Lactacystin has also been prepared in 22 steps and 2% overall yield from

D-glucose (Chida et al, J. Chem. Soc, Chem. Commun. 793 (1995)). The biosynthetic pathway involved in production of the natural product has been investigated in feeding experiments involving ¹³C-enriched compounds (Νakagawa et al, Tetrahedron Lett. 35:5009 (1994)). The reported syntheses of lactacystin are lengthy and proceed in low yield. Furthermore, none of these syntheses is readily adapted for analog synthesis. Thus, there is a need for improved methods for preparing lactacystin, clasto- lactacystin β-lactone, and analogs thereof.

Summary of the Invention

A first aspect of the present invention relates to a process for forming lactacystin or analogs thereof having Formula VI or c/αstø-lactacystin β-lactone or analogs thereof having Formula VII:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxy alkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substituted. A second aspect of the present invention is directed to a method of forming formyl amides of Formula XIV:

where R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7- membered heterocyclic ring, which may be optionally substituted, and which optionally may include an additional oxygen or nitrogen atom.

A third aspect of the present invention relates to forming tri-substituted oxazolines of Formula Ia or lb:

where R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and R⁴ is aryl or heteroaryl, either of which may be optionally substituted. The tri- substituted oxazolines of Formulae Ia and lb are useful as starting materials in forming lactacysin, c/αsto-lactacystin β-lactone or analogs thereof via the process described herein. A fourth aspect of the present invention is directed to lactacysin, clasto- lactacystin β-lactone or analogs of Formulae /and FiZ that possess unexpected biological activity. Lactacystin, c/αsto-lactacystin β-lactone, and analogs thereof possess biological activity as inhibitors of the proteasome. They can be used to treat conditions mediated directly by the function of the proteasome, such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome, such as the transcription factor NF-κB.

A fifth aspect of the present invention relates to pharmaceutical compositions, comprising a compound of Formula VI or Formula VII, and a pharmaceutically acceptable carrier or diluent. A sixth aspect of the present invention relates to methods of inhibiting proteasome function or treating a condition that is mediated directly or indirectly by the function of the proteasome, by administering a compound of Formula VI or Formula VII that possesses unexpectedly high activity in inhibiting the proteasome. Preferred Embodiments are directed to the use of a compound of Formulae VI or VII to prevent or reduce the size of infarct after vascular occlusion for example, for treating neuronal loss following stroke. An additional preferred embodiment is directed to the use of said compounds for treating asthma.

A seventh aspect of the invention relates to enantiomerically-enriched compositions of formyl amides of Formula XIV.

An eighth aspect of the present invention relates to novel individual intermediates, such as aldols of Formula II and aminodiols of Formula III:

and individual steps within the multistep process for forming lactacystin, clasto- lactacystin β-lactone or various analogs thereof.

A ninth aspect of the present invention relates to individual intermediates, such as compounds of Formulae XVII, XVIII and XIX:

XVIIa

XVIIb

XVIIIa

N,

XVIIIb

OH

where X is a halogen, preferably Cl, Br or I, as well as individual steps within the multistep process for forming substituted oxazolines of Formula /.

Other features or advantages of the present invention will be apparent from the following detailed description, and also from the appending claims. Brief Description of the Drawings

FIG. 1. depicts a graph showing the effect of compound 3b, administered i.v., on infarct volume in rats (n=6-8).

FIG. 2. depicts a graph showing the effect of compound 3b, administered i.v. on neurological score in rats (n=6-8).

Detailed Description of the Preferred Embodiments

The present invention relates to an improved multi-step synthesis of lactacystin, c/αsto-lactacystin β-lactone, and analogs thereof, that proceeds in fewer steps and in much greater overall yield than syntheses described in the prior art. A number of individual process steps and chemical intermediates distinguish this synthetic pathway from pathways described in the prior art. For example, this synthetic pathway relies upon a novel stereospeciflc synthesis of an oxazoline intermediate, and a unique stereoselective addition of a formyl amide to the oxazoline. The invention is also directed to novel analogs of Formulae VI and VII that possess unexpected biological activity. Lactacystin, c/ sto-lactacystin β- lactone, and analogs thereof possess biological activity as inhibitors of the proteasome. They can be used to treat conditions mediated directly by the function of the proteasome, such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome, such as the transcription factor

NF-κB. The present invention is also directed to methods of inhibiting proteasome function or treating a condition that is mediated directly or indirectly by the function of the proteasome, by administering a compound of Formula VI or VII that possesses unexpectedly high activity in inhibiting the proteasome. In a preferred aspect of the invention, a pharmaceutical composition that includes a compound of Formula VI or Formula VII is administered to treat ischemic or reperfusion injury. For example, in a preferred embodiment said compounds can be used to treat, prevent or ameliorate neuronal loss following stroke.

Synthetic Processes

A first aspect of the present invention relates to processes for forming lactacystin and analogs thereof having Formula Viand c/αsto-lactacystin β-lactone and analogs thereof having Formula VII:

wherein R^! is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substituted.

The processes for forming these compounds rely upon formation of a common carboxylic acid intermediate of Formula V:

where R¹ and R² are as defined above for Formulae VI and VII. These steps include:

(a) deprotonating a substituted aryl or heteroaryl oxazoline of Formula/:

where R¹ is as defined above, and R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is aryl or heteroaryl, either of which may be optionally substituted; by treating said substituted aryl or heteroary 1 oxazoline with a strong base to form an enolate;

(b) transmetallating said enolate with a metal selected from the group consisting of titanium, aluminum, tin, zinc, magnesium and boron, and thereafter treating with a formyl amide of Formula XIV:

where R² is as defined above for Formulae VI and VII, and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7- membered heterocyclic ring, which may be optionally substituted, and which optionally may include an additional oxygen or nitrogen atom, to form an adduct of Formula II:

where R¹ through R⁶ are as defined above; c) catalytically hydrogenating said adduct of Formula Z/to form a γ- lactam of Formula IV:

where R¹, R² and R³ are as defined above; d) saponifying said γ-lactam of Formula IV to form a lactam carboxylic acid of Formula V:

where R¹ and R² are as defined above.

The carboxylic acid intermediate of Formula V can be cyclized by treatment with a cyclizing reagent to form a c/ ^t -lactacystin-β-lactone or analog thereof of Formula VII, which can be optionally further reacted with a thiol

(R⁷SH), such as N-acetylcysteine, to form lactacystin or an analog thereof having

Formula VI.

Alternatively, the carboxylic acid intermediate of Formula Kcan be directly coupled to a thiol (R⁷SH), such as N-acetylcysteine, to form lactacystin or an analog thereof having Formula VI.

A second aspect of the present invention relates to the formation of enantiomerically-enriched formyl amides of Formula XIV:

XIV

wherein R², R⁵ and R⁶ are as defined above, said method comprising: (a) deprotonating a compound of Formula VIII:

VIII where R⁸ is isopropyl or benzyl, and thereafter acylating the resultant anion with R CH₂COCl to form an acyloxazolidinone of Formula IX:

IX

where R² and R⁸ are as defined above; (b) stereo selectively reacting the acyloxazolidinone of Formula IX with benzyloxymethyl chloride to form a protected alcohol of Formula X:

where R² and R⁸ are as defined above;

(c) hydrolyzing the protected alcohol of Formula X to form a carboxylic acid of Formula XI:

XI

where R² is as defined above; (d) coupling said acid of Formula XI with an amine R⁵R⁶NH₂ to provide an amide of Formula XII:

XII

where R², R⁵ and R⁶ are as defined above;

(e) catalytically hydrogenating, the amide of Formula XII to form an alcohol of Formula XIII:

XIII

where R², R⁵ and R⁶ are as defined above; and

(f) oxidizing the resultant alcohol of Formula XIII to give a formyl amide of Formula XIV.

A third aspect of the invention relates to a process for forming a tri- substituted cis-oxazoline compound of Formula la:

wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is aryl or heteroaryl, either of which may be optionally substituted; said method comprising: (a) asymmetrically dihydroxylating an alkene intermediate of

Formula XV:

C0₂R3 ^XV

to form an optically active diol of Formula XVIa:

(b) reacting said optically active diol of Formula XVIa with an orthoester derived from an aromatic carboxylic acid under acid catalysis (Lewis or Brδnsted acid) to give a mixed orthoester, and thereafter reacting the resulting mixed orthoester intermediate with a reagent selected from the group consisting of lower alkanoyl halides, hydrohalic acids (HX, where X is a halogen), acid chlorides, and halogen-containing Lewis acids (for example BBr₃, SnCl₄

Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX, where X is a halogen, and the like) in the presence of a base to form a derivative of Formula XVIIa:

XVIIa:

wherein X is a halogen, preferably Cl, Br or I;

(c) reacting said derivative of Formula XVIIa with an alkali metal azide to form an azide of Formula XVIIIa:

XVIIIa,

(d) catalytically hydrogenating said azide to form a compound of Formula XlXa:

(e) subjecting the compound of Formula XlXa to ring closing conditions to form said substituted aryl- or heteroary loxazoline of Formula/ with inversion of configuration at the oxygen-substituted carbon to produce a cis- oxazoline of Formula/α; wherein for each of Formulae AT , XVIa, XVIIa,XVIHa and XlXa, R¹, R³ and R⁴ are as defined above for Formula /.

Alternatively, the third aspect of the invention relates to a process for forming a tri-substituted trans-oxazoline compound of Formula lb comprising: (a) asymmetrically dihydroxylating an alkene intermediate of

Formula XV:

XV

C0₂R3 o form an optically active diol of Formula XVIb:

(b) reacting said optically active diol of Formula XVIb with an orthoester derived from an aromatic carboxylic acid under acid catalysis (Lewis or Brόnsted acid) to give a mixed orthoester, and thereafter reacting the resulting mixed orthoester intermediate with a reagent selected from the group consisting of lower alkanoyl halides, hydrohalic acids (HX, where X is halogen), acid chlorides, and halogen-containing Lewis acids (for examples, BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX, where X is a halogen, and the like) in the presence of a base to form a derivative of Formula XVIIb:

XVIIb,

wherein X is a halogen, preferably Cl, Br, or I;

(c) reacting said derivative of Formula XVIIb with an alkali metal azide to form an azide of Formula XVIIIb:

XVIIIb,

(d) catalytically hydrogenating said azide to form a compound of Formula XlXb:

(e) subjecting the compound of Formula XlXb to ring closing conditions to form said substituted aryl- or heteroaryloxazoline of Formula lb, wherein the ring closure reaction proceeds with retention of configuration at the oxygen-substituted carbon to produce a tra -oxazoline of Formula lb; wherein for each of Formulae XV, XVI, XVII, XVIII and XIX, R¹, R³, and R⁴ are as defined above for Formula /.

With respect to the processes described above, the following preferred values are applicable:

Preferred values of R¹ are C,.₁₂ alkyl, especially C,_₈ alkyl, C_3.8 cycloalkyl, especially C_3.6 cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl, C_6.,₄ aryl, especially C_6.10aryl, C_6.10ar(C,_.6)alkyl or C_].₆alk(C₆.,₀)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted. Substituents that can be optionally present on the aryl ring of an R¹ moiety include one or more, preferably one or two, of hydroxy, nitro, trifluoromethyl, halogen, C,_.6 alkyl, C_6.10 aryl, C,_₆ alkoxy, C_1-6 aminoalkyl, C,.₆ aminoalkoxy, amino, C₂_₆ alkoxycarbonyl, carboxy, C,_₆ hydroxyalkyl, C₂.₆ hydroxyalkoxy, C,_₆ alkylsulfonyl, C₆.,₀ arylsulfonyl, C,_.6 alkylsulfinyl, C,.₆ alkylsulfonamido, C₆.₁₀ arylsulfonamido, C_6.I0 ar(C,_.6) alkylsulfonamido, C,.₆ alkyl, C,.₆ hydroxyalkyl, C₆.₁₀ aryl, C_6.]0 aryl(C,_.6)alkyl, C,_.6 alkylcarbonyl, C_2.6 carboxyalkyl, cyano, and trifluoromethoxy.

R¹ is more preferably one of C,._g alkyl such as ethyl, propyl or isopropyl; cycloalkyl, such as cyclohexyl; or C₆.₁₀ aryl, such as phenyl. Most preferred is isopropyl. Preferred values of R² are C,._g alkyl, C₃._g cycloalkyl, especially C_3.6 cycloalkyl, C,._g alkoxy, C₂._g alkenyl, C₂._g alkynyl C_6.14 aryl, especially C_6.10aryl, C₆._]0ar(C,.₆)alkyl or C,.₆alk(C₆._]0)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted with any of the substituents as described for R¹ above.

R² is more preferably C_M alkyl, such as methyl, ethyl, propyl, or butyl; or

C_M alkoxy, such as methoxy, or ethoxy. Most preferred are methyl, ethyl and propyl, and butyl.

With respect to R³, a variety of ester functionalities can be employed at this position. Preferred values are C,._g alkyl, C₃.₈ cycloalkyl, especially C_4.7 cycloalkyl,

C₂._g alkenyl, C₂.₈ alkynyl, C₆.₁₄ aryl, especially C₆.₁₀ aryl, C₆.₁₀ ar(C,_.6) alkyl or

C,_.6alk(C_6.10)aryl, any of which can be optionally substituted. Substituents that can be optionally present on R³ include one or more, preferably one or two, of the substituents as described for R¹ above.

R³ is more preferably C alkyl, C₆._I0 aryl or C₆._I0 ar(C,.₆)alkyl. Most preferred are methyl, ethyl, tert-butyl and benzyl.

R⁴ is preferably C₆.₁₀ aryl, preferably phenyl, or a heteroaryl group selected from the group consisting of thienyl, benzo[b]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, or triazolyl. The phenyl or heteroaryl group can be optionally substituted by one or two of the substituents as described for R¹ above. Most preferred are phenyl, and phenyl substituted by halogen, C,.₆ alkyl, C,.₆ alkoxy, carboxy, amino, C,_₆ alkylamino and or di(C,.₆)alkylamino.

R⁵ and R⁶ are independently one of alkyl, aralkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom. Optional substituents are those listed above for R¹.

R⁵ and R⁶ are preferably C,.₆ alkyl, C₆._]0ar(C,_.6)alkyl or C,_.6alk(C_6.ι₀)aryl or together with the nitrogen atom to which they are attached form a 5- to 7- membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino, diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, and oxazolidinone substituted by halogen, C,_₆ alkyl, C₆.₁₀ ar(C,_.6)alkyl, C,.₆ alkoxy, carboxy, and/or amino. R⁷ is preferably C,._g alkyl, C₃.₈ cycloalkyl, C₆.₁₀ aryl, C_6.10 ar(C,_.6)alkyl,

C,_.6alk(C_6.]0)aryl, any of which can be optionally substituted. Substituents that can be optionally present on either or both of the ring or chain portions of R⁷ include one or more, preferably one or two, of the substituents as described for R¹ above. Preferably, R⁷ together with the sulfur atom to which it is attached is cysteine or a derivative of cysteine such as N-acetyl cysteine, glutathione, and the like.

Scheme 1 is a general scheme for forming lactacystin and c/αstø-lactacystin-β-lactone analogs from substituted oxazoline starting materials.

Scheme 1

VII The starting oxazoline /, which may be of either the cis (Ia) or trans (lb) configuration, is deprotonated with a strong base to form the enolate. Examples of bases suitable for use in this reaction are organic bases, including hindered amide bases such as lithium diisopropylamide (LDA), lithium tetramethylpiperidide (LiTMP), lithium, sodium or potassium hexamethyldisilazide (LiHMDS,

NaHMDS, KHMDS), or the like; or hindered alkyllithium reagents, such as sec- butyllithium, tert-butyllithium, or the like. The reaction is preferably conducted at reduced temperature in an ethereal solvent, such as diethyl ether, tetrahydrofuran (THF), or dimethoxy ethane (DME). Reaction temperatures preferably range from about -100°C to about -30°C, more preferably from -85 °C to -50°C, and most preferably from -85 °C to -75 °C. The reaction temperature is important in determining the stereochemical outcome of the subsequent addition to the aldehyde, with lower temperatures providing better selectivity.

