Optimization of subsite binding to the β5 subunit of the human 20S proteasome using vinyl sulfones and 2-keto-1,3,4-oxadiazoles: Syntheses and cellular properties of potent, selective proteasome inhibitors

Article abstract of DOI:10.1021/jm058289o

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confir

Full text of DOI:10.1021/jm058289o

J. Med. Chem. 2006, 49, 2953-2968
2953
Optimization of Subsite Binding to the â5 Subunit of the Human 20S Proteasome Using Vinyl
Sulfones and 2-Keto-1,3,4-oxadiazoles: Syntheses and Cellular Properties of Potent, Selective
Proteasome Inhibitors
Robert M. Rydzewski,*,† Leland Burrill,† Rohan Mendonca,† James T. Palmer,† Mark Rice,† Ram Tahilramani,†
Kathryn E. Bass,‡ Ling Leung,‡ Erik Gjerstad,§ James W. Janc,§ and Lin Pan|
Departments of Medicinal Chemistry, Cell Biology, Enzymology, and Distribution, Metabolism, and Pharmacokinetics, Celera Genomics,
180 Kimball Way, South San Francisco, California 94080
ReceiVed December 27, 2005
Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones
(VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome.
Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic
group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic
ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere,
N-â-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with
a p-tosyl group without loss of potency. Small L-amino acids were used at P2, where D-substitution was not
tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead,
2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-
fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome
activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects
against the human prostate cancer cell line PC3 at submicromolar concentrations.
Introduction
Controlled proteolysis of cellular proteins is a process vital
to cell survival, proliferation, and even death. Improperly folded
or damaged proteins as well as many intact ones, such as cyclins,
cyclin dependent kinase inhibitors, and MHC class I antigens,
do not generally suffer the fate of either uncatalyzed hydrolysis
or degradation by lysosomal enzymes but instead are destroyed
via the ubiquitin-proteasome pathway.1 Conjugation with ubiq-
uitin, a 76-amino acid protein generally attached through a lysine
side chain of the target protein, begins the ATP-driven process,
which is consummated within the inner chamber of the 26S
proteasome where twin sets of three different catalytically active
subunits carry out the proteolysis.2 The large, barrel-shaped core
of the 26S proteasome, which lacks the 19S regulatory subunits
Figure 1. Structures of Bortezomib, Epoxomicin, PSI, and MB2.
but is otherwise enzymatically competent, is known as the 20S
proteasome or core particle (CP). Elegant work using scanning
electron microscopy,3 X-ray crystallography,4 and mutation
destruction of tumor suppressor protein p53 and may regulate
analysis5 has elucidated structural and functional aspects of the
the concentrations of a number of proteins controlling cell cycle
progression,7 should therefore increase the levels of apoptosis,
thereby exerting anticancer effects. Theory has yielded to current
clinical practice through the approval of the proteasome inhibitor
bortezomib (1), shown in Figure 1, which has demonstrated
statistically significant beneficial effects and is currently used
to treat patients with multiple myeloma.8
The proteasome has also been proposed to play a role in HIV
infectivity,9 and several teams report that a number of HIV
protease inhibitors are also weak proteasome inhibitors.10 Other
therapeutic areas where inhibiting the proteasome could have a
therapeutic effect include inflammatory11 and cerebrovascular12
diseases. Clearly, there is a need for novel, small molecule
proteasome inhibitors to be used as tool compounds in exploring
the many cellular pathways involving this unique and ubiquitous
enzyme.
proteasome. The three catalytic subunits, known as â1, â2, and
â5 utilize N-terminal threonine residues to cleave peptides
having basic, acidic, and hydrophobic residues in their P1
positions, respectively.4a
Interest in interfering with this biologically important process
stems from the potential for therapeutic utility. The proteasome
degrades IκBR, an endogenous inhibitor of nuclear translocation
for the important protein NF-κB, which has antiapoptotic as
well as many other cellular effects.6 In addition, the proteasome
is required for the production of the p50 subunit of NF-κB.
Inhibiting the proteasome, which is also responsible for the
* To whom correspondence should be addressed. Tel: 510 795-8711.
E-mail: brydzewski@sbcglobal.net.
† Department of Medicinal Chemistry.
‡ Department of Cell Biology.
Figure 1 presents a number of known small molecule inhib-
itors of the 20S proteasome. To date, only a limited number of
§ Department of Enzymology.
| Department of Distribution, Metabolism, and Pharmacokinetics.
10.1021/jm058289o CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/21/2006

2954 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
Scheme 1a
a Reagents: (a) NaH, Boc-Leu-H, THF, 0 °C; (b) HCl/dioxane; (c) IBCF, NMM, THF, -10 °C; (d) TFA.
SAR studies aimed at optimizing substituents at various posi-
tions have been reported for proteasome inhibitors. Bogyo13 used
scanning libraries to develop a â2 specific inhibitor. Positional
scanning libraries have also been used to study substrate
interactions at â1,14 whereas Crews15,16 has reported epoxomicin
analogues with â1 and â5 selectivity. SAR optimization,
selectivity data versus various antitargets, and profiles of cellular
properties are all vitally needed to evaluate compounds for in
vitro and in vivo use. We herein report the syntheses, activities,
selectivities, and selected properties for two different series of
20S proteasome inhibitors, the irreversible series of vinyl
sulfones (VS)17 10a-d, 17a-t, and 22a-o as well as the novel,
reversible 2-keto-1,3,4-oxadiazoles (KOD) 29a-l.
protection via tert-butyl ether (11b) was used, and the subse-
quent treatment of 12b with TFA in place of HCl afforded the
bis-deprotected product 13b.
Compounds 17a-t (Scheme 2) in the long, tetrapeptide-like
vinyl sulfone series were prepared by one of two routes. Either
tripeptide 16 was coupled with P1 amine 8 (Method A) or Cbz
dipeptide 14 was coupled with dipeptide amines 13a-c (Method
B). Negligible racemization (<5%) was encountered in the final
coupling by either method, so long as mixed anhydride coupling
was carried out below 0 °C. Requisite dipeptides 14a-t were
either commercially available (14a, 14b, 14t), synthesized by
standard Fmoc chemistry on Wang resin (14c-f, 14i, 14l, 14m),
or prepared by solution phase coupling of the free P3 amino
acid with the succinate ester of Cbz isoleucine (14g, 14h, 14j,
14k, 14n-s). In Method A, dipeptides 14 were converted to
the corresponding succinate esters 15 by reaction with NHS in
the presence of DCC, whereas in Method B, the free acid was
used in the subsequent coupling reaction.
As shown in Scheme 3, where side chain amide derivatives
of aspartic or glutamic acid were required (14o-s), the starting
monomers were prepared by EDC/HOAt coupling of the desired
side chain amine with the corresponding R-N-Boc protected
amino tert-butyl ester followed by global deprotection. The
experimental procedure for the preparation of 14q is an example
of this route. Scheme 3 also shows the synthetic route used to
Chemistry
Vinyl sulfone 8, shown in Scheme 1, has been previously
reported.18 This compound was prepared by the Horner-
Emmons reaction of Boc leucinal with sulfone 619 followed by
deprotection with HCl. Compounds 10a-d were prepared by
mixed anhydride coupling of vinyl sulfone 8 with capped
tripeptides 9a-d, which had been made by standard solid-phase
syntheses on Wang resin. In an analogous fashion, mixed
anhydride coupling of 8 with a Boc-protected amino acid (11a
and c) and subsequent deprotection using HCl gave dipeptide
analogues 13a and c. In the case of P2 serine, side chain

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2955
Scheme 2a
a Reagents: (a) N-Hydroxysuccinimide, DCC, THF; (b) L-alanine, Et3N, aq THF; (c) 8, IBCF, NMM, THF, -10 °C; (d) 13, IBCF, NMM, THF, -10 °C.
Scheme 3a
a Reagents: (a) Neopentylamine, EDC, HOAt, DMF; (b) HCl/dioxane; (c) Cbz-Ile-OSu, Et3N, aq THF; (d) p-Tosyl chloride, NaOH, aq dioxane; (e) 13b,
IBCF, NMM, THF, -10 °C.
prepare tripeptide vinyl sulfones 22a-o. The desired N-terminal
group was attached to the P3 amino acid under Schotten-
Baumann conditions, and the resulting adduct was coupled to
dipeptide vinyl sulfone 13 via the mixed anhydride.
The KODs 29a-l were synthesized according to the route
shown in Scheme 4. Oxadiazole 23 was lithiated, then trans-
metalated at low temperature as previously reported by Nakai.20
The resulting magnesio species reacted with Boc leucinal to
give 24 as a mixture of diastereomers. Deprotection gave 25,
which could be coupled to the P2 Boc amino acids to afford
26. Another deprotection followed by coupling to the desired
N-functionalized P3 acid gave 28. The N-functionalized P3 acids
were, in turn, synthesized by reaction of the corresponding P3
amino acid with the appropriate sulfonyl chloride under Schot-
ten-Baumann conditions. For the P3-substituted benzyl com-
pounds 28i-l, the requisite amino acids were prepared by direct
alkylation of Boc serine using two equiv of NaH and one equiv
of the substituted benzyl bromide in DMF. Oxidation of
diastereomeric mixture 28 using the Dess-Martin periodinane
afforded 29 in good yield.
The R-heteroaryl carbonyl groups of compounds 29a-l were
sufficiently electrophilic that small amounts (∼5%) of hydrates
(m + 18)+ were observed as peaks separate from the main
product in the LCMS spectra, except in the case of the hindered

