PRIORITY CLAIM

This application claims priority from copending U.S. Patent Application Serial No. 10/821,621, filed 9 April 2004.

BACKGROUND OF THE INVENTION

Salinosporamide A (1) was recently discovered by Fenical et al. as a bioactive product of a marine microorganism that is widely distributed in ocean sediments. Feeling, R.H.; Buchanan, G.O.; Mincer, TJ.; Kauffman, C.A.; Jensen, P.R.; Fenical, W., Angew. Chem. Int. Ed., 2003, 42, 355-357.

Structurally Salinosporamide A closely resembles the terrestrial microbial product omuralide (2a) that was synthesized by Corey et al. several years ago and demonstrated to be a potent inhibitor of proteasome function. See, (a) Corey, EJ.; Li, W. D., Z. Chem. Pharm. Bull, 1999, 47, 1-10; (b) Corey, EJ., Reichard, G.A.; Kania, R., Tetrahedron Lett, 1993, 34, 6977-6980; (c) Corey, E. J.; Reichard, G. A., /. Am. Chem. Soc, 1992, 114, 10677-10678; (d) Fenteany, G.; Standaert, R.F.; Reichard, G. A.; Corey, E. J.; Schreiber, S. L., Proc. Natl. Acad. ScL USA, 1994, 91, 3358-3362.

Omuralide is generated by β-lactonization of the N-acetylcysteine thiolester lactacystin (2b) that was first isolated by the Omura group as a result of microbial screening for nerve growth factor-like activity. See, Omura, S., Fujimoto, T., Otoguro, K., Matsuzaki, K., Moriguchi, R., Tanaka, H., Sasaki, Y., Antibiot, 1991, 44, 113-116; Omura, S., Matsuzaki, K., Fujimoto, T., Kosuge, K., Furuya, T., Fujita, S., Nakagawa, A., /. Antibiot, 1991, 44, 117-118.

Salinosporamide A, the first compound Fenical's group isolated from Salinospora, not only had a never-before-seen chemical structure 1, but is also a highly selective and potent inhibitor of cancer-cell growth. The compound is an even more effective proteasome inhibitor than omuralide and, in addition, it displays surprisingly high in vitro cytotoxic activity against many tumor cell lines (IC50 values of 10 nM or less). Fenical et al. first found the microbe, which they've dubbed Salinospora, off the coasts of the Bahamas and in the Red Sea. See, Appl. Environ. Microbiol, 68, 5005 (2002).

Fenical et al. have shown that Salinospora species requires a salt environment to live. Salinospora thrives in hostile ocean-bottom conditions: no light, low temperature, and high pressure. The Fenical group has now identified Salinospora in five oceans, and with 10,000 organisms per cm3 of sediment and several distinct strains in each sample; and according to press reports, they've been able to isolate 5,000 strains. See, Chemical & Engineering News, 81, 37 (2003).

A great percentage of the cultures Fenical et al. have tested are said to have shown both anticancer and antibiotic activity. Like omuralide 2a, salinosporamide A inhibits the proteasome, an intracellular enzyme complex that destroys proteins the cell no longer needs. Without the proteasome, proteins would build up and clog cellular machinery. Fast-growing cancer cells make especially heavy use of the proteasome, so thwarting its action is a compelling drug strategy. See, Fenical et al., U.S. Patent Publication No. 2003-0157695A1, the disclosure of which is hereby incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention is directed to a method for the enantiospecific total synthesis of the compound of structure 1.

One preferred synthetic route to compound 1 is illustrated in the Figure (Scheme 1) accompanying this specification, and as discussed in greater detail below. In summary, that method of the present invention includes the following steps, which are detailed here with the preferred reagents and reaction conditions. The skilled artisan may likely be able to make substitutions of reagents and/or reaction conditions in any one or more of these reaction steps without necessarily departing from scope and teachings of the present invention:

(S)-Threonine methyl ester was N-acylated with 4-methoxybenzoly chloride in CH2Cl2 at 23°C to form the amide 3 (71%) which was then cyclized to oxazoline 4 (80%) by heating at reflux in toluene with p-toluenesulfonic acid.

Deprotonation of 4 with lithium diisopropylamide in THF and alkylation of the resulting enolate with chloromethyl benzyl ether afforded the required tertiary stereocenter of 5 selectively in 69% yield.

Reduction of 5 with NaBH3CN-HOAc gave the N-4-methoxybenzylamine 6 (90%).

Compound 6 was then transformed in 96% yield to the N-acrylyl-N-PMB

derivative 7 (PMB = 4-methoxybenzyl) by the one flask, seqeuence: (1) reaction with

Me3SiCl and Et3N to form the TMS ether (6a - OH is OTMS), followed by (2) acylation with acrylyl chloride at 0°C and (3) acidic work up with aqueous HCl.

Dess-Martin periodinane oxidation of 7 produced the keto amide ester 8 in

96% yield.

Cyclization of 8 to the γ-lactam 9 was accomplished by means of an internal

Baylis-Hillman-aldol reaction using quinuclidine as the catalytic base in

dimethoxyethane at 23°C for 5 h. See, Frank, S. A.; Mergott, D. J., Roush, W. R., /.

Am. Chem. Soc, 2002, 124, 2404-2405. Mergott, D.J., Frank, S. A., Roush, W. R.,

Org. Lett., 2002, 4, 3157-3160. Aggarwal, V. K., Emme, L, Fulford, S. Y., /. Org.

Chem., 2003, 68, 692-700. Yeo, J. E., Yang, X., Kim, HJ., Koo, S., J. Chem. Soc,

Chem. Commun., 2004, 236-237. The cyclization product, obtained in 94% yield

consisted of 9 and the C(β) diastereomer (10) in a ratio of 3:1.

9, H 11 ,R« SsfMeWCHjBr

The N-benzyl analog of 10 (not shown) was obtained in crystalline form mp =

136-7°C, and demonstrated to possess the stereochemistry shown by single crystal X- ray diffraction analysis.

Silylation of 9 with bromomethyldimethylsilyl chloride afforded 11 in 95% yield. Silyl ether 11 and the diastereomeric silyl ether were easily and conveniently separated at this stage by silica gel column chromatography on a preparative scale.

