MARIZOMIB, Salinosporamide A

MARIZOMIB
http://www.ama-assn.org/resources/doc/usan/marizomib.pdf
THERAPEUTIC CLAIM Antineoplastic
CHEMICAL NAMES
1. 6-Oxa-2-azabicyclo[3.2.0]heptane-3,7-dione, 4-(2-chloroethyl)-1-[(S)-(1S)-2-
cyclohexen-1-ylhydroxymethyl]-5-methyl-, (1R,4R,5S)-
2. (1R,4R,5S)-4-(2-chloroethyl)-1-{(S)-[(1S)-cyclohex-2-en-1-yl]hydroxymethyl}-5-methyl-
6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione

MOLECULAR FORMULA C15H20ClNO4
MOLECULAR WEIGHT 313.8

MANUFACTURER Nereus Pharmaceuticals, Inc.

NOTE….Nereus Pharmaceuticals was acquired by Triphase Research and Development in 2012.
CODE DESIGNATION NPI-0052
CAS REGISTRY NUMBER 437742-34-2

Scripps Institution of Oceanography (Originator)

US7183417

mp, 168–170° C. (authentic sample: 168–170° C., 169–171° C. in Angew. Chem. Int. Ed., 2003, 42, 355–357); mixture mp, 168–170C.

[α]²³ _D −73.2 (c 0.49, MeOH), −72.9 (c 0.55, MeOH, in Angew. Chem. Int. Ed., 2003, 42, 355–357);

FTIR (film) ν_max: 3406, 2955, 2920, 2844, 1823, 1701, 1257, 1076, 1012, 785, 691 cm⁻¹;

¹H NMR (CDCl₃, 500 MHz): δ 10.62 (1H, br), 6.42 (1H, d, J=10.5 Hz), 5.88 (1H, m), 4.25 (1H, d, J=9.0 Hz), 4.14 (1H, m), 4.01 (1H, m), 3.17 (1H, t, J=7.0 Hz), 2.85 (1H, m), 2.48 (1H, m), 2.32 (2H, m), 2.07 (3H, s), 1.91 (2H, m), 1.66 (2H, m), 1.38 (1H, 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)⁻ C₁₅H₁₉ClNO₄ 312.1003, found 312.1003.

Marizomib, a highly potent proteasome inhibitor, is in early clinical development at Triphase Research and Development I Corp for the treatment of relapsed or relapsed/refractory multiple myeloma. Phase I clinical trials have also been carried out for the treatment of solid tumors and lymphoma; however, no recent developments have been reported for these studies.

HDAC inhibitors halt tumor cell differentiation and growth, and when combined with marizomib in preclinical in vitro and in vivo studies, show additive and synergistic antitumor activities.

The compound was discovered from a new marine-obligate gram-positive actinomycete (Salinispora tropica). Preclinical studies suggest that this next-generation compound may be superior to other proteasome inhibitors, with broader target inhibition, faster onset and longer duration of action, higher potency, and oral and intravenous availability. By inhibiting proteasomes, marizomib prevents the breakdown of proteins involved in signal transduction, which blocks growth and induces apoptosis in cancer cells.

In 2013, orphan drug designation was assigned in the U.S. for the treatment of multiple myeloma.

The compound was originally developed by Nereus Pharmaceuticals, which was acquired by Triphase Research and Development in 2012.

marizomib is a naturally-occurring salinosporamide, isolated from the marine actinomycete Salinospora tropica, with potential antineoplastic activity. Marizomib irreversibly binds to and inhibits the 20S catalytic core subunit of the proteasome by covalently modifying its active site threonine residues; inhibition of ubiquitin-proteasome mediated proteolysis results in an accumulation of poly-ubiquitinated proteins, which may result in the disruption of cellular processes, cell cycle arrest, the induction of apoptosis, and the inhibition of tumor growth and angiogenesis. This agent more may more potent and selective than the proteasome inhibitor bortezomib

Marizomib (NPI-0052) is an oral, irreversible ββ-lactone derivative that binds selectively to the active proteasomal sites. In vivo studies with marizomib demonstrate reduced tumor growth without significant toxicity in myeloma xenograft models. A phase I trial in refractory and relapsed MM is under way.