2956 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
Scheme 4a
a Reagents: (a) i. n-BuLi, THF, -78 °C, ii. MgBr2‚Et2O, -78 °C, iii. Boc-Leu-H, -78 °C; (b) HCl/dioxane; (c) Boc-Ser(OMe)-OH, EDC/HOBT, NMM,
CH2Cl2; (d) 21, EDC/HOBT, NMM, CH2Cl2; (e) Dess-Martin periodinane, wet CH2Cl2.
dependent process,21 are shown as IC50 values after a 1 h
Table 1. Inhibition Constants for Some Proteasome Inhibitors
compd
valuea
ChT-Lb
Tryp-Lc
PGPHd
CatBe
CatSf
inhibitor/proteasome incubation, whereas Kiapp values are used
to characterize reversible inhibitors. This allows for SAR
comparisons within a given series and selectivities versus
antitargets for a given compound to be derived, but values of
different types should not be directly compared. Inhibition
constants were obtained for chymotrypsin-like, trypsin-like, and
PGPH-like proteasome activities of the human 20S proteasome.
These reflect proteolysis at the active sites of the â5, â2, and
â1 subunits, respectively.22 Experiments using mutations in
active site subunits of the yeast proteasome demonstrated that
of the three activities, chymotrypsin-like activity was the one
most necessary for cell viability and protein processing,23 and
existing proteasome inhibitors target this â5 activity.
2
3
5a
5b
5c
10a
10b
10c
10d
IC50
Kiapp
Kiapp
Kiapp
Kiapp
IC50
IC50
IC50
IC50
0.0037
<0.001
0.0023
0.0037
>450
1.3
0.12
56
0.035
0.16
2.7
110
0.33
nd
nd
nd
nd
>150
2.2
14
48
.47
nd
nd
nd
nd
>150
120
ndg
nd
0.012
18
>150
1.6
>150
0.85
25
nd
nd
nd
0.046
12
6.9
77
0.15
a The type of inhibition constant. b The value (µM) for chymotrypsin-
like proteasome activity. c The value (µM) for trypsin-like proteasome
activity. d The value (µM) for post glutamyl peptide hydrolase-like protea-
some activity. e The value (µM) for cathepsin B activity. f The value (µM)
for cathepsin S activity. g Not determined.
In general, subsite selectivity was not a significant issue for
any of the inhibitors, with the exceptions of epoxide 2 and
aldehyde 5b, where only moderate selectivities versus trypsin-
like and PGPH-like activities were observed. This lack of cross-
reactivity could be rationalized in terms of the varying P1 and
P3 steric and electronic requirements for the different subunits.
Selectivity versus cysteinyl cathepsins, the major sources of
lysosomal proteolysis in cells, was potentially a bigger problem.
Previously described warheads for proteasome inhibitors, in-
cluding vinyl sulfones24 and aldehydes25, are known to also
target enzymes such as cathepsin B (CatB) and cathepsin S
(CatS), where the active site sulfhydryl group of the cysteine
protease can form a covalent bond to the electrophile when the
inhibitor is appropriately substituted. For example, peptidyl vinyl
sulfones, originally reported as inhibitors of cathepsins,24 were
later reported by Bogyo et al18 to be active proteasome inhibitors
when properly extended. Assays for the inhibition of CatB and
CatS, performed as previously described,26 revealed that several
of the compounds in Table 1, including the aldehyde MG-132
P1 Aib17 analogue, 29e. The samples of 29a-d and 29f-l that
had been dissolved in methanol for LCMS analysis also tended
to show the reversibly formed methyl hemiketal, (m + 32)+,
as another separate peak that was more prominent than the
hydrate, but this adduct was entirely absent when ACN was
used as the solvent. In all cases, however, NMR spectra in
DMSO-d6 showed the desired product present as a single species
characterized by the downfield-shifted P1 methine proton around
δ 5.1.
Results and Discussion
The search for novel proteasome inhibitors suitable for testing
in cellular and in vivo models began with a determination of
the enzymatic activities for the known proteasome inhibitors
shown in Table 1. The inhibition constants shown in Table 1
are of two different types. The values for irreversible inhibitors,
where inhibition is by definition a nonequilibrium, time-

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2957
Table 2. SAR of Tetrapeptide Vinyl Sulfones
compd
method
R
ChT-La
Tryp-Lb
PGPHc
CatBd
CatSe
17a
17b
17c
17d
17e
17f
17g
17h
17i
A
A
A
A
A
A
A
B
A
A
B
A
A
B
B
B
B
B
B
B
CH2CH(CH3)2
CH2Ph
CH2CH2Ph
(E)-CH2CHdCHPh
CH2OCH2Ph
CH2SO2CH3
CH2SO2CH2Ph
CH2CO2(t-Bu)
CH2CO2CH2Ph
CH2CH2CO2t-Bu
CH2CH2CH2CO2(t-Bu)
CH2CH2CO2(C6H11)
CH2CH2CONH2
CH2CH2CH2CONH2
CH2CH2CONH(i-Pr)
CH2CH2CONH(t-Bu)
CH2CONHCH2C(CH3)3
CH2CH2CONHCH2C(CH3)3
CH2CH2CONHC(CH3)2CH2CH3
see Figure 2
0.069
0.46
0.027
0.044
0.049
3.3
1.4
0.078
2.5
0.0025
0.024
0.01
0.57
0.12
0.052
0.0032
0.0015
0.049
0.0024
150
39
>150
nd
>150
nd
>150
nd
nd
>150
nd
>150
>150
nd
nd
nd
>150
>150
>150
>150
>150
nd
5.8
27
41
nd
38
140
>150
nd
nd
29
18
nd
54
nd
>150
51
nd
nd
0.7
9
ndf
>150
nd
>150
nd
nd
>150
nd
>150
>150
nd
10
nd
35
13
12
nd
nd
36
7.1
nd
48
nd
41
3.3
nd
nd
nd
13
17j
17k
17l
17m
17n
17o
17p
17q
17r
17s
17t
nd
nd
>150
>150
>150
>150
>150
nd
nd
23
a IC50 (µM) for chymotrypsin-like proteasome activity. b IC50 (µM) for trypsin-like proteasome activity. c IC50 (µM) for post glutamyl peptide hydrolase-
like proteasome activity. d IC50 (µM) for cathepsin B activity. e IC50 (µM) for cathepsin S activity. f Not determined.
(5b), are submicromolar cathepsin inhibitors. The corresponding
peptide nitrile 5c27 was devoid of proteasome activity, possibly
indicating the failure of the enzyme’s active site to accommodate
the resulting sp2 (versus sp3 for other electrophiles) intermediate.
Nitrile 5c, however, was a 46 nM inhibitor of CatS, emphasizing
the importance of electrophile selection to inhibitor selectivity.
Having observed good proteasome inhibition with the P1 keto
epoxide, aldehyde, and boronate but not the nitrile electrophiles,
we decided to produce a focused series of peptidyl vinyl
sulfones, where the effects of substitution at P2 could be
assessed. The P5-P4-P3-P2 sequence used in the case of 10d,
Ac-hPhe-Leu-Phe, stems from an optimized proteasome inhibi-
tor in the keto epoxide series reported by Crews.15 Although
vinyl sulfone 10d was the most potent of the four P2 analogues
against chymotrypsin-like proteasome activity (35 nM), it also
displayed 150 nM potency as a CatS inhibitor. The removal of
its P2 phenyl ring in the Ala analogue 10b resulted in a 3-fold
loss of proteasome activity. The replacement of L-Ala with D-Ala
at P2, potentially of interest to discourage unwanted in vivo
proteolysis, gave 56 µM 10c. The observed 500-fold diminution
of activity in reversing the stereochemistry of the methyl group
at P2 was not easily explained on the basis of sterics but may
reflect a shift in the conformational space away from the bound
conformation for this ligand. The P2 Gly analogue 10a was
about 10-fold less potent than the L-Ala, emphasizing, along
with the 1600-fold difference in activities between the most-
and least-potent members of this small series, that specific
binding requirements at P2 cannot be disregarded in proteasome
inhibitor design.
viable route to improved proteasome inhibitors, the peptide
sequence of 3 seemed to be a good starting point for optimiza-
tion studies using a different P1 electrophile. For reasons
previously noted, Ala was retained at P2, whereas its P3 tert-
butyl glutamic ester, an unusual structural feature and one
suspected to be key to good binding at S3, was replaced with
a number of analogues in a series featuring the robust, readily
accessible vinyl sulfone electrophile. Table 2 summarizes the
resulting SAR.
Compound 17j, the vinyl sulfone analogue of 3, proved to
be a 2.5 nM inhibitor of chymotrypsin-like proteasome activity.
Selectivities versus trypsin-like and PGPH-like proteasome
activities as well as CatB and CatS, were at least 10 000-fold,
and in general, the selectivities tended to be high for all of the
compounds in the series. The replacement of the tert-butyl
glutamic ester with smaller residues such as Leu 17a (69 nM)
or Phe 17b (460 nM) resulted in diminished activity. Although
larger P3 replacements tended to fare well, attesting to the
preference for large hydrophobic interactions at S3, hydropho-
bicity was optimally placed at the distal end of the side chain;
simple, extended linear chains such as Gln (17m, 570 nM) and
hGln17 (17n, 120 nM) were not potent. The trend thus observed
is consistent with modeling predictions. As shown in Figure 3,
the interface between the â5 and â6 subunits, which constitutes
the S3 binding cleft, can accommodate long, linear side chains,
but the best opportunity for hydrophobic interactions lies at the
end of the resulting cleft. That the tert-butyl Glu ester fits this
well is attested to by observing that both the shorter tert-butyl
Asp ester (17h, 78 nM) and the longer tert-butyl hGlu17 ester
(17k, 24 nM) are an order of magnitude less active. Unfavorable
interactions along the linear part of the binding cleft may account
for the micromolar potencies encountered with sulfonyl-contain-
ing analogues 17f and g. The replacement of the tert-butyl Glu
One of the promising, potent, and selective proteasome
inhibitors shown in Table 1 is PSI (3), an inhibitor first reported
by Wilk.28 Although the potential for metabolism, Schiff base
formation, and, perhaps, nonspecific inhibition may be suf-
ficiently troubling to rule out optimization of aldehydes as a