The deprotonation step is followed by transmetallating said enolate with a metal selected from the group consisting of titanium, aluminum, tin, zinc, magnesium and boron. Preferred reagents for this step include titanium or aluminum Lewis acids, for example Me₂AlCl or ( -PrO)₃TiCl or a mixture of the two. Preferably, between one and three molar equivalents of the Lewis acid are used, more preferably between two and three equivalents, and most preferably about 2.2-2.3 equivalents. Subsequent treatment of the enolate with a formyl amide (XIV) affords the adduct//. Excess aldehyde is washed away with sodium bisulfite solution, and the crude material is carried forward to the next step without further purification. The use of 2.2-2.3 equivalents of Me₂AlCl results in selective formation of the (65)-product (lactacystin numbering), in a ratio generally better than about 10:1, whereas the use of 1 equivalent of Me₂AlCl results in selective formation of the (67?)-product, in a ratio of about 5:1.

Catalytic hydrogeno lysis of the adduct //, as a mixture of (65)- and (67?)- epimers, affords the desired γ-lactam (IV), sometimes as a mixture with the aminodiol ///:

Useful catalysts for this reaction include palladium black, palladium on activated carbon, palladium hydroxide on carbon, or the like. Organic solvents suitable for use in this reaction include lower alkanols such as methanol, ethanol, or isopropanol, lower alkanoates such as ethyl acetate, lower alkanoic acids such as acetic acid, or mixtures thereof. The reaction is conducted under an atmosphere of hydrogen, at pressures ranging from about 15 to about 100 p.s.i., more preferably from about 30 to about 50 p.s.i. Alternatively, transfer hydrogenation procedures (R.A.W. Johnstone et al, Chem. Rev. 55:129 (1985)) may be used, in which the adduct // is treated at atmospheric pressure with a catalyst and a hydrogen donor.

Upon heating of the crude product mixture, the aminodiol ///is converted to the γ-lactam IV, which can then be isolated in approximately 60-75% overall yield from//. The heating step is conveniently carried out by first filtering off the catalyst used in the hydrogenation step and then heating the filtrate to reflux.

When no aminodiol /// is present in the crude product mixture, the heating step is omitted. Ester saponification, followed by cyclization, affords the β-lactone VII in 40-90% yield, and generally in greater than 60% yield. Cyclization can be effected with coupling reagents known in the art, including aryl sulfonyl chlorides, benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), O-(lH-benzotriazol-l -yl)-N, N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), alkyl, aryl or alkenyl chloroformates, and the like. Isopropenyl chloroformate is a preferred reagent for this step, since all byproducts are volatile and chromatographic purification of the product is not necessary. c/ wto-Lactacystin β-lactone can be converted to lactacystin by treatment of the β-lactone with N-acetylcysteine according to the reported procedure (Corey et al, Tetrahedron Lett. 34:6977 (1993)). Reactions of the β-lactone //with other thiols proceed analogously. Alternatively, lactacystin analogs are prepared by coupling the carboxylic acid intermediate V with a thiol to form the corresponding thiolester VI. The method of this invention is therefore useful for synthesis of both lactacystin and c/αsto-lactacystin β-lactone, as well as analogs thereof.

The enantiomerically-enriched formyl amides XIV employed in the aldol reaction are new. They can be prepared according to a representative reaction sequence such as that depicted in Scheme 2. For purposes of the present invention, the term "enantiomerically-enriched" means that one enantiomer is present in excess relative to the other; that is, one enantiomer represents greater than 50% of the mixture. The term "stereoselective" is used to mean that a synthesis or reaction step produces one enantiomer or diastereomer in excess relative to the other enantiomer or to other diastereomer(s).

Scheme 2

.R⁵

R2 .

XIV

Acylation of the anion of (<S)-(-)-4-benzyl-2-oxazolidinone ( Villa) or (S)- (-)-4-isopropyl-2-oxazolidinone (VHIb) (where R⁸ is benzyl or isopropyl) affords the acyloxazolidinone IX in greater than 80% yield. Subsequent stereoselective benzyloxymethylation (Evans et al, J. Am. Chem. Soc. 772:8215 (1990)) gives the protected alcohol X in greater than 80% yield, provided that the benzyl chloromethyl ether is freshly prepared (Connor et al, Organic Syntheses 52:16 ( 1974)). Peroxide mediated hydrolysis affords the acid XI, which is coupled with an amine to provide the amide XII, generally in greater than 50% overall yield. Benzyl group hydrogenolysis, followed by oxidation of the resultant alcohol (XIII) then affords the formyl amide XlVin 80-85% yield. Pearlmans catalyst (Pd(OH)₂) is preferably used for the hydrogenolysis step. The final oxidation step is best accomplished with the periodinane reported by Dess and Martin, J. Org. Chem. 48:4156 (1983) or with 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO) free radical, and buffered hypochlorite in the presence of bromide ion (J. Org. Chem. 50:4888 (1985); Org. Synth. Coll. 5:367 (1993)). Other mild oxidants such as tetrapropyl-ammonium perruthenate (TPAP) can also be used. The formyl amide

XIV can be shown to be enantiomerically pure by reducing the aldehyde with sodium borohydride and converting the resultant alcohol to the corresponding Mosher ester using 7?-(+)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (Dale et al, J. Org. Chem. 34:2543 (1969)). Η NMR analysis at 300 MHz reveals a single diastereomer. The aldehydes prepared according to Scheme 2 are configurationally stable, showing no signs of enantiomeric deterioration after one week, when stored at 0 °C. The aldehyde is also configurationally stable under the conditions of the aldol reaction, and the adduct // is formed without epimerization of the substituent R² at C(7). The synthetic methods will work with any substituent at R¹ that is stable to strong base and to hydrogenation. Isopropyl is the preferred substituent for good proteasome inhibiting activity of the final product.

Scheme 3

OH asymmetric

R dihydroxylation

COzR³ CQ-R³

OH

XV XVIa

The invention also relates to a new route to form the oxazoline starting material /. The overall synthesis includes five steps (Scheme 3) and affords the c/5-substituted oxazoline Ia, which is thereafter employed in the method described above. The first step depicted in Scheme 3 is Sharpless asymmetric dihydroxylation (Sharpless et al, J. Org. Chem. 57:2768 (1992); Kolb et al, Chem. Rev. 94:2483 (1994); Shao and Goodman, J. Org. Chem. (57:2582 (1996)) of the alkene XV. If not commercially available, the alkene XV is prepared by Wittig condensation between the aldehyde and carboethoxymethylene triphenylphosphorane (Hale et al, Tetrahedron 50:9181 (1994)). Other olefination procedures are also known in the art. The dihydroxylation reaction is preferably conducted with AD-mix-β (Aldrich Chemical Co.) in the presence of methane sulfonamide and stereoselectively affords the diol XVIa, as predicted by the Sharpless face-selection rule. On a large scale, the dihydroxylation reaction is preferably conducted using N-methylmorpholine-N-oxide (NMO) as the reoxidant in place of K₃Fe(CN)₆ present in AD-mix-β . Although proceeding with somewhat lower enantioselectivity, this procedure allows more concentrated reaction mixtures and greatly simplifies the workup. The enantiomeric purity of the product can be enhanced by recrystallization. In the next step, the diol XVIa is treated with an orthoester under Lewis or Brόnsted acid catalysis to give a mixed orthoester, which is converted in situ to the haloester XVIIa by treatment with an acyl halide (Haddad et al, Tetrahedron Lett. 37:4525 (1996)). Although acyl halides, especially acetyl halides are preferred for this reaction, other acid halides such as HCl, HBr, HI, Me₃SiCl, Me₃SiI, Me₃SiBr and the like may be used. Halogen-containing Lewis acids of the formula ML_nX, such as BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, and the like can also be used. In the previous formula, M is a metal selected from the group consisting of B, Ti, Sn, Al, Zn, and Mg; L is any suitable ligand for the metal, preferably an alkoxide or halogen group; n is an integer that results in a stable complex, and X is a halogen. Preferably acetyl bromide is used to produce the haloester XVIIa. Preferably the orthoester employed in this reaction is derived from an aromatic or heteroaromatic carboxylic acid. More preferably, the orthoester is derived from benzoic acid, e.g., trimethyl orthobenzoate. The use of boron trifluoride etherate as the Lewis acid catalyst in the formation of the mixed orthoester is preferred, but other acids, such as HBr, SnCl₄, TiCl₄, BBr₃, and the like, can also be used.

After workup, the crude halide XVIIa is converted to the azide XVIIIa by treatment with an alkali metal azide in a polar aprotic organic solvent, such as dimethyl sulfoxide (DMSO) or N,N-dimethyl formamide (DMF). Catalytic hydrogenation of the azide XVIIIa over a palladium catalyst in ethyl acetate proceeds with concomitant migration of the aroyl group (Wang et al. , J. Org. Chem. 59:50X4 (1994)) to afford the hydroxyamide XlXa.

Treatment of XlXa with thionyl chloride in methylene chloride effects ring closure with inversion of configuration at the hydroxyl-substituted carbon atom to produce the cw-substituted oxazoline starting material Ia. Other reagents suitable for use in this reaction include sulfuryl chloride, phosphorous trichloride, phosphorous oxychloride, and (methoxycarbonylsulfamoyl)-triethylammonium hydroxide, inner salt (Burgess reagent). Treatment of XlXa under Mitsunobu conditions (Mitsunobu, Synthesis:! (1981) will also effect a ring closure. The oxazoline ring oxygen atom is destined to become the C(9)-hydroxyl group in the final products VI and VII. Under equilibrating conditions (sodium methoxide, methanol), the cw-oxazoline (Ia) is converted to the trflrø-oxazoline (lb) by inversion of configuration of the ester substituent, with the configuration of the

R¹ substituent remaining fixed. The cis- and trarø-oxazolines can both be used in the method depicted in Scheme 1, with equivalent results.

In an alternative route to form the oxazoline starting material /, /?-toluenesulfonic acid ( ?-TsOH) is used to effect ring closure (Scheme 4). In this case, ring closure proceeds with retention of configuration at the hydroxyl-substituted carbon atom to afford the trαπs-oxazoline (lb). In order to obtain the proper stereochemistry at C(9) of the final product, the chiral ligand employed in the dihydroxylation reaction must be selected so as to provide the opposite face selectivity from that depicted in Scheme 3. For example, AD-mix-α is used in place of AD-mix-β . All other steps in the sequence proceed analogously to those described for the synthesis of the -oxazoline la. Scheme 4

Compounds

Many of the compounds described above are novel compounds; the novel compounds are also claimed.

Fourth, fifth and sixth aspects of the invention relate to lactacystin analogs that can be made by the synthetic routes described herein; to pharmaceutical compositions including such compounds; and to methods of treating a subject having a condition mediated by proteins processed by the proteasome by administering to a subject an effective amount of a pharmaceutical composition disclosed herein. These methods include treatments for Alzheimers disease, cachexia, cancer, inflammation (e.g., inflammatory responses associated with allergies, bone marrow or solid organ transplantation, or disease states, including but not limited to arthritis, multiple sclerosis, inflammatory bowel disease and parasitic diseases such as malaria), psoriasis, restenosis, stroke, and myocardial infarction. The compounds of Formulae VI and VII disclosed herein are highly selective for the proteasome, and do not inhibit other proteases such as trypsin, oc-chymotrypsin, calpain I, calpain II, papain, and cathepsin B.

As disclosed by Fenteany et al. (WO 96/32105), hereby incorporated by reference in its entirety, lactacystin, c/αsto-lactacystin β-lactone, and analogs thereof possess biological activity as inhibitors of the proteasome. They can be used to treat conditions mediated directly by the function of the proteasome, such as muscle wasting, or mediated indirectly via proteins which are processed by the proteasome, such as the transcription factor NF-κB. The compounds prepared by the methods of this invention can also be used to determine whether a cellular, developmental, or physiological process or output is regulated by the proteolytic activity of the proteasome.

Those compounds that possess unexpected proteasome function-inhibiting activity are compounds of Formulae VI and VII:

or a salt thereof, wherein:

R¹ is C,_.12 alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂._g alkynyl, C₆.₁₄ aryl, C₆.₁₀ ar(C,_.6)alkyl or C₁.₆alk(C₆.₁₀)aryl;

R² is C₂.₆ alkyl; and R⁷ is C,._g alkyl, C₃._g cycloalkyl, C₆.₁₀ aryl, C_6.10 ar(C,_.6)alkyl,

C_].6alk(C_6.10)aryl, any of which can be optionally substituted. Substituents that can be optionally present on either or both of the ring or chain portions of R⁷ include one or more, preferably one or two, of the substituents as described for R¹ above.

Preferred compounds are those where R¹ is C,_.4 alkyl, more preferably isopropyl. R² is preferably ethyl, n-propyl, n-butyl or isobutyl. Preferably, R⁷ together with the sulfur atom to which it is attached is cysteine or a derivative of cysteine such as N-acetyl cysteine, glutathione, and the like.

A seventh aspect of the present invention is directed to enantiomerically- enriched formyl amides of Formula XIV:

XIV

or salts thereof, wherein

R² is C,._g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl, C_6.14 aryl, C_6.10 ar(C,.₆)alkyl or C,.₆alk(C₆._]0)aryl; and

R⁵ and R⁶ are independently C,.₆ alkyl, C₆.,₀ ar(C,.₆)alkyl or C_1.6alk(C_6.10)aryl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

Preferred compounds are those where R² is C_2.6 alkyl.

An eighth aspect of the present invention is directed to compounds of Formulae // and ///:

or salts thereof wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;

R⁴ is optionally substituted aryl or optionally substituted heteroaryl; and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7- membered heterocyclic ring, which can be optionally substituted, and which optionally include an additional oxygen or nitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino, diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, and oxazolidinone substituted by halogen, C,.₆ alkyl, C₆.₁₀ ar(C,_.6)alkyl, C^ alkoxy, carboxy, and/or amino.

Preferred compounds of Formulae // and /// are those wherein: R¹ is C,.₁₂ alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C_2.8 alkynyl, C_6.14 aryl, C₆._]0 ar (C_j.^alkyl or C,.₆alk(C₆.₁₀)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is C,._g alkyl, C₃.₈ cycloalkyl, C₂._g alkenyl, C_2.g alkynyl, C_6.14 aryl, C_6.10 ar(C,_.6)alkyl or C,.₆alk(C_6.10)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is C,._g alkyl, C₃._g cycloalkyl, C₂._g alkenyl, C₂._g alkynyl, C_6.I4 aryl, C₆.,₀ ar(C,_.6)alkyl or C,.₆alk(C₆.₁₀)aryl, any of which can be optionally substituted;

R⁴ is optionally substituted C_6.10 aryl, or an optionally substituted heteroaryl group selected from the group consisting of thienyl, benzo[β]thienyl, fiiryl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinoliziny 1, isoquinolyl, quinolyl, or triazolyl; and

R⁵ and R⁶ are independently C,_.6 alkyl, C_6.,₀ ar(C,_.6)alkyl or C,_.6alk(C_6.]0)aryl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom. Most preferred values for NR⁵R⁶ are dimethyl ami no, diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, and oxazolidinone substituted by halogen, C,.₆ alkyl, C₆._]0ar(C,.₆)alkyl, C,_₆ alkoxy, carboxy, and/or amino.

A ninth aspect of the present invention is directed to compounds of Formulae XVIIa, XVIIb, XVIIIa, XVIIIb, XlXa or XlXb:

XVIIa XVIIb

X X

XVIIIa XVIIIb

XlXa XlXb

OH

or salts thereof, wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl.

Preferred compounds of Formulae XVII, XVIII or XIX are those wherein

R¹ is C ₂ alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl C₆.₁₄ aryl, C₆.₁₀ ar(C,_.6)alkyl or C,_₆alk(C_6.10)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is C,_.8 alkyl, C_3.g cycloalkyl, C₂.₈ alkenyl, C_2.g alkynyl, C_6.I4 aryl, C, 10 ar(C,.₆)alkyl or C,_.6alk(C₆.₁₀)aryl, any of which can be optionally substituted; and R⁴ is optionally substituted C₆.,₀ aryl, or an optionally substituted heteroaryl group selected from the group consisting of thienyl, benzo[b]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, or triazolyl.