The required stereochemical relationship about C(α) and C(β) of the γ-lactam core was established by tri-zi-butyltin hydride-mediated radical-chain cyclization which transformed 11 cleanly into the cώ-fused γ-lactam 12.

11 12

See, (a) BoIs, M., Skrydstrup, T., Chem. Rev., 1995, 95, 1253-1277. (b) Fleming, L, Barbero, A., Walter, D., Chem. Rev., 1997, 97, 2063-2092. (c) Stork, G., Mook, R., Biller, S.A., Rychnovsky, S. D., /. Am. Chem. Soc, 1983, 105, 3741-3742. (d) Stork, G., Sher, P. M., Chen, H.L., /. Am. Chem. Soc, 1986, 108, 6384-6385.

Cleavage of the benzyl ether of 12 (H2j Pd-C) afforded the primary alcohol (12a - OBn is OH), and Dess-Martin periodinane oxidation of 12a provided the aldehyde 13 in about 90% yield from 12.

12 The next step, the attachment of the 2-cyclohexenly group to the formyl

carbon and the establishment of the remaining two stereocenters was accomplished in

a remarkably simple way.

2-Cyclohexenyl-tri-n-butyltin (from Pd(O)-catalyzed 1,4-addition of

tributyltin hybride to 1,3-cyclohexadiene) was sequentially transmetalated by

treatment with 1 equiv of n-butyllithium and 1 equiv of zinc chloride to form 2- cyclohexenylzinc chloride in THF solution. See, Miyake, H., Yamamura, K., Chem.

Lett., 1992, 507-508. Reaction of this reagent with the aldehyde 13 furnished the

desired formyl adduct 14 stereoselectively (20:1) in 88% yield.

13 14 tfr= 20:1

Tamao-Fleming oxidation of 14 gave the triol 15 in 92% yield.

See, Fleming, T., Chemtracts-Org. Chem., 1996, 9, 1-64, and Jones, G. R., Landais,

Y., Tetrahedron, 1996, 52, 7599-7662.

Ce(IV)-induced oxidative cleavage of the PMB group of 15 afforded the triol ester 16:

Compound 16 was then hydrolyzed to the corresponding γ-lactam-carboxylic acid 16a (CO2Me is CO2H) using 3:1 aqueous 3N-lithium hydroxide and THF at 4°C.

The acid 16a was first cyclized to the beta-lactone 16b (1 where CH2CH2Cl is CH2CH2OH), which is then converted to salinosporamide A (1) by successive reaction with 1.1 equiv of bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BOPCl) and pyridine at 23 "C for 1 h, in 65% overall yield.

16a -» 16b -_> 1

The identity of synthetic 1 and natural Salinosporamide A was established by comparison measurements of 1H and 13C NMR spectra, mp and mixed mp (168- 17O0C), optical rotation, FTIR and mass spectra and chromatographic mobilities in three different solvent systems. Dr. Fenical graciously provided a sample of the natural product for this comparison. - Si -

There are a number of steps in the synthesis of 1 that require comment. The direct conversion of 6 to 7 with acrylyl chloride under a wide variety of conditions gave considerably lower yields than the process shown in Scheme 1 mainly because of competing O-acylation and subsequent further transformations.

In the conversion of 8 to 9, quinuclidine has proved superior to other catalytic bases tried, e.g., l,4-diaza[2.2.2]bicycloocate, for maximizing the formation of 9 over the diastereomer 10. As shown in Scheme 2, better stereoselectivity in the formation of 9 is provided by an alternative two-step procedure, in which compound 8 is first treated with tetraisopropyl titanate, cyclopentylmagnesium chloride, and tert-butyl methyl ether, and then with triethylamine:

As indicated above, the attachment of the 2-cyclohexenyl group to aldehyde 13 to form 14 worked best with the reagent cyclohexenylzinc chloride.

The stereochemistry of the conversion 13 to 14, established by the identity of totally synthetic 1 with naturally foπned salinosporamide A, is that predicted from a cyclic, chair-formed, six-membered transition state involving addition of the organozinc reagent to the sterically more accessible face of the formyl group. The use of 2-cyclo-hexenylzinc chloride may be critical to successful formation of 14 since none of this product is obtained with 2-cyclohexenyllithium (probably because the initial carbonyl adduct undergoes retroaldol cleavage and decomposition; see Corey, E. J., Li, W., Nagamitsu, T., Angew. Chem. Int, Ed., 1998, 37, 1676-1679).

Attempts to form 14 from 13 using Lewis acid-catalyzed reaction with tri-n- butyl-2-cyclohexenyltin have thus far been unsuccessful. The saponification of methyl ester 16 at temperatures above +5°C led to lowered yields of the required carboxylic acid. Finally, the one flask β-lactonization and chlorination reactions leading to 1 were remarkably clean and probably proceed in greater than 90% yield per step.

In addition to the methods of Schemes 1 and 2, preferred embodiments of the invention also include novel synthetic intermediate compounds, intermediate steps of the preferred synthetic process; and the uses of this method and/or intermediate compounds thereof, in the preparation of synthetic analogs or derivatives of the compound Salinosporamide A. Typical substituent modifications for compounds of this type are known to persons having ordinary skill in this art. See, for example, the substituent groups defined for analogs of lactacystin compounds as taught in Corey et al., Chem. Pharm. Bull, 1999, 47, 1-10, the disclosure of which is incorporated herein by reference. Other substituent modifications will be apparent based upon the disclosures in related patents. See, for example, U.S. Patent Nos. 6,645,999; 6,566,553; 6,458,825; 6,335,358; 6,294,560; 6,214,862; 6,147,223; 6,133,308; 5,869,675; 5,756,764; and PCT Publication No. WO 96/32105; the disclosures of which are hereby incorporated herein by reference.

Scheme 2 provides another synthetic route to the compound Salinosporamide A. In this scheme, the actual compound formed is the isopropyl analog of Salinosporamide A, in which the cyclohexene substituent has been replaced by isopropyl.

17 In Step 1, Compound 4 is converted to Compound 17 by reaction with lithium diisopropyl amide and chloromethyl benzyl ether.