2958 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
hydrophobicity at the distal end of the P3 substituent (22k, 22l),
and attempting to prophylactically block potential in vivo
proteolysis with the P2 Aib group (22o) resulted in losses in
potency of several orders of magnitude.
Having simplified the original Cbz-Ile-Glu(OtBu)-Ala se-
quence of 3 to the smaller, more soluble, and presumably more
stable pTs-NeopentylAsn-Ala sequence of VS 22m, we decided
to incorporate this sequence into a new series of reversible
proteasome inhibitors. A number of known drugs, including
aspirin and penicillin, are irreversible enzyme inhibitors,21,30 and
at least a few irreversible inhibitors including a rhinovirus 3C
protease inhibitor31 are reported to be in development. One might
also debate the physiological consequences of using an irrevers-
ible inhibitor versus a reversible, slow off-rate (or functionally
irreversible) inhibitor.32 Still, widespread concern about potential
nonspecific inhibition and immunogenicity suggest that revers-
ibility be preferred for pharmacological intervention. A series
of P1 modifications led to the discovery that the 2-keto-1,3,4-
oxadiazole (KOD) warhead previously known to be active in
inhibitors of neutrophil elastase20 and HepC NS3 protease33
could also function as a potent electrophile for the proteasome
â5 subunit active site.
The reversibility of the inhibition of chymotrypsin-like 20S
proteasome activity was established for KOD 29c through
experiments in which the 20S proteasome was incubated with
the inhibitor at a concentration that fully inhibited enzyme
activity. After incubation, the enzyme-inhibitor complex was
diluted 50-fold into a substrate-containing buffer. The chymo-
trypsin-like activity of the proteasome, initially fully inhibited,
slowly recovered with a first-order rate constant of 0.010 min-1
(t1/2 ) 68 min). The final steady-state enzyme activity was 65%
of the control reaction in which the 20S proteasome was not
exposed to the inhibitor. This value is consistent with the
establishment of a new equilibrium after the dilution of the
inhibitor and enzyme by 50-fold. These results fit with a model
of reversible inhibition with slow decomposition of the tetra-
hedral hemiketal intermediate at the active site.
Figure 2.
ester with the cyclohexyl Glu ester gave another potent
compound, 17l (10 nM).
Uncertainty about whether such tert-butyl esters would
be substrates for esterase-catalyzed in vivo hydrolysis provided
the impetus for the synthesis of several isosteres. The amide
isostere of 17j, tert-butyl Gln 17p (3.2 nM), was about
equipotent to the ester but was presumably more difficult
to hydrolyze. The importance of a single methyl group in
hydrophobic S3 interactions was emphasized by the nearly
20-fold loss of activity observed with corresponding i-propyl
Gln amide 17o (52 nM). Another advantageous isostere, the
neopentyl Asn amide 17q proved to be the most potent
compound in the series at 1.5 nM, and therefore, this tert-butyl
Glu ester analogue was used extensively in subsequent inhibi-
tors.
Although the potencies and selectivities of these compounds
were favorable, considering the size and relatively high mo-
lecular weights of these molecules led us to suspect that other
properties such as solubility and cell permeability might not be
favorable. Our molecular weight loss program began with an
exploration of substituent requirements at P4 and P5. Table 3
summarizes the results for this series, where the Cbz-Ile
N-terminus of compound 17 was replaced with a simple capping
group on the N-terminus of the P3 residue. Although the
replacement of the N-terminus of 17j with a simple acetyl group
(22a) resulted in a 100-fold loss of potency, most of this activity
was restored upon replacement with an phenyl-containing
substituent such as the benzoyl group (22b, 17 nM), the Cbz
group (22c, 6.1 nM), or, better still, the p-tosyl group (22g, 2.1
nM), which was equipotent with 17j but nearly 100-fold lower
in molecular weight. As predicted, and as shown in Table 5,
the kinetic solubility of 22g, at 48 µM, was greater than that of
17j (<10 µM). Despite concerns stemming from a report that
P4 modifications in a series of vinyl sulfone proteasome
inhibitors can affect subunit selectivity,29 all P4 analogues shown
in Table 3, which were tested against the trypsin-like and PGPH-
like activities of the proteasome, were found to be inactive.
Combining the newly found P4 p-tosyl group with the
neopentyl Asn P3 isostere gave 22m, a 4.3 nM proteasome
inhibitor with good (470 µM) solubility. The replacement of
the P2 Ala with Ser, which was expected to improve solubility
still further, gave 22n, a 5.9 nM inhibitor with slightly reduced
(361 µM) solubility relative to the Ala analogue 22m. Other
modifications in this shortened vinyl sulfone series including
using BocDab17 as a P3 isostere (22i), placing still more
Table 4 shows the structures and activities of the KODs.
Grafting the preferred sequence found in VS 22m onto the
LeuKOD P1 moiety gave 29c, a 720 pM inhibitor of chymo-
trypsin-like 20S proteasome activity, which was >50 000-fold
selective against antitargets. As in the VS series, compounds
lacking a phenyl ring at P4, such as 29a and 29f, tended to be
less active. Substituting the meta-tosyl for the para-tosyl group
gave the essentially equipotent compound 29b (650 pM). The
PBS solubility of 29d (37 µM), a P2 methyl Ser compound,
was slightly improved relative to that of the corresponding Ala
compound 29c (13 µM), whereas the potency remained the
same. The solubility was not increased by the substitution of a
p-dimethylamino group on the phenyl ring of the ketooxadiazole
(29g, <10 µM), but potency may have improved slightly (510
pM versus 720 pM), suggesting that further prime side
modifications might prove fruitful. Substituting Aib17 for Leu
at P1 resulted in a 180-fold loss of potency (29e), as might
have been expected in light of the known affinity of the enzyme
subunit for moderately sized, hydrophobic side chains in
substrates at this position.34
A different P3 unit was also pursued in a series of several
analogues. The P3 benzyl Ser residue found in 17e, a VS, gave
reasonable inhibition at 49 nM, although about 30-fold less
potent than the corresponding neopentyl Asn (17q). Compounds
29i-l were synthesized to test whether this was transferable to
the KOD series and, if so, whether potency could by optimized
by substituents on the phenyl ring. At 32 nM, the proteasome

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2959
Figure 3. (a) MB2 top view, (b) 22g top view, (c) MB2 side view, and (d) 22g side view. The catalytic residues are shown as stick figures in c
and d. All inhibitors are docked into the â5 (grey) and â6 (pink) subunits of the bovine proteasome structure 1IRU using the MMF94 force field
in MOE (Chemical Computing Group, Montreal, Quebec) as rendered in Pymol (DeLano Scientific, South San Francisco, CA).
Table 3. SAR of Tripeptide Vinyl Sulfones
compd
P4
P3
P2
ChT-La
Tryp-Lb
PGPHc
CatBd
CatSe
22a
22b
22c
22d
22e
22f
22g
22h
22i
22j
22k
22l
22m
22n
22o
acetyl
benzoyl
Cbz
Cbz
Cbz
PhSO2
p-tosyl
Ph2CHCO
Cbz
acetyl
Cbz
t-butylGlu
t-butylGlu
t-butylGlu
t-butylGlu
t-butylGlu
t-butylGlu
t-butylGlu
t-butylGlu
BocDab17
benzylGlu
CH2CONH-(S)-CH(t-bu)CO2t-bu
CH2CH2CONHC(CH3)2CO2t-bu
neopentylAsn
neopentylAsn
neopentylAsn
Ala
Ala
Ala
Gly
Ser
0.26
ndf
>450
>150
nd
>450
>450
>450
>450
nd
nd
nd
nd
>450
>450
>450
nd
77
nd
20
>150
nd
nd
13
nd
nd
nd
nd
nd
nd
>150
nd
170
nd
3
0.017
0.0061
0.17
0.0029
0.0011
0.0021
0.0067
0.43
21
0.54
2.3
0.0043
0.0059
28
>450
>150
nd
>450
>450
>450
>450
nd
nd
nd
nd
>450
>450
>450
21
nd
nd
67
nd
nd
nd
nd
nd
nd
>150
nd
Ser
Ala
Ala
Ser
Ala
Ala
Ala
Ala
Ser
Cbz
p-tosyl
p-tosyl
p-tosyl
Aib17
a IC50 (µM) for chymotrypsin-like proteasome activity. b IC50 (µM) for trypsin-like proteasome activity. c IC50 (µM) for post glutamyl peptide hydrolase-
like proteasome activity. d IC50 (µM) for cathepsin B activity. e IC50 (µM) for cathepsin S activity. f Not determined.
inhibition observed for parent compound 29i was about 40-fold
less than that of matching neopentyl Asn 29d. Unfortunately,
small substituents in the ortho (29j, 45 nM), meta (29k, 140
nM), and para (29l, 500 nM) positions only served to decrease
potency in this series. Figure 4 models the binding of 29c, a P3
neopentyl Asn compound and the corresponding o-tolyl Ser
compound 29j in the active site of the â5 subunit, and shows
one of the tert-butyl methyl groups reaching down into a part
of the S3 pocket that may not be accessible to phenyl
substituents in the benzyl Ser analogues.
At this point, potent and selective proteasome inhibitors had
been made in both the VS and the KOD series. These
compounds were investigated as potential tool compounds for
purposes of cellular and animal testing. Beyond simple in vitro
potency and selectivity, other parameters must be met for such
compounds to be useful in biological systems. Kinetic solubility
in PBS can be measured to provide insight into the less easily
quantified but the more important property of formulability.
Because the proteasome is found in cytosol, cell permeability
can be assessed to see whether the inhibitors can reach the target.
Finally, because metabolism can occur in plasma, principally
by the action of nonspecific esterases, potentially reducing
available levels of the inhibitor to zero, plasma stabilities needed
to be assessed. Cellular potencies and other physical data for
selected proteasome inhibitors are shown in Table 5.
The remarkable ability of plasma esterases to hydrolyze
hindered esters in buffered systems at neutral pH is demonstrated
by the low plasma stabilities exhibited by all the hindered Glu
esters (17j-l, 22c, 22g-h). After a 2 h incubation in human
plasma, for example, only about half of the tetrapeptide vinyl
sulfone tert-butyl ester 17j and none of the corresponding
cyclohexyl ester 17l remained.35 These results go a long way
toward explaining the observed low cellular potencies of these
compounds because hydrolysis would result in an inactive
species. The success of the isostere approach in circumventing
this problem can be seen by comparing ester 22g, only 7% of

