Bioinspired total synthesis and human proteasome inhibitory activity of (-)-salinosporamide A, (-)-homosalinosporamide A, and derivatives obtained via organonucleophile promoted bis-cyclizations

Article abstract of DOI:10.1021/jo101638r

A full account of concise, enantioselective syntheses of the anticancer agent (-)-salinosporamide A and derivatives, including (-)-homosalinosporamide, that was inspired by biosynthetic considerations is described. The brevity of the synthetic strategy stems from a key bis-cyclization of a β-keto tertiary amide, which retains optical purity enabled by A^1,3-strain rendering slow epimerization relative to the rate of bis-cyclization. Optimization studies of the key bis-cyclization, enabled through byproduct isolation and characterization, are described that ultimately allowed for a gram scale synthesis of a versatile bicyclic core structure with a high degree of stereoretention. An optimized procedure for zincate generation by the method of Knochel, generally useful for the synthesis of salino A derivatives, led to dramatic improvements in side-chain attachment and a novel diastereomer of salino A. The versatility of the described strategy is demonstrated by the synthesis of designed derivatives including (-)-homosalinosporamide A. Inhibition of the human 20S and 26S proteasome by these derivatives using an enzymatic assay are also reported. The described total synthesis of salino A raises interesting questions regarding how biosynthetic enzymes leading to the salinosporamides proceeding via optically active β-keto secondary amides, are able to maintain the stereochemical integrity at the labile C2 stereocenter or if a dynamic kinetic resolution is operative.

Full text of DOI:10.1021/jo101638r

pubs.acs.org/joc
Bioinspired Total Synthesis and Human Proteasome Inhibitory Activity of
(-)-Salinosporamide A, (-)-Homosalinosporamide A, and Derivatives
Obtained via Organonucleophile Promoted Bis-cyclizations
Henry Nguyen,† Gil Ma,†,§ Tatiana Gladysheva,‡ Trisha Fremgen,‡ and Daniel Romo*,†
†Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012,
United States, and Genzyme Corporation, Drug and Biomaterial R & D, 153 Second Avenue, Waltham,
‡
Massachusetts 02451, United States. §Current address: Lundbeck Research USA, Inc., Paramus, NJ 07652.
romo@tamu.edu
Received August 25, 2010
A full account of concise, enantioselective syntheses of the anticancer agent (-)-salinosporamide
A and derivatives, including (-)-homosalinosporamide, that was inspired by biosynthetic con-
siderations is described. The brevity of the synthetic strategy stems from a key bis-cyclization of a
β-keto tertiary amide, which retains optical purity enabled by A1,3-strain rendering slow epimer-
ization relative to the rate of bis-cyclization. Optimization studies of the key bis-cyclization,
enabled through byproduct isolation and characterization, are described that ultimately allowed
for a gram scale synthesis of a versatile bicyclic core structure with a high degree of stereoretention.
An optimized procedure for zincate generation by the method of Knochel, generally useful for the
synthesis of salino A derivatives, led to dramatic improvements in side-chain attachment and a
novel diastereomer of salino A. The versatility of the described strategy is demonstrated by the
synthesis of designed derivatives including (-)-homosalinosporamide A. Inhibition of the human
20S and 26S proteasome by these derivatives using an enzymatic assay are also reported. The
described total synthesis of salino A raises interesting questions regarding how biosynthetic enzymes
leading to the salinosporamides proceeding via optically active β-keto secondary amides, are able to
maintain the stereochemical integrity at the labile C2 stereocenter or if a dynamic kinetic resolution is
operative.
Introduction
great interest due to a desire to further explore biological
activity or to facilitate development of structure-activity
relationship profiles. Inevitably, one of the best ways to
achieve such a practical synthesis involves consideration of
The development of practical, scaleable total syntheses of
complex, bioactive natural products has recently become of
2
J. Org. Chem. 2011, 76, 2–12
Published on Web 11/03/2010
DOI: 10.1021/jo101638r
r
2010 American Chemical Society

Nguyen et al.
JOCFeatured Article
FIGURE 1. Structures of naturally occurring bicyclic β-lactone
proteasome inhibitors.
potential biosynthetic strategies utilized by the produc-
ing organism to assemble such stereochemically complex
structures.1
The potential of human 20S proteasome inhibitors con-
tinues to be of interest for cancer chemotherapy and the
recent FDA approval of bortezomib (Velcade) validates the
proteasome as a target for cancer chemotherapy.2 Isolated
by Fenical and co-workers3 from the marine actinomycete,
Salinispora tropica, salinosporamides A and B (1a,b, salino A
and B) are unique bicyclo[3.2.0] β-lactone-containing natu-
ral products (Figure 1). Phase I human clinical studies for
multiple myeloma followed the finding that salino A exhib-
ited great potential in mouse models toward several cancers
when administered intravenously despite the potentially
labile β-lactone.4 The terrestrial metabolites, cinnabaramides
FIGURE 2. Enantioselective strategy to salino A and derivatives.
(e.g., A, 2),5 lactacystin-β-lactone (omuralide, 3a) derived from
the prodrug lactacystin (4),6 and the recently isolated mixed
congener antiprotealide (3b),7 bear close structural similarities
to salino A and congeners, which have been targets for total8
and formal9 syntheses,10 structure-activity studies,11 biosyn-
thetic engineering,12 and crystallographic studies with the 20S
proteasome.13
Our synthetic strategy to salino A and derivatives utilizes
a nucleophile-catalyzed aldol-lactonization (NCAL) pro-
cess, a reaction we previously developed for the synthesis
of carbocycle-fused β-lactones (Figure 1).14 Indeed, salino A
and related natural products were the initial inspiration for
the development of the NCAL methodology in our group
(Figure 2). In particular, the study of an intramolecular aldol
lactonization was inspired by biosynthetic considerations of
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SCHEME 1. Synthesis of Ketene Homodimers 12/13 and
Heterodimers 14/15
TABLE 1. Synthesis of Simplified, C4-Unsubstituted Salino A
Derivatives 21a-e from Homodimers 12a-e
this class of proteasome inhibitors, and this general biosyn-
thetic pathway has recently been supported by detailed bio-
synthetic studies.12 We previously reported racemic synth-
eses of cinnabaramide A and salino A8e and more recently an
enantioselective synthesis of (-)-salino A and (-)-homosa-
lino A on the basis of these strategies.8j Retention of optical
purity during the bis-cyclization process was made possible
by the A1,3-strain of β-keto tertiary amides.15 Herein, we dis-
close a full account of our bioinspired syntheses of salino A
and derivatives including optimization of the key bis-cycliza-
tion process that simultaneously forms the γ-lactam and
fused β-lactone core. Furthermore, the bioactivity of these
derivatives was assayed against the chymotripsin-like, cas-
pase-like, and trypsin-like activities of the human protea-
some.