Salinosporamide A is a potent proteasome inhibitor used as an anticancer agent that recently entered phase I human clinical trials for the treatment of multiple myeloma only three years after its discovery.^[1][2] This novel marine natural product is produced by the recently described obligate marine bacteria Salinispora tropica and Salinispora arenicola, which are found in ocean sediment. Salinosporamide A belongs to a family of compounds, known collectively as salinosporamides, which possess a densely functionalized γ-lactam-β-lactone bicyclic core.

Salinosporamide A was discovered by William Fenical and Paul Jensen from Scripps Institution of Oceanography in La Jolla, CA. In preliminary screening, a high percentage of the organic extracts of cultured Salinospora strains possessed antibiotic and anticancer activities, which suggests that these bacteria are an excellent resource for drug discovery.Salinospora strain CNB-392 was isolated from a heat-treated marine sediment sample and cytotoxicity-guided fractionation of the crude extract led to the isolation of salinosporamide A. Although salinosporamide A shares an identical bicyclic ring structure with omuralide, it is uniquely functionalized. Salinosporamide A displayed potent in vitro cytotoxicity against HCT-116 human colon carcinoma with an IC50 value of 11 ng mL-1. This compound also displayed potent and highly selective activity in the NCI’s 60-cell-line panel with a mean GI50 value (the concentration required to achieve 50% growth inhibition) of less than 10 nM and a greater than 4 log LC50 differential between resistant and susceptible cell lines. The greatest potency was observed against NCI-H226 non-small cell lung cancer, SF-539 CNS cancer, SK-MEL-28 melanoma, and MDA-MB-435 breast cancer (all with LC50 values less than 10 nM). Salinosporamide A was tested for its effects on proteasome function because of its structural relationship to omuralide. When tested against purified 20S proteasome, salinosporamide A inhibited proteasomal chymotrypsin-like proteolytic activity with an IC50 value of 1.3 nM.^[3] This compound is approximately 35 times more potent than omuralide which was tested as a positive control in the same assay. Thus, the unique functionalization of the core bicyclic ring structure of salinosporamide A appears to have resulted in a molecule that is a significantly more potent proteasome inhibitor than omuralide.^[1]

Salinosporamide A inhibits proteasome activity by covalently modifying the active site threonine residues of the 20S proteasome.

Biosynthesis

Salinosporamide A and B building blocks

Proposed biosynthesis of the nonproteinogenic amino-acid beta-hydroxycyclohex-2′-enylanine (3) (R = H or S~PCP) via a shunt in the phenylalanine biosynthetic pathway

Biosynthesis

It was originally hypothesized that salinosporamide B was a biosynthetic precursor to salinosporamide A due to their structural similarities.

It was thought that the halogenation of the unactivated methyl group was catalyzed by a non-heme iron halogenase.^[4][5]Recent work using ¹³C-labeled feeding experiments reveal distinct biosynthetic origins of salinosporamide A and B.^[4][6]

While they share the biosynthetic precursors acetate and presumed β-hydroxycyclohex-2′-enylalanine (3), they differ in the origin of the four-carbon building block that gives rise to their structural differences involving the halogen atom. A hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway is most likely the biosynthetic mechanism in which acetyl-CoA and butyrate-derived ethylmalonyl-CoA condense to yield the β-ketothioester (4), which then reacts with (3) to generate the linear precursor (5).