2960 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Table 4. SAR of Tripeptide 2-Keto-1,3,4-oxadiazoles
Rydzewski et al.
compd
P4
P3
P2
Ala
Ala
Ala
MeSer
Ala
Ala
P1
R
ChT-La
Tryp-Lb
PGPHc
CatBd
CatSe
29a
29b
29c
29d
29e
29f
29g
29h
29i
mesyl
neopentylAsn
neopentylAsn
neopentylAsn
neopentylAsn
neopentylAsn
neopentylAsn
neopentylAsn
neopentylAsn
benzylSer
Leu
Leu
Leu
Leu
Aib
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Ph
Ph
Ph
Ph
Ph
i-Pr
0.0057
0.00065
0.00072
0.00072
0.13
ndf
nd
nd
nd
72
44
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
39
8.1
nd
nd
nd
nd
nd
nd
nd
nd
m-tosyl
p-tosyl
p-tosyl
p-tosyl
DMS17
p-tosyl
p-tosyl
p-tosyl
p-tosyl
p-tosyl
p-tosyl
>450
>300
>900
>450
>900
290
>300
51
nd
nd
>450
>900
>900
>450
>900
>300
>300
>900
nd
0.038
Ala
p-N(Me)2Ph
p-N(Me)2Ph
0.00051
0.0005
0.032
0.045
0.14
MeSer
MeSer
Ala
Ala
Ala
Ph
Ph
Ph
Ph
29j
29k
29l
o-MebenzylSer
m-ClbenzylSer
p-MebenzylSer
nd
nd
0.5
nd
a Kiapp (µM) for chymotrypsin-like proteasome activity. b Kiapp (µM) for trypsin-like proteasome activity. c Kiapp (µM) for post glutamyl peptide hydrolase-
like proteasome activity. d Kiapp (µM) for cathepsin B activity. e Kiapp (µM) for cathepsin S activity. f Not determined.
Table 5. Properties of Selected Proteasome Inhibitors
PC3
plasma stab.
(%)c
PBS sol
compd
(nM)a
shiftb
(µM)d
2
3
5b
1
0.27
>380
160
860
12
22
9.2
42
nde
nd
nd
100
66
22
83
nd
52
20
0
nd
nd
nd
nd
nd
<10
nd
nd
<10
nd
375
600
3000
800
600
450
3250
2000
nd
10d
17a
17c
17e
17h
17j
17k
17l
800
nd
nd
nd
nd
17o
17p
17q
17s
22a
22b
22c
22e
22f
22g
22h
22m
22n
22o
29b
29c
29d
29e
29g
3000
5000
200
550
7500
1750
6500
3000
4000
500
500
nd
2400
nd
nd
200
nd
nd
58
89
89
90
83
nd
nd
1
nd
nd
7
nd
57
<10
20
nd
nd
<10
nd
1600
130
230
29
100
1100
1000
3600
240
75
nd
410
nd
nd
280
nd
Figure 4. Top and side views of 29c (orange) and 29j (violet) docked
into the â5 and â6 subunits of the bovine proteasome structure 1IRU
using the MMF94 force field in MOE (Chemical Computing Group,
Montreal, Quebec) as rendered in Pymol (DeLano Scientific, South
San Francisco, CA).
nd
48
1
<10
470
381
500
<10
13
37
249
<10
96
99
100
28
86
79
100
72
group provides a potential site for reversible hydration and
subsequent H-bonding with solvent.
nd
nd
A preliminary assessment of potential anticancer effects of
these proteasome inhibitors was undertaken by examining the
growth inhibitory effects of the compounds on PC3 cells, a
human prostate cancer cell line, in a cellular assay from which
a cytotoxic component cannot per se be excluded.36 Whereas
compounds tested had nanomolar or subnanomolar potencies
in the in vitro enzyme assay, cellular IC50 values were often
micromolar with the most notable exception being epoxomicin
2. This compound, remarkably, showed cell growth inhibitory
effects at a concentration below its IC50 value for the isolated
enzyme, the explanation for which requires further study. Other
submicromolar IC50 values were obtained with compounds such
as VS 17q and KOD 29c, both having a value of 200 nM in
this cellular assay.
nd
a IC50 for growth inhibitory effects in PC3 cells. b IC50 for PC3 cells/
Kiapp or IC50 for chymotrypsin-like proteasome activity. c Percent remaining
in human plasma at 120 min, 37 °C. d Kinetic solubility in PBS at pH 7.4.
e Not determined.
which remains after 2 h in plasma, with its neopentyl Asn
isostere 22m, 96% of which remains under the same conditions.
Only two other compounds (17c, 29b) had stability problems
that could not be attributed to ester hydrolysis, and no inherent
differences in plasma stability between VSs and KODs were
observed.
Solubilities, as expected, were poorest for the tetrapeptide
vinyl sulfones and improved somewhat, as previously noted,
for shorter compounds and N-terminal p-tosyl compounds. A
comparison of the solubilities of VS 22m (470 µM) and the
analogous KOD 29c (13 µM) reveals a propensity for lower
solubilities with the latter, more hydrophobic phenyl-containing
P1 electrophiles despite the fact that the electrophilic carbonyl
For some of the compounds (e.g., 17j, 22c), the magnitude
of the resulting shift, defined as the cellular IC50 value divided
by the isolated enzyme inhibition constant, can be accounted
for in terms of plasma instability, which reduces the concentra-
tion of the active component over the long time course of the

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2961
Experimental Section
experiment. In other cases, the reason the inhibitor needed to
be present at such high concentrations to have a cellular effect
was less apparent. Lack of cell permeability is the most likely
explanation, and in that context it was noted that none of the
P3 Ser compounds (22e, 22f, 22n) had submicromolar cellular
activity, perhaps implicating the additional -OH group in cell
impermeability. Cellular efflux by active transporters such as
P-gp, a mechanism which has been previously demonstrated to
extrude one VS from intestinal cells,37 is another possible
explanation for the observed shifts. Additional experiments
would be required to determine whether this is the case.
Attempts to correlate observed cellular IC50 values for plasma
stable compounds with enzyme potency, MW, cLogP, TPSA,
the number of rotatable bonds, or combinations thereof met with
little success. Empirically, the P3 neopentyl Asn isostere fared
relatively well in the cellular assay, with tetrapeptide VS 17q
and KOD 29c, both having IC50 values of 200 nM, being the
best in each class.
General Procedures. Reagents and solvents were obtained from
commercial sources and were used without purification. Flash
chromatography was performed on 230-400 mesh silica gel (E.
M. Science). Melting points were obtained on a Mel-Temp 3.0
melting point apparatus and are uncorrected. NMR spectra were
recorded at the specified frequencies in DMSO-d6 unless otherwise
stated and were referenced to TMS. The following abbreviations
were used: s, singlet; d, doublet; dd, doublet of doublets, etc.; m,
multiplet; q, quartet; and b, broad. Mass spectra were obtained using
PE Sciex API 150EX instruments in electrospray positive and
negative ionization modes. HPLC homogeneities were determined
using the specified reverse phase conditions. System A: Varian
Polaris C18-A column (5 µm, 5 × 20 mm), 10-99% MeOH (+
0.1% HCO2H) in 0.1% HCO2H, linear gradient in 12 min, then 2
min isocratic, and 1.5 mL/min flow rate with UV detection at 214
nM. System B: Phenomenex Prodigy C-18 ODS3 column (5 µm,
4.6 × 100 mm), 1-99% ACN (+ 0.1% TFA) in 0.1% TFA, linear
gradient in 12 min, then 2 min isocratic, and 1.5 mL/min flow rate
with UV detection at 214 nM. Elemental analyses were performed
by Robertson Microlit Laboratories, Madison, NJ. Unless otherwise
stated, the reactions were conducted at room temperature under an
atmosphere of dry nitrogen.
Boc-LeuVSMe (7). Compound 619 (5.48 g, 23.8 mmol) was
dissolved in anhydrous THF (200 mL), cooled in an ice bath, and
vigorously stirred. Sodium hydride was added in portions as a 60%
mineral oil dispersion (1.10 g, 27.5 mmol). When the addition was
complete and no more gas evolution was observed, a solution of
Boc leucinal (5.12 g, 23.8 mmol), which had been made by the
procedure of Falkiewicz, et al.,38 in dry THF (50 mL) was added.
After 30 min of vigorous stirring, 1 M HCl (50 mL) was carefully
added, followed by the addition of EtOAc (100 mL). The phases
were separated. The organic phase was washed with 1 M HCl,
saturated NaHCO3, and brine, and then dried over MgSO4. Filtration
and solvent evaporation left 8.0 g of a yellow oil that was flash
chromatographed on silica gel using 20% EtOAc in hexane. Crude
7 (5.76 g) was obtained. This contained a small amount of 6 by
LCMS analysis, but was suitable for use in the next step without
further purification.
Conclusions
Optimization of binding interactions between the â5 subunit
of the human 20S proteasome and small, peptide-like VS
inhibitors 10a-d, 17a-t, and 22a-o uncovered key interactions
at the S3 and S4 subsites, many of which agreed with modeling
predictions. Most critical for optimal binding was a relatively
linear P3 side chain that terminated with a large, hydrophobic
substituent, such as tert-butyl Glu. Lack of solubility and plasma
instabilities for such esters triggered a search for more favorable
isosteres, which resulted in the selection of the neopentyl Asn
side chain at P3. As shown in Figure 4, this moiety afforded a
relatively tight fit with the cleft that constitutes the S3 subsite
of the proteasome â5 subunit. Attempts to optimize a second
series of P3 substituents, P3 benzyl serine ethers such as 17e,
led to less potent compounds.
Inhibitors could be made smaller by replacing the Cbz
isoleucine N-terminus found in compounds 17 with a simpler
aryl substituent interacting at S4. In practice, the p-toluene-
sulfonyl group or its analogues used in 22m-o were found to
be optimal. Substituent effects at P2 also played a definite role
in compound activity, possibly because of conformational
effects. Out of concern for keeping the molecular weights low,
most inhibitors retained an unoptimized alanine at this position,
although P2 serine and its methyl ether were also used in some
analogues.
Optimal substituents at all positions were grafted onto a newly
discovered warhead for proteasomes, 2-keto-1,3,4-oxadiazoles,
to give remarkably potent, selective, and reversible inhibitors
such as the 720 pM 29c, thus demonstrating just how transfer-
able the SAR was between the VS series and the later KODs.
The vinyl sulfone SAR, in turn, had correlated well with what
had previously been observed for aldehydes, providing evidence
that all three series, as expected, utilize the same mode of
binding with the enzyme, with significant differences in
geometry being limited to the P1 and P1′ positions.
Optimization was accelerated through the concurrent mea-
surement of enzymatic, physical, and cellular properties of the
inhibitors, an approach, which more and more, is found to be
necessary for timely drug discovery and development. Results
from these studies combined with modeling considerations and
a starting point based on a comparison of previously known
proteasome inhibitors led to the discovery of novel, reversible
2-keto-1,3,4-oxadiazoles such as 29c that could serve as key
tool compounds for cellular and animal studies as well as
provide a springboard for further optimization.
H-LeuVSMe Hydrochloride (8). Compound 7 (5.75 g crude)
and 4.0 M HCl in 1,4-dioxane (50 mL) were stirred together. After
1 h, Et2O (100 mL) was added. The reaction mixture was filtered.
The precipitate was rinsed with a little more Et2O and dried.
Compound 8 (4.33 g) was obtained as a white solid, an 80% yield
1
for the two steps. H NMR (400 MHz): δ 8.59 (bs, 3H), 7.14 (d,
J ) 15.2 Hz, 1H), 6.69 (dd, J ) 15.2, 8.0 Hz, 1H), 3.98 (m, 1H),
3.07 (s, 3H), 1.61 (m, 3H), 0.91 (m, 6H). MS m/z: 192 (MH)+.
Ac-hPhe-Leu-Gly-LeuVSMe (10a). Mixed Anhydride Cou-
pling. [2-(S)-(2-(S)-Acetylamino-4-phenyl-butyrylamino)-4-methyl-
pentanoylamino]-acetic acid (117 mg, 0.30 mmol) was dissolved
in 5 mL of anhydrous THF and stirred in an ice/methanol bath.
N-Methylmorpholine (50 µL, 0.45 mmol) and then IBCF17 (39 µL,
0.30 mmol) were added. After 10 min, compound 8 (68 mg, 0.30
mmol) was added as a solid, followed by the addition of NMM
(50 µL, 0.45 mmol). After 30 min, the cold reaction mixture was
poured into 0.2 M HCl and extracted twice with CH2Cl2. The
combined organics were washed with water, saturated NaHCO3,
and brine and then dried over MgSO4. Filtration and solvent
evaporation followed by flash chromatography on silica gel using
5% MeOH/CH2Cl2 gave 88 mg of 10a (52%) as a white solid. 1H
NMR (270 MHz): δ 8.20 (t, J ) 5.7 Hz, 1H), 8.06 (t, J ) 6.9 Hz,
2H), 7.83 (d, J ) 7.9 Hz, 1H), 7.22 (m, 5H), 6.71 (m, 2H), 4.56
(m, 1H), 4.23 (m, 2H), 3.71 (ABX m, 2H), 2.97 (s, 3H), 2.58 (m,
2H), 1.88 (s, 3H), 1.3-1.8 (m, 8H), 0.83 (m, 12H). MS m/z: 565
(MH)+. Anal. (C28H44N4O6S) C, H, N.
Ac-hPhe-Leu-Ala-LeuVSMe (10b). Using the same procedure
described for the preparation of 10a but starting with 9b, a 41%
1
yield of 10b was obtained as a white solid. H NMR (270 MHz):
δ 8.14 (d, J ) 7.4 Hz, 1H), 7.97 (d, J ) 7.7 Hz, 2H), 7.88 (d, J )
8.4 Hz, 1H), 7.27 (m, 2H), 7.18 (m, 3H), 6.64 (m, 2H), 4.52 (m,