Our primary retrosynthetic disconnections, namely the bis-
cyclization (7 f 6) and amide coupling steps (8 þ 9 f 7), which
were inspired by biosynthetic considerations, are key steps in
our retrosynthetic analysis. The optical purity of β-lactone 6
would reflect the diastereomeric purity of β-ketoamide 7
derived from acylation of serine derivatives with racemic
ketene dimer 9. The C4 stereocenter in β-ketoamide 7 is lost
during the bis-cyclization process due to formation of an
ammonium enolate intermediate, thus the integrity of the C2
stereocenter primarily dictates the optical purity of β-lactone 6
which we proposed would be preserved by A1,3 strain.15 Intro-
duction of the C4-side chain initially relied on the strategy
developed by Corey,8a and in this report, we describe the use of
Knochel’s procedure for zincate formation;16 however, the
success of this process and subsequent manipulations was
initially not guaranteed given the potential reactivity of the
β-lactone.17
aYield is for two steps. bYields refer to isolated, purified (SiO2)
product. cDetermined by 1H NMR analysis of crude reaction mixtures.
dObserved diastereomeric ratio (dr) if reaction is allowed to proceed
at 25 °C for 36 h (54% yield). PMB=p-methoxybenzyl, 4-PPY =
4-pyrrolidinopyridine.
good yields (43-65%) following silica gel purification; how-
ever, heterodimers 14/15 provided the expected statistical
mixtures, and further reductions in yields were also noted dur-
ing purification leading to low absolute yields (5-13% yield,
20-52% based on a statistical, theoretical yield of 25%).
Importantly, these dimerizations are readily run on multi-
gram scale from inexpensive, commercially available acid
chlorides, and heterodimers 14/15, despite low absolute
yields, can also be obtained in multigram quantities follow-
ing silica gel purification.
With racemic ketene homodimers 12a-e in hand, coupling
with N-PMB-glycine benzyl ester (16) by the method of
Calter19 proceeded efficiently to provide keto acid substrates
19a-e following hydrogenolysis (Table 1). Bis-cyclization of
these keto acids was achieved using conditions similar to those
developed for carbocycles, with 4-pyrrolidinopyridine (4-
PPY) as nucleophilic promoter at 0 °C and proceeded effi-
ciently (70-93%) inmost cases togivebicyclic lactones21a-e
with moderate diastereoselectivity (dr, 2.2-4:1). Keto acid
Results and Discussion
Initial Studies of the Bis-cyclization Leading to C4-Unsub-
stituted Bicyclic β-Lactones. We first targeted simple C4-
unsubstituted substrates for the bis-cyclization. Ketene
homodimers 12/13 and subsequently employed (vide infra)
heterodimers 14/15 were prepared by dimerization of acid
chlorides 10/11 by the method of Sauer (Scheme 1).8e,j,18 As
expected, homodimers 12/13 were obtained in moderate to
(15) Evans, D. A.; Ennis, M. D.; Le, T.; Mandel, N.; Mandel, G. J. Am.
Chem. Soc. 1984, 106, 1154.
(16) Ren, H.; Dunet, G.; Mayer, P.; Knochel, P. J. Am. Chem. Soc. 2007,
129, 5376.
(17) Jacobsen and co-workers had previously demonstrated the stability
of a related spiro-β-lactone in their studies toward omuralide; see: Balskus,
E. P.; Jacobsen, E. N. J. Am. Chem. Soc. 2006, 128, 6810.
(18) (a) Sauer, J. C. J. Am. Chem. Soc. 1947, 69, 2444. (b) Purohit, V. C.;
Richardson, R. D.; Smith, J. W.; Romo, D. J. Org. Chem. 2006, 71, 4549. (c)
Duffy, R. J.; Morris, K. A.; Romo, D. J. Am. Chem. Soc. 2005, 127, 16754.
(19) Calter, M. A.; Orr, R. K.; Song, W. Org. Lett. 2003, 5, 4745.
4
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SCHEME 2. Total Synthesis of rac-Cinnabaramide A ((()-2)
19c gave high diastereoselectivity (>19:1) but in reduced yield
(54%; Table 1, entry 3); however, monitoring this reaction by
1H NMR at 25 °C indicated that the high selectivity was due to
selective degradation of the minor diastereomer by prolonged
reaction times. The C2-unsubstituted keto acid 19d gave only
25% yield (Table 1, entry 4) which may result from facile
enolization due to the absence of the C2-substitutent (R). This
likely leads to diminished rates of the initial aldol step.
Importantly, keto acid 19e bearing two primary chlorides thus
mimicking the substrate required for the proposed salino A
synthesis also proceeded efficiently (Table 1, entry 5).
SCHEME 3. Total Synthesis of rac-Salino A ((()-1a)
Total Synthesis of rac-Cinnabaramide A (2). Encouraged
by the results with C4-unsubstituted keto acid substrates, we
next studied more sterically demanding substrates (i.e., bear-
ing C4-substititution) by targeting the synthesis of cinnabar-
amide A (2). The synthesis commenced with reductive ami-
nation of commercially available (S)-O-benzylserine ((S)-22)
with p-anisaldehyde (Scheme 2). Subsequent esterification
provided the protected serine derivative 23a in 58% overall
yield (two steps). The required unsymmetrical ketene dimer
14a, derived from heterodimerization of acetyl and octanoyl
chlorides (5% yield),18a was coupled with serine derivative
23a to provide diastereomeric esters 24 (dr 1:1), and a
Me3SnOH-mediated hydrolysis20 provided keto acid 25.
The key bis-cyclization provided diastereomeric β-lactones 26/
27 in 45% yield with moderate diastereoselectivity (dr 3.3:1),
and NOE analysis confirmed that the major diastereomer
corresponded to that found in the cinnabaramides.21 Hydro-
genolysis of the benzyl ether facilitated separation of the major
alcohol diastereomer 28 (79%, dr >19:1), and this was followed
by Parikh-Doering oxidation22 to give an intermediate alde-
hyde, which was used directly in the next step. Applying the
method developed by Corey with zinc reagent 29 gave alcohol
30 in 57% yield (two steps, dr 4.7:1). Oxidative PMB deprotec-
tion with ceric ammonium nitrate gave rac-cinnabaramide
A ((()-2), which could be isolated diastereomerically pure in
48% yield. Spectral data for the synthetic material correlated
with the published data and further verification of the relative
stereochemistry was possible by X-ray analysis.21 This synthesis
demonstrated that several reactions could be performed in the
presence of the β-lactone including organozinc additions to
aldehydes.