The first stereoselective synthesis was reported by Rajender Reddy Leleti and E. J.Corey.^[7] Later several routes to the total synthesis of salinosporamide A have been reported.^{[7][8][9][10]}

In vitro studies using purified 20S proteasomes showed that salinosporamide A has lower EC50 for trypsin-like (T-L) activity than does Bortezomib. In vivo animal model studies show marked inhibition of T-L activity in response to salinosporamide A, whereas bortezomib enhances T-L proteasome activity.

Initial results from early-stage clinical trials of salinosporamide A in relapsed/refractory multiple myeloma patients were presented at the 2011 American Society of Hematology annual meeting.^[11] Further early-stage trials of the drug in a number of different cancers are ongoing.^[12]

1.  Feling RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR, Fenical W (2003). “Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora”. Angew. Chem. Int. Ed. Engl. 42 (3): 355–7.doi:10.1002/anie.200390115. PMID 12548698.
2.  Chauhan D, Catley L, Li G et al. (2005). “A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib”. Cancer Cell 8 (5): 407–19.doi:10.1016/j.ccr.2005.10.013. PMID 16286248.
3.  K. Lloyd, S. Glaser, B. Miller, Nereus Pharmaceuticals Inc.
4.  Beer LL, Moore BS (2007). “Biosynthetic convergence of salinosporamides A and B in the marine actinomycete Salinispora tropica”. Org. Lett. 9 (5): 845–8.doi:10.1021/ol063102o. PMID 17274624.
5.  Vaillancourt FH, Yeh E, Vosburg DA, Garneau-Tsodikova S, Walsh CT (2006). “Nature’s inventory of halogenation catalysts: oxidative strategies predominate”. Chem. Rev.106 (8): 3364–78. doi:10.1021/cr050313i.PMID 16895332.
6.  Tsueng G, McArthur KA, Potts BC, Lam KS (2007). “Unique butyric acid incorporation patterns for salinosporamides A and B reveal distinct biosynthetic origins”. Applied Microbiology and Biotechnology 75 (5): 999–1005. doi:10.1007/s00253-007-0899-7.PMID 17340108.
7.  Reddy LR, Saravanan P, Corey EJ (2004). “A simple stereocontrolled synthesis of salinosporamide A”. J. Am. Chem. Soc. 126 (20): 6230–1. doi:10.1021/ja048613p.PMID 15149210.
8.  Ling T, Macherla VR, Manam RR, McArthur KA, Potts BC (2007). “Enantioselective Total Synthesis of (-)-Salinosporamide A (NPI-0052)”.Org. Lett. 9 (12): 2289–92. doi:10.1021/ol0706051. PMID 17497868.
9.  Ma G, Nguyen H, Romo D (2007). “Concise Total Synthesis of (±)-Salinosporamide A, (±)-Cinnabaramide A, and Derivatives via a Bis-Cyclization Process: Implications for a Biosynthetic Pathway?”. Org. Lett. 9 (11): 2143–6. doi:10.1021/ol070616u. PMC 2518687.PMID 17477539.
10.  Endo A, Danishefsky SJ (2005). “Total synthesis of salinosporamide A”. J. Am. Chem. Soc. 127 (23): 8298–9.doi:10.1021/ja0522783. PMID 15941259.
11.  “Marizomib May Be Effective In Relapsed/Refractory Multiple Myeloma (ASH 2011)”. The Myeloma Beacon. 2012-01-23. Retrieved 2012-06-10.
12.  ClinicalTrials.gov: Marizomib

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IMPORTANT PAPERS

Total synthesis of salinosporamide A
Org Lett 2008, 10(19): 4239

Entry to heterocycles based on indium-catalyzed conia-ene reactions: Asymmetric synthesis of (-)-salinosporamide A
Angew Chem Int Ed 2008, 47(33): 6244

A concise and straightforward total synthesis of (+/-)-salinosporamide A, based on a biosynthesis model
Org Biomol Chem 2008, 6(15): 2782

Formal synthesis of salinosporamide A starting from D-glucose
Synthesis (Stuttgart) 2009, 2009(17): 2983