Step 2:

89% 17 18

In Step 2, Compound 17 is converted, in a two-step procedure, to Compound 18 - first by reaction with sodium cyanoborohydride and second by reaction with trimethylsilyl chloride.

Step 3:

18 19

In Step 3, Compound 18 is converted, in a two-step procedure, to Compound 19 ■ first by reaction with acrylyl chloride, and second by treatment with aqueous hydrogen fluoride.

Step 4:

19 20

In Step 4, Compound 19 is subjected to Dess-Martin oxidation to afford Compound 20.

Step 5:

20 21

In Step 5, Compound 20, in a two-step procedure, is converted into the pyrrole, Compound 21 - first by reaction with tetraisopropyl titanate, cyclopentyl magnesium chloride and tert-butyl methyl ether; followed by triethylamine.

Step 6:

21 22

In Step 6, the free-hydroxyl group in Compound 21 is protected by reaction with bromomethyl dimethyl silylchloride and imidazole, yielding Compound 22. Step 7:

22 23

In Step 7, Compound 22 is reacted with tributylstannane and azoisobutyronitrile

(AIBN) to afford the bicyclic compound 23.

Step 8:

23 24

In Step 8, Compound 23 is converted, in a two-step procedure, to Compound 24 -

first by hydrogenation over Pd-C catalyst, followed by Dess-Martin oxidation.

Step 9:

24 25 26 In Step 9, Compound 24 is reacted with isopropenyl magnesium chloride instead of cyclohexenyl zinc chloride to afford a mixture of Compounds 25 and 26 in a 3:1 ratio. It is believed that cyclohexenyl zinc chloride would also work in this reaction.

Step 10:

25 26 27

Hydrogenation of the mixture of Compounds 25 and 26 over Pd-C catalyst converts the mixture to Compound 27.

Step 11:

27 28

In Step 11, the free hydroxyl group in Compound 27 is protected by reaction with dimethyl isopropyl silyltriflate and 2,6-lutidine, yielding Compound 28.

Step 12:

28 29

In Step 12, the bicyclic compound 28 is oxidized by treatment with hydrogen

peroxide, potassium fluoride and potassium bicarbonate to yield Compound 29,

without disturbing two protecting groups - PMB and dimethyl isopropyl silyl protecting group (DMIPS).

Step 13:

29 30

In Step 13, the amino protecting group PMB in Compound 29 is removed by reaction

with eerie (IV) ammonium nitrate (CAN) affording Compound 30.

Step 14:

30 31 In Step 14, Compound 30 is converted, in a two-step procedure, to the beta-lactone of Compound 31 - first by reaction with dimethylaluminum methyl tellurolate, followed by triphenylphosphine chloride.

Step 15:

31 32

In Step 15, the DMIPS protecting group in Compound 31 is removed by treatment with triethylamine and hydrogen fluoride to afford Compound 32 - the isopropyl analog of Salinosporamide A.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates Scheme 1, a preferred synthetic route used to achieve the enantiospecific total synthesis of Salinosporamide A.

Figure 2 illustrates Scheme 2, another preferred synthetic route to the compound Salinosporamide A.

DETAILED DESCRIPTION OF THE INVENTION

As set forth above, one embodiment of the present invention comprises a simple and effective stereocontrolled synthesis of Salinosporamide A, the compound of Formula (1). Two synthetic schemes are provided herein - Schemes 1 and 2.

Scheme 1 is one preferred pathway to accomplish this synthesis, the details of which are provided in the following Examples.

Experimental Details Part I. Synthesis of the Salinosporamide A Part 2. Synthesis of the Cyclohexenyl Zinc Chloride

General

All moisture sensitive reactions were performed under nitrogen gas in glassware that was flame-dried and equipped with a magnetic stir bar. Tetrahydrofuran (THF) and 1,2-dimethoxyethane (DME) were freshly distilled from sodium benzophenone ketyl before use. Hexanes, pyridine, rriethylamine, pentane and dichloromethane were freshly distilled from CaH2 before use. Toluene was distilled from sodium.

Thin-layer chromatography (TLC) was performed using E. Merck silica gel 60 F254 pre-coated plates (0.25 mm). Flash chromatography was performed using Baker silica gel (40 /wn particle size). All products were purified to homogeneity by TLC analysis (single spot/two solvent systems) using a UV lamp or CAM or PMA or anisaldehyde or basic KMnO4 for detection purposes.

NMR spectra were recorded on 400 MHz, 500 MHz and 600 MHz spectrometers. 1H and 13C NMR chemical shifts are reported as δ using residual solvent as an internal standard. High-resolution mass spectral analyses were performed at Harvard University Mass Spectrometry Center. Part I. Synthesis of the Salinosporamide A (1)

Example 1:

(4S, 5R) Methyl 4,5-dihydro-2 (4-methoxyphenyl)-5-methyloxazole-4-carboxylate (4).

A mixture of (2S, 3R)-methyl 2-(4-methoxybenzamido)-3-hydroxybutanoate (3) (35.0 g, 131 mmol) and p-TsOH-H20 (2.5 g, 13.1 mmol) in toluene (400 mL) was heated at reflux for 12 h. The reaction mixture was diluted with water (200 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with water, brine and dried over Na2SO4. The solvent was removed in vacuo to give crude oxazoline as yellow oil. Flash column chromatography on silica gel (eluent 15% EtOAc-Hexanes) afforded the pure oxazoline (26.1 g, 80%) as solid.

Rf = 0.51 (50% ethyl acetate in hexanes), mp, 86-870C; [α]23D +69.4 (c 2.0, CHCl3); FTIR (film) vmax: 2955, 1750, 1545, 1355, 1187, 1011, 810 cm"1; 1H NMR (CDCl3, 400 MHz): δ 7.87 (2H, d, / = 9.2 Hz), 6.84 (2H, d, / = 8.8 Hz), 4.90 (1 H, m), 4.40 (1 H, d, / = 7.6 Hz), 3.79 (3H,s), 3.71 (3H, s), 1.49 (3H, d, /= 6.0 Hz); 13C NMR (CDCl3, 100 MHz): δ 171.93, 165.54, 162.64, 130.52, 119.80, 113.85, 78.91, 75.16, 55.51, 52.73, 21.14; HRMS (ESI) calcd for C13H16NO4 (M + H)+.250.1079, found 250.1084. Example 2:

(4R, 5R)-Methyl 4-{(benzyloxy) methyl) }-4,5-dihydro-2- (4-methoxyphenyl)-5- methyloxazole-4-carboxylate (5).