Total Synthesis of rac-Salinosporamide A ((()-1a). In a
highly analogous manner to that described for cinnabara-
mide A, the synthesis of racemic salino A was accomplished,
and thus only key reactions are highlighted (Scheme 3). The
required heteroketene dimer 14b derived from acetyl chlo-
ride and commercially available 4-chlorobutanoyl chloride
could be obtained in multigram quantities by the method of
Sauer.18 The keto amide substrate 31 was then prepared by
coupling heteroketene dimer 14b and serine derivative 23b,
bearing an allyl ester which aided subsequent deprotection.
In this case, the unoptimized bis-cyclization proceeded to give
β-lactone 32 in 25-35% yield (dr 2-3:1); however, the relative
stereochemistry of the major diastereomer corresponded to
ꢀ
(20) Furlam, R. L. E.; Emesto, G.; Mata, E. G.; Masearetti, O. A.
Tetrahedron 1998, 54, 13023.
(21) See the Supporting Information for experimental details.
(22) Parikh, J. P.; Doering, W. E. J. Am. Chem. Soc. 1967, 89, 5505.
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SCHEME 4. Proposed Mechanism for the Bis-cyclization with Two Diastereomeric Keto Acid Substrates (a) (2R,4R)-34 from
(R)-Serine (b) (2R,4S)-40 from (S)-Serine
salino A as confirmed by subsequent conversion to the natural
product. Bicyclic β-lactone 32 was processed to racemic salino
A (1a) using a sequence identical to that described for cinna-
baramide A (cf. Scheme 2). Once again, it was gratifying
to verify that the potentially labile β-lactone and primary
chloride were compatible with several final reaction steps in
the synthesis.
β-lactone 39. While a [2 þ 2] cycloaddition mechanism via an
intermediate ketene has not been excluded,24 evidence to date
including reduced conversions upon varying nucleophilic
promoters (vide infra) is suggestive of nucleophile involve-
ment in the rate-determining or prior step. Indirect evidence
comes from previously reported highly enantioselective bis-
cyclization routes to carbocycle-fused β-lactones employing
chiral nucleophiles.14a,c
Optimization Studies of the Bis-cyclization. Our racemic
syntheses of cinnabaramide A and salino A demonstrated
the viability of our synthetic approach. However, with an
ultimate goal of developing an efficient, enantioselective
route to a versatile salino A core (i.e., β-lactone 32) amenable
to the synthesis of derivatives, we set out to identify reaction
conditions that would minimize formation of observed by-
products and most critically, epimerization of the C2 stereo-
center of β-keto acid 34 during the bis-cyclization. Our
working mechanism for this bis-cyclization invokes a nu-
cleophile catalyzed aldol-lactonization process and follows
from our previous studies with carbocycle-fused β-lactones
(Scheme 4).14 Activation of the carboxylic acid is followed by
transacylation with 4-PPY to provide acyl ammonium 36,
and deprotonation provides ammonium enolate 37. Equili-
bration to the reactive conformer 3700 enables subsequent
aldol reaction leading to aldolate 38. Lactonization,
which may occur via an “SN2” process,14c,23 then delivers
At the outset of our studies, we recognized that several
energetically favored conformers dictated by A1,3-strain,
e.g., conformers 37 and 370, while unproductive for aldol
lactonization since the ketone is not proximal to the ammo-
nium enolate,25 would preserve the enantiopurity of the
single stereocenter (C2). This would be possible since the
R-proton is nearly in plane with the amide carbonyl in these
conformations due to A1,3-strain (Scheme 4a).15 Further-
more, an expectedly higher energy conformation such as that
represented by 3700 is required to achieve bis-cyclization, and
because of the proximity and favorable formation of a
(24) For previous reports of β-lactones from keto acid derivatives via
proposed [2 þ 2] mechanisms, see: (a) Boswell, G. A.; Dauben, W. G.;
Ourisson, G.; Rull, T. Bull. Soc. Chim. Fr. 1958, 1598. (b) Kagan, H. B.;
Jacques, J. Bull. Soc. Chim. Fr. 1958, 1600. (c) Brady, W. T.; Gu, Y. Q. J. Org.
Chem. 1988, 53, 1353. (d) Reddy, L. R.; Corey, E. J. Org. Lett. 2006, 8,
1717For a previous report of an aldol-lactonzation pathway, see:. (e) Merlic,
C. A.; Marlog, B. C J. Org. Chem. 2003, 68, 6056. For an anionic version, see:
(f) Shindo, M.; Matsumoto, K.; Sato, Y.; Shishido, K. Org. Lett. 2001, 3,
2029.
(23) For lead references to this mechanistic possibility, see: (a) Adler, M.;
Adler, S.; Boche, G. J. Phys. Org. Chem. 2005, 18, 193. (b) Fox, J. M.;
Dmitrenko, O.; Liao, L.; Bach, R. D. J. Org. Chem. 2004, 69, 7317.
(25) The alternate N-C1 amide rotamer, which is also accessible energet-
ically but also unproductive for aldol lactonization, is not considered in this
analysis.
6
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TABLE 2. Optimization of the Bis-cyclization Leading to Bicyclic β-Lactone (()-32
reaction conditionsa
d
entry
act. agentb (equiv)
base (equiv)
nucc (equiv)
solvent
% yield 32/320
% yield 43-45e
1
2
3
4
5
20 (1.5)
20 (1.5)
20 (1.5)
MsCl (1.5)
MsCl (1.5)
i-Pr2NEt (3.0)
i-Pr2NEt (1.0)
4-PPY (1.5)
4-PPY (4.0)
4-PPY (7.0)
4-PPY (3.5)
DMAP (3.5)
CH2Cl2
CH2Cl2
CH2Cl2
PhMe
10
33
42
61
54
23 (43/44)
25 (43/44)
19 (43/45)
10f (rec SM 52)
8f (rec SM 52)
c
-
-
-
PhMe
aAll reactions were performed at -5 to -10 °C except entry 1, which was performed at 23 °C. bCarboxylic acid activating agent. Nucleophilic
promoter. dYields are given for two steps (bis-cyclization/allyl deprotection) and refer to isolated, purified (SiO2) products. eByproducts were not easily
separated, and thus, approximate yields are provided on the basis of 1H NMR (500 MHz) integration. fRecovered starting material, keto acid 52.