Stereoselective functionalization of pyrrolidinone moiety towards the synthesis of salinosporamide A
Tetrahedron 2012, 68(32): 6504

………………

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, T. J.; 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, E. J.; Li, W. D., Z. Chem. Pharm. Bull., 1999, 47, 1–10; (b) Corey, E. J., Reichard, G. A.; Kania, R., Tetrahedron Lett., 1993, 34, 6977–6980; (c) Corey, E. J.; Reichard, G. A., J. 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. Sci. 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., J. 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 (IC₅₀values 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 Salinosporain 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

PATENTS

WO 2005113558

http://www.google.com/patents/US7183417

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.H₂O (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 (3×200 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.

R_f=0.51 (50% ethyl acetate in hexanes), mp, 86–87° C.; [α]²³ _D+69.4 (c 2.0, CHCl₃); FTIR (film) ν_max: 2955, 1750, 1545, 1355, 1187, 1011, 810 cm⁻¹; ¹HNMR(CDCl₃, 400 MHz): δ 7.87 (2H, d, J=9.2 Hz), 6.84 (2H, d, J=8.8 Hz), 4.90 (1H, m), 4.40 (1H, d, J=7.6 Hz), 3.79 (3H,s), 3.71 (3H, s), 1.49 (3H, d, J=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 −78° C. 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 23° C. for 30 min. Then the mixture was extracted with ethyl acetate (3×50 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%).

R_f=0.59 (50% ethyl acetate in hexanes). [α]²³ _D−6.3 (c 1.0, CHCl₃); FTIR (film) (ν_max; 3050, 2975, 1724, 1642, 1607, 1252, 1027, 745, 697 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz): δ 7.96 (2H, d, J=9.2 Hz), 7.26 (5H, m), 6.90 (2H, J=8.8 Hz), 4.80 (1H, m), 4.61 (2H, s), 3.87 (3H, m), 3.81 (3H, s), 3.73 (3H, s), 1.34 (3H, d, J=6.8 Hz); ¹³C NMR (CDCl₃, 100 MHZ): 6171.23, 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 C₂₁H₂₄O₅ (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 23° C. was added in portions NaCNBH₃ (9.3 g, 150 mmol). The reaction mixture was then stirred at 40° C. 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 (3×100 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%).

R_f=0.50 (50% ethyl acetate in hexanes). [α]²³ _D−9.1(c 1.0, CHCl₃); FTIR (film) ν_max; 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 (1H, q, J=6.4 Hz), 3.85 (2H, dd, J=17.2, 10.0 Hz), 3.81 (3H, s,), 3.77 (3H, s), 3. 69 (2H, dd, J=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 C₂₁H₂₈NO₅ (M+H)⁺ 374.1967, found 374.1974.

EXAMPLE 4

(2R,3R)-Methyl-2-(N-(4-methoxybenzyl)acrylamido)-2-(benzyloxy)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 23° C. 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 0° C. Acryloyl chloride (6.64 mL, 82.2 mmol) was added dropwise with vigorous stirring and the reaction temperature was maintained at 0° C. 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 23° C. 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.

R_f=0.40 (50% ethyl acetate in hexanes), [α]²³ _D−31.1 (c 0.45, CHCl₃), FTIR (film) ν_max; 3435, 2990, 1725, 1649, 1610, 1512, 1415, 1287, 1242, 1175, 1087, 1029, 732, 698 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.25 (5H, m), 7.15 (2H, d, J=6.0 Hz), 6.85 (2H, d, J=7.5 Hz), 6.38 (2H, d, J=6.0 Hz), 5.55 (1H, t, J=6.0 Hz), 4.81 (2H, s), 4.71 (1H, q, J=6.5 Hz), 4.35 (2H, s), 4.00 (1H, d, J=10.0 Hz), 3.80 (1H, d, J=10.0 Hz), 3.76 (3H, s), 3.75 (3H, s), 3.28 (1H, bs), 1.22 (3H, d, J=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

(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 (3×50 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%).