2962 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
1H), 4.23 (m, 3H), 2.98 (s, 3H), 2.56 (m, 2H), 1.88 (s, 3H), 1.3-
1.8 (m, 8H), 1.22 (d, J ) 6.9 Hz, 3H), 0.83 (m, 12H). MS m/z:
579 (MH)+. Anal. (C29H46N4O6S) C, H, N.
(0.442 g, 3.84 mmol) were dissolved in 30 mL of dry THF and
stirred under nitrogen at 5 °C. Molten DCC (0.792 g, 3.84 mmol)
was poured in. After 14 h, the reaction mixture was chilled and
filtered, and the filtrate was evaporated. Trituration with ether gave
2.06 g of a hard white solid (98% yield). Proton NMR analysis
indicates that the product contained 6% (by weight) dicyclohexy-
lurea (DCU), but the material was suitable for use without further
Ac-hPhe-Leu-(D)-Ala-LeuVSMe (10c). Using the same pro-
cedure described for the preparation of 10a but starting with 9c, a
1
31% yield of 10c was obtained as a white solid. H NMR (270
MHz): δ 8.10 (m, 3H), 7.91 (d, J ) 8.4 Hz, 1H), 7.22 (m, 5H),
6.72 (m, 2H), 4.53 (m, 1H), 4.22 (m, 3H), 2.95 (s, 3H), 2.58 (m,
2H), 1.88 (s, 3H), 1.3-1.8 (m, 8H), 1.21 (d, J ) 7.2 Hz, 3H), 0.82
(m, 12H). MS m/z: 579 (MH)+. Anal. (C29H46N4O6S) C, H, N.
Ac-hPhe-Leu-Phe-LeuVSMe (10d). Using the same procedure
described for the preparation of 10a but starting with 9d, a 61%
1
purification. H NMR (270 MHz): δ 8.64 (d, J ) 7.2 Hz, 1H),
7.35 (m, 7H), 5.03 (s, 2H), 4.72 (m, 1H), 3.92 (t, J ) 8.4 Hz, 1H),
2.81 (s, 4H), 2.50 (t, J ) 2.0 Hz, 2H), 1.8-2.15 (m, 2H), 1.5-1.8
(m, 2H, includes DCU), 1.43 (s, 9H), 0.83 (m, 6H).
Cbz-Ile-Glu(OtBu)-Ala-OH (16j). Succinate Ester Coupling
Procedure. Compound 15j (2.01 g, 3.67 mmol) was dissolved in
40 mL of dry THF. Triethylamine (0.51 mL, 7.34 mmol) was added,
followed by L-alanine (0.327 g, 3.67 mmol) as a cloudy suspension
in 5 mL of THF plus 5 mL of water. The addition of another 5 mL
of water gave a clear, homogeneous reaction mixture at pH 5.5.
Triethylamine was periodically added in 0.10 mL portions until
the pH stabilized at 8.5. After 14.5 h, the reaction was poured into
200 mL of 0.5 M HCl and extracted twice with CH2Cl2. The
combined organic phase was washed with water and brine and then
dried over anhyd Mg2SO4. Filtration and solvent evaporation gave
1.9 g of a sticky white foam. Trituration with ether gave 1.35 g of
16j as a white solid (71% yield). 1H NMR (270 MHz): δ 8.17 (d,
J ) 7.2 Hz, 1H), 7.96 (d, J ) 7.9 Hz, 1H), 7.35 (m, 6H), 5.02 (s,
2H), 4.30 (m, 1H), 4.16 (m, 1H), 3.90 (t, J ) 8.4 Hz, 1H), 2.24 (t,
J ) 8.2 Hz, 2H), 1.55-1.95 (m, 3H), 1.39 (s, 9H), 1.25 (d, J )
7.2 Hz, 3H), 1.12 (m, 2H), 0.81 (m, 6H). MS m/z: 520 (M - H)-.
Cbz-Ile-Glu(OtBu)-Ala-LeuVSMe (17j). Mixed anhydride cou-
pling was carried out using the general method given for the
synthesis of 10a. Starting with 16j (261 mg, 0.50 mmol) and 8
(114 mg, 0.50 mmol), 17j was obtained as a white solid (239 mg,
1
yield of 10d was obtained as a white solid. H NMR (270 MHz):
δ 8.09 (t, J ) 7.9 Hz, 2H), 7.97 (d, J ) 8.7 Hz, 1H), 7.92 (d, J )
7.9 Hz, 1H), 7.15 (m, 10H), 6.56 (dd, J ) 15.2, 4.7 Hz, 1H), 6.25
(d, J ) 15.2 Hz, 1H), 4.48 (m, 2H), 4.21 (m, 2H), 2.90 (s, 3H),
2.75-3.0 (m, 2H), 2.54 (m, 2H), 1.87 (s, 3H), 1.81 (m, 2H), 1.51
(m, 2H), 1.34 (m, 4H), 0.78 (m, 12H). MS m/z: 655 (MH)+. Anal.
(C35H50N4O6S) C, H, N.
Boc-Ala-LeuVSMe (12a). Boc-L-alanine (1.66 g, 8.77 mmol)
and NMM (0.96 mL, 8.77 mmol) were dissolved in 50 mL of
anhydrous THF and cooled to -10 °C under an inert atmosphere.
IBCF (1.14 mL, 8.77 mmol) was added via syringe. After 5 min,
8 (2.00 g, 8.77 mmol) was added as a solid, followed by the addition
of more NMM (0.96 mL, 8.77 mmol). After 1 h, the reaction was
quenched with 1 M HCl and extracted three times with EtOAc.
The combined extract was washed with 1 M HCl, saturated
NaHCO3, and brine. Drying, filtration, and solvent evaporation,
followed by trituration with Et2O/hexane gave 2.1 g of the product
as a white solid (66% yield). 1H NMR (400 MHz): δ 7.93 (d, J )
8.0 Hz, 1H), 6.94 (d, J ) 7.2 Hz, 1H), 6.69 (dd, J ) 15.6, 5.2 Hz,
1H), 6.60 (d, J ) 15.6 Hz, 1H), 4.57 (m, 1H), 3.91 (m, 1H), 2.98
(s, 3H), 1.62 (m, 1H), 1.45 (m, 2H), 1.38 (s, 9H), 1.24 (d, J ) 8.0
Hz, 3H), 0.85 (m, 6H). MS m/z: 385 (m + Na)+.
1
69% yield). H NMR (270 MHz): δ 8.03 (m, 3H), 7.35 (m, 6H),
6.66 (m, 2H), 5.04 (s, 2H), 4.55 (m, 1H), 4.25 (m, 2H), 3.91 (t, J
) 7.7 Hz, 1H), 2.99 (s, 3H), 2.22 (t, J ) 8.2 Hz, 2H), 1.55-1.95
(m, 5H), 1.38 (s, 9H), 1.23 (d, J ) 6.9 Hz, 3H), 1.0-1.2 (m, 2H),
0.83 (m, 12H). MS m/z: 696 (M + H)+. HPLC homogeneity 97.0%
(System A); 100% (System B).
H-Ala-LeuVSMe Hydrochloride (13a). Compound 12a (1.80
g, 50 mmol) was stirred with 4 M HCl in 1,4-dioxane (125 mL,
500 mmol) for 1 h. Ether (200 mL) was added with vigorous
stirring. The reaction mixture was filtered and the precipitate washed
with more ether. The product (1.45 g) was obtained as a white
Other Compounds Made Using Method A. Cbz-Ile-Leu-Ala-
LeuVSMe (17a). 1H NMR (270 MHz): δ 7.95 (m, 3H), 7.35 (m,
6H), 6.65 (m, 2H), 5.03 (s, 2H), 4.53 (m, 1H), 4.26 (m, 2H), 3.88
(t, J ) 7.9 Hz, 1H), 2.99 (s, 3H), 1.3-1.8 (m, 7H), 1.22 (d, J )
7.2 Hz, 3H), 1.0-1.25 (m, 2H), 0.82 (m, 18H). MS m/z: 624
(MH)+. Anal. (C31H50N4O7S) C, H, N.
1
solid (97% yield). H NMR (400 MHz): δ 8.82 (d, J ) 8.0 Hz,
1H), 8.31 (broad s, 3H), 6.72 (s, 2H), 4.57 (m, 1H), 3.91 (m, 1H),
3.03 (s, 3H), 1.69 (m, 1H), 1.35-1.54 (m, 2H), 1.44 (d, J ) 6.8
Hz, 3H), 0.93 (d, J ) 6.8 Hz, 3H), 0.89 (d, J ) 6.8 Hz, 3H). MS
m/z: 263 (MH)+.
H-Ser-LeuVSMe Trifluoroacetate (13b). 1H NMR (400
MHz): δ 8.57 (d, J ) 8.0 Hz, 1H), 8.16 (broad s, 3H), 6.72 (s,
2H), 4.59 (broad q, J ) 6.8 Hz, 1H), 3.6-3.9 (m, 2H), 3.77 (t, J
) 6.8 Hz, 1H), 2.99 (s, 3H), 1.65 (m, 1H), 1.45 (m, 2H), 0.90 (d,
J ) 6.8 Hz, 3H), 0.87 (d, J ) 6.8 Hz, 3H). MS m/z: 279 (MH)+.
Cbz-Ile-Phe-Ala-LeuVSMe (17b). 1H NMR (400 MHz): δ 8.14
(d, J ) 7 Hz, 1H), 7.97 (m, 2H), 7.32 (m, 6H), 7.22 (m, 5H), 6.65
(m, 2H), 5.02 (s, 2H), 4.54 (m, 2H), 4.24 (m, 1H), 3.80 (t, J ) 8
Hz, 1H), 3.01 (m, 4H), 2.77 (m, 1H), 1.62 (m, 2H), 1.39 (m, 2H),
1.23 (d, J ) 7 Hz, 4H), 1.02 (m, 1H), 0.83 (m, 6H), 0.74 (t, J )
7 Hz, 3H), 0.65 (d, J ) 7 Hz, 3H). MS m/z: 658 (MH)+. Anal.
(C34H48N4O7S) C, H, N.
1
H-Aib-LeuVSMe Hydrochloride (13c). H NMR (400 MHz):
δ 8.45 (d, J ) 8.4 Hz, 1H), 8.23 (broad s, 3H), 6.70 (dd, J ) 15.2,
4.8 Hz, 1H), 6.63 (d, J ) 15.2 Hz, 1H), 4.65 (m, 1H), 3.04 (s,
3H), 1.60 (s, 3H), 1.47 (s, 3H), 1.4-1.7 (m, 3H), 0.93 (d, J ) 5.6
Hz, 3H), 0.88 (d, J ) 6.0 Hz, 3H). MS m/z: 299 (m + Na)+.
Tetrapeptide Vinyl Sulfone Syntheses. Method A. Cbz-Ile-
Glu(OtBu)-OH (14j). Succinate Ester Coupling. Cbz-(L)-isoleu-
cine succinate ester (2.17 g, 6.00 mmol) and (L)-glutamic acid
γ-tert-butyl ester (1.22 g, 6.00 mmol) were dissolved in a mixture
of THF (98 mL) and water (9 mL). Triethylamine (1.67 mL, 12.00
mmol) was added. The solution was stirred at ambient temperature
for 14 h, then poured into 0.2 N aqueous HCl (200 mL), and
extracted twice with CH2Cl2. The combined organic phase was
washed with water and brine and then dried over anhyd Mg2SO4.
Filtration and solvent evaporation afforded 2.5 g of foam. Trituration
with ether/hexane gave 1.84 g of 14j as a white powder (68% yield).
1H NMR (270 MHz): δ 8.15 (d, J ) 7.6 Hz, 1H), 7.35 (m, 6H),
5.02 (s, 2H), 4.22 (m, 1H), 3.89 (m, 1H), 2.26 (t, J ) 7.9 Hz, 2H),
1.94 (m, 1H), 1.72 (m, 3H), 1.39 (s, 9H), 1.09 (m,1H), 0.83 (m,
6H). MS m/z: 449 (M - H)-.
1
Cbz-Ile-hPhe-Ala-LeuVSMe (17c). H NMR (400 MHz): δ
8.09 (d, J ) 7.2 Hz, 1H), 8.00 (d, J ) 10.0 Hz, 2H), 7.47 (d, J )
7.2 Hz, 1H), 7.2-7.4 (m, 10H), 6.65 (m, 2H), 5.03 (s, 2H), 4.53
(m, 1H), 4.26 (m, 2H), 3.93 (t, J ) 7.2 Hz, 1H), 2.98 (s, 3H), 2.56
(m, 2H), 1.8-2.0 (m, 3H), 1.58 (m, 1H), 1.3-1.5 (m, 2H), 1.24
(d, J ) 6.8 Hz, 3H), 1.15 (m, 2H), 0.85 (m, 12H). MS m/z: 671
(MH)+. Anal. (C35H50N4O7S) C, H, N.
1
Cbz-Ile-trans-StyrylAla-Ala-LeuVSMe (17d). H NMR (270
MHz, CD3OD): δ 7.05-7.3 (m, 10H), 6.72 (dd, J ) 15.4, 4.5 Hz,
1H), 6.54 (d, J ) 15.4 Hz, 1H), 6.42 (d, J ) 15.8 Hz, 1H), 6.14
(m, 1H), 4.94 (ABq, 2H), 4.55 (m, 1H), 4.32 (m, 1H), 4.21 (q, J )
7.2 Hz, 1H), 3.83 (d, J ) 6.2 Hz, 1H), 2.87 (s, 3H), 2.65 (m, 1H),
2.53 (m, 1H), 1.2-1.8 (m, 5H), 1.30 (d, J ) 7.4 Hz, 3H), 1.08 (m,
1H), 0.81 (m, 12H). MS m/z: 683 (MH)+. HPLC homogeneity
95.0% (System A); 93.7% (System B).
Cbz-Ile-Ser(OBn)-Ala-LeuVSMe (17e). 1H NMR (400 MHz):
δ 8.07 (m, 2H), 7.81 (d, J ) 8 Hz, 1H), 7.41 (d, J ) 7 Hz, 1H),
7.30 (m, 10H), 6.62 (m, 2H), 5.02 (ABq, 2H), 4.56 (m, 1H), 4.50
(m, 1H), 4.48 (s, 2H), 4.24 (t, J ) 7 Hz, 1H), 3.95 (t, J ) 7 Hz,
1H), 3.62 (broad s, 2H), 2.97 (s, 3H), 1.75 (m, 1H), 1.56 (m, 1H),
Cbz-Ile-Glu(OtBu)-OSu (15j). Succinate Ester Preparation.
Compound 14j (1.73 g, 3.84 mmol) and N-hydroxysuccinimide