To a solution of LDA (50 mmol, 1.0 M stock solution in THF) was added HMPA (24 mL, 215 mmol) at -780C and then oxazoline 4 (12.45 g, 50 mmol, in 20 mL THF) was added dropwise with stirring at -78°C for 1 h to allow complete enolate formation. Benzyloxy chloromethyl ether (8.35 mL, 60 mmol) was added at this temperature and after stirring the mixture at -78°C for 4 h, it was quenched with water (50 mL) and warmed to 230C for 30 min. Then the mixture was extracted with ethyl acetate (3 x 50 mL) and the combined organic phases were dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:4 then 1:3) to give the benzyl ether 5 (12.7 g, 69%).

Rf= 0.59 (50% ethyl acetate in hexanes). [α]23D -6.3 (c 1.0, CHCl3); FTIR (film) (vmax: 3050, 2975, 1724, 1642, 1607, 1252, 1027, 745, 697 cm"1; 1H NMR (CDCl3, 400 MHz): δ 7.96 (2H, d, / = 9.2 Hz), 7.26 (5H, m ), 6.90 (2H, / = 8.8 Hz), 4.80 (1 H,m), 4.61 (2H,s), 3.87 (3H, m), 3.81 (3H, s), 3.73 (3H, s), 1.34 (3H, d, / = 6.8 Hz); 13C NMR (CDCl3, 100MHz) : δ 171.23, 165.47, 162.63, 138.25, 130.64, 128.52, 127.87, 127.77, 120.15, 113.87, 81.40, 79.92, 73.91, 73.43, 55.58, 52.45, 16.92; HRMS (ESI) calcd for C2IH24O5 (M + H)+ 370.1654, found 370.1644. Example 3:

(2R,3R)-Methyl 2-(4-methoxybenzylamino)-2-((benzyloxy)methyl)- 3hydroxybutanoate (6).

To a solution of oxazoline 5 (18.45 g, 50 mmol) in AcOH (25 mL) at 230C was added in portions NaCNBH3 (9.3 g, 150 mmol). The reaction mixture was then stirred at 4O0C for 12 h to allow complete consumption of the starting material. The reaction mixture was diluted with water (100 mL), neutralized with solid Na2CO3 and the aqueous layer was extracted with ethyl acetate (3 x 100 mL). The combined organic phases were dried over NaSO4 and concentrated in vacuo. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:5) to give the N-PMB amino alcohol 6 (16.78 g, 90%).

Rf= 0.50 (50% ethyl acetate in hexanes). [α]23D -9.1(c 1.0, CHCl3); FTIR (film) vmax: 3354, 2949, 1731, 1511, 1242, 1070, 1030, 820,736, 697 cm"1; 1H NMR (CDCl3, 400 MHz): δ 7.32 (7H, m), 6.87 (2H, d, J= 8.8 Hz), 4.55 (2H, m), 4.10 (1 H, q, J= 6.4 Hz), 3.85 (2H, dd, / = 17.2, 10.0 Hz), 3.81 (3H, s,), 3.77 (3H, s), 3. 69 (2H, dd, / = 22.8, 11.6 Hz), 3.22 (2H, bs), 1.16 (3H, d, J = 6.0 Hz); 13C NMR (CDCl3, 100 MHz): δ 173.34, 159.03, 137.92, 132.51, 129.78, 128.67, 128.07, 127.98, 114.07, 73.80, 70.55, 69.82, 69.65, 55.51, 55.29, 47.68, 18.15; HRMS (ESI) calcd. for C2iH2SNO5 (M + H)+ 374.1967, found 374.1974. Example 4:

1OBn

(2R,3R)-Methyl-2-(N-(4-methoxybenzyl)acrylamido)-2-(benzy loxy)methyl)-3- hydroxybutanoate (7).

A solution of amino alcohol 6 (26.2 g, 68.5 mmol) in Et2O (200 mL) was treated with Et3N (14.2 mL, 102.8 mmol) and trimethylchlorosilane (10.4 mL, 82.2 mmol) at 230C and stirred for 12 h. After completion, the reaction mixture was diluted with ether (200 mL) and then resulting suspension was filtered through celite. The solvent was removed to furnish the crude product (31.2 g, 99%) in quantitative yield as viscous oil. A solution of this crude trimethylsilyl ether (31.1 g) in CH2Cl2 (200 mL) was charged with diisopropylethylamine (14.2 mL, 81.6 mmol) and then cooled to 00C. Acryloyl chloride (6.64 mL, 82.2 mmol) was added dropwise with vigorous stirring and the reaction temperature was maintained at 00C until completion (1 h). The reaction mixture was then diluted with CH2Cl2 (100 mL) and the organic layer was washed with water and brine. The organic layer was separated and dried over Na2SO4. The solvent was removed to afford the crude acrylamide 7 as a viscous oil. The crude product was then dissolved in Et2O (200 mL) and stirred with 6N HCl (40 mL) at 230C for 1 h. The reaction mixture was diluted with water (100 mL) and concentrated to provide crude product. The residue was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:5 to 1 : 1) to give pure amide 7 (28.3 g, 96%) as colorless solid, mp 88-89°C.