Definitions

The term "alkyl" as employed herein includes both straight and branched chain radicals of up to 12 carbons, preferably 1 -8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.

The term "substituted alkyl" as employed herein, includes alkyl groups as defined above that have one, two or three halo, hydroxy, nitro, trifluoromefhyl, halogen, C,.₆ alkyl, C₆.₁₀ aryl, C,.₆ alkoxy, C,_₆ aminoalkyl, C,_.6 aminoalkoxy, amino, C₂.₆ alkoxycarbonyl, carboxy, C,.₆ hydroxyalkyl, C_2.6 hydroxyalkoxy, C,_.6 alkylsulfonyl, C₆.₁₀ arylsulfonyl, C,_₆ alkylsulfinyl, C,.₆ alkylsulfonamido, C₆.₁₀ arylsulfonamido, C₆.₁₀ ar(C,.₆) alkylsulfonamido, C,.₆ alkyl, C,_.6 hydroxyalkyl, C_6.!0 aryl, C_6.10 aryl(C,.₆)alkyl, C,.₆ alkylcarbonyl, C_2.6 carboxyalkyl, cyano, and trifluoromethoxy and/or carboxy substituents.

The term "cycloalkyl" as employed herein includes saturated cyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8 carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of which groups may be substituted with substituents such as halogen, C,.₆ alkyl, C,_.6 alkoxy and/or hydroxy group.

The term "heteroaryl" as employed herein refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; 6, 10 or 14 π electrons shared in a cyclic array; and containing carbon atoms and 1 , 2 or 3 oxygen, nitrogen or sulfur heteroatoms (where examples of heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 47^-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, tetrazolyl, quinazolinyl, cinnolinyl, pteridinyl, 4αH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups).

The term "aryl" as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl. The term "aralkyl" or "arylalkyl" as employed herein by itself or as part of another group refers to C,.₆ alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.

The term "alkaryl" or "alkylaryl" as employed herein by itself or as part of another group refers to an aryl group as discussed above having a C,_₆ alkyl substituent, such as toluyl, ethylphenyl, or methylnaphthyl.

The term "optionally substituted" when used with respect to aryl, aralkyl, alkaryl or 5-, 6-, 9- or 10- membered heteroaryl groups means that the ring portion of said groups can be optionally substituted by one or two substituents independently selected from C,_₆ alkyl, C₃.₈ cycloalkyl, C,_₆ alkyl(C₃.₈)cycloalkyl, C_2.g alkenyl, C₂._g alkynyl, cyano, amino, C,_₆ alkylamino, di(C,.₆)alkylamino, benzylamino, dibenzylamino, nitro, carboxy, carbo(C,_.6)alkoxy, trifluoromethyl, halogen, C,.₆ alkoxy, C₆.₁₀aryl, C^oary C^alkyl, C_6.I0aryl(C,.₆)alkoxy, hydroxy, C,.₆ alkylthio, C,.₆ alkylsulfmyl, C_1-6 alkylsulfonyl, C₆.₁₀ arylthio, C_6.10 arylsulfinyl, C₆.,₀ arylsulfonyl, C₆.₁₀ aryl, C,.₆ alkyl(C₆.₁₀)aryl, and halo(C_6.I0)aryl. The term "alkoxy" refers to the above alkyl groups linked to oxygen.

The term "halogen" or "halo" as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.

The term "amido" as employed herein refers to formylamino, alkylcarbonylamino or arylcarbonylamino. Uses

Pharmacological data for c/αsto-lactacystin β-lactone analogs prepared by the methods of this invention are provided in Table 1. These compounds are all irreversible inactivators of the 20S proteasome, acylating the N-terminal threonine residue of the X/MBl subunit. The value K_obs/[I] is a measure of the rate of enzyme inactivation. Several compounds show improved activity, i.e., more rapid rates of inactivation, when compared to clasto- lactacystin β-lactone itself (2). The compound that is most potent in the enzyme assay is the 7-methoxy derivative 3f. However, when assayed in cell culture, 3f is less potent than 2. The lactone ring is subject to nucleophilic attack not only by the threonine residue of the proteasome X/MBl subunit, but also by water. Hydrolysis results in formation of the hydroxy acid V, which is not active as an inhibitor of the proteasome. Relative potency in cell culture is a composite of many factors, including enzyme potency, cell penetration, and hydrolysis rate. Although more potent than 2 against the enzyme, 3f is also more rapidly hydrolyzed, resulting in much weaker activity in cell culture. By contrast, the analogs 3a-3d show unexpectedly improved potency not only in the enzyme assay, but also in cell culture.

The disclosed compounds are used to treat conditions mediated 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 ΝF-κB. The proteasome participates in the rapid elimination and post-translational processing of proteins involved in cellular regulation (e.g., cell cycle, gene transcription, and metabolic pathways), intercellular communication, and the immune response (e.g., antigen presentation). Specific examples include β-amyloid protein and regulatory proteins such as cyclins and transcription factor NF-κB. Treating as used herein includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize the subject's condition. Other embodiments of the invention relate to cachexia and muscle-wasting diseases. 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. Proteasome inhibitors are 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. Pat. No. 5,340,736 (1994).

Embodiments of the invention therefore encompass methods for reducing the rate of muscle protein degradation in a cell, and reducing the rate of intracellular protein degradation. Each of these methods includes the step of contacting a cell (in vivo or in vitro, e.g., a muscle in a subject) with an effective amount of a compound (e.g., pharmaceutical composition) of a formula disclosed herein.

Proteasome inhibitors block processing of ubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors also block IκB-α degradation and NF-κB activation. (Palombella, et al; and Traenckner, et al, EMBO J. 73:5433-5441 (1994)). One embodiment of the invention is a method for inhibiting IκB-α degradation, including contacting the cell with a compound of a formula described herein. A further embodiment is a method for reducing the cellular content of NF- KB in a cell, muscle, organ, or subject, including contacting the cell, muscle, organ, or subject with a compound of a formula described herein. Additional embodiments encompass methods for treating inflammatory responses associated with allergies, bone marrow or solid organ transplantation, or disease states, including but not limited to arthritis, inflammatory bowel disease, asthma, and multiple sclerosis by administering a compound of a formula disclosed herein. A preferred embodiment of the invention is directed to treating asthma by administering a compound of Formula VI or Formula VII, most preferably compound 3b.

Proteasome inhibitors are also useful for treatment of ischemic or reperfusion injury, particularly for preventing or reducing the size of infarct after vascular occlusion such as occurs during a stroke or heart attack, as described in

Brand, U.S. patent application Serial No. (ProScript Docket No.

102.603.173), filed February 17, 1998, U.S. patent application Serial No. 08/988,339, filed December 3, 1997, and U.S. patent application Serial No. 08/801,936, filed February 15, 1998. Proteasome inhibitors also block proteasome-dependent transformation of protazoan parasites (Gonzalez et al , J.

Exp. Med. 184:1909 (1996). Further embodiments of the invention therefore encompass methods for treating an infarct or a protazoan parasitic disease by administering a compound of a formula disclosed herein. In a preferred aspect of the invention, a compound of Formula VI or Formula VII is administered to prevent or reduce the size of the infarct after vascular occlusion. Said compounds can be administered from about 0 to about 10 hours after the occurrence of a stroke in order to treat or reduce neuronal loss following an ischemic event.

Compounds 3b is the most preferred compound in this aspect of the invention.

Proteasome inhibitors also block degradation of cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinase inhibitors, and tumor suppressor proteins, such as p53. Other embodiments of the invention therefore encompass methods for blocking the cell cycle and for treating cell proliferative diseases such as cancer, psoriasis, and restenosis with a compound of a formula described herein. The term "inhibitor" is meant to describe a compound that blocks or reduces the activity of an enzyme (e.g., the proteasome, or the X/MB 1 subunit of the 20S proteasome). An inhibitor may act with competitive, uncompetitive, or noncompetitive inhibition. An inhibitor may bind reversibly or irreversibly, and therefore the term includes compounds which are suicide substrates of an enzyme. An inhibitor may modify one or more sites on or near the active site of the enzyme, or it may cause a conformational change elsewhere on the enzyme.

Amounts and regimens for the administration of proteasome inhibitors and compositions of the invention can be determined readily by those with ordinary skill in the clinical art. Generally, the dosage of the composition of the invention will vary depending upon considerations such as: type of composition employed; age; health; medical conditions being treated; kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired; extent of tissue damage; gender; duration of the symptoms; and, counter indications, if any, and other variables to be adjusted by the individual physician.

A desired dosage can be administered in one or more applications to obtain the desired results. Pharmaceutical compositions containing the proteasome inhibitors of the invention can be provided in unit dosage forms.

Compositions within the scope of this invention include all compositions wherein the compounds of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typically, the compounds may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for a proteosome-mediated condition such as a stroke or asthma. For intramuscular injection, the dose is generally about one-half of the oral dose.

In the method of prevention or reduction of infarct size the compound can be administered by intravenous injection at a dose of about 0.01 to about 10 mg/kg, preferably about 0.025 to about 1 mg/kg.

The unit oral dose may comprise from about 0.01 to about 50 mg, preferably about 0.1 to about 10 mg of the compound. The unit dose may be administered one or more times daily as one or more tablets each containing from about 0.1 to about 10, conveniently about 0.25 to 50 mg of the compound or its solvates. For use in treating stroke, it is preferred that a single dosage be administered, 0 to about 10 hours post-event, preferably 0 to about 6 hours post- event.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.

The preparation of formyl amides XIV according to the synthetic scheme depicted in scheme 2 as exemplified in Examples 1 -6.

Example 1: Acyl Oxazolidinones (IX)

a. Acyl oxazolidinone IXb (R² = w-Pr; R⁸ = CH₂Ph): A cooled (-78 °C) solution of (5)-(-)-4-benzyl-2-oxazolidinone (4.0 g, 22.6 mmol) in 75 mL anhydrous THF was treated with a 2.5 M solution of n-BuLi in hexane (9.1 mL, 22.6 mmol) over 15 min. After 5 min, neat valeryl chloride (2.95 mL, 24.9 mmol) was added dropwise and the mixture was stirred for another 45 min. at -78 °C.

The mixture was then allowed to reach room temperature, stirred for another 90 min, and then treated with 50 mL saturated NH₄C1 solution. Dichloromethane (50 mL) was then added and the organic layer was washed with brine (2 x 30 mL), dried over MgSO₄ and concentrated in vacuo. This afforded 5.94 g (100 %) of the desired acyl oxazolidinone IXb as a clear colorless oil. 'H NMR (300 MHz,

CDC1₃) δ 7.36-7.20 (m, 5H), 4.71-4.64 (m, 1H), 4.23-4.14 (m, 1H), 3.40 (dd, J= 13.3, 3.2 Hz, 1H), 3.04-2.84 (m, 2H), 2.77 (dd, J=13.3, 9.6 Hz, 1H), 1.74-1.63 (m, 2H), 1.46-1.38 (m, 2H), 0.96 (t, J=7.3 Hz, 3H). b. Acyl oxazolidinone IXa (R² = Et; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone IXb, the lithium anion of (S)-(-)-4-benzyl-2-oxazolidinone was treated with butyryl chloride to provide acyl oxazolidinone IXa in 94% yield. 'H NMR (300 MHz, CDC1₃) δ 7.37-7.20 (m, 5H), 4.68 (ddd, J= 13.1, 7.0, 3.4 Hz, 1H), 4.23-4.13 (m, 2H), 3.30 (dd,J= 13.3, 9.6 Hz, 1H), 3.02-2.82 (m, 2H), 2.77 (dd, J=13.3, 9.6 Hz, 1H), 1.73 (q, J= 7.3 Hz, 2H), 1.01 (t, J= 7.3 Hz, 3H). c. Acyl oxazolidinone IXc (R² = n-Bu; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone IXb, the lithium anion of (S)-(-)-4-benzyl-2-oxazolidinone was treated with hexanoyl chloride to provide acyl oxazolidinone IXc in 96% yield. Η NMR (300 MHz, CDC1₃) δ 7.36-7.20 (m, 5H), 4.68 (m, 1H), 4.23-4.14 (m, 2H), 3.30 (dd, J= 13.3, 3.3 Hz, 1H), 3.02-2.83 (m, 2H), 2.76 (dd, J=13.3, 9.6 Hz, 1H), 1.70 (m, 2H), 1.43-1.34 (m, 4H), 0.92 (t, J= 3.3 Hz, 3H). d. Acyl oxazolidinone IXd (R² = i-Bu; R⁸ = CH₂Ph): i. 4-Methylvaleryl chloride

4- Methylvaleryl chloride was prepared from commercially available 4-methylvaleric acid in the following way : a cold (0 ° C) solution of 4-methy Ivaleric acid (1.85 mL, 15.0 mmol) in 50 mL anhydrous CH₂C1₂ containing 10 mL of DMF was treated with 1.95 μL oxalyl chloride (22.5 mmol). The mixture was then stirred for 3 h at room temperature, concentrated in vacuo and filtered to affords 1.65 g (100%>) of the desired acid chloride as a colorless liquid. ii. Acyl oxazolidinone IXd (R² = -Bu; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone IXb, the lithium anion of (S)-(-)-4-benzyl-2-oxazolidinone was treated with 4-methy Ivaleryl chloride to provide acyl oxazolidinone IXd in 85% yield. Η NMR (300 MHz, CDC1₃) δ 7.37-7.20 (m, 5H), 4.70-4.63 (m, 1H), 4.23-4.15 (m, 2H), 3.30 (dd, J= 13.2, 3.2 Hz, 1H), 2.98-2.90 (m, 2H), 2.76 (dd, J=13.3, 9.6 Hz, 1H), 1.68-1.54 (m, 3H), 0.94 (d, J= 6.2 Hz, 3H). e. Acyl oxazolidinone IXe (R² = CH₂Ph; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone IXb, the lithium anion of (S)-(-)-4-benzyl-2-oxazolidinone was treated with hydrocinnamoyl chloride to provide acyl oxazolidinone IXe in 82% yield. Η NMR (300 MHz, CDC1₃) δ 7.35-7.16 (m, 10H), 4.70-4.63 (m, 1H), 4.21-4.14 (m, 2H), 3.38-3.19 (m, 3H), 3.08-2.98 (m, 2H), 2.75 (dd, J=13.4, 9.5 Hz, 1H).