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2963
1.0-1.45 (m, 4H), 1.23 (d, J ) 7 Hz, 3H), 0.82 (m, 12H). MS
m/z: 687 (MH)+. HPLC homogeneity 94.7% (System A); 96.3%
(System B).
4.55 (q, J ) 6.8 Hz, 1H), 3.92 (m, 1H), 2.88 (d, J ) 5.6 Hz, 2H),
2.61 (dd, overlaps with HOD, J ) 15.2, 5.6 Hz, 2H), 1.0-1.8 (m,
3H), 0.84 (m, 15H). MS m/z: 448 (M - H)-.
1
Cbz-Ile-MetO2-Ala-LeuVSMe (17f). H NMR (400 MHz): δ
Cbz-Ile-NeopentylAsn-Ala-LeuVSMe (17q). Mixed anhydride
coupling of 14q with 13a using the general procedure outlined for
8.18 (d, J ) 8.0 Hz, 1H), 8.13 (d, J ) 6.8 Hz, 1H), 8.08 (d, J )
8.4 Hz, 1H), 7.45 (d, J ) 8.4 Hz, 1H), 7.35 (m, 5H), 6.68 (dd, J
) 15.2, 4.4 Hz, 1H), 6.62 (d, J ) 15.2 Hz, 1H), 5.03 (s, 2H), 4.55
(m, 1H), 4.42 (q, J ) 6.0 Hz, 1H), 4.22 (m, 1H), 3.90 (t, J ) 7.6
Hz, 1H), 3.11 (m, 2H), 3.00 (s, 3H), 2.94 (s, 3H), 2.00 (m, 2H),
1.73 (m, 1H), 1.62 (m, 1H), 1.41 (m, 3H), 1.25 (d, J ) 7.2 Hz,
3H), 1.13 (m, 1H), 0.83 (m, 12H). MS m/z: 674 (MH)+. Anal.
(C30H48N4O9S2) C, H, N.
1
10a gave 17q in a 71% yield. H NMR (400 MHz): δ 8.35 (d, J
) 7.2 Hz, 1H), 8.17 (d, J ) 7.6 Hz, 1H), 8.10 (d, J ) 8.4 Hz, 1H),
8.07 (t, J ) 6.4 Hz, 1H), 7.41 (d, J ) 8.0 Hz, 1H), 7.36 (m, 5H),
6.65 (dd, J ) 15.2, 4.0 Hz, 1H), 6.60 (d, J ) 15.2 Hz, 1H), 5.03
(ABq, 2H), 4.55 (m, 2H), 4.10 (m, 1H), 3.90 (t, J ) 8.0 Hz, 1H),
2.92 (s, 3H), 2.87 (m, 2H), 2.70 (m, 1H), 2.55 (m, 1H), 1.5-1.75
(m, 3H), 1.41 (m, 2H), 1.25 (d, J ) 7.2 Hz, 3H), 1.12 (m, 1H),
0.89 (d, J ) 6.4 Hz, 3H), 0.84 (d, J ) 6.4 Hz, 3H), 0.81 (m, 15
H). gCOSY NMR shows coupling between peaks at δ 1.25 (Ala
CH3) and δ 4.10 (Ala CH), δ 3.90 (Ile CH) and δ 7.41 (Ile NH),
and between peaks at δ 2.87 (HNCH2t-Bu) and δ 8.07 (HNCH2t-
Bu). MS m/z: 694 (MH)+. Anal. (C34H55N5O8S) C, H, N.
1
Compound 17g. H NMR (400 MHz): δ 8.65 (d, J ) 8.0 Hz,
1H), 8.31 (d, J ) 6.8 Hz, 1H), 7.75 (d, J ) 8.0 Hz, 1H), 7.52 (d,
J ) 7.6 Hz, 1H), 7.3-7.5 (m, 10H), 6.66 (dd, J ) 15.2, 4.0 Hz,
1H), 6.61 (d, J ) 15.2 Hz, 1H), 4.99 (ABq, 2H), 4.90 (m, 1H),
4.53 (m, 3H), 4.18 (m, 1H), 3.87 (t, J ) 7.2 Hz, 1H), 3.61 (dd, J
) 14.8, 6.0 Hz, 1H), 3.39 (m, 1H), 2.97 (s, 3H), 1.73 (m, 1H),
1.59 (m, 1H), 1.40 (m, 3H), 1.25 (d, J ) 6.8 Hz, 3H), 1.12 (m,
1H), 0.83 (m, 12H). MS m/z: 736 (MH)+. Anal. (C35H50N4O9S2)
C, H, N.
Other Compounds Made Using Method B. Cbz-Ile-Asp-
1
(OtBu)-Ala-LeuVSMe (17h). H NMR (400 MHz): δ 8.32 (d, J
) 7.6 Hz, 1H), 7.90 (d, J ) 6.8 Hz, 1H), 7.85 (d, J ) 8.4 Hz, 1H),
7.42 (d, J ) 8.0 Hz, 1H), 7.35 (m, 5H), 6.67 (dd, J ) 15.2, 4.4 Hz,
1H), 6.60 (d, J ) 15.2 Hz, 1H), 5.02 (ABq, 2H), 4.56 (m, 2H),
4.20 (m, 1H), 3.87 (t, J ) 7.0 Hz, 1H), 2.98 (s, 3H), 2.73 (dd, J )
16.8, 5.6 Hz, 1H), 2.51 (m, overlaps with DMSO, 1H), 1.5-1.8
(m, 2H), 1.42 (m, 2H), 1.37 (s, 9H), 1.22 (d, J ) 6.8 Hz, 3H), 1.12
(m, 2H), 0.83 (m, 12H). MS m/z: 682 (MH)+. Anal. (C33H52N4O9S)
C, H, N.
Cbz-Ile-Asp(OBn)-Ala-LeuVSMe (17i). 1H NMR (400 MHz):
δ 8.40 (d, J ) 8.0 Hz, 1H), 7.94 (d, J ) 6.8 Hz, 1H), 7.83 (d, J )
8.8 Hz, 1H), 7.42 (d, J ) 8.0 Hz, 1H), 7.34 (m, 10H), 6.68 (dd, J
) 15.4, 4.4 Hz, 1H), 6.60 (dd, J ) 15.4, 1.2 Hz, 1H), 5.01 (m,
4H), 4.66 (q, J ) 7.6 Hz, 1H), 4.54 (m, 1H), 4.19 (m, 1H), 3.88 (t,
J ) 7.6 Hz, 1H), 2.99 (s, 3H), 2.90 (dd, J ) 16.4, 6.4 Hz, 1H),
2.70 (dd, J ) 16.4, 8.0 Hz, 2H), 1.65 (m, 2H), 1.41 (m, 3H), 1.22
(d, J ) 6.8 Hz, 2H), 1.11 (m, 1H), 0.83 (m, 12H). MS m/z: 716
(MH)+. Anal. (C36H50N4O9S) C, H, N.
Cbz-Ile-t-ButylhomoGlu-Ala-LeuVSMe (17k). 1H NMR (270
MHz): δ 7.97 (m, 3H), 7.35 (m, 6H), 6.65 (m, 2H), 5.03 (s, 2H),
4.53 (m, 1H), 4.24 (m, 2H), 3.89 (t, J ) 7.9 Hz, 1H), 2.99 (s, 3H),
2.14 (broad t, J ) 6.0 Hz, 2H), 1.0-1.8 (m, 10H), 1.41 (s, 9H),
1.24 (d, J ) 7.0 Hz, 3H), 0.83 (m, 12H). MS m/z: 709 (MH)+.
Anal. (C35H56N4O9S) C, H, N.
Cbz-Ile-CyclohexylGlu-Ala-LeuVSMe (17l). 1H NMR (300
MHz): δ 8.07 (m, 3H), 7.44 (d, J ) 9 Hz, 1H), 7.37 (s, 5H), 6.64
(m, 2H), 5.04 (s, 2H), 4.61 (m, 2H), 4.28 (m, 2H), 3.91 (t, J ) 9
Hz, 1H), 3.00 (s, 3H), 2.30 (m, 2H), 1.55-2.0 (m, 8H), 1.1-1.55
(m, 10H), 1.22 (d, J ) 7 Hz, 3H), 0.83 (m, 12H). MS m/z: 722
(MH)+. Anal. (C36H56N4O9S) C, H, N.
1
Cbz-Ile-hGln-Ala-LeuVSMe (17n). H NMR (400 MHz): δ
8.00 (m, 4H), 7.36 (m, 5H), 7.21 (broad s, 1H), 6.73 (broad s, 1H),