R_f=0.80 (50% ethyl acetate in hexanes), mp 85 to 86° C.; [α]²³ _D−12.8 (c 1.45, CHCl₃); FTIR (film) ν_max: 3030, 2995, 1733, 1717, 1510, 1256, 1178, 1088, 1027, 733, 697 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.30 (2H, d, J=8.0), 7.25 (3H, m), 7.11 (2H, m), 6.88 (2H, d, J=9.0 Hz), 6.38 (2H, m), 5.63 (1H, dd, J=8.5, 3.5 Hz), 4.93 (1H, d, J=18.5 Hz), 4.78 (1H, d, J=18.5, Hz), 4.27 (2H, m), 3.78 (3H, s), 3.76 (3H, s), 2.42 (3H, 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

(2R,3S)-Methyl-1-(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 mL) was stirred for 5 h at 23° C. After completion, the reaction mixture was diluted with ethyl acetate (50 mL) 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]²³ _D−37.8 (c 0.51, CHCl₃); FTIR (film) v_max: 3450, 3055, 2990, 1733, 1683, 1507, 1107, 1028, 808,734 cm⁻¹; 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 (1H, s), 5.57 (1H, s), 4.81 (1H, d, J=14.5 Hz), 4.45(1H, d, J=15.0 Hz), 4.20 (1H, d, J=12.0 Hz), 4.10 (1H, d, J=12.0 Hz) 3.75 (3H, s), 3.70 (1H, d, J=10.5 Hz), 3.64 (3H, s), 3.54 (1H, d, J=10.5 Hz), 2.55 (1H, 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]²³ _D−.50.1 (c 0.40, CHCl₃); FTIR (film) ν_max: 3450, 3055, 2990, 1733, 1683, 1507, 1107, 1028, 808, 734 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.29 (5H, m), 7.12 (2H, d, J=7.5 Hz), 6.73 (2H, d, J=8.5 Hz), 6.12 (1H, s), 5.57 (1H, s), 4.88 (1H, d, J=15.5 Hz), 4.31 (1H, d, J=15.0 Hz), 4.08 (3H, m), 3.99 (1H, d, J=12.0 Hz) 3.73 (3H, s), 3.62 (3H, s), 3.47 (1H, bs, OH), 3.43 (1H, 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 0° C., and then bromomethyl-dimethylchlorosilane (5.05 g, 27 mmol) (added dropwise). After stirring the mixture for 30 min at 0° C., it was quenched with aq NaHCO₃ and the resulting mixture was extracted with ethyl acetate (3×50 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).

R_f=0.80 (30% ethyl acetate in hexanes). [α]²³ _D−58.9 (c 0.55, CHCl₃); FTIR (film) ν_max; 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, J=8.5 Hz), 6.18 (1H, s), 5.53 (1H, s), 4.95 (1H, d, J=15.5 Hz), 4.45 (1H, d, J=15.0 Hz), 4.02 (1H, J=12.0 Hz), 3.86 (1H, d, J=11.5 Hz) 3.72 (3H, s), 3.68 (3H, s), 3.65 (1H, d, J=10.5 Hz), 3.30 (1H, d, J=10.0 Hz), 2.34 (2H, d, J=2.0 Hz), 1.58 (3H, s), 0.19 (3H, s), 0.18 (3H, 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 80° C. under nitrogen was added a mixture of tributyltin hydride (4.03 ml, 15 mmol) and AIBN (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 80° C. and the solvent was removed in vacuo. The residue was dissolved in hexanes (20 mL) and washed with saturated NaHCO₃ (3×25 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%).

