Nitrone [2+3]-cycloadditions in stereocontrolled synthesis of a potent proteasome inhibitor: (-)-Omuralide

Article abstract of DOI:10.1021/jo701968d

(Chemical Equation Presented) A new stereocontrolled synthetic route to omuralide has been developed from methyl pyroglutamate. This route involves regio- and stereoselective N-methylnitrone 1,3-dipolar cycloadditions to appropriate pyrrolinones, β-eliminations, and highly selective hydrogenations as the main steps.

Full text of DOI:10.1021/jo701968d

Nitrone [2+3]-Cycloadditions in Stereocontrolled Synthesis of a
Potent Proteasome Inhibitor: (-)-Omuralide
Jean-Christophe Legeay and Nicole Langlois*
Institut de Chimie des Substances Naturelles, CNRS, AVenue de la Terrasse, 91198 Gif-sur-YVette, France
nicole.langlois@icsn.cnrs-gif.fr
ReceiVed September 8, 2007
A new stereocontrolled synthetic route to omuralide has been developed from methyl pyroglutamate.
This route involves regio- and stereoselective N-methylnitrone 1,3-dipolar cycloadditions to appropriate
pyrrolinones, â-eliminations, and highly selective hydrogenations as the main steps.
Introduction
Indeed, while bortezomib is the first proteasome inhibitor
approved by the FDA for the treatment of multiple myeloma,6
salinosporamide A (NPI-0052) is currently in clinical trials, due
to its oral activity against bortezomib-resistant multiple myeloma
cells,7 and the discovery of its biological activities renders
omuralide analogues attractive synthetic targets.
These small but complex molecules are composed of a highly
functionalized pyrrolidinone-â-lactone bicyclic core. The pres-
ence of three chiral centers on the γ-lactam ring, with a
quaternary asymmetric carbon at the â-lactone-ring junction
in the R-position to the nitrogen, constitutes structural features
common to 1-3 and related compounds. Since the pioneering
work of E. J. Corey,8 numerous lactacystin or omuralide
syntheses have been described to date, and several reviews have
been devoted to them.9,10 To synthesize salinosporamide A,
different strategies have already been developed by five
groups.11 Most of them involve an introduction of the cyclo-
hexenyl side chain from an aldehyde intermediate at a late stage,
Lactacystin (1) was first isolated from a culture broth of
Streptomyces sp. OM-6519.1 It generates, by loss of N-
acetylcysteine and lactonization, omuralide (2) [(-)-clasto-
lactacystin], which is well-known to specifically inhibit the
proteolytic activity of the proteasome 20S and represents the
cell-permeable and biologically active form of 1.2 The protea-
some, participating in a wide range of cellular processes, is
responsible for the normal turnover of cellular proteins and
recently became a novel target for cancer therapy.3 Structurally
related salinosporamide A (3), isolated more recently from a
marine actinomycete, Salinispora tropica,4 is a still more
(5) (a) Macherla, V. R.; Mitchell, S. S.; Manam, R. R.; Reed, K. A.;
Chao, T.-H.; Nicholson, B.; Deyanat-Yazdi, G.; Mai, B.; Jensen, P. R.;
Fenical, W. F.; Neuteboom, S. T. C.; Lam, K. S.; Palladino, M. A.; Potts,
B. C. M. J. Med. Chem. 2005, 48, 3684-3687. (b) Groll, M.; Huber, R.;
Potts, B. C. M. J. Am. Chem. Soc. 2006, 128, 5136-5141.
(6) (a) Adams, J. Drug DiscoVery Today 2003, 8, 307-315. (b)
Rajkumar, S. V.; Richardson, P. G.; Hideshima, T.; Anderson, K. C. J.
Clin. Oncol. 2005, 23, 630-639.
(7) (a) Chauhan, D.; Catley, L.; Li, G.; Podar, K.; Hideshima, T.;
Velankar, M.; Mitsiades, C.; Mitsiades, N.; Yasui, H.; Letai, A.; Ovaa, H.;
Berkers, C.; Nicholson, N.; Chao, T.-H.; Neuteboom, S. T. C.; Richardson,
P.; Palladino, M. A.; Anderson, K. C. Cancer Cell 2005, 8, 407-419. (b)
Joazeiro, C. A. P.; Anderson, K. C.; Hunter, T. Cancer Res. 2006, 66, 7840-
7842.
effective proteasome inhibitor and a promising anticancer drug.5
(1) (a) Omura, S.; Fujimoto, T.; Otoguro, K.; Matsuzaki, K.; Moriguchi,
R.; Tanaka, H.; Sasaki, Y. J. Antibiot. 1991, 44, 113-116. (b) Omura, S.;
Matsuzaki, K.; Fujimoto, T.; Kosuge, K.; Furuya, T.; Fujita, S.; Nakagawa,
A. J. Antibiot. 1991, 44, 117-118.
(2) (a) Fenteany, G.; Standaert, R. F.; Reichard, G. A.; Corey, E. J.;
Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3358-3362. (b)
Dick, L. R.; Cruikshank, A. A.; Grenier, L.; Melandri, F. D.; Nunes, S. L.;
Stein, R. L. J. Biol. Chem. 1996, 271, 7273-7275.
(3) (a) Borissenko, L.; Groll, M. Chem. ReV. 2007, 107, 687-717. (b)
Voorhees, P. M.; Dees, E. C.; O’Neil, B.; Orlowski, R. Z. Clin. Cancer
Res. 2003, 9, 6316-6325. (c) Spano, J.-P.; Bay, J.-O.; Blay, J.-Y.; Rixe,
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(4) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem., Int. Ed. 2003, 42, 355-357.
(8) (a) Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114,
10677-10678. (b) Corey, E. J.; Reichard, G. A.; Kania, R. Tetrahedron
Lett. 1993, 34, 6977-6980. (c) Corey, E. J.; Li, W.; Nagamitsu, T. Angew.
Chem., Int. Ed. 1998, 37, 1676-1679.
10.1021/jo701968d CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/29/2007
10108
J. Org. Chem. 2007, 72, 10108-10113

Synthesis of a Potent Proteasome Inhibitor
SCHEME 1. General Retrosynthetic Route
SCHEME 2. Synthesis of (S)-7
relying on Corey et al. strategies.11a,b These pathways, indeed,
allow structural modifications of the side chain R to the nitro-
gen, and this advantage has been exemplified by this group in
several syntheses of salinosporamide A as well as omuralide
analogues.8c,12
SCHEME 3. Preparation of Dipolarophile 14 and
Cycloaddition of N-Methylnitrone
Results and Discussion
We planned to develop a general approach to 1-3 and to
several natural or synthetic analogues from particularly inex-
pensive methyl pyroglutamate, with the aim to synthesize the
key advanced intermediates 4 and 5 reported by Corey,
following the retrosynthetic Scheme 1. Accordingly, the densely
substituted pyrrolidinone 4 would be generated by stereoselec-
tive hydrogenation of 4-exo-methylene precursor A (R ) H).
4-exo-methylene-5-oxopyrrolidines of general structure A would
be obtained in a few steps through â-elimination of an amino
group formed from B, via the reductive opening of the
isoxazolidine ring. Indeed, we previously reported preliminary
studies of this new strategy leading to salinosporamide A
precursor 5,13 and we expected that versatile exo-methylene
derivative A (R ) H) could also be converted into 3-hydroxy-
4-methylpyrrolidin-2-ones 4 with the cis configuration present
in omuralide (2). We anticipated that requisite functionalities
of B could be introduced with appropriate configurations through
regio- and stereoselective N-alkylnitrone cycloadditions to
unsaturated γ-lactams C, already bearing the quaternary ste-
reogenic center C-2. The preparation of C could take advantage
of the original access to (S)-methyl 2-(hydroxymethyl)pyro-
glutamate [(S)-6], previously developed in our laboratory to
synthesize deoxydysibetaine.14 Thus, we selected as a synthetic
target compound 4, which has been converted into 2 by Corey
et al.,8c Pattenden et al.,10a and very recently Kobayashi et al.10i
We reported therein the pathways and results to achieve this
goal. The scope, efficiency, and stereoselectivity of this new
route were investigated starting with methyl 2-[(benzyloxy)-
methyl]pyroglutamate (7), and we showed that the results
depend to a large extent on the protection of functional groups
and on the order in which the main steps are performed. Racemic
7 was directly obtained from methyl pyroglutamate13 and used
to check the validity and efficiency of our routes.
(S)-Methyl 2-[(benzyloxy)methyl]pyroglutamate [(S)-7] was
prepared from bicyclic [(tert-butyldimethylsilyl)oxy]pyrrole 8
derived from (S)-pyroglutaminol as summarized in Scheme 2.14
This pathway involved the formation of the nitrile 10 with
control of the stereogenic quaternary center by a selective
addition of trimethylsilyl cyanide to the acyliminium ion
generated from tertiary carbinolamide 9.15 Acidic hydrolysis of
10, followed by methylation with diazomethane, gave rise to
(S)-6, which was selectively O-benzylated to (S)-7 (75%) with
2-(benzyloxy)-1-methylpyridinium triflate (Dudley’s reagent) in
the presence of MgO.16 It is worthy of note that neither
N-benzylation nor transesterification with the methoxycarbonyl
(9) For reviews see: (a) Corey, E. J.; Li, W.-D. Z. Chem. Pharm. Bull.
1999, 47, 1-10. (b). Masse, C. E.; Morgan, A. J.; Adams, J.; Panek, J. S.
Eur. J. Org. Chem. 2000, 2513-2528. (c) Kang, S. H.; Kang, S. Y.; Lee,
H.-S.; Buglass, A. J. Chem. ReV. 2005, 105, 4537-4558. (d) Shibasaki,
M.; Kanai, M.; Fukuda, N. Chem. Asian J. 2007, 2, 20-38.
(10) For recent syntheses see: (a) Brennan, C. J.; Pattenden, G.;
Rescourio, G. Tetrahedron Lett. 2003, 44, 8757-8760. (b) Ooi, H.;
Ishibashi, N.; Iwabuchi, Y.; Ishihara, J.; Hatakeyama, S. J. Org. Chem.
2004, 69, 7765-7768. (c) Donohoe, T. J.; Sintim, H. O.; Sisangia, L.; Ace,
K. W.; Guyo, P. M.; Cowley, A.; Harling, J. D. Chem.sEur. J. 2005, 11,
4227-4238. (d) Wardrop, D. J.; Bowen, E. G. Chem. Commun. 2005,
5106-5108. (e) Hayes, C. J.; Sherlock, A. E.; Selby, M. D. Org. Biomol.
Chem. 2006, 4, 193-195. (f) Fukuda, N.; Sasaki, K.; Sastry, T. V. R. S.;
Kanai, M.; Shibasaki, M. J. Org. Chem. 2006, 71, 1220-1225. (g) Balskus,
E. P.; Jacobsen, E. N. J. Am. Chem. Soc. 2006, 128, 6810-6812. (h) Yoon,
C. H.; Flanigan, D. L.; Yoo, K. S.; Jung, K. W. Eur. J. Org. Chem. 2007,
37-39. (i) Gilley, C. B.; Buller, M. J.; Kobayashi, Y. Org. Lett 2007, 9,
3631-3636.
(11) (a) Reddy, L. R.; Saravanan, P.; Corey, E. J. J. Am. Chem. Soc.
2004, 126, 6230-6231. (b) Reddy, L. R.; Fournier, J.-F.; Reddy, B. V. S.;
Corey, E. J. Org. Lett. 2005, 7, 2699-2701. (c) Endo, A.; Danishefsky, S.
J. J. Am. Chem. Soc. 2005, 127, 8298-8299. (d) Mulholland, N. P.;
Pattenden, G.; Walters, I. A. S. Org. Biomol. Chem. 2006, 4, 2845-2846.
(e) Ma, G.; Nguyen, H.; Romo, D. Org. Lett. 2007, 9, 2143-2146. (f) Ling,
T.; Macherla, V. R.; Manam, R. R.; McArthur, K. A.; Potts, B. C. M. Org.
Lett. 2007, 9, 2289-2292.
(12) Corey, E. J.; Li, W.-D. Z.; Nagamitsu, T.; Fenteany, G. Tetrahedron
1999, 55, 3305-3316.
(13) (a) Caubert, V.; Langlois, N. Tetrahedron Lett. 2006, 47, 4473-
4475. (b) Caubert, V.; Masse´, J.; Retailleau, P.; Langlois, N. Tetrahedron
Lett. 2007, 48, 381-384. (c) Legeay, J.-C.; Langlois, N. XI Symposium
ICSN, Gif-sur-Yvette, France, June 7, 2007.
(14) Langlois, N.; Le Nguyen, B. K. J. Org. Chem. 2004, 69, 7558-
7564.
(15) Langlois, N.; Choudhury, P. K. Tetrahedron Lett. 1999, 40, 2525-
2528.
J. Org. Chem, Vol. 72, No. 26, 2007 10109

Legeay and Langlois
SCHEME 4. Synthesis of 4
group was observed under these particular conditions. Com-
pound 7 was transiently N-protected as a tert-butyl carbamate
(11) (100%) to enhance the reactivity of lactam carbonyl,
allowing efficient deprotonation at C-4 and introduction of the
conjugate double bond of 12 (89%, Scheme 3).
OTf and 2,6-lutidine (not optimized, 73% yield), followed by
highly stereoselective hydrogenation furnished 19, the direct
precursor of 4, as the sole observed diastereomer (dr > 95:5).
The expected compound 4, an intermediate in several syntheses
of omuralide (2),8c,10a,i was isolated in 89% yield after quantita-
tive O-debenzylation. Direct conversion of 18 into 4 was also
obtained (87%) under the same conditions (10% Pd/C, EtOH).
In a first route, the N-Boc group of (()-12 was quantitatively
removed, providing 13, and the PMB group present in the target
molecule was introduced ((PMB)Br, Cs2CO3, DMF, rt) to give
the dipolarophile 14 in 88% yield. The 1,3-dipolar cycloaddition
of N-methylnitrone to 14 proceeded by heating in toluene with
high regioselectivity but rather moderate stereoselectivity. This
addition was complete after 3 h, affording two main adducts,
15 and 16, isolated in 85% combined yield and a ratio near
2.1:1. In the NMR spectra of 15 and 16, proton and carbon
chemical shifts at the bridgehead positions 3a (CDCl3; 1H, 3.52
and 3.46 ppm; 13C, 51.9 and 51.0 ppm, respectively) support
the same regiochemistry in the cycloaddition.17 An NOESY
experiment performed with the major diastereomer 15 showed
a weak correlation between H-6a and one proton of the
oxymethylene group at C-6. The configurations consequently
attributed were confirmed later by further chemical correlations.
The isoxazolidine 15 was hydrogenolyzed using Pearlman’s
catalyst,18 with complete chemoselectivity, leaving the benzy-
loxy protecting group untouched. The cleavage product was
converted into trimethylammonium iodide as the leaving group
by MeI in MeOH and directly submitted to a â-elimination in
a basic biphasic mixture (CH2Cl2-aqueous 10% (w/v) Na2CO3)
to afford compound 17 in 67% yield for this three-step procedure
(Scheme 4). Conversion of 17 into its TBS ether 18 with (TBS)-
Despite the excellent stereoselectivity of the hydrogenation
step, the efficiency of the general Scheme 1 could be improved
by increasing the stereoselectivity of the N-methylnitrone
cycloaddition to the flat pyrrolinone ring. The modest facial
selectivity observed with 14 could be explained by a relatively
weak steric hindrance exerted by the side chain at C-2. Indeed,
the 2-(benzyloxy)methyl group could adopt a conformation
minimizing the congestion and allowing an attack of the nitrone
from the same face, plausibly due to implication of the N-PMB
group in intramolecular π-stacking.
Accordingly, we investigated a second route starting with
N-Boc-lactam (S)-12 as the dipolarophile. The 1,3-dipolar
cycloaddition is effectively much more selective in this case,
leading to the adduct (3aR,6R,6aS)-20 in 89% yield. The
stereostructure 20 was assigned on the basis of NMR data,
including an NOESY experiment. Only ca. 2% of another
diastereoisomer was isolated, whose spectral data support
structure 21. After hydrogenolysis of the isoxazolidine moiety,
the corresponding trimethylammonium iodide obtained with
excess MeI in THF was treated in one pot with anhydrous
NaHCO3, affording the 4-exo-methylene derivative 22 in 89%
yield for the three steps (from 20, Scheme 4).
Hydrogenation of the exo-methylene double bond of 22, with
PtO2 as the catalyst, occurred quantitatively but without any
selectivity, leading to 23 and 24 in a ratio near 1:1 determined
by 1H NMR (Table 1, entry 1). The lack of selectivity could be
due to the much decreased bulk of the vicinal C-3 substituent,
(16) Poon, K. W. C.; Dudley, G. B. J. Org. Chem 2006, 71, 3923-
3927.
(17) Langlois, N.; Van Bac, N.; Delcroix, J.-M.; Deyine, A.; Griffart-
Brunet, D.; Chiaroni, A.; Riche, C. Tetrahedron 1995, 51, 3571-3586.
(18) Pearlman, W. M. Tetrahedron Lett. 1967, 1663-1664.
10110 J. Org. Chem., Vol. 72, No. 26, 2007

Synthesis of a Potent Proteasome Inhibitor
TABLE 1. Hydrogenation of 4-exo-Methylene Derivatives
methylene moiety. This strategy could also be broadened to the
synthesis of structural analogues using substituted nitrones.
Along with elaboration of the side chain R to the nitrogen at a
late stage of the synthesis according to the versatile Corey
procedures,8c,11a,b,12 it could also offer additional possibilities
for variation of substituent R to the lactam carbonyl. It opens
the way to other members of the salinosporamide-cinnabara-
mide family,22,23 as well as to the design of synthetic related
compounds interesting to improve the selectivity of proteasome
inhibition.10g,24
a Determined by 1H NMR of the crude diastereomeric mixture obtained
in 99-100% yield, except for entry 2 (40%).
Experimental Section
(S)-Methyl 2-[(Benzyloxy)methyl]-5-oxopyrrolidine-2-carbox-
ylate ((S)-7) from (S)-Methyl 2-(Hydroxymethyl)-5-oxopyrro-
lidine-2-carboxylate [(S)-6]. A mixture of (S)-6 (320 mg, 1.85
mmol),14 2-(benzyloxy)-1-methylpyridinium triflate (1.40 g, 4.01
mmol), and dried MgO (163.4, 4.05 mmol) in (trifluoromethyl)-
benzene (3.8 mL) was stirred at 83 °C for 21 h before filtration
through Celite. The Celite was washed with CH2Cl2, and the
solvents were evaporated. The product was purified by column
chromatography (eluent heptane-EtOAc, 3:7) to give O-benzylated
but also to its directing effect. Indeed, hydroxyl groups are
known to enforce addition of hydrogen from their own side of
the molecule.19 This result indicated that the cleavage of the
isoxazolidine moiety of cycloadduct 20 has to be carefully
controlled since some â-elimination of methylamine and
subsequent nonstereoselective hydrogenation could occur in situ
after a prolonged time under the hydrogenolysis conditions
(Table 1, entry 2). It led us also to change the chronology of
the O-protection step by tert-butyldimethylsilylation. Although
N-Boc protective groups are known to be generally sensitive to
(TBS)OTf under O-silylation conditions,20 sequential N-depro-
tection followed by O-silylation was preferred. In this case,
N-deprotection of 22 under mild conditions using ZnBr221 was
less efficient than classical treatment with TFA in CH2Cl2,
affording quantitatively compound 25, which was O-silylated
into 26 (97%, Scheme 4). Hydrogenation of 26 (PtO2, EtOAc)
proceeded with good selectivity (9:1; see Table 1, entry 3),
providing the requisite diasterereomer 27, which was isolated
in 77% yield. However, this compound was shown to be poorly
reactive toward N-p-methoxybenzylation into 19 ((PMB)Br, Cs2-
CO3, DMF, rt), and only 40% conversion was obtained after
24 h. Consequently, this step was performed at an early stage,
with the flattened and less hindered 4-exo-methylenepyrrolidi-
none 26, leading to (2R,3S)-18 (92%), the precursor of (-)-
omuralide via (2R,3S,4R)-19 and (2R,3S,4R)-4, as shown
previously. Enantiomeric purity of (2R,3S,4R)-4 (96% ee) was
verified by chiral HPLC and indicated no significative lowering
during the synthesis. Thus, by this second route, the overall
yield of the key intermediate 4 from N-Boc-2-[(benzyloxy)-
methyl]-2-(methoxycarbonyl)pyrrolin-2-one [(S)-12] was dra-
matically improved and reached 64%, compared to 22%
obtained in the first route.
25
compound (S)-7 (363 mg, 75%). [R]D +25.7 (c 1.72, CHCl3).
For spectral data see ref 13b. Anal. Calcd for C14H17NO4: C, 63.86;
H, 6.51; N, 5.32. Found: C, 63.56; H, 6.52; N, 5.37.
(S)-1-tert-Butyl 2-Methyl 2-[(Benzyloxy)methyl]-5-oxo-pyr-
rolidine-1,2-dicarboxylate [(S)-11]. A mixture of (S)-7 (342 mg,
1.3 mmol), DMAP (158.6 mg, 1.3 mmol), and Boc2O (437 mg,
2.0 mmol) in MeCN (1.6 mL) was stirred at rt under argon for 1
h and then concentrated under vacuum (without heating). The crude
orange oil was purified by column chromatography (eluent petro-
leum ether-EtOAc, 65:35) to yield (S)-11 as a colorless oil (472
25
mg, 100%). [R]D -40.2 (c 1.93, CHCl3). IR (cm-1): 2978, 1788,
1742, 1713, 1454, 1368, 1300, 1256. MS (ESI, MeOH, m/z): 386
[(MNa)+, 100], 286. 1H NMR (500 MHz, CDCl3): δ 7.33 (m, 2H),
7.28 (m, 3H), 4.55 (d, 1H, J ) 12.2 Hz), 4.53 (d, 1H, J ) 12.2
Hz), 4.07 (d, 1H, J ) 10 Hz), 3.86 (d, 1H, J ) 10 Hz), 3.73 (s,
3H), 2.75 (m, 1H), 2.50 (m, 1H), 2.29 (m, 1H), 2.08 (m, 1H), 1.43
(s, 9H). 13C NMR (75 MHz, CDCl3): δ 174.4, 171.8, 149.2, 137.7,
128.6, 127.9, 127.7, 83.7, 73.6, 71.2, 68.1, 52.5, 31.4, 28.0, 27.2.
Anal. Calcd for C19H25NO6: C, 62.80; H, 6.93; N, 3.85. Found:
C, 62.41; H, 6.84; N, 3.74.