6.65 (ABq, 2H), 5.03 (ABq, 2H), 4.54 (m, 1H), 4.22 (m, 2H), 3.89
(t, J ) 7.6 Hz, 1H), 2.99 (s, 3H), 2.02 (broad t, J ) 6.4 Hz, 2H),
1.3-1.8 (m, 10H), 1.22 (d, J ) 7.2 Hz, 3H), 1.0-1.3 (m, 2H),
0.83 (m, 12H). MS m/z: 652 (MH)+. Anal. (C31H49N5O8S‚H2O)
C, H, N.
1
Cbz-Ile-Gln-Ala-LeuVSMe (17m). H NMR (270 MHz): δ
8.14 (J ) 7.4 Hz, 1H), 7.95 (d, J ) 6.9 Hz, 2H), 7.37 (m, 6H),
7.23 (broad s, 1H), 6.78 (broad s, 1H), 6.65 (m, 2H), 5.03 (d, J )
2.5 Hz, 2H), 4.55 (m, 1H), 4.22 (m, 2H), 3.89 (t, J ) 7.7 Hz, 1H),
2.99 (s, 3H), 2.11 (t, J ) 7.7 Hz, 2H), 1.3-1.9 (m, 7H), 1.22 (d,
J ) 6.9 Hz, 3H), 1.0-1.2 (m, 1H), 0.85 (m, 12H). MS m/z: 639
(MH)+. Anal. (C30H47N5O8S) C, H, N.
Cbz-Ile-iPrGln-Ala-LeuVSMe (17o). 1H NMR (300 MHz): δ
8.26 (d, J ) 8 Hz, 1H), 8.04 (m, 2H), 7.85 (d, J ) 8 Hz, 1H), 7.49
(d, J ) 7 Hz, 1H), 7.36 (m, 5H), 6.67 (m, 2H), 5.05 (s, 2H), 4.54
(m, 1H), 4.23 (m, 2H), 3.88 (m, 1H), 2.97 (s, 3H), 2.11 (broad t,
J ) 7 Hz, 2H), 1.3-2.0 (m, 9H), 1.24 (d, J ) 7 Hz, 3H), 1.03 (d,
J ) 6 Hz, 6H), 0.86 (m, 12H). MS m/z: 681 (MH)+. HPLC
homogeneity 96.1% (System A); 91.0% (System B).
Method B. Boc-NeopentylAsn-O-t-Bu (19q). Side Chain Cou-
pling with EDC/HOAt. To a stirred solution of Boc-Asp-R-tert-
butyl ester (3.00 g, 10.4 mmol) and N-methylmorpholine (2.3 mL,
21 mmol) in dry DMF (50 mL) were added EDC (2.00 g, 10.4
mmol) and HOAt (0.14 g, 1.04 mmol). After 15 min, neopenty-
lamine (1.40 mL, 12.0 mmol) was added. After 4 h, 1 M HCl (150
mL) was added, and the mixture plus 100 mL of ether was
transferred to a separatory funnel. The organic phase was separated,
and the aqueous phase was washed with more ether. The combined
organic phase was washed with water, saturated NaHCO3, and brine.
Drying over MgSO4 followed by concentration gave 3.21 g, a 90%
yield, of 19q as a colorless syrup that slowly crystallized. 1H NMR
(400 MHz, CDCl3): δ 5.72 (broad d, 2H), 4.36 (m, 1H), 3.05 (d,
J ) 6.6 Hz, 2H), 2.77 (ABm, 2H), 1.48 (s, 9H), 1.46 (s, 9H), 0.92
(s, 9H). MS m/z: 359 (MH)+.
Cbz-Ile-t-ButylGln-Ala-LeuVSMe (17p). 1H NMR (300 MHz):
δ 8.16 (d, J ) 8 Hz, 1H), 8.09 (m, 1H), 7.97 (m, 2H), 7.45 (d, J
) 8 Hz, 1H), 7.33 (m, 5H), 6.66 (m, 2H), 5.05 (s, 2H), 4.56 (m,
1H), 4.23 (m, 2H), 3.90 (t, J ) 7 Hz, 1H), 2.98 (s, 3H), 2.09 (broad
t, J ) 7 Hz, 2H), 1.5-2.0 (m, 3H), 1.40 (m, 3H), 1.21 (m, 14H),
0.83 (m, 12H). MS m/z: 695 (MH)+. Anal. (C34H55N5O8S) C, H,
N.
Cbz-Ile-NeopentylGln-Ala-LeuVSMe (17r). 1H NMR (400
MHz): δ 8.16 (d, J ) 7.6 Hz, 1H), 7.96 (d, J ) 8.0 Hz, 2H), 7.69
(t, J ) 6.4 Hz, 1H), 7.43 (d, J ) 8.4 Hz, 1H), 7.36 (m, 5H), 5.04
(ABq, 2H), 4.54 (m, 1H), 4.23 (m, 2H), 3.89 (t, J ) 7.2 Hz, 1H),
2.99 (s, 3H), 2.87 (m, 2H), 2.18 (broad t, J ) 7.2 Hz, 2H), 1.65-
1.95 (m, 4H), 1.60 (m, 1H), 1.41 (m, 3H), 1.22 (d, J ) 7.2 Hz,
3H), 1.13 (m, 2H), 0.82 (m, 21H). MS m/z: 709 (MH)+. Anal.
(C35H57N5O8S) C, H, N.
â-Neopentylasparagine Hydrochloride (20q). Compound 19q
(1.20 g, 3.48 mmol) was dissolved in 4 M HCl in 1,4-dioxane (10
mL, 40 mmol). After 2 h, the reaction mixture was concentrated
by rotary evaporation in the presence of several portions of toluene.
Ether was added, and the white solid was filtered and vacuum-
dried. Compound 20q (0.82 g, 99%) was obtained. 1H NMR (400
MHz, CDCl3): δ 8.32 (broad s, 3H), 8.15 (s, 1H), 4.13 (broad s,
1H), 2.80 (m, 2H), 2.52 (s, 2H), 0.83 (s, 9H). MS m/z: 203 (MH)+.
Cbz-Ile-NeopentylAsn-OH (14q). Coupling of 20q with Cbz-
Ile-OSu using the procedure described for the preparation of 16j
1
Compound 17s. H NMR (300 MHz): δ 8.16 (d, J ) 8 Hz,
1H), 7.95 (m, 2H), 7.45 (d, J ) 8 Hz, 1H), 7.38 (s, 5H), 7.20 (s,
1H), 6.65 (m, 2H), 5.03 (s, 2H), 4.57 (m, 1H), 4.23 (m, 2H), 3.91
(t, J ) 7 Hz, 1H), 3.00 (s, 3H), 2.12 (bt, J ) 7 Hz, 2H), 1.5-1.9
(m, 6H), 1.42 (m, 4H), 1.22 (d, J ) 7 Hz, 3H), 1.15 (d, J ) 5 Hz,
6H), 0.83 (m, 15H). MS m/z: 709 (MH)+. Anal. (C35H57N5O8S)
C, H, N.
1
afforded dipeptide 14q in a 47% yield. H NMR (400 MHz): δ
12.6 (broad s, 1H), 8.12 (d, J ) 7.6 Hz, 1H), 7.83 (broad t, J )
5.6 Hz, 1H), 7.52 (d, J ) 8.4 Hz, 1H), 7.35 (m, 5H), 5.04 (s, 2H),
Cbz-Ile-Pro-Ala-LeuVSMe (17t). 1H NMR (400 MHz): δ 7.99
(d, J ) 7.6 Hz, 1H), 7.87 (d, J ) 8.4 Hz, 1H), 7.54 (d, J ) 8.8 Hz,