SCHEME 5. Proposed Mechanisms for Formation of Bypro-
ducts 43 and 44
5-membered lactam ring, aldol reaction might be expected
to be facile. What we did not anticipate was the impact of the
C4 absolute stereochemistry on the diastereoselectivity of the
bis-cyclization. In our early optimization studies, we deter-
mined that diastereoselectivity was highly dependent on
whether (S)- or (R)-serine-derived keto acids were employed
leading to different diastereoselectivities, 1:2 vs 8:1, respec-
tively. We suggest that the moderate diastereoselectivity
observed for the bis-cyclization is governed by developing
torsional strain between the methyl group (C3) of the ketone
and the C2 substituent (cf. 41, RSMe) during the initial aldol
reaction, leading to the preferred relative stereochemistry
following lactonization (cf. 39). Note that only a moderate
preference for this conformation (cf. 3700) over the alterna-
tive (cf. 41) is expected,26 and this is reflected in the maxi-
mum observed diastereoselectivity for the bis-cyclization (dr
4-7:1, vide infra). Regarding the C4 absolute stereochemis-
try dependence, this is likely a result of required conforma-
tions for deprotonation imposed by the N-C4 bond rotamer
of the two diastereomeric, acyl ammonium species 36 and
40 leading to ammonium enolates 37 and 41 and suggests
the intriguing possibility of a memory of chirality effect
which we are currently studying in related systems. With
acyl ammonium 36 derived from (R)-serine, deprotonation
leads directly to a N-C4 conformer that can access reactive
conformer 3700 leading to major diastereomer 39. Alterna-
tively, deprotonation of the diastereomeric acyl ammonium
40 derived from (S)-serine leads directly to reactive conformer
41 that delivers the diastereomeric β-lactone 42. This analysis
assumes a rapid bis-cyclization since enantiopurity could in-
deed be maintained in a non-Curtin-Hammett situation, if
once the higher energy reactive conformation is achieved, bis-
cyclization immediately takes place. For these reasons, we
utilized (R)-serine for the enantioselective synthesis (vide infra).
In early optimization studies of the bis-cyclization of keto
acid 52 directed toward salino A, we isolated and identified
three byproducts: enol lactone 43, cyclopropyl ketoamide 44,
and γ-lactam 45 (Table 1). Only the former two byproducts
€
were observed when both 4-PPY and Hunig’s base were
employed accompanied by low yields of β-lactones 32 and
320 (Table 1, entries 1 and 2). Enol lactone 43 is likely derived
from intramolecular acylation of enol 46 of the β-ketoamide
with the activated ester (Scheme 5). Cyclopropyl ketoamide
44 may be derived from a condensation reaction of enol 46
onto the activated acid leading to β-diketone 47, followed
by a retro-Claisen process induced by 4-PPY via 48, and
finally cyclization via enol 49 leading to the cyclopropane
(Scheme 5, red mechanism arrows). We next studied the use
of 4-PPY (7.0 equiv) as both base and nucleophilic promoter,
which reduced the amount of byproduct 43 and 44 with
attendant dramatic improvements in yield of β-lactone but
formation of a new byproduct, unsaturated γ-lactam 45
(Table 2, entry 3). Consideration of possible mechanisms
for formation of this byproduct suggested control experi-
ments to determine if lactam 45 was derived from decom-
position of β-lactones 32 and 320 via a decarboxylation
pathway. Subjecting β-lactones 32/320 to 4-PPY at 23 °C
indeed led to lactam 45 (31%) and interestingly recovered
β-lactones 32 and 320 with improved diastereomeric ratio
(dr, 5:1f9:1, Scheme 6). Decomposition was much slower at
(26) The preferred conformation of 2-butanone places the methyl and
carbonyl oxygen nearly in plane (eclipsed conformation). However, the energy
difference is low (∼1 kcal/mol); see: Eliel; E. L.; Wilen, S. H. Stereochemistry
of Oganic Compounds; Wiley & Sons: New York, 1994; pp 616-617.
€
-5 to -10 °C and was not observed if only Hunig’s base was
added to β-lactones 32 and 320. The increased diastereomeric
J. Org. Chem. Vol. 76, No. 1, 2011
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Nguyen et al.
JOCFeatured Article
SCHEME 6. Differential Decomposition of β-Lactones 32/320 Leading to Unsaturated γ-Lactam 45
TABLE 3. Variation of the C2-Side Chain: Inductive Effects on Effi-
ciency of Bis-cyclization
ratio suggests a differential rate of decomposition for the two
diastereomers. This may be explained by a lower barrier syn-
elimination (endothermic, late transition state) for β-lactone
320 due to a more accessible proton and release of torsional
strain (RSMe) which is absent in diastereomeric β-lactone
32. Finally, decarboxylation and protonation leads to the
observed unsaturated γ-lactam 45.
These studies highlighted the importance of minimizing
reaction time to avoid decomposition of β-lactone and to
minimize C2-deprotonation which avoids these byproducts
and maintains optical purity during the bis-cyclization pro-
cess. Dramatic improvements were therefore realized when
alternative activating agents and most importantly a less
polar solvent (e.g., toluene), which may slow C2-deprotona-
tion, were employed. Use of MsCl in toluene avoided
formation of all byproduct and yields of the β-lactones 32,
320 increased to 61% (Table 2, entry 4). Employing the less
nucleophilic dimethylaminopyridine (DMAP) also led to
β-lactone 32, 320 but in slightly reduced yields (54%) under
similar conditions suggestive of nucleophile involvement in
the rate-determining or prior step (Table 2, entry 5).
c
entry
R1
β-lactone
% yielda,b
δ Ha, Hb
1
2
3
(CH2)2Cl
(CH2)2OTBS
(CH2)3Cl
32/320
51a/51a0
51b/51b0
40
42
62
3.92, 3.94
3.92, 3.95
3.52, 3.57
aYield for two steps (deprotection and bis-cyclization). bYields refer
to isolated, purified β-lactones. cChemical shift of C2 protons (Ha/b) of
diastereomeric ketoamide esters 31, 50a,b.
entries 3 and 5) in comparison to other C2-alkyl substrates in
that series (90-93% yield).