(S)-1-tert-Butyl 2-Methyl 2-[(Benzyloxy)methyl]-5-oxo-1H-
pyrrole-1,2-(2H,5H)-dicarboxylate [(S)-12]. LiHMDS (1 M in
THF, 1.4 mL) was added dropwise to a solution of (S)-11 (421
mg, 1.16 mmol) in THF (13.3 mL) and the mixture stirred under
argon at -78 °C. After 30 min of stirring, PhSeCl (244.2 mg, 1.28
mmol) in THF (1.16 mL) was added. The mixture was stirred for
1.5 h at -78 °C, and satd NH4Cl was added. Back at rt, the reaction
mixure was extracted four times with EtOAc. The organic phase
was dried on MgSO4 and evaporated under vacuum. The residue
was taken up in CH2Cl2 (7.5 mL) and pyridine (283 µL), and H2O2
(30% aq, 1.24 mL) was added after the mixture was cooled at 0
°C. The mixture was stirred at 0 °C for 30 min and then at rt for
1.75 h. After addition of aqueous Na2CO3 (10%, w/v) and CH2Cl2
under stirring, the organic phase was separated and washed by
water. The aqueous phase was extracted three times with CH2Cl2,
and the organic layers were washed twice with H2O, gathered, dried
Conclusions
We have developed a new synthetic route to (-)-omuralide
(2) from cheap methyl pyroglutamate. N-Methylnitrone 1,3-
dipolar cycloaddition to the N-Boc-pyrrolinone already bearing
the R,R-disubstituted stereogenic center, as described in the
second route, was shown to be remarkably efficient, allowing
achievement of the synthesis of the key omuralide precursor 4
with excellent stereoselectivities and high yields, provided that
the N-Boc dipolarophile was used and that N-p-methoxyben-
zylation was performed before hydrogenation of the 4-exo-
(22) Stadler, M.; Bitzer, J.; Mayer-Bartschmid, A.; Mu¨ller, H.; Benet-
Buchholz, J.; Gantner, F.; Tichy, H.-V.; Reinemer, P.; Bacon, K. B. J. Nat.
Prod. 2007, 70, 246-252.
(23) (a) Williams, P. G.; Buchanan, G. O.; Feling, R. H.; Kauffman, C.
A.; Jensen, P. R.; Fenical, W. J. Org. Chem. 2005, 70, 6196-6203. (b)
Reed, K. A.; Manam, R. R.; Mitchell, S. S.; Xu, J.; Teisan, S.; Chao, T.-
H.; Deyanat-Yazdi, G.; Neuteboom, S. T. C.; Lam, K. S.; Potts, B. C. M.
J. Nat. Prod. 2007, 70, 269-276.
(24) Soucy, F.; Grenier, L.; Behnke, M. L.; Destree, A. T.; McCormack,
T. A.; Adams, J.; Plamondon, L. J. Am. Chem. Soc. 1999, 121, 9967-
9976.
(19) Thompson, H. W.; Rashid, S. Y. J. Org. Chem. 2002, 67, 2813-
2825 and references therein.
(20) Kocienski, P. J. Protecting Groups, 3rd ed.; Thieme: Stuttgart, New
York, 2005.
(21) (a) Nigam, S. C.; Mann, A.; Taddei, M.; Wermuth, C.-G. Synth.
Commun. 1989, 19, 3139-3142. (b) Donohoe, T. J.; Chiu, J. Y. K.; Thomas,
R. E. Org. Lett. 2007, 9, 421-424.
J. Org. Chem, Vol. 72, No. 26, 2007 10111

Legeay and Langlois
over MgSO4, and concentrated under vacuum. The residue was
without further purification. To a solution of this product in dry
THF (3.0 mL) was added under Ar MeI (0.69 mL, 11.0 mmol).
The solution was stirred at rt for 24 h. Then MeI (0.69 mL, 11.0
mmol) and NaHCO3 (66.9 mg, 0.80 mmol) were added to the
mixture. The reaction medium was stirred at rt for 3 days. After
elimination of the solvent under reduced pressure, the crude product
was purified by filtration over silica gel (eluent Et2O) to give
filtered on silica gel (eluent heptane-Et2O, 1:9) to give (S)-12 as
27
a colorless oil (373.5 mg, 89%). [R]D +56 (c 1.36, CHCl3). IR
(cm-1): 1782, 1739, 1711, 1319, 1254, 1158, 1102, 1049, 821.
MS (ESI, MeCN, m/z): 745 (2MNa)+, 384 [(MNa)+, 100]. 1H (500
MHz, CDCl3): δ 7.37-7.24 (m, 5H), 6.99 (d, 1H, J ) 6.1 Hz,),
6.24 (d, 1H, J ) 6.1 Hz), 4.53 (d, 1H, J ) 12.2 Hz), 4.51 (d, 1H,
J ) 12.2 Hz), 4.17 (d, 1H, J ) 10.4 Hz), 4.14 (d, 1H, J ) 10.4
Hz), 3.73 (s, 3H), 1.47 (s, 9H). 13C NMR (75 MHz, CDCl3): δ
169.0, 167.8, 148.6, 147.0, 137.5, 128.6, 128.4, 128.0, 127.7, 83.8,
73.8, 73.2, 68.6, 53.0, 28.0. Anal. Calcd for C19H23NO6: C, 63.15;
H, 6.41, N, 3.88. Found: C, 63.21; H, 6.41, N, 3.71.
27
(2R,3S)-22 as a clear oil (136.7 mg, 89% for three steps). [R]D
-32.6 (c 1.13, CHCl3). IR (cm-1): 3448, 2943, 1772, 1746, 1454,
1369, 1304, 1248, 1151, 1116, 1048. MS (ESI, MeCN + CH2Cl2,
1
m/z): 414 (MNa)+, 314 [[(M - Boc)Na]+, 100]. H NMR (500
MHz, CDCl3): δ 7.38-7.25 (m, 5H), 6.41 (d, 1H, J ) 2.7 Hz),
5.83 (d, 1H, J ) 2.1 Hz), 4.95 (m, 1H), 4.57 (d, 1H, J ) 12.2 Hz),
4.54 (d, 1H, J ) 12.2 Hz), 4.10 (d, 1H, J ) 10.0 Hz), 4.07 (d, 1H,
J ) 10.0 Hz), 3.75 (s, 3H), 2.57 (br m, 1H), 1.47). 13C NMR (75
MHz, CDCl3): δ 168.9, 165.0, 149.8, 140.6, 137.5, 128.7, 128.1,
127.8, 123.5, 84.3, 73.7, 71.5, 70.3, 69.3, 52.7, 28.0. HRMS (ESI,
MeCN + CH2Cl2, m/z): calcd for C20H25NO7Na (MNa)+ 414.1529,
found 414.1536.