Rf= 0.40 (50% ethyl acetate in hexanes), [α]23D -31.1 (c 0.45, CHCl3), FTIR (film) vmax: 3435, 2990, 1725, 1649, 1610, 1512, 1415, 1287, 1242, 1175, 1087, 1029, 732, 698 cm4; 1H NMR (CDCl3, 500 MHz): δ 7.25 (5H, m), 7.15 (2H, d, / = 6.0 Hz), 6.85 (2H, d, J = 7.5 Hz), 6.38 (2H, d, /= 6.0 Hz), 5.55 (1 H, t, J = 6.0 Hz), 4. 81 (2H, s), 4.71 (1 H, q, J = 6.5 Hz), 4.35 (2H, s), 4.00 (1 H, d, /= 10.0 Hz), 3.80 (1 H, d, J = 10.0 Hz), 3.76 (3H, s), 3.75 (3H, s), 3.28 (1 H, bs), 1.22 (3H, d, /= 6.0 Hz); 13C NMR (CDCl3, 125 MHz): δ 171.87, 168.74, 158.81, 137.73, 131.04, 129.68, 128.58, 128.51, 127.94, 127.72, 127.20, 127.14, 114.21, 73.71, 70.42, 69.76, 67.65, 55.45, 52.52, 49.09, 18.88; HRMS (ESI) calcd. for C24H30NO6 (M + H)+428.2073, found 428.2073.

Example 5:

Dfsss Martin [0}

7 S

(R)-Methyl-2-(N-(4-methoxybenzyl)acrylamido)-2-(benzyloxy )methyl)-3- oxybutanoate (8).

To a solution of amide 7 (10.67 g, 25.0 mmol) in CH2Cl2 (100 mL) was added Dess-Martin periodinane reagent (12.75 g, 30.0 mmol, Aldrich Co.) at 23°C. After stirring for 1 h, the reaction mixture was quenched with aq NaHCO3-Na2S2O3 (1:1, 50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic phase was dried and concentrated in vacuo to afford the crude ketone. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes) to give pure keto amide 8 (10.2 g, 96%).

Rf= 0.80 (50% ethyl acetate in hexanes), mp 85 to 860C; [α]23D -12.8 (c 1.45, CHCl3); FTIR (film) vmax: 3030, 2995, 1733, 1717, 1510, 1256, 1178, 1088, 1027, 733, 697 cm"1; 1H NMR (CDCl3, 500 MHz): δ 7.30 (2H, d, /= 8.0), 7.25 (3H, m), 7.11 (2H, m), 6.88 (2H, d, /= 9.0 Hz), 6.38 (2H, m), 5.63 (1 H, dd, J= 8.5, 3.5 Hz ), 4. 93 (1 H, d, /= 18.5 Hz), 4.78 (1 H, d, /= 18.5, Hz), 4.27 (2H, m), 3.78 (3H, s), 3.76 (3H, s), 2.42 (3 H, s); 13C NMR (CDCl3, 125 MHz): δ 198.12, 169.23, 168.62, 158.01, 136.95, 130.64, 130.38, 128.63, 128.13, 127.77, 127.32, 114.33, 77.49, 73.97, 70.66, 55.49, 53.09, 49.03, 28.24; HRMS (ESI) calcd. for C24H28NO6 (M + H)+ 426.1916, found 426.1909.

Example 6:

r

(2R,3S)-Methyl-l-(4-methoxybenzyl)-2-((benzyloxy)methyl)- 3-hydroxy-3-methyl-4- methylene-5-oxopyrrolidine-2-carboxylate (9 + 10).

A mixture of keto amide 8 (8.5 g, 20.0 mmol) and quinuclidine (2.22 g, 20.0 mmol) in DME (10 lnL) was stirred for 5 h at 230C. After completion, the reaction mixture was diluted with ethyl acetate (50 lnL) washed with 2N HCl, followed by water and dried over Na2SO4. The solvent was removed in vacuo to give the crude adduct (8.03 g, 94.5%, 3:1 ratio of 9 to 10 dr) as a viscous oil. The diastereomeric mixture was separated at the next step, although small amounts of 9 and 10 were purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:10 to 1:2) for analytical purposes. Major Diastereomer (9).

[α]23D -37.8 (c 0.51, CHCl3); FTIR (film) V013x: 3450, 3055, 2990, 1733, 1683, 1507, 1107, 1028, 808,734 cm4; 1H NMR (CDCl3, 500 MHz): δ 7.29 (5H, m), 7.15 (2H, d, J = 7.5 Hz), 6.74 (2H, d, J = 8.5 Hz), 6.13 (1 H, s), 5.57 (1 H,s ), 4.81 (1 H, d, J = 14.5 Hz), 4.45(1 H, d, J = 15.0 Hz ), 4.20 (1 H, d, J= 12.0 Hz), 4.10 (1 H, d, J = 12.0 Hz) 3.75 (3H, s), 3.70 (1 H, d, J= 10.5 Hz), 3.64 (3H, s), 3.54 (1 H, d, J = 10.5 Hz), 2.55 (1 H, bs, OH), 1.50 (3H, s); 13C NMR (CDCl3, 125 MHz): δ 169.67, 168.42, 158.97, 145.96, 137.57, 130.19, 130.12, 128.53, 127.83, 127.44, 116.79, 113.71, 76.32, 76.00, 73.16, 68.29, 55.45, 52.63, 45.36, 22.64; HRMS (ESI) calcd. for C24H28NO6 (M + H)+ 426.1916, found 426.1915.

Minor Diastereomer (10).

[α]23D .50.1 (c 0.40, CHCl3); FTIR (film) vmax: 3450, 3055, 2990, 1733, 1683, 1507, 1107, 1028, 808, 734 cm4; 1H NMR (CDCl3, 500 MHz): δ 7.29 (5H, m), 7.12 (2H, d, J = 7.5 Hz), 6.73 (2H, d, / = 8.5 Hz), 6.12 (1 H, s), 5.57 (1 H, s), 4.88 (1 H, d, J = 15.5 Hz), 4.31 (1 H, d, J - 15.0 Hz ), 4.08 (3H, m), 3.99 (1 H, d, J= 12.0 Hz) 3.73 (3H, s), 3.62 (3H, s), 3.47 (1 H, bs, OH), 3.43 (1 H, d, J= 10.0 Hz), 1.31 (3H, s); 13C NMR (CDCl3, 125 MHz): δ 169.65, 167.89, 159.13, 147.19, 136.95, 130.29, 129.76, 128.74, 128.19,127.55, 116.80, 113.82, 76.21, 75.66, 73.27, 68.02, 55.45, 52.52, 45.24, 25.25; HRMS (ESI) calcd. for (M + H)+ C24H28NO6 426.1916, found 426.1915.