Total Synthesis of (-)-Salinosporamide A. Having identi-
fied bis-cyclization conditions that improved yields and dia-
stereoselectivity, we turned our attention to the asymmetric
version of the bis-cyclization. The synthesis of salino A
began with reductive amination of commercially available
(R)-O-benzyl serine ((R)-22) (99% ee) with p-anisaldehyde
and subsequent esterification provided allyl ester (þ)-23b
(Scheme 7). After extensive optimization, the desired ester
(þ)-23b could be obtained with minimal racemization (98%
ee) in 79% yield (two steps) by careful control of reaction
temperature and duration.27 Acylation of serine derivative
(þ)-23b with unsymmetrical ketene dimer (()-14b under
microwave conditions gave diastereomeric β-ketoamides
31/310 (dr 1:1) in 80% yield. Separation of the diastereomers
by MPLC on mulitgram scale (3-4 g/run) provided the re-
quired (R,R)-β-ketoamide 31 (dr 30:1, 98% ee, 46%). Under
acidic conditions, the undesired diastereomer (2S,4R)-310
could be transformed to a 1:1 mixture of diastereomers via
epimerization of the C2 but not the C4 stereocenter (verified
by chiral HPLC), thus achieving an effective resolution of
ketene dimer (()-14b28 and greater material throughput.29
The impact of the chlorine atom on the acidity of the C2-
proton was investigated under the original bis-cyclization
conditions with a dual purpose of studying inductive effects
on conversion and byproduct formation, and demonstrating
the versatility of this synthetic strategy for C2-derivative
synthesis. Ketoamide substrate 50a was prepared from a
β-keto thioester while 50b was prepared from heteroketene
dimer 14c (not shown).21 Bis-cyclization of the keto acid
derived from allyl ester 50a bearing a γ-siloxy group (Table 3,
entry 2) led to similar results as those from ketoamide 31
bearing a γ-chloro substituent (salino core shown for compar-
ison, Table 3, entry 1); however the δ-chloro keto acid derived
from allyl ester 50b led to a significant increase in yield (Table 3,
1
entry 3). The yields obtained appear to correlate with the H
NMR chemical shifts of the C2 protons of β-ketoamides 31 and
50a,b (Table 3, entries 1-3). Comparison of Ha,b chemical
shifts of the γ-heteroatom substituted ketoamide 31 (Table 3,
entry 1) leading to the salino A core with that of the
δ-substituted ketoamides 50a-b (Table 3, entries 2, 3) shows a
downfield shift (Δδ ∼ 0.4) pointing to the inductive effects
and attendant increased acidity leading to byproduct forma-
tion imposed by the heteroatoms. Inductive effects leading to
reduced yields (70-85%) and greater byproduct formation
were also observed in simpler C4-unsubstituted substrates
bearing R-phenyl and β-chloro C2 substituents (Table 1,
(27) Optical purities were determined by chiral HPLC analysis (ref 21).
(28) Attempts to prepare the required optically active heteroketene dimer
14b by the method of Calter (ref 19) led to very low yields.
(29) Chiral HPLC analysis verified that epimerization only occurred at
C2 within limits of detection (ref 21).
8
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SCHEME 7. Nine-Step Enantioselective Synthesis of (-)-Salino A (1a) from (R)-OBn Serine ((þ)-22)
SCHEME 8. Mechanism of Inhibition of the Proteasome (CT
Site) with Salino A (1a13a) and Proposed for Homosalino A (56)
SCHEME 9. Varying the C2-Side Chain: Synthesis of
(-)-Homosalino A (54)
€
modified Mukaiyama’s reagent but without Hunig’s base
led to similar yields and diastereoselectivity (40%, dr 2.5:1)
obtained previously, with an attendant erosion of optical purity
of β-lactone 32 (85% ee). As described in our optimization
studies above, we ultimately found that MsCl at lower tempera-
ture with less polar solvents led to increased diastereoselectivity.
When these conditions were utilized, the diasteroeselectivity
improved (7:1) and minimal epimerization (∼3%) was observed
providing bicyclic β-lactone 32 in 35% yield and 92% ee.
Further improvements in yield up to 60% were realized with
longer reaction times under these same conditions; however, this
led to reduced diastereoselectivity and enantiopurity (dr 4:1,
88% ee). Importantly, the bis-cyclization could be performed on
Optimization of the Pd(0)-mediated allyl deprotection of
ester (R,R)-31 was also required to minimize epimerization
of the C2 stereocenter and ultimately delivered keto acid 52
with neglibile erosion of the diastereomeric purity.
At the outset of the studies toward an asymmetric process, it
was unclear whether the A1,3-strain induced conformational
bias in β-keto tertiary amides (∼4 kcal/mol) would be sufficient
to avoid epimerization of this center in the time frame and under
the basic conditions of the bis-cyclization. In our initial studies,
application of bis-cyclization conditions developed for the
racemic series8e to keto acid 52 (dr ∼29:1, 98% ee) using
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Nguyen et al.
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SCHEME 10. Varying the C4-Side Chain of Salino A
TABLE 4. IC50 Values for Inhibition of the Human 20S and 26S Proteasome by Salino A Derivatives in a Luminogenic Enzymatic Assaya
IC50
entry
compd
CT-L activity 20S/26S (nM)
C-L activity 20S/26S (nM)
T-L activity 20S/26S (nM)
260 ( 29b/412 ( 83
39 ( 7/37 ( 16
1
2
3
4
5
6
rac-cinnabaramide A, (()-2
(-)-salino A (1a) (92% ee)
rac-phenyl deriv (61)
rac-diMePh (63)
(-)-homosalino (54) (88% ee)
velcade
2.8 ( 0.6/2.2 ( 0.04
0.8 ( 0.08/2.5 ( 1.3
29 ( 0.7/57 ( 20
inactiveb,c
0.7 ( 0.04/2.3 ( 1.1
2.6 ( 0.7/7.4 ( 1.9
125 ( 12b/230 ( 24b
111 ( 22/156 ( 18
788 ( 50/1585 ( 633
inactiveb,c
144 ( 12/188 ( 3
25 ( 7/72 ( 17
702 ( 235/589 ( 120
inactiveb,c
118 ( 28/125 ( 30
254 ( 24/680 ( 150
aIC50 values are the mean ( standard deviation of 4 or more experiments. bThe average of two experiments. cEssentially inactive at >99,000 nM.
(CT-L: chymotrypsin-like activity; C-L: caspase-like activity; T-L: trypsin-like activity)
gram scale with comparable diastereoselectivity and retention of
enantiopurity (52% over two steps, dr 5:1, 90% ee).
could be formed at the active site of the proteasome in analogy
to the known formation of a tetrahydrofuran.
The synthesis followed an identical route as for salino A
but employed heteroketene dimer (()-14c with serine deri-
vative (þ)-23b to provide the homologous ketoester (-)-58
(Scheme 9). Bis-cyclization following ester deprotection
provided the bicyclic-β-lactone (-)-59 in 60% yield (dr
3.5:1). Using an identical sequence as described for salino
A, the β-lactone core was converted to (-)-homosalino A
((-)-54, 88% ee).21
Variation of the C4-Side Chain of the Salino Core. Our
synthetic strategy also enables facile variation of the C4-side
chain by addition of organometallic reagents to the ver-
satile aldehyde obtained by oxidation of alcohol (()-53
(Scheme 10). Addition of Grignard reagents (dr ∼3:1) fol-
lowed by PMB deprotection provided the known phenyl12c
and novel dimethylphenyl salino derivatives (()-61 and
(()-63, respectively. The relative stereochemistry of the major
diastereomer of phenyl derivative (()-61 was verified by single-
crystal X-ray analysis.21
Biological Studies. The salino A derivatives synthesized
were analyzed for their ability to inhibit the chymotrypsin-like
(CT-L), caspase-like (C-L), and trypsin-like (T-L) activities of
the 20S and 26S human proteasome in a luminogenic enzy-
matic assay (Table 4). Both velcade (entry 6) and synthetic
salino A (92% ee, entry 2) were included as controls and data
obtained correlates well with previous analyses.13 As expected
on the basis of known structure-activity relationships,7,13a
changes made to the C5-side chain in general led to lower
activity with drastic loss of activity observed for the rac-
dimethylphenyl derivative 63. The potency of the racemic
phenyl derivative against the human 20A proteasome was
much greater than that reported previously for the optically
active phenyl derivative against the yeast 20S proteasome.12c
However, as previously noted, changes in the C2-side chain
are more tolerable as indicated by (-)-homosalino A (54,
Table 4, entry 5), and indeed, this derivative exhibited very
similar activity to the natural product with the exception of
decreased inhibition of the trypsin-like activity of the 20S
proteasome (∼3ꢀ decrease).
Completion of the salino A synthesis entailed hydrogeno-
lysis to provide alcohol (-)-53, which could be separated at
this stage from the minor diastereomer produced during the
bis-cyclization process (75% yield). Modified Moffatt oxida-
tion30 and addition of the zinc reagent 29 derived from the
reaction of n-butyllithium with cyclohexenyltributyltin and
ZnCl2 following the procedure of Corey8a gave a mixture of
C5,C6-diastereomeric alcohols (dr 11:3:1:1) in 62% yield
(twosteps, Scheme7, procedureA (“proc A”)) withthe desired
diastereomer 33 as the major adduct. Alternately, zinc rea-
gent 290 could be prepared directly from commercially avail-
able 3-bromocyclohexene and activated zinc by the method of
Knochel,16 which greatly simplified product purification since
tin byproducts were avoided (Scheme 7, proc B). Most im-
portantly, this protocol gave only two diastereomers 33/330
(dr 4:1), improved the yield to 74%, and avoided the use of
toxic tin reagents. This procedure also enabled isolation of a
novel salino A diastereomer 1a0 (C5, C6-bis-epi-salino A, not
shown) whose relative stereochemistry was verified by X-ray
analysis.21 Finally, deprotection of PMB-protected lactam
(-)-33 led to pure (-)-salino A (1a) which correlated well
with spectroscopic data reported previously (syn [R]23
D
-71.3; lit. [R]25D -72.9).3
Total Synthesis of (-)-Homosalinosporamide A (Homosalino
A). In our optimization studies described above, we altered the
C2-side chain with the dual purpose of studying inductive
effects (cf. Table 3) and providing the one carbon homologated
side chain that could ultimately lead to (-)-homosalino A (54).
Groll and co-workers have proposed that acylation of the
proteasome by the β-lactone of salino A at the catalytic
N-terminus (Thr 1) is followed by cyclization of the incipient
alcohol with the pendant chloroethyl group leading to the
formation of a tetrahydrofuran as verified by X-ray analysis
(Scheme 8).13a This led us to consider the synthesis of homo-
salino A (54) to determine if a tetrahydropyran (cf. 57, n=2)
(30) (a) Pfitzner, K. E.; Moffatt, J. G. J. Am. Chem. Soc. 1965, 87, 5661.
(b) Coulton, S.; Southgate, R. J. Chem. Soc., Perkin Trans. 1 1992, 961.
10 J. Org. Chem. Vol. 76, No. 1, 2011

Nguyen et al.
JOCFeatured Article
In summary, we developed a concise, nine-step enantiose-
lective route to (-)-salino A and derivatives from O-benzyl
serine. The key bis-cyclization of a β-ketoamide, amenable to
gram scale, constructs both the γ-lactam and the fused-β-
lactoneinone operationleading to the brevityof the synthesis.
The flexibility of the described strategy derives from the
versatility of the bicyclic core (-)-32 enabling attachment of
various C4-side chains, even in the presence of the β-lactone,
and the ability to use alternative ketene dimers 14 to vary the
C2side chain. Several derivativesincluding(-)-homosalino A
were synthesized, and their activity toward proteasome in-
hibition was measured. The ability of the described β-keto
tertiary amide substrates to maintain stereochemical integrity
by virtue of A1,3 strain raises interesting questions regarding
how such integrity is maintained with β-keto secondary
amides, known salino A precursors,4c or if a dynamic kinetic
resolution is operative during related biosynthetic aldol
lactonizations.31 The synthesis of additional hypothesis-dri-
ven salino derivatives by the described strategy, their bioac-
tivity, and crystallographic studiesof their interaction withthe
proteasome will be reported in due course.
129.2(2C), 128.6, 128.5(2C), 128.2, 128.0(2C), 113.9(2C), 83.4,
79.3, 73.5, 61.6, 55.2, 45.0, 44.3, 42.5, 28.4, 19.2; HRMS (ESI)
Calcd. For C24H27ClNO5 [M þ H] 444.1578, found 444.1590. The
enantiomeric excess of (-)-32 was determined to be 92% by chiral
HPLC (CHIRALPAK IA, 250 ꢀ 4.6 mm (L ꢀ I.D.), solvent
(isocratic) 87:13 hexanes/2-propanol, flow rate 1.0 mL/min, λ=
230 nm). Retention times: (-)-32, 13.68 min; (þ)-32, 16.12 min.