R_f=0.80 (30% ethyl acetate in hexanes). [α]²³ _D−38.8 (c 0.25, CHCl₃); FTIR (film) ν_max; 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, J=7.0 Hz), 6.73 (2H, d, J=9.0 Hz), 4.96(1H, d, J=15.0 Hz), 4.35 (1H, d, J=15.5 Hz), 3.97 (1H, d, J=12.5 Hz), 3.86 (1H, d, J=12.0 Hz), 3.80 (1H, d, J=10.0 Hz), 3.72 (3H, s), 3.65 (3H, s), 3.27 (1H, d, J=10.5 Hz), 2.67 (1H, t, J=4.0 Hz), 2.41 (1H, m), 1.79 (1H, m), 1.46 (3H, s), 0.77 (1H, m), 0.46 (1H, 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 23° C. 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 23° C. 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. R_f=0.41 (50% ethyl acetate in hexanes).

mp, 45–47° C.; [α]²³ _D−30.9 (c 0.55, CHCl₃); FTIR (film) ν_max: 3432, 3020, 2926, 1735, 1692, 1512, 1244, 1174, 1094, 1024, 870, 795 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz): δ 7.36 (2H, d, J=8.5 Hz), 6.83 (2H, d, J=8.5 Hz), 5.16 (1H, d, J=15.0 Hz), 4.29 (1H, d, J=15.0 Hz), 3.92 (1H, m), 3.78 (3H, s), 3.68 (3H, s), 3.45 (1H, m), 2.53 (1H, t, J=4.0 Hz), 2.42 (1H, m), 1.82 (1H, m), 1.50 (3H, s), 1.28 (1H, m), 0.75 (1H, m), 0.47 (1H, 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 23° C., the reaction mixture was quenched with aq NaHCO₃—Na₂S₂O₃ (1:1, 50 mL) and extracted with ethyl acetate (3×50 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%). R_f=0.56 (50% ethyl acetate in hexanes).

mp, 54–56° C.; [α]²³ _D−16.5 (c 0.60, CHCl₃); FTIR (film) ν_max: 3015, 2925, 1702 1297, 1247, 1170, 1096, 987, 794 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 9.62 (1H, s), 7.07 (2H, d, J=8.0 Hz), 6.73 (2H, d, J=8.5 Hz), 4.49 (1H, quart, J=8.5 Hz), 3.70 (3H, s), 3.67 (3H, s), 2.36 (2H, m), 1.75 (1H, m), 1.37 (3H, s), 0.73 (1H, m), 0.48 (1H, 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 (ESD calcd. for C₂₀H₂₈NO₆Si (M+H)⁺ 406.1686, found 406.1692.

EXAMPLE 11

Conversion of (13) to (14).

To a solution of freshly prepared cyclohexenyl zinc chloride (10 mL, 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 −78° C. reaction mixture was quenched with water (10 mL) then extracted with ethyl acetate (3×10 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: R_f=0.56 (50% ethyl acetate in hexanes).

mp, 79–81° C.; [a]²³ _D−28.5 (c 1.45, CHCl₃); FTIR (film) ν_max: 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, J=9.0 Hz), 5.84 (1H, m), 5.73 (1H, m), 4.88 (1H, d, J=15.5 Hz), 4.39 (1H, d, J=14.5 Hz), 4.11 (1H, t, J=6.5 Hz), 3.77 (3H, s), 3.58 (3H, s), 3.00 (1H, m), 2.95 (1H, d, J=9.0 Hz), 2.83 (1H, t, J=3.5 Hz), 3.36 (1H, m), 2.27 (1H, m), 1.98 (2H, m), 1.74 (3H, m), 1.62 (3H, s), 1.14 (2H, m), 0.59 (1H, m), 0.39 (11H, 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

Tamao-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 23° C. 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 18 h, the reaction mixture was quenched carefully with NaHSO₃ solution (15 mL). The mixture was extracted with ethyl acetate (3×25 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%).