Example 7:

Silylation of 9 and 10 and Purification of 11.

To a solution of lactams 9 and 10 (7.67 g, 18 mmol) in CH2Cl2 (25 ml) was added Et3N (7.54 ml, 54 mmol), and DMAP (2.2 g, 18 mmol) at O0C, and then bromomethyl-dimethylchlorosilane (5.05 g, 27 mmol) (added dropwise). After stirring the mixture for 30 min at O0C, it was quenched with aq NaHCO3 and the resulting mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with water, brine and dried over Na2SO4. The solvent was removed in vacuo to give a mixture of the silated derivatives of 9 and 10 (9.83 g, 95%). The diastereomers were purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:5 to 1:4) to give pure diastereomer 11 (7.4 g, 72%) and its diastereomer (2.4 g, 22%).

Silyl Ether (11).

Rf= 0.80 (30% ethyl acetate in hexanes). [α]23D -58.9 (c 0.55, CHCl3); FTIR (film) vmax: 3050, 2995, 1738, 1697, 1512, 1405, 1243, 1108, 1003, 809, 732 cm"1; 1H NMR (CDCl3, 500 MHz): δ 7.27 (5H, m), 7.05 (2H, d, J = 7.0 Hz), 6.71 (2H, d, / = 8.5 Hz), 6.18 (1 H, s), 5.53 (1 H,s ), 4.95 (1 H, d, J = 15.5 Hz), 4.45 (1 H, d, J = 15.0 Hz ), 4.02 (1 H, J = 12.0 Hz), 3.86 (1 H, d, / = 11.5 Hz) 3.72 (3H, s), 3.68 (3H, s), 3. 65 (1 H, d, / = 10.5 Hz), 3.30 (1 H, d, J = 10.0 Hz), 2.34 (2H, d, / = 2.0 Hz), 1.58 (3H, s), 0.19 (3 H, s), 0.18 (3 H, s); 13C NMR (CDCl3, 125 MHz): δ 168.62, 168.12, 158.93, 145.24, 137.53, 130.32, 130.30, 128.49, 127.76,127.22, 117.26, 113.60, 78.55, 78.03, 72.89, 68.45, 55.43, 52.37, 45.74, 21.87, 17.32, -0.72, -0.80; HRMS (ESI) Calcd. for C27H35BrNO6Si (M + H)+ 576.1417, found 576.1407.

Example 8:

Conversion of (11) to (12).

To a solution of compound 11 (5.67 g 10 mmol) in benzene (250 mL) at 8O0C under nitrogen was added a mixture of tributyltin hydride (4.03 ml, 15 mmol) and AEBN (164 mg, 1 mmol) in 50 ml benzene by syringe pump over 4 h. After the addition was complete, the reaction mixture was stirred for an additional 4 h at 8O0C and the solvent was removed in vacuo. The residue was dissolved in hexanes (20 mL) and washed with saturated NaHCO3 (3 x 25 mL), water and dried over Na2SO4. The solvent was removed in vacuo to give crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1 : 5) to afford the pure 12 (4.42 g, 89%).

Rf= 0.80 (30% ethyl acetate in hexanes). [α]23D -38.8 (c 0.25, CHCl3); FTIR (firm) vmax: 3025, 2985, 1756, 1692, 1513, 1247, 1177, 1059, 667 cm"1; 1H NMR (CDCl3, 500 MHz): δ 7.28 (5H, m), 7.09 (2H, d, / = 7.0 Hz), 6.73 (2H, d, / = 9.0 Hz), 4.96(lH,d, /=15.0Hz), 4.35 (lH,d, /=15.5Hz), 3.97 (IH, d, J=12.5Hz), 3.86 (IH, d, J = 12.0 Hz), 3.80 (1 H, d, /= 10.0 Hz), 3.72 (3H, s), 3.65 (3H, s), 3.27 (1 H, d, / = 10.5 Hz), 2.67 (1 H, t, J = 4.0 Hz), 2.41 (1 H, m), 1.79 (1 H, m), 1.46 (3H, s), 0.77 (1 H, m), 0.46 (IH, m), 0.10 (3H, s), 0.19 (3H, s); 13C NMR (CDCl3, 125 MHz): δ 175.48, 169.46, 158.76, 137.59, 131.04, 129.90, 128.58, 127.88,127.52, 113.59, 113.60, 81.05, 78.88, 73.12, 69.03, 55.45, 51.94, 48.81, 45.50, 22.79, 17.06, 7.76, 0.54; HRMS (ESI) calcd. for (M + H)+C27H36NO6Si 498.2312, found 498.2309.

Example 9:

Debenzylation of (12).

A solution of 12 (3.98 g, 8 mmol) in EtOH (50 ml) at 230C was treated with 10% Pd-C (~1 g) under an argon atmosphere. The reaction mixture was evacuated and flushed with H2 gas (four times) and then stirred vigorously under an atmosphere of H2 (1 atm, H2 balloon) at 230C. After 12 h, the reaction mixture was filtered through Celite and concentrated in vacuo to give the crude debenzylation product (3.08 g, 95%) which was used for the next step. A small amount crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1: 3) for analytical purposes. Rf= 0.41 (50% ethyl acetate in hexanes).

mp, 45 - 47°C; [α]23D-30.9 (c 0.55, CHCl3); FTIR (film) vmax: 3432, 3020, 2926, 1735, 1692, 1512, 1244, 1174, 1094, 1024, 870, 795 cm"1; 1H NMR (CDCl3, 400 MHz): δ 7.36 (2H, d, /= 8.5 Hz), 6.83 (2H, d, J = 8.5 Hz), 5.16 (1 H, d, /= 15.0 Hz), 4.29 (1 H, d, / = 15.0 Hz ), 3.92 (1 H, m), 3.78 (3H, s), 3.68 (3H, s), 3.45 (1 H, m), 2.53 (1 H, t, J = 4.0 Hz), 2.42 (1 H, m), 1.82 (1 H, m), 1.50 (3H, s), 1.28 (1 H, m), 0.75 (1 H, m), 0.47 (1 H, m), 0.11 (3H, s), 0.02 (3H, s) ; 13C NMR (CDCl3, 125 MHz): δ 175.82, 169.51, 159.32, 131.00, 129.72, 114.52, 80.79, 80.13, 61.85, 55.48, 51.99, 49.29, 45.06, 23.11, 17.03, 7.44, 0.54; HRMS (ESI) calcd. for C20H30NO6Si (M + H)+ 408.1842, found 408.1846.

Example 10:

Oxidation to Form Aldehyde (13).

To a solution of the above alcohol from debenzylation of 12 (2.84 g, 7 mmol) in CH2Cl2 (30 mL) was added Dess-Martin reagent (3.57 g, 8.4 mmol) at 23°C. After stirring for 1 h at 230C, the reaction mixture was quenched with aq NaHCO3-Na2S2O3 (1:1, 50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic phase was dried and concentrated in vacuo to afford the crude aldehyde. The crude product was purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:5) to give pure aldehyde 13 (2.68 g, 95%). Rf = 0.56 (50% ethyl acetate in hexanes).

mp, 54-560C; [α]23D -16.5 (c 0.60, CHCl3); FTIR (film) vmax: 3015, 2925, 1724, 1702, 1297, 1247, 1170, 1096, 987, 794 cm"1; 1H NMR (CDCl3, 500 MHz): δ 9.62 (1 H, s), 7.07 (2H, d, / = 8.0 Hz), 6.73 (2H, d, / = 8.5 Hz), 4.49 (1 H, quart, 7= 8.5 Hz), 3.70 (3H, s), 3.67 (3H, s), 2.36 (2H, m), 1.75 (IH, m), 1.37 (3H, s), 0.73 (1 H, m), 0.48 (1 H, m), 0.07 (3H, s), 0.004 (3H, s); 13C NMR (CDCl3, 125 MHz): δ 197.26, 174.70, 167.36, 158.07, 130.49, 128.96, 113.81, 83.97, 82.36, 55.34, 52.43, 47.74, 46.32, 23.83, 16.90, 7.52, 0.56, 0.45; HRMS (ESI) calcd. for C20H28NO6Si (M + H)+ 406.1686, found 406.1692.

Example 11:

13 14

Conversion of (13) to (14).

To a solution of freshly prepared cyclohexenyl zinc chloride (10 niL, 0.5 M solution in THF, 5 mmol) (see Example 15 below) at -78°C under nitrogen was added a -78°C solution of aldehyde 13 (1.01 g, in 3 ml of THF, 2.5 mmol). After stirring for 5 h at -780C reaction mixture was quenched with water (10 mL) then extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4 and solvent was removed in vacuo to give crude product (20 : 1 dr). The diastereomers were purified by column chromatography (silica gel, ethyl acetate/hexanes, 1:10 to 1:2 affords the pure major diastereomer 14 (1.0 g, 83%) and a minor diastereomer (50 mg 5%). For 14: Rf= 0.56 (50% ethyl acetate in hexanes).

mp, 79-810C; [α]23D -28.5 (c 1.45, CHCl3); FTIR (film) vmax: 3267, 2927, 2894, 2829, 1742, 1667, 1509, 1248, 1164, 1024, 795 cm"1; 1H NMR (CDCl3, 500 MHz): δ 7.34 (2H, d, J = 8.5 Hz), 6.81 (2H, d, / = 9.0 Hz), 5.84 (1 H, m), 5.73 (1 H, m ), 4.88 (1 H, ■ d, J = 15.5 Hz), 4.39 (1 H, d, J = 14.5 Hz), 4.11 (1 H, t, / = 6.5 Hz), 3.77 (3H, s), 3.58 (3H, s), 3.00 (1 H, m), 2.95 (1 H, d, 7 = 9.0 Hz), 2.83 (1 H, t, / = 3.5 Hz), 3.36 (1 H, m), 2.27 (IH, m), 1.98 (2H, m), 1.74 (3H, m), 1.62 (3H, s), 1.14 (2H, m), 0.59 (IH, m), 0.39 (HH, m), 0.13 (3H, s), 0.03 (3H, s) ; 13C NMR (CDCl3, 125 MHz): δ 176.80, 170.03, 158.27, 131.86, 131.34, 128.50, 126.15, 113.40, 83.96, 82.45, 77.17, 55.45, 51.46, 48.34, 48.29, 39.08, 28.34, 25.29, 22.45, 21.09, 17.30, 7.75, 0.39, 0.28; HRMS (ESI) calcd. for C26H38NO6Si (M + H)+ 488.2468, found 488.2477.

Example 12:

Tainao-Fleming Oxidation of (14) to (15).

To a solution of 14 (0.974 g, 2 mmol) in THF (5 mL) and MeOH (5 mL) at 230C was added KHCO3 (0.8 g, 8 mmol) and KF (0.348 g, 6 mmol). Hydrogen peroxide (30% in water, 5 mL) was then introduced to this mixture. The reaction mixture was vigorously stirred at 23°C and additional hydrogen peroxide (2 ml) was added after 12 h. After 18h, the reaction mixture was quenched carefully with NaHSO3 solution (15 mL). The mixture was extracted with ethyl acetate (3 x 25 mL) and the combined organic layers were washed with water and dried over Na2SO4. The solvent was removed in vacuo to give the crude product. The crude product was purified by column chromatography (silica gel, ethyl acetate) to give the pure triol 15 (0.82 g, 92%).

Rf= 0.15 (in ethyl acetate), mp, 83-840C; [α]23D: +5.2 (c 0.60, CHCl3); FTIR (film) vmax: 3317, 2920, 2827, 1741, 1654, 1502, 1246, 1170, 1018, 802 cm-1; 1H NMR (CDCl3, 500 MHz): δ 7.77 (2H, d, / = 8.0 Hz), 6.28 (2H, ά, J= 8.0 Hz), 5. 76 (1 H, m), 5.63 (IH, d, J = 10.0 Hz), 4.74 (IH, d, J= 15.5 Hz), 4.54 (IH, d, /= 15.0 Hz), 4.12 (IH, d, , / = 2.5 Hz), 3.80 (1 H, m), 3.76 (3H, s), 3.72 (1 H, m), 3.68 (3H, s), 3.00 (1 H, m), 2.60 (1 H, br), 2.20 (1 H, m), 1.98 (2H, s), 1.87 (1 H, m), 1.80 (1 H, m), 1.71 (2H, m), 1.61 (3 H, s), 1.14 (2 H, m); 13C NMR (CDCl3, 125 MHz): δ 178.99, 170.12, 158.27, 131.30, 130.55, 128.13, 126.39, 113.74, 81.93, 80.75, 76.87, 61.61, 55.45, 51.97, 51.32, 48.07, 39.17, 27.71, 27.13, 25.22, 21.35, 21.22; HRMS (ESI) calcd. for C24H34NO7 (M + H)+ 448.2335, found 448.2334.

Example 13:

Deprotection of (15) to (16).

To a solution of 15 (0.670 g, 1.5 mmol) in acetonitrile (8 mL) at 00C was added a pre-cooled solution of eerie ammonium nitrate (CAN) (2.46 g 4.5 mmol in 2 mL H2O). After stirring for 1 h at 00C the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated NaCl solution (5 mL) and organic layers was dried over Na2SO4. The solvent was removed in vacuo to give the crude product which was purified by column chromatography (silica gel, ethyl acetate) to give the pure 16 (0.4 g, 83%).

Rf= 0.10 (5% MeOH in ethyl acetate), mp, 138 to 1400C; [α]23D +14.5 (c 1.05, CHCl3); FTlR (film) vmax 3301, 2949, 2911, 2850, 1723, 1673, 1437, 1371, 1239, 1156, 1008, 689 cm"1; 1H NMR (CDCl3, 600 MHz): δ 8.48 (IH, br), 6.08 (IH, m), 5. 75 (1 H, d, / = 9.6 Hz), 5.29 (1 H, br), 4.13 (1 H, d, /= 6.6 Hz), 3.83 (3H, m), 3.79 (1 H, m), 3.72 (1 H, m), 2.84 (1 H, d, /= 10.2 Hz), 2.20 (1 H, m), 2.16 (1 H, br), 1.98 (3H, m), 1.77 (3H, m), 1.59 (1 H, m), 1.54 (3H, s), 1.25 (1 H, m). 13C NMR (CDCl3, 125 MHz): δ 180.84, 172.95, 135.27, 123.75, 82.00, 80.11, 75.56, 62.39, 53.14, 51.78, 38.95, 28.79, 26.48, 25.04, 20.66, 19.99; HRMS (ESI) calcd. (M + H)+ for C16H26NO6 328.1760, found 328.1752.

Example 14:

Conversion of (16) to Salinosporamide A (1).

A solution of triol ester 16 (0.164 g, 0.5 mmol) in 3 N aq LiOH (3 mL) and THF (1 mL) was stirred at 50C for 4 days until hydrolysis was complete. The acid reaction mixture was acidified with phosphoric acid (to pH 3.5). The solvent was removed in vacuo and the residue was extracted with EtOAc, separated, and concentrated in vacuo to give the crude trihydroxy carboxylic acid 16a (not shown). The crude acid was suspended in dry CH2Cl2 (2 mL), treated with pyridine (0.5 mL) and stirred vigorously at 230C for 5 min. To this solution was added BOPCl (152 mg, 0.6 mmol) at 230C under argon, and stirring was continued for Ih. The solvent was removed under high vacuum and the residue was suspended in dry CH3CN (1 mL) and treated with pyridine (1 mL). To this solution was added PPh3Cl2 (333 mg, 1.0 mmol) at 230C under argon with stirring. After 1 h the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel, ethyl acetate- CH2Cl2, 1 : 5) to give the pure β -lactone 1 (100 mg, 64%) as a colorless solid.

Rf = 0.55 (50% ethyl acetate in hexane). mp, 168-17O0C (authentic sample: 168- 1700C, 169-1710C in Angew. Chem. Int. Ed., 2003, 42, 355-357); mixture mp, 168- 170°C. [α ir23 D -73.2 (c 0.49, MeOH) , -72.9 (c 0.55, MeOH, in Angew. Chem. Int. Ed, 2003, 42, 355-357); FTIR (film) vmax: 3406, 2955, 2920, 2844, 1823, 1701, 1257, 1076, 1012, 785, 691 cm"1; 1H NMR (CDCl3, 500 MHz): δ 10.62 (1 H, br), 6.42 (1 H, d, J= 10.5 Hz), 5. 88 (1 H, m), 4.25 (1 H, d, /= 9.0 Hz), 4.14 (1 H, m), 4.01 (1 H, m), 3.17 (1 H, t, J = 7.0 Hz), 2.85 (1 H, m), 2.48 (1 H, m), 2.32 (2H, m), 2.07 (3H, s), 1.91 (2H, m), 1.66 (2H, m), 1.38 (1 H, m); 13C NMR (CDCl3, 125 MHz): δ 176.92, 169.43, 129.08, 128.69, 86.32, 80.35, 70.98, 46.18, 43.28, 39.31, 29.01, 26.47, 25.35, 21.73, 20.00; HRMS (ESI) calcd. for (M - H)" C15Hi9ClNO4 312.1003, found 312.1003.

Part 2. Synthesis of the 2-Cyclohexenylzinc chloride

Example 15:

SnEJy3 ZnCt 1, «EaU,THF,-78βC, 3OmIiI

Synthesis of the Cyclohexenyl Zinc Chloride.

To a solution of cyclohexenyltributyl tin (1.85 g 5 mmol) in THF (5ml) at - 78°C under nitrogen was added /iBuLi (2 ml, 2.5M solution in hexaαe, 5 πimol). See Miyake, H., Yamamura, K., Chan. Lett., 1992, 507-508. After an additional 30 min stirring, ZnCl2 (5 ml, 1 M solution in THF, 5 mmol) was added and stirring was continued at this temperature for 30 min at -780C to give a 0.5M solution of 2- cyclohexenyl zinc chloride for reaction with the aldehyde 13.

The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled iu the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention and still be within the scope of this invention as set forth in the following claims.