Hydroxy β-Lactone (-)-53. To a mixture of β-lactones (-)-32
and 320 (260 mg, 0.586 mmol, dr 7:1) in THF was added
palladium on carbon (52 mg, 20 wt %). After the flask was
evacuated twice by aspirator vacuum and refilled with H2, a
balloon of H2 was attached to the flask and the heterogeneous
solution was stirred vigorously at 23 °C for 12 h. The reaction
mixture was then diluted with Et2O and dried over MgSO4. The
organics were filtered through a pad of Celite, concentrated, and
purified by MPLC (SiO2, 5:95 EtOAc/CH2Cl2) to give the desired
alcohol(-)-53in75%yield (155 mg, dr>19:1, 92% ee) as a waxy
solid: Rf=0.29 (5:95 EtOAc/CH2Cl2); [R]23D=-67.0 (c=0.95,
1
CHCl3). Data for (-)-53: IR (neat) 3449, 1831, 1687 cm-1; H
NMR (500 MHz, CDCl3) δ 7.30 (d, J = 8.5 Hz, 2H), 6.89 (d, J =
8.5 Hz, 2H), 5.13 (d, J = 15.0 Hz, 1H), 4.06 (d, J = 15.5 Hz, 1H),
4.03 (ddd, J = 5.5, 7.5, 12.5 Hz, 1H), 3.92 (dd, J = 9.0, 13.5 Hz,
1H), 3.85 (dd, J = 4.5, 13.5 Hz, 1H), 3.80 (s, 3H), 3.78-3.82 (m,
1H), 2.94 (t, J=7.0 Hz, 1H), 2.32-2.38 (m, 1H), 2.01-2.18 (m,
1H), 1.77 (s, 3H), 0.86 (dd, J = 5.0, 9.5 Hz, 1H); 13C NMR (125
MHz, CDCl3) δ 174.2, 166.7, 159.6, 129.0(2C), 128.7, 114.7(2C),
83.6, 80.2, 55.3, 55.1, 44.9, 44.1, 42.4, 28.4, 19.1; HRMS (ESI)
Calcd. For C17H21ClNO5 [M þ H] 354.1108, found 354.1098.
Representative Procedure for Modified Moffatt Oxidation and
Zincate Formation/Addition As Described for N-PMB-salino A,
(-)-33. To a solution of alcohol (-)-53 (42 mg, 0.119 mmol, dr
>19:1) in DMSO/toluene (0.9 mL/0.9 mL) were added EDCI
(0.114 mg, 0.595 mmol) and dichloroacetic acid (5 μL, 0.060
mmol) at 23 °C. The reaction mixture was stirred for 6 h and then
diluted with EtOAc (100 mL). The reaction mixture was acidified
using 0.1 N HCl to pH 3. The organic layer was then washed with
brine (60 mL), dried over MgSO4, filtered, and concentrated. The
crude aldehyde was used for the next step without further puri-
fication due to some instability of the aldehyde observed upon
flash column chromatography.
Preparation of a stock solution of zincate 290: Zinc dust
(1.64 g, 25.0 mmol, Baker, 20 mesh) was activated by the addition
of an HCl solution (5 mL, 1.0 M) in a 25 mL round-bottomed flask.
After the mixture was stirred vigorously for 15 min at 23 °C, the acid
solution was decanted and the zinc was repeatedly washed with dry
THF (3 ꢀ 5 mL). The zinc was then dried under high vacuum for 2
h. Addition of dry THF (5 mL) was followed by slow addition of
3-bromocyclohexene (0.806 g, 5.0 mmol) over 15 min. The resulting
mixture was stirred at 23 °C for 18 h and then transferred via syringe
to a dry centrifuge tube to remove the excess zinc suspension (10
min, 80 rpm). The resulting clear solution was then utilized directly,
following titration as described below, being careful not to
disturb the pellet at the bottom during transfers. The concentra-
tion of the zincate 290 solution was determined as follows: A
stock solution of iodine in THF was prepared by addition of
iodine (254 mg, 1.0 mmol) in 10 mL of THF leading to a 100 μM
solution. A portion (500 μL) of this iodine solution was placed
into a dry 5 mL round-bottomed flask equipped with a magnetic
stir bar. The zincate solution, after being centrifuged, was added
dropwise into the iodine stock solution until the red color
disappeared. Using this procedure, the prepared zincate solu-
tion was determined to be ∼200 μM and was used (in excess)
directly in the following reaction.
Experimental Section
(2R,4R)-β-Keto Acid, 52. To a solution of ketoamide (2R,4R)-
31 (2.30 g, 4.55 mmol, ∼32:1 dr) in THF (91.0 mL) at -5 °C (ice
and saturated NaCl solution) was added Pd(PPh3)4 (526 mg,
0.455 mmol), followed by immediate addition of morpholine
(0.475 mL, 5.46 mmol). The reaction mixture was stirred at -5 °C
for 70 min and then diluted with ice-cold Et2O (800 mL). A 0.02 N
HCl solution was added until the pH was measured to be ∼3. The
layers were separated, and the organic layer was washed with brine
(400 mL), dried over MgSO4 ,and concentrated. The crude keto acid
(2R,4R)-52 (∼ 29:1 dr according to 500 MHz 1H NMR) was used
directly in the subsequent step without further purification. (Note:
longer reaction times led to epimerization).
Gram-Scale Bis-cyclization Leading to Benzyloxy β-Lactone
(-)-32/320. To a solution of 4-pyrrolidinopyridine (2.60 g, 18.2
mmol, 4.0 equiv) in toluene (84 mL) at -5 °C (ice and saturated
NaCl solution) was added MsCl (0.53 mL, 6.83 mmol, 1.5 equiv).
Immediately, a solution of the freshly prepared keto acid (R,R)-5a
(total amount from above, ∼4.55 mmol) in toluene (25 mL) was
added to the resulting suspension via syringe pump over 45 min,
and 5 mL of additional toluene was used to ensure complete
transfer. After 3 h, the reaction mixture was diluted with ice-cold
Et2O (700 mL) and washed with 20% CuSO4 solution (500 mL) to
remove most of the 4-pyrrolidinopyridine and saturated NH4Cl
(500 mL), and then washed with water (2ꢀ500 mL). The organic
layer was dried over MgSO4, filtered, and concentrated. The resi-
due was purified by flash chromatography (1:9 f 3:7 EtOAc/
hexanes) to give a mixture of two inseparable β-lactones (-)-32/
320 (1.05 g, 52%, dr 5:1, 500 MHz 1H NMR) as a yellow oil and
recovered keto acid (10%, 4:1 dr): Rf = 0.36 (20% EtOAc/
hexanes). The enantiomeric excess of β-lactone 32 was determined
to be 90%. The diasteromeric β-lactones were carried directly
forward to the deprotection at which point they could be sepa-
rated. Data for (-)-32: IR (neat) 1830, 1703 cm-1; 1H NMR (500
MHz, CDCl3) δ 7.32-7.36 (m, 3H), 7.13-7.15 (m, 4H), 6.80 (d,
J=8.5Hz, 2H), 4.73 (d, J=15.5 Hz, 1H), 4.31 (d, J=15.5 Hz, 1H),
4.17 (d, J = 12.0 Hz, 1H), 4.13 (d, J = 11.5 Hz, 1H), 4.01 (ddd,
J = 5.0, 7.5, 12.5 Hz, 1H), 3.77-3.81 (m, 1H), 3.77 (s, 3H), 3.73
(d, J = 11.5 Hz, 1H), 3.57 (d, J = 11.5 Hz, 1H), 2.91 (t, J =
7.5 Hz, 1H), 2.31-2.38 (m, 1H), 2.10-2.16 (m, 1H), 1.72 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 173.8, 166.1, 159.2, 136.4,
To a solution of the crude aldehyde (∼0.119 mmol) in THF
(1.0 mL) at -78 °C was added ∼1.2 mL (0.238 mmol, ∼200 μM
in THF, ∼2.0 equiv) of zincate 290 via syringe pump over 30 min.
The resulting mixture was slowly warmed to -20 °C for 1 h,
quenched with water and diluted with Et2O (120 mL). Saturated
(31) The C2-epimer of salino A has been isolated; see ref 11.
J. Org. Chem. Vol. 76, No. 1, 2011 11

Nguyen et al.
JOCFeatured Article
NH4Cl was added until a pH of 7 was achieved. The layers were
separated, and then the organic layer was washed with brine
(80 mL). The filtrate was dried over MgSO4, filtered, and con-
centrated. The residue was purified by flash chromatography
(95:5 f 85:5 EtOAc/hexanes) to give a mixture of two insepar-
able diastereomers of N-PMB salino A (-)-33 and C5, C6-bis-
epi-N-PMB salino A (-)-330 (38 mg, 74%, dr =4:1 according to
500 MHz 1H NMR) as a colorless oil, which was carried directly
to the next step without further purification.
Proteasome Inhibition Assay. The Proteasome-Glo Assay
(Promega) was used, which is a homogeneous coupled-enzyme
system that utilizes luminogenic substrates to measure the three
proteolytic activities (chymotrypsin-like(CT-L), trypsin-like
(T-L), and caspase-like (C-L)) associated with the proteasome.
Purified human 20S and 26S proteasome (BioMol/Enzo Life
Sciences, Inc.) at 0.5-2 μg/mL was incubated in 10 μL of
assay buffer (10 mM Hepes, pH 7.6) at room temperature for
20-30 min in the presence of the salino A derivatives at various
concentrations or equivalent volume of solvent DMSO as a con-
trol along with salino A and Velcade. The reaction was initiated
by addition of 10 μL of 2X solution of various substrates. Reac-
tions were performed at room temperature. After 1 h, inhibition
of each proteasomal activity was measured on a multilabel
microtiter plate reader.
Salino A ((-)-1a) and C5,C6-Bis-epi-salino A ((-)-1a0). To a
methanolic solution (0.8 mL) containing the mixture of alcohols
(-)-33/330 (38 mg, 0.088 mmol) was added an aqueous solution
of CAN (0.240 g, 0.44 mmol) in H2O (0.2 mL) at 0 °C dropwise.
After being stirred at 0 °C for 6 h, the reaction mixture was
diluted with EtOAc (100 mL) and washed with saturated solu-
tion of NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered, and concentrated. The residue was purified by
flash chromatography (5:95 f 15:85 EtOAc/CH2Cl2) to give
salino A (-)-1a (14 mg, 51%, dr >19:1, 500 MHz 1H NMR) as a
white solid (previously described, see ref 8j) in addition to a
novel diastereomeric salino A 1a0 (3.3 mg, 12%, dr >19:1, 500
Acknowledgment. We thank the NIH (GM069784) and
the Welch Foundation (A-1280) for support. We thank
Drs. Joe Reibenspies and Nattamai Bhuvanesh (TAMU)
for X-ray analysis. We thank Mr. Nicholas Krudy (TAMU
NSF REU, CHE-0755207) for technical assistance. We thank
Mr. Ranjith C. Perumalil and Mr. Mikail C. Abbasov for
graphics assistance for journal cover art. The U.S. Department
of Energy Genomics: GTL Program, http://doegenomestolife.
org, is acknowledged for the proteasome graphic used in the
Table of Contents entry and Abstract graphic.
1
MHz H NMR). Data for C5,C6-bis-epi-salino A (1a0): Rf =
0.56 (33% EtOAc/hexanes); IR (neat) 2931, 1830, 1702 cm-1
;
1H NMR (500 MHz, CDCl3) δ 6.28 (s, 1H), 6.10 - 6.14 (m, 1H),
5.85 - 5.88 (m, 1H), 3.99 (ddd, J=5.0, 7.5, 12.5 Hz, 1H), 3.83
(dd, J=7.5, 9.0 Hz, 1H), 3.78 (ddd, J=5.0, 7.5, 12.5 Hz, 1H),
2.83 (t, J = 7.5 Hz, 1H), 2.48-2.50 (m, 1H), 2.27-2.34 (m, 1H),
2.11-2.18 (m, 1H), 2.06-2.08 (m, 2H), 1.98 (d, J=9 Hz, 1H),
1.80 - 1.91 (m, 2H), 1.87 (s, 3H), 1.60 - 1.67 (m, 2H); 13C NMR
(125 MHz, CDCl3) δ 175.8, 165.0, 135.2, 123.5, 85.4, 78.7,
71.8, 44.9, 42.7, 38.4, 28.5 26.6, 25.2, 21.8, 19.4; HRMS
(MALDI) calcd for C15H20ClNO4Na [M þ Na] 336.0973, found
336.0957.
Supporting Information Available: General procedures for
synthesis and the human proteasome inhibiton assay and char-
acterization data including 1H and 13C NMR spectra (for com-
pounds 1a0, 21e, 44, 45, 61, 63) and X-ray analysis (61, 1a0). This
material is available free of charge via the Internet at http://
pubs.acs.org.
12 J. Org. Chem. Vol. 76, No. 1, 2011