R_f=0.15 (in ethyl acetate). mp, 83–84° C.; [α]²³ _D: +5.2 (c 0.60, CHCl₃); FTIR (film) ν_max; 3317, 2920, 2827, 1741, 1654, 1502, 1246, 1170, 1018, 802 cm⁻¹; ¹HNMR(CDCl₃, 500 MHz): δ 7.77 (2H, d, J=8.0 Hz), 6.28 (2H, d, J=8.0 Hz), 5. 76 (1H, m), 5.63 (1H, d, J=10.0 Hz), 4.74 (1H, d, J=15.5 Hz), 4.54 (1H, d, J=15.0 Hz), 4.12 (1H, d, J=2.5 Hz), 3.80 (1H, m), 3.76 (3H, s), 3.72 (1H, m), 3.68 (3H, s), 3.00 (1H, m), 2.60 (1H, br), 2.20 (1H, m), 1.98 (2H, s), 1.87 (1H, m), 1.80 (1H, m), 1.71 (2H, m), 1.61 (3H, s), 1.14 (2H, 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 0° C. was added a pre-cooled solution of ceric ammonium nitrate (CAN) (2.46 g 4.5 mmol in 2 mL H₂O). After stirring for 1 h at 0° C. 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%).

R_f=0.10 (5% MeOH in ethyl acetate). mp, 138 to 140° C.; [α]²³ _D+14.5 (c 1.05, CHCl₃); FTIR (film) ν_max 3301, 2949, 2911, 2850, 1723, 1673, 1437, 1371, 1239, 1156, 1008, 689 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz): δ 8.48 (1H, br), 6.08 (1H, m), 5. 75 (1H, d, J=9.6 Hz), 5.29 (1H, br), 4.13 (1H, d, J=6.6 Hz), 3.83 (3H, m), 3.79 (1H, m), 3.72 (1H, m), 2.84 (1H, d, J=10.2 Hz), 2.20 (1H, m), 2.16 (1H, br), 1.98 (3H, m), 1.77 (3H, m), 1.59 (1H, m), 1.54 (3H, s), 1.25 (1H, 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 5° C. 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 23° C. for 5 min. To this solution was added BOPCl (152 mg, 0.6 mmol) at 23° C. under argon, and stirring was continued for 1 h. 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 23° C. 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.

R_f=0.55 (50% ethyl acetate in hexane). mp, 168–170° C. (authentic sample: 168–170° C., 169–171° C. in Angew. Chem. Int. Ed., 2003, 42, 355–357); mixture mp, 168–170C. [α]²³ _D −73.2 (c 0.49, MeOH), −72.9 (c 0.55, MeOH, in Angew. Chem. Int. Ed., 2003, 42, 355–357); FTIR (film) ν_max: 3406, 2955, 2920, 2844, 1823, 1701, 1257, 1076, 1012, 785, 691 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 10.62 (1H, br), 6.42 (1H, d, J=10.5 Hz), 5.88 (1H, m), 4.25 (1H, d, J=9.0 Hz), 4.14 (1H, m), 4.01 (1H, m), 3.17 (1H, t, J=7.0 Hz), 2.85 (1H, m), 2.48 (1H, m), 2.32 (2H, m), 2.07 (3H, s), 1.91 (2H, m), 1.66 (2H, m), 1.38 (1H, 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)⁻ C₁₅H₁₉ClNO₄ 312.1003, found 312.1003.

 

 

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Name:		Marizomib
Synonyms:		6-Oxa-2-azabicyclo[3.2.0]heptane-3,7-dione, 4-(2-chloroethyl)-1-[(S)-(1S)-2-cyclohexen-1-ylhydroxymethyl]-5-methyl-, (1R,4R,5S)-;  Other Names: (-)-Salinosporamide A; ML 858; Marizomib; NPI 0052; Salinosporamide A
CAS Registry Number:		437742-34-2
Molecular Formula:		C15H20ClNO4
Molecular Weight:		313.1
Molecular Structure: