α,β-unsaturated β-silyl lmide substrates for catalytic, enantioselective conjugate additions: A total synthesis of (+)-lactacystin and the discovery of a new proteasome inhibitor

Article abstract of DOI:10.1021/ja061970a

Chiral (salen)Al μ-oxo dimer 1 catalyzes the highly enantioselective conjugate addition of carbon-centered nucleophiles to α,β-unsaturated silyl imides. Allyldimethylsilane-substituted imide 4 was identified as an optimal substrate, undergoing addition re

Full text of DOI:10.1021/ja061970a

Published on Web 05/06/2006
r,â-Unsaturated â-Silyl Imide Substrates for Catalytic, Enantioselective
Conjugate Additions: A Total Synthesis of (+)-Lactacystin and the Discovery
of a New Proteasome Inhibitor
Emily P. Balskus and Eric N. Jacobsen*
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received March 22, 2006; E-mail: jacobsen@chemistry.harvard.edu
Enantioenriched â-silyl carboxylic acid derivatives are versatile
building blocks for organic synthesis,1 allowing subsequent dias-
tereoselective transformations2 and the possibility of stereospecific
Si-C bond oxidation.3 To date, enantioselective routes to these
compounds have relied mainly on auxiliary-based methods.4
Asymmetric, catalytic conjugate addition of C-centered nucleophiles
to â-silyl-R,â-unsaturated acid derivatives represents a most at-
tractive, but relatively undeveloped, alternative.5,6 We describe here
the application of a new â-silyl unsaturated imide in highly
enantioselective, catalytic Michael additions. This methodology
serves as the basis for a concise total synthesis of the natural product
(+)-lactacystin and of a new, synthetic 26S proteasome inhibitor
Figure 1. Retrosynthesis of (+)-lactacystin.
with comparable activity to omuralide.
Aluminum salen complexes, such as µ-oxo dimer 1, have proven
effective for a variety of asymmetric catalytic conjugate additions,7
including Michael reactions of electron-deficient nitrile nucleophiles
with R,â-unsaturated imides.8 Addition of aminocyanoacetate
derivatives can result in direct formation of γ-lactam derivatives
with high enantio- and diastereoselectivity. We recognized the
potential applicability of this methodology to the preparation of
important γ-lactam-containing natural products, such as the pro-
teasome inhibitor lactacystin (2).9 Over the last 14 years since Corey
first accomplished the total synthesis of 2,10 there have been
numerous approaches to lactacystin reported, including several
elegant asymmetric catalytic routes.11,12 One of the key challenges
in any lactacystin synthesis is the efficient, stereoselective construc-
tion of the functionalized γ-lactam. In evaluating this target, we
considered methods by which asymmetric catalysis could allow
quick access to a core lactam structure amenable to further
elaboration. In particular, use of a â-silyl imide substrate in an
asymmetric 1,4-addition (Figure 1) could provide an appropriately
functionalized intermediate for the synthesis of 2 and related
structures, and might further allow general access to a variety of
useful â-silyl carboxylic acid derivatives in enantioenriched form.
Initial screening of (N-p-methoxybenzylamino) cyanoacetate
addition to various R,â-unsaturated â-silyl imides in the presence
of 10 mol % of (R,R) µ-oxo dimer 1 led to the identification of
allyldimethylsilyl imide 413 as a promising substrate (95% ee, 5:1
dr, 28% yield).14 Increasing the amount of tert-butyl alcohol (from
1.2 to 2.0 equiv relative to imide) and lowering reaction concentra-
tion (from 0.5 to 0.1 M) led to substantially improved product yields
while maintaining high enantioselectivity (Table 1, entry 1). The
synthesis of lactam 5a was performed on either milligram or
multigram scale with similar results. Gratifyingly, a variety of other
nitrile-bearing nucleophiles also underwent reaction with 4 with
high enantioselectivity under the optimized conditions (entries 2-8).
Both unsubstituted and aryl-substituted cyanoacetate derivatives are
useful substrates, in the latter case affording enantioenriched
products bearing quaternary stereocenters with good diastereose-
lectivity (entries 5-7).
Table 1. Enantioselective Conjugate Additions to Silyl Imide 4
a Isolated yield, after chromatography, of diastereomerically pure material
unless noted otherwise (0.2 mmol scale). b Determined by HPLC using
commercial chiral columns unless noted otherwise. c Determined by 1H
NMR. d Inseparable from unreacted nucleophile and minor diastereomer;
yield determined by 1H NMR. e Determined by SFC after reduction of the
ester with NaBH4. f 25 mmol scale. g Inseparable from unreacted nucleo-
phile, yield calculated by 1H NMR analysis. h Determined by SFC. i 5.0
mol % of catalyst was used.