Example 2: Acyl Oxazolidinones (X)

a. Acyl oxazolidinone Xb (R² = «-Pr; R⁸ = CH₂Ph) : A cold (0 ° C) solution of acyl oxazolidinone IXb (5.74 g, 22.0 mmol) in 110 mL anhydrous CH₂C1₂ was treated with 2.52 mL TiCl₄ (23.1 mmol) resulting in the formation of an abundant precipitate. After 5 min, diisopropylethylamine (4.22 mL, 24.2 mmol) was added slowly and the resulting dark brown solution was stirred at room temperature for 35 min. Benzyl chloromethyl ether (6.0 mL, 44.0 mmol) was rapidly added and the mixture was stirred for 5 h at room temperature. 50 mL CH₂C1₂ and 75 mL of

10% aqueous NH₄C1 were then added, resulting in the formation of yellow gummy material. After stirring the suspension vigorously for 10 min, the supernatant was transferred in a separatory funnel and the gummy residue was taken up in 100 mL 1:1 10% aqueous NH₄C1/CH₂C1₂. The combined organic layers were then washed successively with IN aqueous HCl, saturated NaHCO₃ and brine, dried over

MgSO₄ and concentrated in vacuo. The crude solid material was recrystallized from EtOAc/hexane affording 6.80 g of desired acyl oxazolidinone Xb as a white solid in 81%yield. ¹HNMR(300 MHz, CDCl₃) δ 7.34-7.18 (m, 1 OH), 4.77-4.69 (m, 1H), 4.55 (s, 2H), 4.32-4.23 (m, 1H), 4.21-4.10 (m, 2H), 3.80 (t, J= 9.0 Hz, 1H), 3.65 (dd, J= 9.0, 5.0 Hz, 1H), 3.23 (dd, J= 13.5, 3.3 Hz, 1H), 2.69 (dd, J=

13.5, 9.3 Hz, 1H), 1.74-1.64 (m, 1H), 1.54-1.44 (m, 1H), 1.40-1.28 (m, 2H), 0.91 (t, J= 7.3 Hz, 3H). LRMS (FAB) m/e 382 (M+H⁺) b. Acyl oxazolidinone Xa (R² = Et; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone Xb, acyl oxazolidinone Xa was obtained in 80% yield. 'H NMR (300 MHz, CDC1₃) δ 7.36-7.18 (m, 10H), 4.55 (s, 2H), 4.21-4.11 (m, 3H), 3.81 (t, J- 9.0 Hz, 1H), 3.66 (dd, J= 9.0, 5.0 Hz, 1H), 3.23 (dd, J= 13.5, 3.2 Hz, 1H), 2.70 (dd, J= 13.5, 9.3 Hz, 1H), 1.78-1.57 (m, 2H), 0.94 (t, J= 7.5 Hz, 3H). c. Acyl oxazolidinone Xc (R² = w-Bu; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone Xb, acyl oxazolidinone Xc was obtained in 91% yield. 'H NMR (300 MHz, CDC1₃) δ

7.38-7.17 (m, 10H), 4.72 (m, 1H), 4.54 (s, 2H), 4.27-4.10 (m, 2H), 3.79 (t,J= 8.7 Hz, 1H), 3.65 (dd, J= 9.1, 5.0 Hz, 1H), 3.23 (dd, J= 13.5, 3.3 Hz, 1H), 2.68 (dd, J= 13.5, 9.3 Hz, 1H), 1.75-1.68 (m, 1H), 1.31-1.26 (m, 4H), 0.87 (t, J= 6.8 Hz, 3H). d. Acyl oxazolidinone Xd (R² = -Bu; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone Xb, acyl oxazolidinone Xd was obtained in 98% yield. Η NMR (300 MHz, CDC1₃) δ 7.38-7.17 (m, 10H), 4.75-4.67 (m, 1H), 4.57 (d, J= 12.0 Hz, 1H), 4.51 (d, J= 12.0 Hz, 1H), 4.41-4.36 (m, 1H), 4.20-4.09 (m, 2H), 3.74 (t,J= 9.0 Hz, 1H), 3.65 (dd, J= 9.0, 5.1 Hz, 1H), 3.23 (dd, J= 13.5, 3.2 Hz, 1H), 2.63 (dd, J= 13.5, 9.5

Hz, 1H), 1.74-1.52 (m, 2H), 1.35 (dd, J= 13.1, 6.1 Hz, 1H), 0.92 (d, J= 2.9 Hz, 3H), 0.90 (d, J= 2.9 Hz, 3H). e. Acyl oxazolidinone Xe (R² = CH₂Ph; R⁸ = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone Xb, acyl oxazolidinone Xe was obtained in 84% yield. Η NMR (300 MHz, CDC1₃) δ

7.38-7.15 (m, 15H), 4.62-4.50 (m, 4H), 4.03 (dd, J= 9.0, 2.7 Hz, 1H), 3.93-3.82 (m, 2H), 3.66 (dd, J= 9.2, 4.8 Hz, 1H), 3.19 (dd, J- 13.5, 3.2 Hz, 1H), 2.98 (dd, J= 13.4, 8.2 Hz, 1H), 2.88 (dd, J= 13.4, 7.3 Hz, 1H), 2.68 (dd, J= 13.5, 9.3 Hz, 1H).

Example 3: Carboxylic Acids (XI)

a. Carboxylic acid Xlb (R² = n-Pr): A cold (0°C) solution of 6.60 g (17.3 mmol) of acyl oxazolidinone Xb in 320 mL THF/H₂O was treated successively with 6.95 mL 35% aqueous H₂O₂ and a solution of lithium hydroxide monohydrate (1.46 g, 34.6 mmol) in 20 mL H₂O. The mixture was stirred for 16 h at 0°C and then treated carefully first with a solution Na^O_j (10.5 g) in 55 mL H₂O and then with a solution of NaHCO₃ (4.35 g) in 100 mL H₂O. The mixture was stirred for 30 min at room temperature and concentrated in vacuo to remove the THF. The resulting aqueous mixture was then washed with CH₂C1₂ (4 x 75 mL), cooled to 0°C, acidified with 6N aqueous HCl and extracted with CH₂C1₂ (1 x 200 mL and 3 x 100 mL). The combined organic layers were then dried over MgSO₄and concentrated in vacuo affording 3.47 g (90%) of desired acid Xlb as a clear colorless oil. Η NMR (300 MHz, CDC1₃) δ 7.38-7.26 (m, 5H), 4.55 (s, 2H), 3.67 (m, 1H), 3.57 (dd, J= 9.2, 5.2 Hz, 1H), 2.75 (m, 1H), 1.72-1.31 (m,

4H), 0.93 (t, J= 7.2 Hz, 3H). LRMS (FAB) m/e 223 (M+H⁺) b. Carboxylic acid XIa (R² = Et): By a procedure analogous to that described for preparing acyl oxazolidinone Xlb, acyl oxazolidinone XIa was obtained in 48% yield. 'H NMR (300 MHz, CDC1₃) δ 7.36-7.27 (m, 5H), 4.55 (s, 2H), 3.68 (dd, J= 9.2, 7.9 Hz, 1H), 3.59 (dd, J= 9.2, 5.4 Hz, 1H), 2.68-2.65

(m, 1H), 1.71-1.62 (m, 2H), 0.97 (t, J= 7.5 Hz, 3H). c. Carboxylic acid XIc (R² = H-BU): By a procedure analogous to that described for preparing acyl oxazolidinone Xlb, acyl oxazolidinone XIc was obtained in 96% yield. ^!H NMR (300 MHz, CDC1₃) δ 7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.67 (dd, J= 9.1, 8.1 Hz, 1H), 3.57 (dd, J- 9.2, 5.3 Hz, 1H), 2.72 (m,

1H), 1.67-1.51 (m, 2H), 1.36-1.27 (m, 4H), 0.89 (t, J= 6.9 Hz, 3H). d. Carboxylic acid Xld (R² = -Bu): By a procedure analogous to that described for preparing acyl oxazolidinone Xlb, acyl oxazolidinone Xld was obtained in 80% yield. Η NMR (300 MHz, CDC1₃) δ 7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.64 (t, J= 9.1 Hz, 1H), 3.54 (dd, J= 9.1, 5.1 Hz, 1H), 2.81 (m, 1H),

1.68-1.54 (m, 2H), 1.36-1.27 (m, 1H), 0.92 (d, J= 4.9 Hz, 3H), 0.90 (d, J= 4.9 Hz, 3H). e. Carboxylic acid Xle (R² = CH₂Ph): By a procedure analogous to that described for preparing acyl oxazolidinone Xlb, acyl oxazolidinone Xle was obtained in 92% yield. 'H NMR (300 MHz, CDC1₃) δ 7.38-7.16 (m, 10H), 4.53 (d, J= 12.1 Hz, 1H), 4.50 (d, J= 12.1 Hz, 1H), 3.68-3.57 (m, 2H), 3.09-2.85 (m, 3H).

Example 4: Diethyl Amides (XII)

a. Diethylamide Xllb (R² = #ι-Pr; R⁵ = R⁶ = Et): A cooled solution (0 °C) of carboxylic acid Xlb (3.40 g, 15.3 mmol) in 1 :1 MeCN/CH₂Cl₂ (150 mL), containing diethylamine (2.36 mL , 23 .0 mmol) and 2-( 1 H-benzotriazol- 1 -yl)- 1,1,3 ,3 -tetramethy luronium tetrafluoro borate (TBTU, 5.89 g, 18.4 mmol), was treated with diisopropylethylamine (6.7 mL, 38.2 mmol) over 1.5 h (syringe pump). The mixture was then concentrated in vacuo and partitioned between ether (200 mL) and H₂O (100 mL). The aqueous layer was extracted with more ether (2 x 100 mL) and the combined organic layers were washed with aqueous IN HCl (3 x 50 mL), saturated aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated in vacuo. Chromatographic purification (230-400 mesh SiO₂, elution with 1 :3 AcOEt/hexane) afforded 4.24 g (97%) of diethyl amide Xllb as a clear colorless oil. Η NMR (300 MHz, CDC1₃) δ

7.35-7.23 (m, 5H), 4.52 (d, J= 12.0 Hz, 1H), 4.44 (d, J= 12.0 Hz, 1H), 3.67 (t, J= 8.6 Hz, 1H), 3.51 (dd, J= 8.7, 5.5 Hz, 1H), 3.46-3.27 (m, 4H), 2.96 (m, 1H), 1.67-1.57 (m, lH), 1.48-1.22 (m, 4H), 1.20-1.10 (m, 6H), 0.90 (t,J= 7.2 Hz, 3H). LRMS (FAB) m/e 278 (M+H⁺) b. Diethylamide Xlla (R² = Et; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing diethylamide Xllb, diethylamide Xlla was obtained in 73% yield. Η NMR (300 MHz, CDC1₃) δ 7.33-7.26 (m, 5H), 4.52 (d, J= 12.0 Hz, 1H), 4.44 (d, J- 12.0 Hz, 1H), 3.68 (t, J= 8.6 Hz, 1H), 3.53-3.33 (m, 5H), 2.90 (m, 1H), 1.75-1.50 (m, 2H), 1.18 (t, J= 7.1 Hz, 3H), 1.13 (t, J= 7.1 Hz, 3H), 0.89 (t, J= 7.4 Hz, 3H). c. Diethylamide XIIc (R² = «-Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing diethylamide Xllb, diethylamide XIIc was obtained in 94% yield. Η NMR (300 MHz, CDC1₃) δ 7.35-7.25 (m, 5H), 4.51 (d, J= 12.0 Hz, 1H), 4.44 (d, J= 12.0 Hz, 1H), 3.67 (t, J- 8.6 Hz, 1H), 3.51 (dd, J= 8.8, 5.5 Hz, 1H), 3.46-3.29 (m, 1H), 2.94 (m, 1H), 1.66-1.62 (m, 2H), 1.33-1.10 (m, 9H), 0.85 (t, J= 7.0 Hz, 3H). d. Diethylamide Xlld (R² = i-Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing diethylamide Xllb, diethylamide Xlld was obtained in 95% yield. Η NMR (300 MHz, CDC1₃) δ 7.35-7.23 (m, 5H), 4.51 (d, J= 12.0 Hz, 1H), 4.44 (d, J= 12.0 Hz, 1H), 3.65 (t, J= 8.7 Hz, 1H), 3.54-3.28 (m, 5H), 3.03 (m, 1H), 1.63-1.49 (m, 2H), 1.33-1.24 (m, 1H), 1.18 (t, J= 7.1 Hz, 3H), 1.12 (t, J= 7.1 Hz, 3H), 0.90 (t, = 6.4 Hz, 3H). e. Diethylamide Xlle (R² = CH₂Ph; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing diethylamide Xllb, diethylamide Xlle was obtained in 89% yield. Η NMR (300 MHz, CDC1₃) δ 7.35-7.16 (m, 10H), 4.53 (d, J= 12.1 Hz, 1H), 4.47 (d, J= 12.1 Hz, 1H), 3.77 (t, J= 8.5 Hz, 1H), 3.59 (dd, J= 8.8, 5.7 Hz, 1H), 3.40 (m, 1H), 3.22-2.89 (m, 5H), 2.79 (dd, J= 13.0, 5.1 Hz, 3H), 1.01 (t, J= 7.1 Hz, 3H), 0.85 (t, J= 7.2 Hz, 3H).

Example 5: Alcohols (XIII)

a. Alcohol XHIb (R² = n-Pr; R⁵ = R⁶ = Et): To a solution of diethylamide

Xllb (4.08 g, 14.7 mmol) in 140 mL MeOH was added 20% Pd(OH)₂/C (400 mg) and the suspension was hydrogenated at atmospheric pressure and room temperature for 15 h. Filtration of the catalyst and concentrating the filtrate in vacuo afforded 2.84 g (100%) of the desired primary alcohol Xlllb. Η NMR (300 MHz, CDC1₃) δ 3.74 (br. d, J=4.2 Hz, 1H), 3.61-3.15 (m, 5H), 2.71 (m, 1H), 1.69-1.24 (m, 4H), 1.20 (t, J= 7.1 Hz, 3H), 1.12 (t, J= 7.1 Hz, 3H), 0.92 (t, J= 7.2 Hz, 3H). LRMS (FAB) m/e 188 (M+H⁺). b. Alcohol Xllla (R² = Et; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol Xlllb, alcohol XHIa was obtained in 100% yield. 'H NMR (300 MHz, CDC1₃) δ 3.76 (m, 2H), 3.58-3.19 (m, 4H), 2.64 (m, 1H), 1.71-1.65 (m, 2H), 1.21 (t, J= 7.1 Hz, 3H), 1.13 (t, J= 7.1 Hz, 3H), 0.96 (t, J= 7.4 Hz, 3H). c. Alcohol XIIIc (R² = n-Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol Xlllb, alcohol XIIIc was obtained in 100% yield. 'HNMR (300 MHz, CDC1₃) δ 3.76 (d, J= 4.5 Hz, 2H), 3.58-3.19 (m, 4H),

2.72-2.65 (m, 2H), 1.68-1.55 (m, 2H), 1.40-1.24 (m, 4H), 1.20 (t,J= 7.1 Hz, 3H), 1.12 (t, J= 7.1 Hz, 3H), 0.90 (t, J= 6.9 Hz, 3H). d. Alcohol Xllld (R² = i-Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol Xlllb, alcohol Xllld was obtained in 100% yield. Η NMR (300 MHz, CDC1₃) δ 3.78-3.68 (m, 2H), 3.57-3.15 (m, 4H),

2.81-2.73 (m, 1H), 1.70-1.60 (m, 2H), 1.40-1.28 (m, 1H), 1.21 (t,J= 7.1 Hz, 3H), 1.12 (t, J= 7.1 Hz, 3H), 0.92 (m, 6H). e. Alcohol Xllle (R² = CH₂Ph; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol Xlllb, alcohol Xllle was obtained in 100% yield. ΗNMR(300 MHz, CDCl₃) δ 7.29-7.16 (m, 5H), 3.81-3.71 (m, 2H),

3.61-3.50 (rn, 1H), 3.15-2.87 (m, 6H), 1.05 (t, J= 7.1 Hz, 3H), 0.98 (t, J= 7.1 Hz, 3H).

Example 6: Aldehydes (XIV)

a. Aldehyde XlVb (R² = «-Pr; R^s = R⁶ = Et): To a solution of alcohol Xlllb (2.34 g, 12.7 mmol) in wet CH₂C1₂ (125 mL, prepared by stirring CH₂C1₂ with water and separating the organic layer) was added Dess-Martin periodinane (8.06 g, 19.0 mmol). The mixture was stirred at room temperature for 40 min and was then poured into a mixture of 5% aqueous Na₂S₂O₃ (250 mL) containing 5.2 g NaHCO₃, and ether (200 mL). The biphasic mixture was stirred vigorously for 5 min and the aqueous layer was extracted with 15% CH₂Cl₂/Et₂O (2 x 100 mL).