Cycloaddition of N-Methylnitrone to (()-14: Methyl 6-[(Ben-
zyloxy)methyl]-5-(4-methoxybenzyl)-2-methyl-4-oxohexahydro-
2H-pyrrolo[3,4-d]isoxazole-6-carboxylates (()-15 and (()-16. A
solution of N-methylnitrone (44.3 mg, 0.75 mmol) in dry toluene
(2.5 mL) was added to (()-14 (111.7 mg, 0.29 mmol) placed under
Ar. The mixture was stirred at 110 °C for 3 h. The solvent was
evaporated under reduced pressure and the crude material purified
by chromatography (eluent EtOAc) to give a mixture of isomers
(127.7 mg, 99%). The diastereomeric ratio (58:28:8:5) was deter-
mined by HPLC (SunFire, 3 × 150 mm; eluent MeCN-H2O, 1:1).
The two main diastereomers were separated by preparative TLC
(eluent CH2Cl2-EtOAc, 9:1) to afford (3aR*,6R*,6aS*)-15 (75.0
mg, 58%) and (3aS*,6R*,6aR*)-16 (35 mg, 27%), described in the
Supporting Information). (()-15 was obtained as a colorless gum.
IR (cm-1): 2953, 2848, 1753, 1692, 1512. MS (ESI, MeOH +
(2R,3S)-Methyl 2-[(Benzyloxy)methyl]-3-hydroxy-4-methyl-
ene-5-oxopyrrolidine-2-carboxylate (25). To a solution of com-
pound 22 (133.9 mg, 0.34 mmol) in CH2Cl2 (1.3 mL) was added
TFA (250 µL, 3.37 mmol) at rt, and the mixture was stirred for 50
min. After dilution with CH2Cl2 and addition of saturated aqueous
Na2CO3, the aqueous phase was extracted with CH2Cl2. The organic
phases were dried over MgSO4, and the solvent was evaporated to
afford (2R,3S)-25 (100.1 mg, 100%) as a colorless oil. [R]D25 -7.0
(c 1.10, CHCl3). IR (cm-1): 3290, 2866, 1737, 1694, 1666, 1433,
1314, 1233. MS (ESI, MeOH + CH2Cl2, m/z): 605 [(2MNa)+, 100],
1
CH2Cl2, m/z): 463 [(MNa)+, 100]. H NMR (500 MHz, CDCl3):
δ 7.30-7.24 (m, 5H), 7.08 (d, 2H, J ) 7.4 Hz), 6.75 (2H, d, 2H,
J ) 8.5 Hz), 5.19 (br d, 1H, J ) 15.7 Hz), 4.66 (br d, 1H, J ) 6.3
Hz), 4.12 (d, 1H, J ) 15.7 Hz), 3.94 (d, H, J ) 11.4 Hz), 3.86 (d,
H, J ) 11.4 Hz), 3.81 (masked d, 1H), 3.79 (s, 3H), 3.74 (s, 3H),
3.63 (m, 1H), 3.52 (m, 1H), 3.25 (d, 1H, J ) 9.8 Hz), 2.65 (s, 3H),
2.56 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 175.0, 168.2, 159.0,
137.6, 130.1, 129.9, 128.5, 127.8, 127.3, 113.7, 79.8, 76.0, 72.9,
70.3, 60.1, 55.4, 52.5, 51.9, 45.3, 44.9. HRMS (ESI, MeOH + CH2-
Cl2, m/z): calcd for C24H28N2O6Na (MNa)+ 463.1845, found
463.1882.
1
314 (MNa)+. H NMR (500 MHz, CDCl3): δ 7.36-7.26 (2 m,
5H), 6.47 (br d, 1H exch., J ) 7.9 Hz), 6.26 (d, 1H, J ) 2.1 Hz),
5.75 (br s, 1H), 4.59 (m, 1H), 4.56 (d, 1H, J ) 12.2 Hz), 4.53 (d,
1H, J ) 12.2 Hz), 4.01 (d, 1H, J ) 9.1 Hz,), 3.81 (s, 3H), 3.41 (d,
1H, J ) 9.1 Hz), 2.71 (m, 1H exch.). 13C NMR (75.0 MHz,
CDCl3,): δ 170.6, 168.3, 141.2, 137.3, 128.6, 128.0, 127.7, 122.0,
73.7, 73.2, 71.9, 70.8, 53.1. HRMS (ESI, MeOH + CH2Cl2, m/z):
calcd for C15H17NO5Na (MNa)+ 314.1004, found 314.1000.
(2R,3S)-Methyl 2-[(Benzyloxy)methyl]-3-[(tert-butyldimeth-
ylsilyl)oxy]-4-methylene-5-oxopyrrolidine-2-carboxylate (26). To
a stirred solution of 25 (97.5 mg, 0.33 mmol) in CH2Cl2 (2.0 mL)
were successively added under argon at rt 2,6-lutidine (218 µL,
1.87 mmol) and (TBS)OTf (285 µL, 1.22 mmol). The mixture was
stirred at rt for 24 h. Then a solution of Na2CO3 (10% w/v) was
added until pH 8, and the product was extracted with CH2Cl2.
Drying on MgSO4 and evaporation of the organic phase under
reduced pressure afforded (2R,3S)-26 as a white solid (131.9 mg,
97%). Mp: 99 °C. [R]D25 +15.0 (c 1.28, CHCl3). IR (cm-1): 3192,
2926, 2854, 1741, 1702, 1674, 1452, 1428, 1359, 1343, 1250, 1235,
1109, 835. MS (ESI, MeOH + CH2Cl2 m/z): 428 [(MNa)+, 100].
1H NMR (500 MHz, CDCl3): δ 7.38-7.28 (m, 5H), 6.18 (br s,
1H, NH), 6.16 (d, 1H, J ) 2.2 Hz), 5.51 (br s, 1H), 4.63 (m, 1H,
J ) 2.2 Hz), 4.56 (d, 1H, J ) 12.2 Hz), 4.54 (d, 1H, J ) 12.2 Hz),
4.06 (d, 1H, J ) 9.2 Hz), 3.73 (s, 3H), 3.42 (d, 1H, J ) 9.2 Hz),
0.87 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 169.5, 167.9, 141.9, 137.3, 128.6, 128.1, 127.9, 119.4,
73.8, 73.0, 72.2, 69.6, 52.6, 25.7, 17.9, -4.2, -4.6. HRMS (ESI,
MeOH + CH2Cl2, m/z): calcd for C21H31NO5SiNa (MNa)+
428.1869, found 428.1858.
Cycloaddition of N-Methylnitrone to (S)-12: 5-tert-Butyl
6-Methyl 6-[(Benzyloxy)methyl]-2-methyl-4-oxohexahydro-5H-
pyrrolo[3,4-d]isoxazole-5,6-dicarboxylates (3aR,6R,6aS)-20 and
(3aS,6R,6aR)-21. A solution of N-methylnitrone (58.0 mg, 0.98
mmol) in dry toluene (2.0 mL) was added to (S)-12 (167.4 mg,
0.46 mmol) placed under Ar. The mixture was stirred at 110 °C
for 2.2 h. The solvent was evaporated under reduced pressure, and
the crude material was purified by chromatography (eluent hep-
tane-EtOAc, 4:6) to give the main cycloadduct (3aR,6R,6aS)-20
(173.3 mg, 89%) as a colorless gum and diastereomer (3aS,6R,-
6aR)-21 (4.3 mg, 2.2% described in the Supporting Information).
24
The following are data for (3aR,6R,6aS)-20. [R]D +5.6 (c 1.55,
CHCl3). IR (cm-1): 2922, 1787, 1718, 1454, 1368, 1298, 1233,
1152, 1124, 1076. MS (ESI, MeOH), m/z): 443 (MNa)+, 343 [[(M
- Boc)Na]+, 100], 321 [M - Boc)H]+. 1H NMR (500 MHz,
CDCl3): δ 7.36-7.23 (m, 5H), 4.64 (d, 1H, J ) 6.4 Hz), 4.53 (d,
1H, J ) 12.2 Hz), 4.49 (d, 1H, J ) 12.2 Hz), 4.12 (d, 1H, J ) 9.8
Hz), 4.03 (d, 1H, J ) 9.8 Hz), 3.77 (s, 3H), 3.62 (m, 1H), 3.56 (m,
1H), 2.63 (s, 3H), 2.58 (m, 1H), 1.44 (s, 9H). 13C NMR (75 MHz,
CDCl3): δ 174.0, 167.5, 148.7, 137.4, 128.7, 128.1, 127.7, 83.8,
78.7, 74.2, 73.7, 70.8, 60.7, 53.2, 52.4, 44.8, 28.0. Anal. Calcd for
C21H28N2O7: C, 59.99, H, 6.71; N, 6.66. Found: C, 59.71; H, 6.58;
N, 6.27.
(2R,3S)-Methyl 2-[(Benzyloxy)methyl]-3-[(tert-butyldimethyl-
silyl)oxy]-1-(4-methoxybenzyl)-4-methylene-5-oxopyrrolidine-2-car-
boxylate [(2R,3S)-18]. Cs2CO3 (151.5 mg, 0.46 mmol) was added
to a solution of 26 (123.4 mg, 0.30 mmol) in anhydrous DMF (1.5
mL) under Ar. The mixture was stirred for 10 min at rt and cooled
to 0 °C before dropwise addition of (PMB)Br (67.0 µL, 0.46 mmol).
The reaction mixture was stirred for 0.5 h at 0 °C and then 16 h at
rt. After addition of H2O and extraction with EtOAc, the organic
solution was dried on MgSO4, filtered, and evapored under reduced
pressure. The crude product was purified by preparative TLC (eluent
heptane-EtOAc, 7:3) to give (2R,3S)-18 (146.7 mg, 92%) as a
(2R,3S)-1-tert-Butyl 2-Methyl 2-[(Benzyloxy)methyl]-3-hy-
droxy-4-methylene-5-oxopyrrolidine-1,2-dicarboxylate (22). Pd-
(OH)2 (22.4 mg) was added to a stirred solution of (3aR,6R,6aS)-
20 (165.1 mg, 0.39 mmol) in EtOAc (3.0 mL) placed under N2.
The mixture was stirred under H2 at rt for 8 h. The catalyst was
filtered through Celite and washed with EtOAc. Evaporation under
reduced pressure afforded the crude product as a colorless foam
(163.5 mg), which was directly engaged in the following step
10112 J. Org. Chem., Vol. 72, No. 26, 2007

Synthesis of a Potent Proteasome Inhibitor
colorless oil. [R]D25 +49.2 (c 1.28, CHCl3). IR (cm-1): 2950, 2929,
2857, 1740, 1699, 1612, 1512, 1398, 1245, 1125, 836. MS (ESI,
MeOH + CH2Cl2, m/z): 548 [(MNa)+, 100]. 1H NMR (300 MHz,
CDCl3): δ 7.40-7.25 (m, 5H), 7.10 (d, 2H, J ) 8.7 Hz), 6.73 (d,
2H, J ) 8.7 Hz), 6.13 (d, 1H, J ) 2.9 Hz), 5.40 (d, 1H, J ) 2.6
Hz), 5.04 (dd, 1H, J ≈ J′ ≈ 2.7 Hz), 4.70 (d, 1H, J ) 15.1 Hz),
4.43 (d, 1H, J ) 11.9 Hz), 4.32 (d, 1H, J ) 11.9 Hz), 4.12 (d, 1H,
J ) 15.1 Hz), 3.83 (2 d, 2H), 3.74 (s, 3H), 3.30 (s, 3H), 0.85 (s,
9H), 0.11 (s, 3H), 0.00 (s, 3H). 13C NMR (75 MHz, CDCl3): δ
169.3, 168.0, 159.0, 142.3, 137.3, 130.5, 128.6, 128.5, 128.4, 128.2,
116.4, 113.6, 73.4, 71.6, 71,1, 65.0, 55.3, 51.8, 43.8, 25.7, 17.9,
-4.2, -4.8. HRMS (ESI, MeOH + CH2Cl2, m/z): calcd for C29H39-
NO6SiNa (MNa)+ 548.2444, found 548.2417.
(2R,3S,4R)-Methyl 3-[(tert-Butyldimethylsilyl)oxy]-2-(hy-
droxymethyl)-1-(4-methoxybenzyl)-4-methyl-5-oxopyrrolidine-
2-carboxylate [(2R,3S,4R)-4]. A 14.7 mg portion of 10% Pd/C
was added to a solution of (2R,3S,4R)-19 (52.3 mg, 0.099 mmol)
in EtOH (0.90 mL) placed under N2. Then the mixture was flushed
with H2 gas and stirred under H2 at rt for 24 h. Filtration of the
catalyst through Celite and washing with EtOH afforded pure
27
product (2R,3S,4R)-4 (43.3 mg, 100%) as a colorless oil. [R]D
-6.9 (c 1.23, CHCl3) [lit. -7.3 (c 0.16, CHCl3),10a -4.4 (c 2.8,
CHCl3)].10i IR (cm-1): 3377, 2930, 2856, 1738, 1669, 1612, 1512,
1436, 1409, 1245, 1177, 1029, 832. MS (ESI, MeOH + CH2Cl2,
1
m/z): 460 [(MNa)+, 100]. H NMR (500 MHz, CDCl3): δ 7.29
(d, 2H, J ) 8.4 Hz), 6.85 (d, 2H, J ) 8.4 Hz), 5.00 (d, 1H, J )
15.1 Hz), 4.56 (d, 1H, J ) 9.1 Hz), 3.83 (dd, 1H, J ) 12.9, J′ )
10.1 Hz), 3.79 (s, 3H), 3.78 (d, 1H, J ) 15.1 Hz), 3.74 (dd, 1H, J
) 12.9, J′ ) 4.7 Hz), 3.64 (s, 3H), 2.71 (dq, 1H, J ) 9.1, J′ ) 7.7
Hz), 1.23 (d, 3H, J ) 7.7 Hz), 1.15 (dd, 1H, J ) 10.1, J′ ) 4.7
Hz), 0.88 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 178.1, 170.6, 159.4, 129.8, 129.7, 114.5, 75.8, 70.0,
61.7, 55.4, 52.2, 44.4, 40.8, 25.8, 18.1, 11.3, -4.7, -5.0. HRMS
(ESI, MeOH + CH2Cl2, m/z): calcd for C22H35NO6SiNa (MNa)+
460.2131, found 460.2137.
(2R,3S,4R)-Methyl 2-[(Benzyloxy)methyl]-3-[(tert-butyldim-
ethylsilyl)oxy]-1-(4-methoxybenzyl)-4-methyl-5-oxopyrrolidine-
2-carboxylate [(2R,3S,4R)-19]. A solution of (2R,3S)-18 (144.3
mg, 0.275 mmol) in EtOAc (5.0 mL) was stirred under H2 in the
presence of PtO2 (15.5 mg) at rt for 1.5 h. Filtration through Celite
and washing with EtOAc afforded the crude product (144.5 mg,
100%), which was purified by preparative thin layer chromatog-
raphy (eluent heptane-EtOAc, 6:4) to give the diastereomer
(2R,3S,4R)-19 (128.4 mg, 89%) as a colorless oil. [R]D25 +18.5 (c
1.43, CHCl3). IR (cm-1): 2948, 2928, 2856, 1738, 1693, 1612,
1512, 1400, 1243, 1076, 832. MS (ESI, MeOH + CH2Cl2, m/z):
Acknowledgment. We are grateful to Prof. G. Pattenden
for providing the NMR spectra and [R]D value of 4 and to ICSN
(CNRS, Gif-sur-Yvette) for a grant (J.-C. L.).
1
550 [(MNa)+, 100]. H NMR (500 MHz, CDCl3): δ 7.36-7.30
(m, 3H), 7.21 (d, 2H, J ) 7 Hz), 7.14 (d, 2H, J ) 8.5 Hz), 6.75 (d,
2H, J ) 8.5 Hz), 4.62 (d, 1H, J3,4 ) 9.2 Hz), 4.40 (d, 1H, J ) 15.3
Hz), 4.32 (d, 1H, J ) 15.3 Hz), 4.28 (d, 1H, J ) 11.9 Hz), 4.21 (d,
1H, J ) 11.9 Hz), 3.77 (masked d, 1H), 3.75 (s, 3H), 3.69 (d, 1H,
J ) 10.4), 3.42 (s, 3H), 2.71 (dq, 1H), 1.22 (d, 3H, J ) 7.6 Hz),
0.84 (s, 9H), -0.03 (s, 3H), -0.04 (s, 3H). 13C NMR (75.0 MHz,
CDCl3): δ 177.7, 170.0, 158.9, 137.4, 130.2, 129.3, 128.5, 128.1,
128.0, 113.6, 73.9, 73.3, 70.2, 67.2, 55.4, 51.8, 44.2, 40.8, 25.7,
18.0, 11.3, -4.7, -5.1. HRMS (ESI, MeOH + CH2Cl2, m/z): calcd
for C29H41NO6SiNa (MNa)+ 550.2601, found 550.2578.
Supporting Information Available: Experimental procedures
for compounds (()-7, 13, 14, 16, 17, 18, 19, (3aS,6R,6aR)-21, (()-
23, 24, 27, and 28 and 1H and 13C NMR spectra of all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO701968D
J. Org. Chem, Vol. 72, No. 26, 2007 10113