With lactam 5a in hand, we undertook the total synthesis of
lactacystin (Scheme 1). Separation of the diastereomers of 5a proved
9
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10.1021/ja061970a CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Enantioselective Total Synthesis of (+)-Lactacystina
Figure 2. Inhibition of the proteasome by omuralide (3).
of the carboxylic acid toward eventual displacement by N-acetyl
cysteine. In attempts to activate the C-6 hydroxyl toward displace-
ment, it was discovered that treatment of 9 with various sulfonyl
chloride and anhydride derivatives led instead to facile spiro
â-lactone formation.17,18 With prior hydrogenation of 9 to the
corresponding isopropyl derivative, sulfonylation with a large excess
of Tf2O resulted in â-lactone construction and triflate formation in
one pot to provide 10. All of the electrophilic functionality needed
for the final steps of the synthesis was thereby established in an
efficient manner.
Invertive triflate displacement was accomplished cleanly in the
presence of the â-lactone by treating 10 with NaNO2 in DMF.19
Removal of the N-PMB group provided a mixture of lactam 11
and p-anisaldehyde. The stability of the spiro â-lactone during these
steps was particularly notable, with no decomposition of the strained
bicyclic framework detected at any stage. Reaction of 11 with
N-acetyl-L-cysteine in the presence of dimethylethylamine afforded
(+)-lactacystin (2).
Interest in lactacystin is tied to its important biological activity:
it is a potent, yet selective inhibitor of the 26S proteasome, the
protein complex involved in ubiquitin-mediated protein degrada-
tion.20 This complex has emerged as an important clinical target,
especially in the context of cancer treatment.21 Studies by Corey
and Schreiber led to identification of the proteasome as the target
of lactacystin and implicated clasto-lactacystin â-lactone (omuralide,
3, Figure 1) as the active agent in the inhibition mechanism.9,22
Omuralide, generated by intramolecular attack of the C-6 hydroxyl
on the adjacent thioester, is cell-permeable and serves as an
acylating agent toward the catalytic N-terminal threonine residue
of the proteasome’s chymotrypsin-like site (Figure 2).23 The
resulting acyl enzyme complex has been characterized by X-ray
crystallography.24
SAR studies carried out by the Corey group were key in
identifying the structural features responsible for the activity of
lactacystin.9a,25 Most relevant to our own synthetic efforts, analogues
that were epimeric at C-6 or lacked the C-6 hydroxyl displayed
either significant or complete loss of activity. Corey ascribed these
observations to the inability of such compounds to form the requisite
â-lactone.
Our results show that C-6 epimeric intermediates readily form
spiro â-lactones, which undergo ring opening with thiol nucleophiles
in a manner similar to that of omuralide. This reactivity pattern
observed with 11 led us to consider whether it might extend to
interaction with the proteasome. Though a spiro â-lactone inter-
mediate has been postulated to explain the weak activity of 6-epi-
lactacystin,9a to our knowledge, such compounds have not been
examined in the context of proteasome inhibition.
a Reagents and conditions: (a) LHMDS, THF, -78 °C, 30 min, then
MeI, -78 to 0 °C over 4 h, 9:1 dr; (b) NaBH4, MeOH, 0 °C, 6 h; 60%
from 4 (3 steps); (c) Dess-Martin periodinane, pyridine, CH2Cl2, rt, 1.5 h;
(d) isopropenyl MgBr, THF, -78 °C, 4 h; 73% (2 steps), >95:5 dr (addition
to major isomer); (e) Red-Al, toluene, -78 to 0 °C over 2 h, then 1.0 M
tartaric acid; (f) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH, H2O, rt,
35 min; (g) H2O2, KF, KHCO3, DMF, H2O, rt, 11 h; 49% (3 steps) of a
single diastereomer; (h) H2, 10% Pd/C, AcOH, MeOH, rt, 2 h; 94%; (i)
Tf2O, DMAP, pyridine, CH2Cl2, 0 °C, 11 h; (j) NaNO2, DMF, rt, 1.5 h;
80% (2 steps); (k) CAN, MeCN, H2O, 0 °C, 10 h; (l) N-acetyl-L-cysteine,
Me2NEt, CH2Cl2, rt, 4 h; 68% (2 steps).
difficult, so the enriched mixture was employed through the early
steps. R-Methylation proceeded with complete diastereoselectivity,
and two-step transformation of the ethyl ester to the corresponding
aldehyde afforded 6 in 60% overall yield from â-silyl imide 4.
Treatment of 6 with isopropenylmagnesium bromide at -78 °C
provided alcohol 7 with excellent diastereoselectivity (>95:5).
Similarly selective addition reactions have been noted in closely
related systems.11a,b X-ray structural analysis of 7 allowed confirma-
tion of both the configuration at C-9 and the relative stereochemical
outcomes of the conjugate addition and methylation steps.
Formal hydrolysis of nitrile 7 was envisaged using a mild two-
step reduction/oxidation sequence. Unexpectedly, reduction of 7
with Red-Al was accompanied by intramolecular allyl group
displacement by the alkoxide intermediate and formation of cyclic
silyl ether 8,15 thereby imparting the beneficial effect of activating
the C-6-Si bond for subsequent oxidation. Furthermore, the
diastereomeric impurity that had been advanced from the original
conjugate addition step failed to undergo analogous cyclization.
Unpurified aldehyde 8 was subjected to oxidation to the corre-
sponding acid, and Si-C bond cleavage was accomplished under
standard Tamao oxidative conditions to provide diol 9 as a single
diastereomer after aqueous workup.16 With this protocol, the unacti-
vated diastereomeric impurity failed to undergo oxidation and was
removed simply by selective extraction from the reaction mixture.
At this point, completion of the synthesis required resolution of
two key issues: inversion of the C-6 stereocenter and activation
Spiro lactacystin â-lactone 11 along with omuralide (3) and 6-epi-
spiro-lactacystin â-lactone 1226 were assayed for inhibition of rabbit
muscle 26S proteasome using fluorogenic peptide substrates (Table
2).27 Analogue 12 was accessed from intermediate 9 by a route
analogous to that used in the lactacystin synthesis, omitting triflate
formation and invertive substitution by a hydroxyl equivalent.
Interestingly, spiro â-lactone 11 was found to inhibit all three
9
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C O M M U N I C A T I O N S
Table 2. Effect of Inhibitors on Peptidase Activities of Rabbit
Am. Chem. Soc. 1998, 120, 10270-10271. (c) Barnhart, R. W.; Wang,
X.; Nobeda, P.; Bergens, S. H.; Whelan, J.; Bosnich, B. J. Am. Chem.
Soc. 1994, 116, 1821-1830. (d) Naasz, R.; Arnold, L. A.; Minnaard, A.
J.; Feringa, B. L. Angew. Chem., Int. Ed. 2001, 40, 927-930. (e) Evans,
D. A.; Janey, J. M. Org. Lett. 2001, 3, 2125-2128.
Muscle 26S Proteasomea
substrate
3 (10
µ
M)b
11 (10
µ
M)b
12 (200 µM)
% inhibition of
chymotrypsin-like site
87.4 ( 3.7
15.5 ( 3.9
35.6 ( 0.2
83.1 ( 3.2
20.5 ( 5.0
24.6 ( 2.8
19.1
(5) For reviews on enantioselective conjugate addition reactions, see: (a)
Tomioka, K.; Nagaoka, Y.; Yamaguchi, M. In ComprehensiVe Asymmetric
Catalysis; Jacobsen, E. N., Pfalz, A., Yamamoto, H., Eds.; Springer:
Berlin, 1999; Vol. 3, Chapters 31.1 and 31.2. (b) Sibi, M. O.; Manyem,
S. Tetrahedron 2000, 56, 8033-8061.
(6) For catalytic, enantioselective conjugate additions using silicon-containing
substrates, see: (a) Masumoto, Y.; Hayashi, T.; Ito, Y. Tetrahedron 1994,
50, 335-346 and references therein. (b) Shintani, R.; Okamoto, K.;
Hayashi, T. Org. Lett. 2005, 7, 4757-4759. (c) Lipshutz, B. H.; Tanaka,
N.; Taft, B. R.; Lee, C.-T. Org. Lett. 2006, ASAP.
% inhibition of
caspase-like site
no inhibition
no inhibition
% inhibition of
trypsin-like site
a Purified 26S proteasome was used at a concentration of 1 µg/mL.
Peptidase activity was measured using fluorogenic substrates specific for
each site, in the presence of inhibitors or 1% DMSO as a control. b The
results shown represent the mean ( the range of two experiments, each
run in duplicate.
(7) Gandelman, M.; Jacobsen, E. N. Angew. Chem., Int. Ed. 2005, 44, 2393-
2397 and references therein.
(8) Taylor, M. S.; Jacobsen, E. N. J. Am. Chem. Soc. 2003, 125, 11204-
11205.
(9) For studies concerning the biological activity of lactacystin, see: (a)
Fenteany, G. A.; Standaert, R. F.; Reichard, G. A.; Corey, E. J.; Schreiber,
S. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3358-3362. (b) Fenteany,
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L. Science 1995, 268, 726-731.
proteolytic subunits at similar levels as omuralide (3) under identical
conditions. In contrast, the epimeric spiro â-lactone 12 was inactive
at concentrations below 200 µM. Though the possibility that spiro
â-lactone 11 could undergo isomerization into omuralide under the
assay conditions must also be considered, this appears unlikely given
the observed stability of 11.28 Rather, these data indicate that the
position of the â-lactone is not important for activity, and that the
configuration at C-6 is critical for reasons other than â-lactone
formation. Recent work has demonstrated the importance of a
hydrogen bond between the C-6 hydroxyl and the N-terminal amino
group of the proteasome subunit in stabilizing the omuralide-
proteasome complex (Figure 2); the presence of the C-6 hydroxyl
prevents the binding of the water molecule necessary for hydroly-
sis.29 An analogous hydrogen bond is revealed in the crystal
structure of the proteasome bound to the dipeptide boronic acid
inhibitor bortezomib.30 These results with 11 and 12 lend additional
support to the importance of this H-bond interaction as a guiding
principle in the design of new proteasome inhibitors.
The total synthesis of lactacystin was accomplished in 13 steps
and 11.0% overall yield from silyl imide 4. The route is efficient,
requiring a single protecting group and only five chromatographic
purifications. The pursuit of lactacystin as a target inspired the
design of a new imide substrate for aluminum salen-catalyzed
conjugate additions. Moreover, an unusual spiro â-lactone was
employed for the first time as an intermediate in total synthesis,
allowing for a novel lactacystin end-game strategy. Spiro â-lactone
11 has proven interesting in a completely different context, as well,
as an inhibitor of the 26S proteasome with similar potency to the
known inhibitor omuralide.
(10) Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc. 1992, 114, 10677-10678.
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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. For
recent total syntheses, see: (c) Donohoe, T. J.; Sintim, H. O.; Sisangia,
L.; Harling, J. D. Angew. Chem., Int. Ed. 2004, 43, 2293-2296. (d) Ooi,
H.; Ishibashi, N.; Iwabuchi, Y.; Ishihara, J.; Hatakeyama, S. J. Org. Chem.
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(12) For approaches to lactacystin involving asymmetric catalysis in key steps,
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Tanaka, H.; Omura, S.; Sprengler, P. A.; Smith, A. B., III. J. Am. Chem.
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(14) For details of imide synthesis, see Supporting Information.
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Acknowledgment. This work was supported by the NIH (GM-
59316) and by a Predoctoral Fellowship from the National Science
Foundation to E.P.B. We thank Prof. Alfred L. Goldberg and Alice
Callard from the Department of Cell Biology at the Harvard Medical
School for materials and assistance in carrying out the 26S
proteasome assay. Dr. R. Staples carried out the X-ray structural
analysis.
(22) Corey, E. J.; Reichard, G. A.; Kania, R. Tetrahedron Lett. 1993, 34, 6977-
6980.
(23) (a) Dick, L. R.; Cruikshank, A. A.; Grenier, L.; Melandri, F. D.; Nunes,
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Cruikshank, A. A.; Destree, A. T.; Grenier, L.; McCormack, T. A.;
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Supporting Information Available: Experimental procedures, ee
analyses, characterization data for all new compounds, details of
proteasome assay experiments, and X-ray coordinates for 7. This
material is available free of charge via the Internet at http://pubs.acs.org.
(26) For details of the synthesis of 12, see Supporting Information.
(27) (a) Kisselev, A. F.; Goldberg, A. L. In Methods of Enzymology; Deshaies,
R. J., Ed.; Elsevier: Amsterdam, 2005; Vol. 398, pp 364-378. (b)
Kisselev, A. F.; Callard, A.; Goldberg, A. L. J. Biol. Chem. 2006, 281,
8582-8590.
(28) â-Lactone 11 does not undergo conversion to 3 upon long-term storage
as a stock solution in DMSO; we are currently investigating its behavior
under assay conditions.
(29) (a) Groll, M.; Huber, R.; Potts, B. C. J. Am. Chem. Soc. 2006, 128, 5136-
5141. (b) Groll, M.; Larionov, O. V.; Huber, R.; de Meijere, A. Proc.
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