The combined organic layers were then washed with H₂O (3 x 75 mL) and brine, dried over MgSO₄, filtered and concentrated in vacuo to afford 2.06 g (88%) of desired aldehyde XlVb, a clear colorless oil. Η NMR (300 MHz, CDC1₃) δ 9.60 (d, J= 3.5 Hz, 1H), 3.49-3.30 (m, 5H), 1.96-1.85 (m, 2H), 1.39-1.31 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.13 (t, J= 7.1 Hz, 3H), 0.95 (t, J= 7.3 Hz, 3H). b. Aldehyde XlVb (R² = n-Pr; R^s = R⁶ = Et): To a solution of crude Xlllb (1.25 g, 6.68 mmol) in a mixture of toluene (20 mL), ethyl acetate (20 mL), and water (3 mL) was added 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO), free radical (9 mg). The mixture was cooled to 0°C and a sodium hypochlorite solution, prepared by adding 4.3 mL of aqueous sodium hypochlorite (10-13%) available chlorine) to 1.6 g of NaHCO₃ in 20 mL of water, was added by portions over a period of 30 min. Sodium bromide (660 mg) was added and the solution turned pale orange. Within a few minutes the color of the reaction mixture returned to off-white. Additional sodium hypochlorite (4.7 mL) was added in several portions to drive the reaction to completion. The aqueous layer was separated and extracted with toluene (20 mL) and ethyl acetate (2 x 20 mL). The combined organic extract was washed with a solution of KI (70 mg) in 10% aqueous KHSO₄. The organic layer was then washed with 5% Na₂S₂O₃ and pH

7 phosphate buffer, dried (NajSO , and concentrated to give XlVb as a pale yellow oil (1.1 g). Spectral data for this compound matched that for the product from Example 6a above. c. Aldehyde XI Va (R² = Et; R^s = R⁶ = Et): By a procedure analogous to that described for preparing alcohol XlVb, aldehyde XlVa was obtained in 80% yield. 'HNMR (300 MHz, CDC1₃) δ 9.61 (d, J= 3.6 Hz, 1H), 3.48-3.29 (m, 5H), 2.02-1.90 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.14 (t, J= 7.1 Hz, 3H), 0.96 (t, J= 7.4 Hz, 3H). d. Aldehyde XI Vc (R² = «-Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol XlVb, aldehyde XIVc was obtained in

98% yield. Η NMR (300 MHz, CDC1₃) δ 9.59 (d, J= 3.6 Hz, 1H), 3.48-3.29 (m, 5H), 1.97-1.87 (m, 2H), 1.39-1.22 (m, 4H), 1.18 (t,J=7.2 Hz, 3H), 1.13 (t,J= 7.2 Hz, 3H), 0.90 (t, J= 7.0 Hz, 3H). e. Aldehyde XI Vd (R² = -Bu; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol XI Vb, aldehyde XlVd was obtained in 96% yield. Η NMR (300 MHz, CDC1₃) δ 9.57 (d, J= 3.7 Hz, 1H), 3.51-3.27 (m, 5H), 1.83 (t, J=7.1 Hz, 3H), 1.66-1.55 (m, 1H), 1.20 (t, J= 7.1 Hz, 3H), 1.13 (t, J= 7.1 Hz, 3H), 0.93 (d, J= 6.6 Hz, 6H). f. Aldehyde XlVe (R² = CH₂Ph; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing alcohol XlVb, aldehyde XlVe was obtained in

97% yield. 'HNMR (300 MHz, CDC1₃) δ 9.69 (d, J= 2.9 Hz, 1H), 7.29-7.16 (m, 5H), 3.65 (m, 1H), 3.53-3.42 (m, 1H), 3.30 (dd, J= 13.5, 9.3 Hz, 1H), 3.23-3.13 (m, 2H), 3.06-2.91 (m, 2H), 1.04 (t, J=7.1 Hz, 3H), 0.93 (t, J= 7.1 Hz, 3H).

The preparation of clasto-lactacystin β-lactone and analogs thereof according to the synthetic scheme outlined in Scheme 1 as exemplified in

Examples 7-9.

Example 7: Aldol adducts (II)

a. Aldol adduct lib (R² = «-Pr; R¹ = -Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ =

Et): To a cold (-78 °C) solution of trαrø-oxazoline Ia (R¹ = /-Pr; R⁴ = Ph) in ether (35 mL) was added lithium bis(trimethylsilyl)amide (2.17 of a 1 M solution in hexane, 2.17 mmol). After 30 min, the orange solution was treated dropwise with a 1 M solution of dimethylaluminum chloride in hexane (4.55 mL, 4.55 mmol) and the mixture was stirred for another 60 min before being cooled down to -85 °C (liquid N₂ was added to the dry ice/acetone bath). A solution of aldehyde XI Vb (420 mg, 2.27 mmol) in ether (4 mL) was then added over 10 min along the side of the flask. The mixture was then allowed to warm up to - 40° C over 2.5 h and then quenched by adding 35 mL of saturated aqueous NH₄C1 and 25 mL AcOEt. Enough 2 N HCl was then added until 2 clear phases were obtained (ca. 15 mL added). The aqueous layer was extracted with AcOEt (2 x 20 mL) and the combined organic layers were washed successively with 0.5 N aqueous HCl (20 mL), H₂O (20 mL), 0.5 M aqueous NaHSO₃ (2 x 15 mL), saturated aqueous NaHCO₃ and finally with brine, then dried over Na₂SO₄ and concentrated in vacuo affording 879 mg (> 100%) of crude Aldol product lib which was pure enough to be used directly in the subsequent step. "H NMR (300 MHz, CDC1₃) δ 8.02-7.97 and 7.53-7.39 (m, 5H), 6.58 (d, J= 9.9 Hz, 1H), 4.82 (d, J= 2.4 Hz, 1H), 3.73 (s, 3H), 3.69-3.61 (m, 2H), 3.49-3.39 (m, 2H), 3.24-3.16 (m, 1H), 3.05 (m, 1H), 2.89 (m, 1H), 2.28-2.23 (m, 1H), 1.98-1.91 (m, 1H), 1.37-1.20 (m, 6H),

1.19-1.06 (m, 6H), 0.87 (t, J= 7.1 Hz, 3H), 0.70 (d, J= 6.7 Hz, 3H).

Aldol product lib was also obtained in 100% yield by a procedure analogous to that described above but using cis-oxazoline lb (see below) instead of trøπs-oxazoline Ia. b. Aldol adduct lib (R² = n-Pr; R¹ = /-Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ =

Et): To a cold (-78 °C) solution of tr ra-oxazoline la (R¹ = /-Pr; R⁴ = Ph) (20.74 g) in THF (280 mL) was added lithium bis(trimethylsilyl)amide (92.4 mL of a 1 M solution in hexane) over 75 min. After 30 min, the orange solution was treated dropwise with a IM solution of dimethylaluminum chloride in hexane (202 mL) and the mixture was stirred for another 40 min before being cooled down to

-85 °C (liquid N₂ was added to the dry ice/acetone bath). A solution of aldehyde XlVb (19.43 g) in THF (50 mL) was then added over 45 min. The mixture was then allowed to warm to -50°C over 40 min and then to - 20°C over 25 min. The yellow reaction mixture was again cooled to -78 ° C and then quenched by cautious addition of 40 mL of saturated aqueous NH₄C1. The reaction mixture was poured slowly into 460 mL of saturated aqueous NH₄C1. AcOEt (500 mL) was added, and with good stirring the reaction mixture was acidifed with 6 N HCl to produce two clear phases. The aqueous layer was extracted with AcOEt (2 x 200 mL), and the combined organic layers were washed successively with H₂O (2 x 200 mL), saturated aqueous NaHCO₃ (2 x 200 mL), and brine (2 x 300 mL). The organic extract was dried over Na₂SO₄ and MgSO₄ and concentrated in vacuo to afford 41.55 g of crude Aldol product lib which was pure enough to be used directly in the subsequent step. Spectral data for this compound matched that for the product from Example 7a above. c. Aldol adduct Ila (R² = Et; R¹ = /-Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ = Et):

By a procedure analogous to that described for preparing Aldol adduct lib, the lithium anion of trørcs-oxazoline Ia (R¹ = /-Pr; R⁴ = Ph) was treated successively with dimethylaluminum chloride and aldehyde XlVa to provide Aldol adduct Ila in 95% yield. 'H NMR (300 MHz, CDC1₃) δ 8.00-7.97 and 7.51-7.39 (m, 5H),

6.50 (d, J= 9.9 Hz, 1H), 4.80 (d, J= 2.4 Hz, 1H), 3.81-3.64 (m, 2H), 3.74 (s, 3H), 3.45 (m, 2H), 3.19 (m, 2H), 2.93-2.84 (m, 2H), 2.24 (m, 1H), 1.89 (m, 1H), 1.73-1.64 (m, 4H), 1.29 (t, J= 7.2 Hz, 3H), 1.12 (d, J= 6.9 Hz, 3H), 1.07 (d, J= 7.2 Hz, 3H), 0.70 (d, J- 6.7 Hz, 3H). d. Aldol adduct lie (R² = n-Bu; R¹ = /-Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ =

Et). By a procedure analogous to that described for preparing Aldol adduct lib, the lithium anion of trans-oxazoline Ia (R¹ = /-Pr; R⁴ = Ph) was treated successively with dimethylaluminum chloride and aldehyde XIVc to provide Aldol adduct He in 100% yield. Η NMR (300 MHz, CDC1₃) δ 8.02-7.98 and 7.53-7.33 (m, 5H), 6.57 (d, J= 10.0 Hz, 1H), 4.81 (d, J= 2.3 Hz, 1H), 3.73 (s, 3H),

3.68-3.60 (m, 2H), 3.49-3.17 (m, 2H), 3.00 (m, 1H), 2.90 (m, 1H), 1.98-1.87 (m, 2H), 1.38-0.83 (m, 16H), 0.70 (d, J= 6.7 Hz, 3H). e. Aldol adduct Hd (R² = -Bu; R¹ = -Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing Aldol adduct lib, the lithium anion of trø/«-oxazoline Ia (R¹ = /-Pr; R⁴ = Ph) was treated successively with dimethylaluminum chloride and aldehyde XlVd to provide Aldol adduct lid in 100% yield. Η NMR (300 MHz, CDC1₃) 68.01-7.80 and 7.55-7.20 (m, 5H), 4.87 (d, J- 2.3 Hz, 1H), 3.73 (s, 3H), 3.69-3.58 (m, 2H), 3.51-3.32 (m, 2H), 2.98-2.87 (m, 1H), 2.33-2.24 (m, 1H), 2.12-2.02 (m, 1H), 1.83 (t,J= 7.1 Hz, 1H), 1.35 (t, J= 7.1 Hz, 3H), 1.25-1.05 (m, 5H), 0.93 (d, J= 6.6 Hz, 3H), 0.89 (d,

J= 6.5 Hz, 3H), 0.80 (d, = 6.5 Hz, 3H), 0.69 (d, J= 6.7 Hz, 3H). f. Aldol adduct He (R² = CH₂Ph; R¹ = /-Pr; R³ = Me; R⁴ = Ph; R⁵ = R⁶ = Et): By a procedure analogous to that described for preparing Aldol adduct lib, the lithium anion of trαns-oxazoline Ia (R¹ = /-Pr; R⁴ = Ph) was treated successively with dimethylaluminum chloride and aldehyde XI Ve to provide Aldol adductllein l00%yield. 'HNMR (300 MHz, CDCl₃) δ 8.01-7.93 and 7.54-7.10 (m, 10H), 4.71 (d,J= 2.5 Hz, 1H), 3.73 (s, 3H), 3.68-3.58 (m, 2H), 3.48-2.79 (m, 6H), 2.17 (m, 1H), 1.12-0.91 (m, 9H), 0.68 (d, J= 6.7 Hz, 3H).

Example 8: γ-Lactams (IV)

a. γ-Lactam IVb (R² = w-Pr; R¹ = /-Pr; R³ = Me): A solution of Aldol adduct lib (4.72 g, 10.9 mmol) in 100 mL 1 :9 AcOH MeOH, to which was added 4.8 g 20% Pd(OH)₂/C, was vigorously shaken under 55 p.s.i. H₂ for 60 h. The mixture was brought down to atmospheric temperature before being filtered and concentrated in vacuo. The solid obtained was purified by flash chromatography (SiO₂, elution with 1 % AcOH in 1 : 1 AcOEt/hexane) affording 2.23 g (75%) of desired γ-lactam IVb as a white solid. 'H NMR (300 MHz, CDC1₃) δ 7.89 (br. s, 1H), 4.77 (br. d, J= 11.5 Hz, 1H), 4.47 (dd, J= 11.5, 5.6 Hz, 1H), 4.08 (dd, J= 9.4, 5.0 Hz, 1H), 3.83 (s, 3H), 2.93 (m, 1H), 1.78-1.39 (m, 6H), 1.02-0.88 (m, 9H). b. γ-Lactam I Va (R² = Et; R¹ = -Pr; R³ = Me): By a procedure analogous to that described for preparing γ-lactam IVb, Aldol adduct Ila was hydrogenated at 55 p.s.i. for 48 h to provide γ-lactam IVa in 72% yield. 'H NMR (300 MHz, CDC1₃) δ 7.79 (br. s, 1H), 4.62 (br. d, J- 11.2 Hz, 1H), 4.51 (dd, J= 11.2, 5.4 Hz, 1H), 3.83 (s, 3H), 2.85 (m, 1H), 1.77-1.64 (m, 3H), 1.01 (t, J= 7.4 Hz, 3H), 0.98 (d, J= 6.9 Hz, 3H), 0.95 (d, J= 6.9 Hz, 3H). c. γ-Lactam IVc (R² = /i-Bu; R¹ = /-Pr; R³ = Me): A solution of Aldol adduct He (361 mg, 0.80 mmol) in 6 mL 1 :9 AcOH/MeOH, to which was added 250 mg 20% Pd(OH)₂/C, was vigorously shaken under 50 p.s.i. H₂ for 24 h. More catalyst (100 mg) was then added and the mixture was again shaken at 50 p.s.i. for another 24 h after which time it brought down to atmospheric temperature before being filtered. The filtrate was then heated to reflux for 30 min, cooled to room temperature and concentrated in vacuo. The solid obtained was co-evaporated once with toluene and purified by flash chromatography (SiO₂, elution with 4% MeOH/CHCl₃) affording 140 mg (61%) of desired γ-lactam IVc as a white solid. »H NMR (300 MHz, CDC1₃) δ 8.02 (br. s, 1H), 4.93 (br. d, J= 11.3 Hz, 1H), 4.46 (dd, J= 11.3, 5.5 Hz, 1H), 4.15-4.08 (m, 1H), 3.83 (s, 3H), 2.94-2.87 (m, 1H), 1.80-1.34 (m, 6H), 0.94 (d, J= 6.9 Hz, 3H), 0.89 (t, J= 7.2 Hz, 3H). d. γ-Lactam IVd (R² = /-Bu; R¹ = -Pr; R³ = Me): By a procedure analogous to that described for preparing γ-lactam IVc, Aldol adduct lid was hydrogenated at 50 p.s.i. for 40 h and heated to reflux for 30 min providing γ-lactam IVd in 61% yield. *H NMR (300 MHz, CDCl₃) δ 7.92 (br. s, 1H), 4.81 (br. d, J= 11.5 Hz, 1H), 4.46 (m, 1H), 4.09 (m, 1H), 3.83 (s, 3H), 3.04-2.98 (m, 1H), 1.78-1.73 (m, 2H), 1.66-1.47 (m, 3H), 1.00-0.90 (m, 12H). e. γ-Lactam IVe (R² = CH₂Ph; R¹ = /-Pr; R³ = Me): By a procedure analogous to that described for preparing γ-lactam IVc, Aldol adduct He was hydrogenated at 50 p.s.i. for 24 h and heated to reflux for 30 min providing γ-lactam IVe in 71% yield. Η NMR (300 MHz, CDC1₃) δ 8.01 (br. s, 1H), 7.35-7.15 (m, 5H), 5.02 (br. d,J= 11.7 Hz, 1H), 4.40-4.34 (m, lH), 4.06-4.01 (m,

1H), 3.84 (s, 3H), 3.34-3.27 (m, 1H), 3.10-3.04 (m, 2H), 1.84-1.72 (m, 1H), 0.98 (d, J= 6.7 Hz, 3H), 0.93 (d, J= 6.9 Hz, 3H).

Example 9: β-Lactones (VII)

a. β-Lactone VHb (R² = i-Pr; R¹ = i-Pr): To a cold (0°C) solution of γ-lactam IVb (2.20 g, 8.06 mmol) in EtOH (100 mL) was added 0.1N aqueous

NaOH (100 mL, 10.0 mmol). The mixture was stirred at room temperature for 15 h after which time H₂O (50 mL) and AcOEt (100 mL) were added. The aqueous layer was then washed with AcOEt (2 x 50 mL), acidified with 6N aqueous HCl and concentrated in vacuo to a volume of co 60 mL. This solution was then frozen and lyophilized. The obtained solid was suspended in THF, filtered to get rid of sodium chloride and concentrated in vacuo affording 2.05 g (98%) of the desired dihydroxyacid as white solid. Η NMR (300 MHz, CD₃OD) δ 4.42 (d, J= 5.8 Hz, 1H), 3.90 (d, J= 6.5 Hz, 1H), 2.84 (m, 1H), 1.70-1.24 (m, 6H), 0.95-0.84 (m, 9H).

To a solution of the dihydroxyacid (1.90 g, 7.33 mmol) in anhydrous THF (36 mL) was added a solution of 2-(lH-benzotriazol-l-yl)-l,l,3, 3-tetramethyluronium tetrafluoroborate (TBTU, 2.59, 8.06 mmol) in anhydrous

MeCN (36 mL) followed by triethylamine (0.72 mL, 22.0 mmol). After stirring for 70 min at room temperature, some toluene was added and the mixture was concentrated in vacuo and co-evaporated 2 more times with toluene. Purification by flash chromatography (SiO₂, elution with 2:3 AcOEt/hexane) afforded 1.44 g (81%) of desired β-lactone Vllb as a white solid. Η NMR (300 MHz, CDC1₃) δ 6.07 (br. s, 1H), 5.26 (d, J= 6.1 Hz, 1H), 3.97 (dd, J= 6.4, 4.4 Hz, 1H), 2.70-2.63 (m, 1H), 2.03 (d, J= 6.4 Hz, 3H), 1.93-1.44 (m, 5H), 1.07 (d, J= 7.0 Hz, 3H), 0.99 (d, J= 7.3 Hz, 3H), 0.91 (d, J= 6.7 Hz, 3H). LRMS (FAB) m/e 242 (M+H⁺). b. β-LactoneVIIa (R² = Et; R' = /-Pr): Hydrolysis oflVa, as described for

IVb above, afforded the corresponding dihydroxyacid in 100% yield. Η NMR (300 MHz, CD₃OD) δ 4.45 (d, J= 5.8 Hz, 1 H). 3.90 (d, J= 6.4 Hz, 1H), 2.74 (m, 1H), 1.71-1.53 (m, 3H), 0.94 (t, J= 7.4 Hz, 3H), 0.92 (d, J= 6.8 Hz, 3H), 0.88 (d, J= 6.8 Hz, 3H). By a procedure analogous to that described for preparing β-lactone Vllb, β-lactone Vila was obtained in 79% yield. "H NMR (300 MHz, CDC1₃) δ 6.17 (br. s, 1H), 5.30 (d, J= 6.0 Hz, 1H), 3.98 (dd, J= 6.4, 4.4 Hz, 1H), 2.60 (m, 1H), 2.08 (d, J= 6.4 Hz, 3H), 1.97 (m, 2H), 1.75 (m, 1 H), 1.12 (t, J= 7.5 Hz, 3H), 1.07 (d, J= 6.8 Hz, 3H), 0.92 (d, J= 6.8 Hz, 3H). c. β-Lactone VIIc (R² = n-Bu; R¹ = /-Pr): Hydrolysis of IVc, as described for IVb above, afforded the corresponding dihydroxyacid in 100% yield. Η NMR (300 MHz, CD₃OD) δ 4.42 (d, J= 5.8 Hz, 1H), 3.90 (d, J= 6.4 Hz, 1H), 2.86-2.79 (m, 1H), 1.70-1.24 (m, 8H), 0.97-0.86 (m, 9H).

By a procedure analogous to that described for preparing β-lactone Vllb, β-lactone VIIc was obtained in 40% yield. 'H NMR (300 MHz, CDC1₃) δ 6.14 (br. s, 1H), 5.27 (d, J= 6.1 Hz, 1H), 3.97 (d, J- 4.4 Hz, 1H), 2.68-2.61 (m, 1H), 1.94-1.86 (m, 2H), 1.72-1.36 (m, 7H), 1.07 (d, J= 7.0 Hz, 3H), 0.93 (t, J= 7.1 Hz, 3H), 0.91 (d, J= 6.8 Hz, 3H). LRMS (FAB) m/e 256 (M+H⁺) d. β-Lactone Vlld (R² = -Bu; R¹ = /-Pr): Hydrolysis of IVd, as described for IVb above, afforded the corresponding dihydroxyacid in 100% yield. Η

NMR (300 MHz, CD₃OD) δ 4.50 (d, J= 5.8 Hz, 1H), 4.00 (d, J= 6.5 Hz, 1H), 3.09-3.02 (m, 1H), 1.90-1.61 (m, 3H), 1.49-1.40 (m, 2H), 1.02 (d, J= 6.7 Hz, 3H), 0.98 (d, J= 6.5 Hz, 3H), 0.97 (d, J= 6.7 Hz, 3H).

By a procedure analogous to that described for preparing β-lactone Vllb, β-lactone Vlld was obtained in 62% yield. Η NMR (300 MHz, CDC1₃) δ 6.16

(br. s, 1H), 5.25 (d, J= 6.1 Hz, 1H), 3.97 (d, J= 4.4 Hz, 1H), 2.71 (dd, J= 15.1,

6.2 Hz, 1H), 1.95-1.66 (m, 5H), 1.08 (d, J= 6.9 Hz, 3H), 0.99 (d, J= 6.3 Hz, 3H),

0.98 (d, J= 6.3 Hz, 3H), 0.92 (d, J= 6.7 Hz, 3H). LRMS (FAB) m/e 256 (M+H⁺). e. β-Lactone Vile (R² = CH₂Ph; R¹ = /-Pr): Hydrolysis of IVe, as described for IVb above, afforded the corresponding dihydroxyacid in 88%) yield.

'H NMR (300 MHz, CD₃OD) δ 7.25-7.04 (m, 5H), 4.29 (d, J= 5.7 Hz, 1H), 3.83 (d, J= 6.4 Hz, 1H), 3.01-2.82 (m, 3H), 1.65 (m, 1H), 0.90 (d, J= 6.6 Hz, 3H), 0.86 (d, J= 6.8 Hz, 3H).

By a procedure analogous to that described for preparing β-lactone Vllb, β-lactone Vile was obtained in 77% yield. 'H NMR (300 MHz, CDC1₃) δ

7.36-7.20 (m, 5H), 6.57 (br. s, 1H), 5.08 (d, J= 5.4 Hz, 1H), 3.94 (d, J= 4.5 Hz,

1H), 3.25 (d,J= 10.1 Hz, 1H), 3.01-2.89 (m, 2H), 1.92-1.81 (m, 1H), 1.05 (d, J=

6.9 Hz, 3H), 0.86 (d, J- 6.7 Hz, 3H). LRMS (FAB) m/e 290 (M+H⁺).

The preparation of cis-oxazolines and trans-oxazolines according to the synthetic schemes illustrated in Schemes 3 and 4 as illustrated by Examples 10 and

11. Example 10: cis-Oxazoline (Ia)

a. Ethyl 3-(isopropyl)propenoate (XV; R¹ = -Pr; R³ = Me): To a stirred solution of carbomethoxymethylene triphenylphosphorane (56.04 g, 167.6 mmol) in dry CH₂C1₂ (168 mL) at 0°C was added dropwise isobutyraldehyde (17.4 mL, ' 191.6 mmol). After 5 min, the reaction mixture was warmed to room temperature and stirred for 24h. The solvent was removed in vacuo and pentane was added to the white oily solid to precipitate triphenylphosphine oxide. The solid was filtered off and the filtrate concentrated in vacuo. The procedure was repeated one more time and the crude olefin (20.00 g, 93%) was obtained as a yellow oil that was sufficiently pure for the next step. Η NMR (300 MHz, CDC1₃) δ 6.95

(dd, J= 15.7, 6.6 Hz, 1H), 5.77 (dd, J= 15.7, 1.5 Hz), 3.72 (s, 3H), 2.44 (m, 1H), 1.06 (d, J= 6.7 Hz, 6H). b. (2S, 3R)-Methyl 2,3-dihydroxy-3- [isopropyl] propionate (XVIa; R¹ = -Pr; R³ = Me): A mixture of AD-mix-β (100.00 g,), methanesulfonamide (6.78 g, 71.3 mmol) and tert-butanol-water (1 :1, 720 mL) was stirred vigorously at room temperature for 5 min. The reaction mixture was then cooled to 0°C and α,β-unsaturated ester XV (R¹ = /-Pr; R³ = Me) (9.14 g, 71.3 mmol) was added dropwise via a Pasteur pipette. After stirring at 0 °C for 96 h, Na₂SO₃ (3.0 g) was added, and stirring continued at room temperature for 1 h. The mixture was diluted with ethyl acetate (200 mL) and transferred to a separatory funnel. The organic layer was removed and the aqueous phase extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. The yellow oil obtained was passed through a silica gel pad using 1:1 hexane/ethyl acetate affording diol XVIa (R¹ = /-Pr; R³ = Me) (11.48 g, 94%) as a yellow solid. 'HNMR (300 MHz, CDC1₃) δ 4.28 (dd, J= 5.6,

1.8 Hz, 1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33 (d, j= 9.3 Hz, 1H), 1.87 (m, 1H), 1.02 (d, J= 6.7 Hz, 3H), 0.95 (d, J= 6.7 Hz, 3H). c. (2R,3R)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate

(XVIIa; R¹ = /-Pr; R³ = Me): (2S,37?)-Methyl 2,3-dihydroxy-3- [isopropyljpropionate XVIa (R¹ = /-Pr; R³ = Me) (1.0 g, 6.17 mmol) and trimethylorthobenzoate (1.02 mL, 80.1 mmol) were dissolved in CH₂C1₂ (20 mL) and treated with BF₃-OEt₂ (40.0 μL, 0.32 mmol). After stirring for 75 min, the mixture was concentrated under full vacuum (0.05 mm Hg) for 35 min. The mixture was redissolved in CH₂C1₂ (20.0 mL), cooled to 0°C and treated sequentially with Et₃N (43.0 μL, 0.31 mmol) and acetyl bromide (0.48 mL, 6.49 mmol). After stirring for 4 h at 0°C, the reaction mixture was treated with saturated NaHCO₃ solution (12 mL) and allowed to warm up to room temperature. The layers were separated and the aqueous layer was extracted with CH₂C1₂ (2 x 20 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo affording the crude -bromo β-benzoate XVIIa (R¹ = /-Pr; R³ = Me) (1.36 g, 85%) as a clear colorless oil. 'H NMR (300 MHz, CDC1₃) 6 8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H), 5.57 (dd, J= 8.8, 3.9 Hz, 1H), 4.47 (d, J= 8.8 Hz, 1H), 3.67 (s, 3H), 2.45 (m, 1H), 1.01 (d, J= 6.8 Hz, 6H). d. (2S3R)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate (XVIIIa;

R¹ = /-Pr; R³ = Me): A solution of (27?, 37?)-Mefhyl 2-bromo-3-dihydroxy- 3-[isoρropyl]propionate XVIIa (R¹ = /-Pr; R³ = Me) (2.00 g, 6.07 mmol) in 15 mL DMSO was treated with sodium azide (790.0 mg, 12.2 mmol). After stirring for 12 h at room temperature, the mixture was partitioned between H₂O and ethyl acetate (50 mL each). The aqueous layer was extracted with more ethyl acetate and the combined organic layers were dried over MgSO₄ and concentrated in vacuo affording the desired α-azo β-benzoate (1.55 g, 87%) as a yellow oil. Η NMR (300 MHz, CDC1₃) δ 8.07-8.02 (m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J= 8.8, 2.8 Hz, 1H), 3.73 (s, 3H), 2.24 (m, 1H), 1.04 (d, J= 5.8 Hz, 3H), 0.98 (d, J= 5.8 Hz, 3H).

Repeating the same procedure but using DMF as the solvent instead of DMSO afforded the desired α-azo β-benzoate in 85% yield. e. Benzamide XlXa (R¹ = -Pr; R³ = Me): A solution of (25, 37?)-Methyl

2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIa (R¹ = /-Pr; R³ = Me) (1.50 g, 5.15 mmol) in ethyl acetate (25 mL) was treated with 200 mg of 20% Pd(OH)₂/C and the suspension was stirred vigorously in a H₂ atmosphere under balloon pressure. After 12 hours, the mixture was filtered and refluxed for 4 hours to complete the migration of the benzoyl group. The mixture was then cooled to room temperature and concentrated in vacuo affording the desired benzamide (1.25 g, 92%) as a yellow oil. 'H NMR (300 MHz, CDC1₃) δ 7.85-7.83 (m, 2H),

7.46-7.40 (m, 3H), 6.99 (br. d, J= 9.1 Hz, 1H), 5.05 (dd, J= 9.1, 1.9 Hz, 1H), 3.77 (s, 3H), 1.79 (m, 1H), 1.03 (d, J= 6.7 Hz, 3H), 0.99 (d, J= 6.7 Hz, 3H). f. c/s-OxazoIine Ia (R¹ = -Pr; R³ = Me): A solution of 500 mg of benzamide XlXa (R¹ = /-Pr; R³ = Me) (18.8 mmol) in CH₂C1₂ (20 mL) was treated with 4.50 mL thionyl chloride (61.7 mmol). After stirring at room temperature for

24 h, the mixture was diluted with CH₂C1₂ and washed with saturated NaHCO₃ solution, dried (Na₂SO₄), concentrated in vacuo and chromatographed (silica gel, 1 : 1 hexane/ethyl acetate) affording the desired -oxazoline (248 mg, 53%) as a pale yellow oil. 'H NMR (300 MHz, CDC1₃) δ 8.01-7.97 (m, 2H), 7.52-7.38 (m, 3H), 4.94 (d, J= 9.8 Hz, 1H), 4.53 (dd, J= 9.8, 7.8 Hz, 1H), 3.76 (s, 3H), 2.09

(m, 1H), 1.05 (d, J= 6.5 Hz, 3H), 1.01 (d, J= 6.7 Hz, 3H).

Example 11: trans-Oxazoline (lb)

a. Ethyl 3-(isopropyI)propenoate (XV; R¹ = /-Pr; R³ = Me): To a stirred solution of carbomethoxymethylene triphenylphosphorane (56.04 g, 167.6 mmol) in dry CH₂C1₂ (168 mL) at 0°C was added dropwise isobutyraldehyde (17.4 mL,

191.6 mmol). After 5 min, the reaction mixture was warmed to room temperature and stirred for 24h. The solvent was removed in vacuo and pentane was added to the white oily solid to precipitate triphenylphosphine oxide. The solid was filtered off and the filtrate concentrated in vacuo. The procedure was repeated one more time and the crude olefin (20.00 g, 93%) was obtained as a yellow oil that was sufficiently pure for the next step. Η NMR (300 MHz, CDC1₃) δ 6.95 (dd, J= 15.7, 6.6 Hz, 1H), 5.77 (dd, J= 15.7, 1.5 Hz), 3.72 (s, 3H), 2.44 (m, 1H), 1.06 (d, J= 6.7 Hz, 6H). b. (2R, 35)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb; R¹ = -Pr; R³ = Me): To a clear yellow solution of K₂OsO₂(OH)₄ (246.1 mg, 0.67 mmol, 0.95 mol %), hydroquinine 1 ,4-phthalazinediyl diether (555.1 mg, 0.71 mmol, 1.01 mol %), N-methylmorpholine N-oxide (50 wt % in water, 25.0 mL, 0.106 mol, 1.51 equiv.), t-BuOH (84 mL), and H₂O (58 mL) was added at 25°C the neat olefin XV (R¹ = z-Pr; R³ = Me) (9.0 g, 70.2 mmol) via a syringe pump over a period of 48 h (the syringe was connected to tubing, whose tip was immersed in the solution throughout the reaction time) . The resulting clear orange solution was then stirred for another 60 min, after which time ethyl acetate (200 mL) and a solution of Na₂SO₃ (15.0 g) in H₂O (150 mL) were added, and the resulting mixture was stirred for 4 h. The phases were separated, and the aqueous layer was extracted with more ethyl acetate (2x). The organic layers were then combined and the chiral ligand was extracted from the organic phase with a solution of 0.3 M H₂SO₄ in saturated Na₂SO₄ (2 x 100 mL). The phases were once again separated and the aqueous layer was extracted with more ethyl acetate ( 1 x) .

The organic layers were combined and dried over Na₂SO₄, filtered and concentrated in vacuo. This afforded 11.4 g (ca. 100%) of a white oily solid which was shown to be 70% e.e (determined by 'H NMR from a 1 :1 molar solution of diol and Europium tris[3-(heptafluoropropy lhydroxymethylene)-(-)-camphorate] in C₆D₆). Recrystallisation from 35-60°C petroleum ether afforded 6.8 g (60%) of (27?,3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb; R¹ - /-Pr; R³ = Me) that was ca. 100%) e.e., obtained as white crystals, mp = 32-34°C; = -110.6° (c 1.04, CHClj)]. 'H NMR (300 MHz, CDC1₃) δ 4.28 (dd, J= 5.6, 1.8 Hz, 1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33 (d, J= 9.3 Hz, 1H), 1.87 (m, 1H), 1.02 (d, J- 6.7 Hz, 3H), 0.95 (d, J= 6.7 Hz, 3H). c. (2S,35)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate

(XVIIb; R¹ = -Pr; R³ = Me): (27?, 3S)-Methyl 2,3-dihydroxy-3-[isopropyl] propionate XVIb (R¹ = /-Pr; R³ = Me) (30.0 g, 185.2 mmol) and trimethylorthobenzoate (41.3 mL, 240.7 mmol) were dissolved in CH₂C1₂ (400 mL) and treated with BF₃OEt₂ (1.16 mL, 9.25 mmol). After 2 h, triethylamine (1.8 mL, 13 mmol) was added, and the mixture was concentrated in vacuo and placed under full vacuum (0.05 mm Hg) for 70 min. The residue was redissolved in CH₂C1₂ (400 mL), cooled to 0°C and treated dropwise with acetyl bromide (14.3 mL, 194.5 mmol). After 2 h, additional acetyl bromide (0.68 mL, 9.25 mmol) was added. After 30 min, saturated NaHCO₃ solution (500 mL) was added and the mixture was stirred vigorously for 5-10 min. The layers were separated and the aqueous layer was extracted with CH₂C1₂ (2 x 20 mL). The combined organic layers were dried (Na₂SO₄), filtered and concentrated in vacuo affording the crude α-bromo β-benzoate XVIIb (R^! = z-Pr; R³ = Me) (66.23 g) as a clear colorless oil, containing -9.3% by wt. methyl benzoate. For product: 'H NMR

(300 MHz, CDC1₃) δ 8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H), 5.57 (dd, J= 8.8, 3.9 Hz, 1H), 4.47 (d, J= 8.8 Hz, 1H), 3.67 (s, 3H), 2.45 (m, 1H), 1.01 (d, j= 6.8 Hz, 6H). d. (2R,35)-Methyl2-azo-3-dihydroxy-3-[isopropyl]propionate (XVIIIb; R¹ = -Pr; R³ = Me): Sodium azide (24 g, 370 mmol) was added to 230 mL of

DMSO and the mixture was stirred at room temperature overnight. To the resultant solution was added a solution of (2S, 35)-Methyl 2-bromo-3-dihydroxy-3-[isopropyl] propionate (XVIIb; R¹ = z-Pr; R³ = Me) (61 g, 185 mmol) in 20 mL DMSO. After stirring for 11 h at room temperature, the mixture was poured into water (1.5 L) and ether (200 mL) and stirred vigorously for 10-15 min. Ether (100 mL) was added and the layers were separated. The aqueous layer was extracted with ether (2 x 100 mL) and the combined organic layers were washed with water (2 x 100 mL) and brine (100 mL), dried over MgSO₄, and concentrated in vacuo affording the crude product (57.5 g), containing approximately 3% starting material and 8% elimination byproduct. For product: 'H NMR (300 MHz, CDC1₃) δ 8.07-8.02 (m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J= 8.8, 2.8 Hz, 1H), 3.73 (s, 3H), 2.24 (m, 1H), 1.04 (d, J= 5.8 Hz, 3H), 0.98 (d, J= 5.8 Hz, 3H). e. Benzamide XlXb (R¹ = -Pr; R³ = Me): To a cold (0-5 °C) solution of (27?,35)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIb (R¹ = z-Pr; R³ = Me) (55 g) in methanol (300 mL) was added 94 mL of 4 M HCl/dioxane and 2.75 g of Pd(OH)₂/C. The mixture was purged with hydrogen and stirred at room temperature. The mixture was purged with hydrogen every 30 min to remove the liberated nitrogen. After 4 h, the reaction mixture was purged with nitrogen and additional Pd(OH)₂/C (1.3 g) was added. The reaction mixture was purged with hydrogen and again purged every hour for 4 h. The mixture was filtered and concentrated in vacuo. The residue was dissolved in water and extracted with EtOAc. The aqueous layer was basified with Na₂CO₃ and again extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated to give a mixture of N- and O-benzoylated products, which was used directly in the next step. f. frα/ts-Oxazoline lb (R¹ = /-Pr; R³ = Me): The crude product IXb obtained in Example lOe above (37.3 g, 141 mmol) was dissolved in toluene (350 mL). j9-Toluenesulfonic acid (2.68 g, 14.1 mmol) was added and the mixture was heated to reflux. Water was removed using a Dean Stark trap. After 3 h, -2.5 mL of water had been collected. The reaction mixture was cooled, diluted with EtOAc (100 mL), washed successively with saturated ΝaHCO₃ (2 x 100 mL) and brine (100 mL), dried over MgSO₄, and concentrated. The residue was purified over a pad of silica gel (-400 g), eluting with 25-30%) EtOAc-hexanes to provide the trazw-oxazoline lb (R¹ = z-Pr; R³ = Me). Η NMR (300 MHz, CDC1₃) δ

8.01-7.97 (m, 2H), 7.52-7.38 (m, 3H), 4.68 (apparent t, J= 7 Hz, 1H), 4.57 (d, J= 7 Hz, 1H), 3.81 (s, 3H), 2.00-1.93 (m, 1H), 1.04 (d, J= 6.7 Hz, 3H), 1.00 (d, = 6.8 Hz, 3H).

Example 12 Inactivation of Proteasome Activity

Purification of 20S proteasome and proteasome activator PA28 was performed as previously described (Dick etal, J. Biol. Chem. 271:7273 (1996)). 2 mL of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) and Suc- Leu-Leu- Val-Tyr-AMC in dimethyl sulfoxide were added to a 3 mL fluorescent cuvette, and the cuvette was placed in the jacketed cell holder of a Hitachi F-2000 fluorescence spectrophotometer. The temperature was maintained at 37 °C by a circulating water bath. 0.34 mg of PA28 were added and the reaction progress was monitored by the increase in fluorescence at 440 nm (λ_ex = 380 nm) that accompanies production of free AMC. The progress curves exhibited a lag phase lasting 1-2 min resulting from the slow formation of the 20S-PA28 complex. After reaching a steady state of substrate hydrolysis, lactacystin was added to a final concentration of 1 mM, and the reaction was monitored for 1 h. The fluorescence (F) versus time (t) data were collected on a microcomputer using LAB CALC (Galactic) software. &_ιnact values were estimated by a nonlinear least- squares fit of the data to the first order equation:

F = A(\ - e ^kl) + C where C ~ F_t, ₀ and A = F_{l = oo}- F_{l = 0}.

Example 13 Inhibition of Intracellular Protein Degradation in C2C12 Cells

C2C12 cells (a mouse myoblast line) were labeled for 48 hrs with ³⁵S- methionine. The cells were then washed and preincubated for 2 hrs in the same media supplemented with 2 mM unlabelled methionine. The media was removed and replaced with a fresh aliquot of the preincubation media containing 50%) serum, and a concentration of the compound to be tested. The media was then removed and made up to 10% TCA and centrifuged. The TCA soluble radioactivity was counted. Inhibition of proteolysis was calculated as the percent decrease in TCA soluble radioactivity. From this data, an IC₅₀ for each compound was calculated. Example 14 Lactone Hydrolysis

The half-lives (t_1/2) for hydrolysis of β-lactone analogs to the corresponding dihydroxy acids were measured at 37 °C at a concentration of 200 mM in 20 mM HEPES, 0.5 mM EDTA, pH 7.8. Absorbance was measured for at least five half-lives (approximately 1 hour) at 230 nm, the wavelength at which there is the greatest difference in extinction coefficients for the lactone and dihydroxy. Half-lives were calculated using Guggenheim analysis (Gutfreund Enzymes: Physical Principles; Wiley and Sons: New York, 1975, pp 118-119). The results of Examples 12- 14 are reported in Table 1.

Table 1

Kinetics of Inhibition of20S Proteasome and Inhibition of Intracellular Protein Degradation

Compound R² Kobs/[I] (M-¹ s-')^a IC₅₀ (μM)^b tι ₂ min^c

2 Me 20,000 0.7-1.1 13

3a Et 39,000 0.32 15.3

3b n-Pr 46,500 0.29 15.3

3c n-Bu 38,000 0.33 17

3d i-Bu 17,000 0.51 16.8

3e CH₂Ph 6,400 - 6.8

3f OMe 82,200 86 3.7

"Inactivation of the Chymotrypsin-like activity of PA28-activated 20S proteasome. ^bInhibition of intracellular protein degradation in C2C12 cells.

Ηydrolysis half-life

The results indicate that the compounds of the present invention are potent inhibitors of the proteasome.

Example 15: Reduction of Infarct Size and Neuronal Loss

Methods

Male Sprague Dawley rats (250-400 g) were anesthetized with haloethane and subjected to middle cerebral artery (MCA) occlusion using a nylon filament for 2 h. Subsequently, the filament was removed and reperfusion of the infarcted tissue occurred for 24 hours before the rat was sacrificed. Immediately after the filament was withdrawn, the animals were evaluated using a neurological scoring system. Neurological scores were expressed on a scale from 0 to 10, with 0 representing no neurological deficit and 10 representing severe neurological deficit. After 24 hours and before sacrifice, animals were evaluated a second time using the same neurological scoring system.

Staining of coronal sections (2.0 mm x 7-8) with triphenyltetrazolium chloride (TTC) taken throughout the brain were evaluated under blinded conditions using image analysis to determine infarct size.

Dosing Regimen Rats were given i.v. bolus injections (1.0 mL/kg) of either vehicle (50% propylene glycol/saline; n=8) or 7-n-propyl-c/α.ytø-lactacystin β-lactone (3b) (0.3 mg/kg; n=7) at 2 hours after the start of the occlusion. Two additional groups of rats were given i.v. bolus injections (1.0 mL/kg) of 3b at 0 minutes, 2 hours, and 6 hours after the start of the occlusion. One group (0.1 mg/kg x 3 ; n=6) received 0.1 mg/kg at each of these times, while another group (0.3 mg/kg x 3; n=7) received 0.3 mg/kg at each of the three timepoints.

Results

In animals treated with a single dose of 7-n-propyl-c/ .stø-lactacystin β- lactone (3b), infarct volume was decreased by 50% (FIG. 1, 0.3 x 1). Infarct volume was not significantly decreased in either the 0.1 mg/kg x 3 dosage group or the 0.3 mg/kg x 3 dosage group (FIG. 1).

All animals had a neurological score of 10 ± 0 immediately after the 2 hour ischemic episode. At 24 hours, the vehicle-treated rats had a mean score of 8.7 ± 0.6, whereas rats treated with a single 0.3 mg/kg dose of 7-n-propyl-c/cwtø- lactacystin β-lactone (3b) had a mean score of 4 ± 1 (FIG. 2). These data represent a 60% neurological improvement for the drug-treated animals. No significant improvement in neurological score was observed in either the 0.1 mg/kg x 3 dosage group of the 0.3 mg/kg x 3 dosage group (FIG. 2). Conclusion

7-n-propyl-c/αstø-lactacystin β-lactone, given once post-ischemia, provides significant protection in both the degree of neurological deficit and infarcted brain damage. From these preliminary data, it appears that a single-dose regimen is preferred over a multiple-dose regimen.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety .

Claims

What Is Claimed Is:

1. A process for forming a γ-lactam carboxylic acid of Formula V:

or a salt thereof, wherein R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and said process comprising:

(a) deprotonating a substituted aryl or heteroaryl oxazoline of Formula/:

where R¹ is as defined above; R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and R⁴ is aryl or heteroaryl, either of which may be optionally substituted; by treating said substituted aryl or heteroaryl oxazoline with a strong base to form an enolate; (b) transmetallating said enolate with a metal selected from the group consisting of titanium, aluminum, tin, zinc, magnesium and boron, and thereafter treating with a formyl amide of Formula XIV:

XIV

where R² is as defined above, and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which may be optionally substituted, and which optionally may include an additional oxygen or nitrogen atom, to form an adduct of Formula //:

where R¹ through R⁶ are as defined above; c) catalytically hydrogenating said adduct of Formula //to form a γ- lactam of Formula IV:

where R¹, R² and R³ are as defined above; and d) saponifying said γ-lactam of Formula/ to form a carboxylic acid of Formula V.

2. The process of claim 1 , further comprising treating the carboxylic acid of Formula Fwith a cyclizing reagent to form a c/αsto-lactacystin β-lactone of Formula VII:

wherein R¹ and R² are as defined in claim 1.

3. The process of claim 2, wherein said cyclizing is effected with a reagent selected from the group consisting of aryl sulfonyl chlorides, benzotriazol-

1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate, 0-(\H- benzotriazol- 1 -yl)-NN,N' N'-tetramethyluronium tetrafluoroborate and alkyl- aryl- or alkenyl chlorofomates.

4. The process of claim 2, further comprising reacting the clasto- lactacystin β-lactone of Formula VII with a thiol, R⁷SH, to form lactacystin having

Formula VI:

wherein R¹ and R² are as defined in claim 1 ; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substiuited.

5. The process of claim 4, wherein c/osto-lactacystin β-lactone is converted to lactacystin by treating the β-lactone with N-acetylcysteine.

6. The process of claim 1 , wherein the carboxylic acid intermediate of Formula F is directly coupled to a thiol, R⁷SH, to form a lactacystin having Formula VI:

wherein R¹ and R² are as defined in claim 1 ; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substituted.

7. The process of claim 1, wherein in step (a) said strong base is selected from the group consisting of hindered amide bases; alkali metal hexamethyldisilazides; or hindered alkyllithium reagents.

8. The process of claim 1, wherein in step (a) the reaction is conducted at reduced temperature in an ethereal solvent.

9. The process of claim 8, wherein in step (a) said ethereal solvent is selected from the group consisting of diethyl ether, tetrahydrofuran, and dimethoxy ethane, and said reaction temperature is from about -100°C to about -30°C.

10. The process of claim 1, wherein in step (b) said enolate is transmetallated with titanium or aluminum or a mixture thereof.

11. The process of claim 1 , wherein in step (b), said enolate is transmetallated by reaction with Me₂AlCl.

12. The process of claim 10, wherein between one and three molar equivalents of said metal are used.

13. The process of claim 1, wherein in step (c) said catalytic hydrogenolysis of the adduct //, affords the desired γ-lactam (IV) as a mixture with an aminodiol ///:

wherein R -R are as defined in claim 1.

14. The process of claim 13, wherein said hydrogenolysis is conducted in the presence of a catalyst selected from the group consisting of palladium black, palladium on activated carbon, and palladium hydroxide on carbon; and in the presence of an organic solvent selected from the group consisting of lower alkanols, lower alkanoates, lower alkanoic acids and mixtures thereof.

15. The process of claim 14, wherein said organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, ethyl acetate, acetic acid, and mixtures thereof.

16. The process of claim 13, wherein the crude product mixture is heated to convert aminodiol /// to the γ-lactam IV.

17. The process of claim 1 , wherein

R¹ is C,.₁₂ alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl C_6.!4 aryl, C_6.10 ar(C,_.6)alkyl or C,_.6alk(C₆.₁₀)aryl;

R² is Cj._g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl C_6.]4 aryl, C₆.₁₀ ar(C_M)alkyl or C₁.₆alk(C₆.₁₀)aryl;

R³ is C,._g alkyl, C₃.₈ cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl, C_6.]4 aryl, C_6.,₀ ar(C,_.6)alkyl or C,_.6alk(C₆._l0)aryl;

R⁴ is C₆._]0 aryl, or a heteroaryl group selected from the group consisting of thienyl, benzo[b]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,

4H-quinolizinyl, isoquinolyl, quinolyl, or triazolyl; and

R⁵ and R⁶ are independently C,_.6 alkyl, C_6.10 ar(C,_.6)alkyl or

C_].₆alk(C₆.₁₀)aryl or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

18. The process of claim 17, wherein R¹ is C,_₆ alkyl, C₃.₆ cycloalkyl, or C₆.₁₀ aryl; R² is methyl, ethyl, propyl, butyl, methoxy, or ethoxy;

R³ is methyl, ethyl, tert-butyl or benzyl; R⁴ is phenyl or phenyl substituted by halogen, C,.₆ alkyl, C,_.6 alkoxy, carboxy, or amino; and

NR⁵R⁶ is one of dimethylamino, diethylamino, pyrrolidino, piperidino, morpholino, or oxazolidinone substituted by halogen, C,.₆ alkyl, C₆._!0ar(C_].₆)alkyl, C,_.6 alkoxy, carboxy, or amino.

19. A process for forming a substituted oxazoline compound of Formula //:

said method comprising: (a) deprotonating a substituted aryl or heteroaryl oxazoline of Formula /:

by treating said substituted aryl or heteroaryl oxazoline with a strong base to form an enolate; and (b) transmetallating said enolate with a metal selected from the group consisting of titanium, aluminum tin, zinc, magnesium and boron, and thereafter reacting with a formyl amide of Formula Λ7F

XIV

wherein for each of Formulae /, // and XIV.

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;

R⁴ is optionally substituted aryl or optionally substituted heteroaryl; and R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a

5- to 7-membered heterocyclic ring, which can be optionally substituted, and which optionally include an additional oxygen or nitrogen atom.

20. The process of claim 19, further comprising catalytically hydrogenating said oxazoline compound of Formula//, and thereafter optionally refluxing the resulting reaction mixture, whereby a β-lactam of Formula TV is formed:

wherein R , R and R are as defined in claim 19.

21. The process of claim 20, further comprising saponifying a compound of Formula IV and thereafter cyclizing to form a c/αstø-lactacystin β- lactone compound having Formula VII:

wherein R¹ and R² are as defined in claim 19.

22. A process for forming a substituted aryl oxazoline compound of Formula la:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of said aryl, aralkyl, or alkaryl can be optionally substituted; R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl; said method comprising: (a) asymmetrically dihydroxylating an alkene intermediate of Formula

XV:

XV

CO2R³

to form an optically active diol of Formula XVIa:

(b) reacting said optically active diol of Formula XVIa with an orthoester under acid catalysis to give a mixed orthoester, and thereafter reacting the resulting mixed orthoester intermediate with a reagent selected from the group consisting of acyl halides, HCl, HBr, HI,Me₃SiCl, Me₃SiI, Me₃SiBr and halogen- containing Lewis acids to form a haloester derivative of Formula XVIIa:

X XVIIa;

wherein X is Cl, Br, or I; (c) reacting said haloester derivative with an alkali metal azide to form an azide of Formula XVIIIa:

XVIIIa;

(d) hydrogenating said azide to form a compound of Formula XlXa:

(e) subjecting the compound of Formula XlXa to ring closing conditions to form said substituted phenyloxazoline of Formula la; wherein for each of Formulae XVa, XVIa, XVIIa, XVIIIa and XlXa, R¹ , R³ and R⁴ are as defined above for Formula Ia.

23. The process of claim 22, wherein in step (a) the dihydroxylation reaction is conducted with AD-mix-β in the presence of methane sulfonamide to stereoselectively afford the diol of Formula XVIa.

24. The process of claim 22, wherein in step (a) the dihydroxylation reaction is conducted using an N-oxide as a reoxidant.

25. The process of claim 22, wherein in step (b) said diol of Formula XVIa is treated with an orthoester under Lewis or Brόnsted acid catalysis to give a mixed orthoester, which is converted in situ to a haloester of Formula XVIIa wherein X is Br by treatment with acetyl bromide.

26. The process of claim 25, wherein the orthoester employed in this reaction is an aromatic carboxylic acid orthoester.

27. The process of claim 26, wherein the orthoester is trimethyl orthobenzoate.

28. The process of claim 25, wherein said acid catalyst is HBr, SnCl₄, TiCl₄, BBr₃ or boron trifluoride.

29. The process of claim 22, wherein in step (c) crude haloester of Formula FWα is converted to the azide of Formula XVIIIa by treatment with an alkali metal azide in a polar aprotic organic solvent.

30. The process of claim 22, wherein in step (d) said catalytic hydrogenation of the azide of Formula XVIIIa is conducted over a palladium catalyst in ethyl acetate.

31. The process of claim 30, wherein said catalytic hydrogenation proceeds with concomitant migration of the aroyl group to afford the hydroxyamide of Formula XlXa.

32. The process of claim 22, wherein in step (e) the hydroxyamide of Formula AΪXα is treated with thionyl chloride in methylene chloride to effect ring closure with inversion of the hydroxyl to produce the cz^'s-substituted oxazoline of Formula Ia.

33. The process of claim 32, wherein the c/s-oxazoline is converted to the trø '-oxazoline under equilibrating condition by inversion of configuration of the ester substituents.

34. A process for forming a substituted aryl oxazoline compound of Formula lb:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl; said method comprising:

(a) asymmetrically dihydroxylating an alkene intermediate of Formula XV:

o form an optically active diol of Formula XVIb:

(b) reacting said optically active diol of Formula XVIb with an orthoester under acid catalysis to give a mixed orthoester, and thereafter reacting the resulting mixed orthoester intermediate with a reagent selected from the group consisting of acyl halides, HCl, HBr, HI,Me₃SiCl, Me₃SiI, Me₃SiBr and halogen- containing Lewis acids to form a haloester derivative of Formula XVIIb:

X

XVIIb;

V-°

R A

wherein X is Cl, Br, or I; (c) reacting said haloester derivative with an alkali metal azide to form an azide of Formula XVIIIb:

XVIIIb;

(d) hydrogenating said azide to form a compound of Formula XlXb:

(e) subjecting the compound of Formula XlXb to ring closing conditions to form said substituted phenyloxazoline of Formula lb; wherein for each of Formulae XVb, XVIb, XVIIb, XVIIIb and XlXb, R¹ , R³ and

R⁴ are as defined above for Formula lb.

35. The process of clam 34, wherein in step (a) the dihydroxylation reaction is conducted with AD-mix-α in the presence of methane sulfonamide to stereoselectively afford the diol of Formula XVIb.

36. The process of claim 34, wherein in step (a) the dihydroxylation reaction is conducted using an N-oxide as a reoxidant.

37. The process of claim 34, wherein in step (b) said diol of Formula XVIb is treated with an orthoester under Lewis or Brόnsted acid catalysis to give a mixed orthoester, which is converted in situ to a haloester of Formula XVIIb wherein X is Br by treatment with acetyl bromide.

38. The process of claim 37, wherein the orthoester employed in this reaction is an aromatic carboxylic acid orthoester.

39. The process of claim 38, wherein the orthoester is trimethyl orthobenzoate.

40. The process of claim 37, wherein said acid catalyst is HBr, SnCl₄, TiCl₄, BBr₃ or boron trifluoride.

41. The process of claim 34, wherein in step (c) crude haloester of Formula XVIIb is converted to the azide of Formula XVIIIb by treatment with an alkali metal azide in a polar aprotic organic solvent.

42. The process of claim 34, wherein in step (d) said catalytic hydrogenation of the azide of Formula XVIIIb is conducted over a palladium catalyst in ethyl acetate.

43. The process of claim 42, wherein said catalytic hydrogenation proceeds with concomitant migration of the aroyl group to afford the hydroxyamide of Formula XlXb.

44. The process of claim 34, wherein in step (e) the hydroxyamide of Formula.XZΛ7j is treated with thionyl chloride in methylene chloride to effect ring closure with inversion of the hydroxyl to produce the trans-substituted oxazoline of Formula lb.

45. A process for forming a compound of Formula XlXa:

said method comprising: hydrogenating an azide compound having Formula XVIIIa: N₃ RL

C0₂R³

XVIIIa

0=K

R4

wherein for each of Formulae XlXa and XVIIIa:

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and R⁴ is aryl or heteroaryl, either of which may be optionally substituted.

46. The process of claim 45, further comprising subjecting a compound of Formula XlXa to ring closing conditions to form a substituted oxazoline compound of Formula la:

wherein R¹, R³ and R⁴ are as defined in claim 45.

47. A process for forming a compound of Formula XlXb:

said method comprising: hydrogenating an azide compound having Formula XVIIIb:

XVIIIb

wherein for each of Formulae XlXb and XVIIIb: R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and R⁴ is aryl or heteroaryl, either of which may be optionally substituted.

48. The process of claim 47, further comprising subjecting a compound of Formula XlXb to ring closing conditions to form a substituted oxazoline compound of Formula lb:

wherein R , R and R are as defined in claim 47.

49. A compound of Formula F/or VII:

or a salt thereof, wherein: R¹ is C,.,₂ alkyl, C₃._g cycloalkyl, C₂._g alkenyl, C₂._g alkynyl, C₆.₁₄ aryl, C₆._]0 ar(C_!.6)alkyl or C,_.6alk(C₆.₁₀)aryl; R² is C₂.₆ alkyl; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl, aralkyl or alkaryl can be optionally substituted.

50. A compound of claim 49, wherein R¹ is C,_₄ alkyl.

51. A compound of claim 50, wherein R¹ is isopropyl.

52. A compound of claim 49, wherein R² is ethyl, n-propyl, n-butyl or isobutyl.

53. A compound of claim 52, wherein R² is ethyl.

54. A compound of claim 52, wherein R² is n-propyl.

55. A compound of claim 52, wherein R² is n-butyl.

56. A compound of claim 52, wherein R² is isobutyl.

57. An enantiomerically-enriched formyl amide of Formula XIV:

XIV

or a salt thereof, wherein

R² is C,_.g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂_₈ alkynyl, C_6.14 aryl, C_6.]0 ar(C,_.6)alkyl or C,_.6alk(C_6.10)aryl; and

R⁵ and R⁶ are independently C,.₆ alkyl, C₆.,₀ ar(C,_.6)alkyl or C_1.6alk(C_6.10)aryl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

58. A pharmaceutical composition comprising a compound according to any one of claims 49-56 and a pharmaceutically acceptable carrier or diluent.

59. A compound of Formula //:

or a salt thereof, wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;

R⁴ is optionally substituted aryl or optionally substituted heteroaryl; and R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which can be optionally substituted, and which optionally include an additional oxygen or nitrogen atom.

60. A compound of claim 59, wherein

R¹ is C,.₁₂ alkyl, C₃.₈ cycloalkyl, C₂.₈ alkenyl, C_2.8 alkynyl, C_6.14 aryl, C_6.10 ar(C,.₆)alkyl or C,.₆alk(C₆.₁₀)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is C,._g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C_2.8 alkynyl, C_6.14 aryl, C_6.10 ar(C,.₆)alkyl or C,.₆alk(C₆._]0)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R³ is C,_.g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C_2.g alkynyl, C₆.₁₄ aryl, C₆.₁₀ ar(C,_.6)alkyl or C,.₆alk(C₆__]0)aryl, any of which can be optionally substitiited;

R⁴ is optionally substituted C₆.₁₀ aryl, or an optionally substituted heteroaryl group selected from the group consisting of thienyl, benzo[β]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, or triazolyl; and R⁵ and R⁶ are independently C,_₆ alkyl, C_6.10 ar(C,.₆)alkyl or

C,_.6alk(C₆._]0)aryl or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

61. A compound of Formula ///:

lt thereof wherein:

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which can be optionally substituted, and which can optionally include an additional oxygen or nitrogen atom.

62. A compound of claim 61, wherein

R¹ is C,.₁₂ alkyl, C₃.₈ cycloalkyl, C₂.₈ alkenyl, C_2.8 alkynyl, C_6.14 aryl, C_6.10 ar(C,_.6)alkyl or C,.₆alk(C₆.₁₀)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is C,._g alkyl, C₃._g cycloalkyl, C₂.₈ alkenyl, C₂._g alkynyl, C₆.₁₄ aryl, C_6.10ar (C,_.6)alkyl or C,_.6alk(C₆._]0)aryl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; and R³ is C,._g alkyl, C₃.₈ cycloalkyl, C₂._g alkenyl, C₂._g alkynyl, C₆.₁₄ aryl, C₆.₁₀ar (C,_.6)alkyl or C,_.6alk(C₆.₁₀)aryl, any of which can be optionally substituted; and R⁵ and R⁶ are independently C,.₆ alkyl, C₆.₁₀ ar(C,_.6)alkyl or C,.

₆alk(C_6.10)aryl, or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

63. A compound of Formula XVIIa or XVIIb:

XVIIa

%/°

R' 4 XVIIIb;

or a salt thereof, wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted; R² is Cl, Br or I;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substituted; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl.

64. A compound of Formula XVIIIa or XVIIIb

XVIIIa

N, XVIIIb

or a salt thereof, wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherein the ring portion of any of said aryl, aralkyl or alkaryl can be optionally substiuited;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of which can be optionally substiuited; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl.

65. A process for forming an enantiomerically-enriched formyl amide of Formula XIV:

XIV

or a salt thereof, wherein R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portion of any of said aryl, aralkyl, or alkaryl can be optionally substituted;

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ when taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring, which can be optionally substituted, and which optionally include an additional oxygen or nitrogen atom; said method comprising:

(a) acylating an anion of a compound of Formula VIII:

VIII

where R⁸ is isopropyl or benzyl, with R²CH₂COCl to form an acyloxazolidinone of Formula IX:

IX

where R² and R⁸ are as defined above;

(b) stereoselectively reacting the acyloxazolidinone of Formula IX with benzyloxymethyl chloride to form a protected alcohol of Formula X:

O UBBnΠ Q υ O υ

R2

R*>

where R² and R⁸ are as defined above;

(c) hydrolyzing the protected alcohol of Formula X to form a carboxylic acid of Formula^./:

XI where R² is as defined above; (d) coupling said acid of Formula XI with an amine R⁵R⁶NH₂ to provide an amide of Formula XII:

OBn O

R2 R6

XII

where R², R⁵ and R⁶ are as defined above; (e) catalytically hydrogenating, the amide of Formula XII to form an alcohol of Formula XIII:

OH O

R2 R6

XIII

where R², R⁵ and R⁶ are as defined above; and

(f) oxidizing the resultant alcohol of Formula XIII to give a formyl amide of Formula XIV.

66. The process of claim 65, wherein:

R² is C_j.g alkyl, C₃.₈ cycloalkyl, C₂.₈ alkenyl, C₂.₈ alkynyl C_6.]4 aryl, C_6.10 ar(C,.₆)alkyl or C₁.₆alk(C₆.₁₀)aryl; and

R⁵ and R⁶ are independently C,.₆ alkyl, C₆.₁₀ ar(C,.₆)alkyl or C₁.₆alk(C₆.₁₀)aryl or together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle which can be optionally substituted, and which optionally can include an additional oxygen or nitrogen atom.

67. A method of inhibiting proteasome function in a cell, comprising contacting said cell with a compound of claim 49.

68. A method of inhibiting proteasome function in a mammal, comprising administering to said mammal a compound of claim 49 in an amount effective to inhibit proteasome function.

69. A method of treating inflammation, comprising administering to a subject an effective anti-inflammatory amount of a compound of claim 49.

70. A method of treating cancer, comprising administering to a subj ect an effective antitumor or antimetastic amount of a compound of claim 49.

71. A method of treating ischemic or reperfusion injury in a mammal comprising administering to said mammal an effective amount of a compound of claim 49.

72. The method of claim 71, wherein the ischemia is the result of vascular occlusion.

73. The method of claim 71 , wherein said vascular occlusion occurs during a stroke.