2964 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
1H), 7.33 (m, 6H), 6.66 (s, 2H), 5.01 (ABq, 2H), 4.55 (m, 1H),
4.32 (m, 1H), 4.15 (m, 1H), 4.07 (t, J ) 8.4 Hz, 1H), 3.74 (m,
1H), 3.60 (m, 1H), 2.97 (s, 3H), 2.04 (m, 1H), 1.65-1.95 (m, 4H),
1.30-1.65 (m, 3H), 1.26 (d, J ) 6.8 Hz, 3H), 1.0-1.3 (m, 1H),
0.83 (m, 12H). MS m/z: 608 (MH)+. Anal. (C30H46N4O7S‚0.5 H2O)
C, H, N.
1H), 4.03 (m, 1H), 3.79 (m, 1H), 3.39 (m, 2H), 2.95 (s, 3H), 2.09
(m, 2H), 1.5-1.8 (m, 3H), 1.34 (s, 9H), 1.25-1.5 (m, 2H), 0.85
(m, 6H). MS m/z: 602 (M - H)-. HPLC homogeneity 97.7%
(System A); 97.1% (System B).
pTosyl-Glu(OtBu)-Ala-LeuVSMe (22g). 1H NMR (400
MHz): δ 8.10 (d, J ) 6.0 Hz, 1H), 7.98 (d, J ) 6.8 Hz, 1H), 7.89
(d, J ) 6.8 Hz, 1H), 7.62 (d, J ) 8.0 Hz, 2H), 7.32 (d, J ) 8.0 Hz,
2H), 6.60 (m, 2H), 4.51 (m, 1H), 3.97 (m, 1H), 3.73 (m, 1H), 2.99
(s, 3H), 2.36 (s, 3H), 2.11 (m, 2H), 1.5-1.8 (m, 3H), 1.36 (s, 9H),
1.2-1.45 (m, 2H), 1.08 (d, J ) 6.8 Hz, 3H), 0.83 (m, 6H). MS
m/z: 600 (M - H)-. Anal. (C27H43N3O8S2) C, H, N.
Tripeptide Vinyl Sulfone Syntheses. pTosyl-NeopentylAsn-
OH (21). Compound 20q (1.50 g, 6.28 mmol) was dissolved in
THF (15 mL) and 1.0 N NaOH (15 mL) and then cooled in an ice
bath. p-Toluenesulfonyl chloride (1.20 g, 6.28 mmol) was added
portionwise to the vigorously stirred solution. The pH of the reaction
mixture was frequently adjusted to >8 by the addition of 4 M
NaOH. After 2 h, the mixture was acidified with 6 M HCl and
extracted thrice with EtOAc. The combined organic phase was
washed with 1 M HCl, water, and brine and then dried over MgSO4.
Filtration and solvent evaporation followed by vacuum-drying gave
1
Compound 22h. H NMR (400 MHz): δ 8.47 (d, J ) 8.0 Hz,
1H), 8.11 (d, J ) 7.2 Hz, 1H), 8.03 (d, J ) 8.0 Hz, 1H), 7.24
(m, 10H), 6.64 (m, 2H), 5.10 (s, 1H), 4.52 (m, 1H), 4.29 (m, 1H),
4.19 (m, 1H), 2.99 (s, 3H), 2.14 (m, 2H), 1.83 (m, 1H), 1.50-1.75
(m, 2H), 1.36 (s, 9H), 1.3-1.5 (m, 2H), 1.21 (d, J ) 7.2 Hz, 3H),
0.84 (m, 6H). MS m/z: 642 (MH)+. Anal. (C34H47N3O7S) C, H,
N.
1
21 (2.24 g, 60%) as a white solid, mp 163.5-165 °C. H NMR
(400 MHz): δ 12.55 (broad s, 1H), 7.89 (d, J ) 8.4 Hz, 1H), 7.71
(t, J ) 6.0 Hz, 1H), 7.62 (d, J ) 8.0 Hz, 2H), 7.31 (d, J ) 8.0 Hz,
2H), 4.09 (q, J ) 7.2 Hz, 1H), 2.79 (m, 2H), 2.50 (m, overlaps
with DMSO, 1H), 2.36 (s, 3H), 2.28 (dd, J ) 15.2, 6.0 Hz, 1H),
0.78 (s, 9H).
Cbz-BocDab-Ser-LeuVSMe (22i). 1H NMR (400 MHz): δ 8.02
(d, J ) 8.4 Hz, 1H), 7.94 (d, J ) 8.0 Hz, 1H), 7.54 (d, J ) 7.2 Hz,
1H), 7.34 (m, 5H), 6.80 (broad t, J ) 7.0 Hz, 1H), 6.69 (m, 2H),
5.07 (m, 1H), 5.02 (s, 2H), 4.57 (m, 1H), 4.27 (m, 1H), 4.04 (m,
1H), 3.60 (m, 2H), 2.99 (s, 5H), 1.77 (m, 1H), 1.61 (m, 2H), 1.36
(s, 9H), 1.2-1.5 (m, 2H), 0.86 (m, 6H). MS m/z: 611 (M - H)-.
Anal. (C27H41N3O8S) C, H, N.
pTosyl-NeopentylAsn-Ser-LeuVSMe (22n). Compound 21 was
coupled with 13b using the standard mixed anhydride protocol
described above for the preparation of 10a. For 22n, a white solid:
1H NMR (400 MHz): δ 8.22 (d, J ) 6.0 Hz, 1H), 8.05 (m, 3H),
7.63 (d, J ) 7.8 Hz, 2H), 7.31 (d, J ) 7.8 Hz, 2H), 6.59 (dd, J )
15.2, 4.0 Hz, 1H), 6.52 (d, J ) 15.2 Hz, 1H), 4.50 (m, 1H), 4.10
(m, 1H), 3.62 (m, 2H), 3.35 (m, obscured by H2O, 2H), 2.86 (s,
3H), 2.7-2.9 (m, 2H), 2.61 (m, 1H), 2.40 (m, 1H), 2.38 (s, 3H),
1.62 (m, 1H), 1.55 (m, 1H), 1.37 (m, 1H), 0.85 (d, J ) 6.8 Hz,
3H), 0.81 (d, J ) 6.8 Hz, 3H), 0.78 (s, 9H). MS m/z: 617 (MH)+.
Anal. (C27H44N4O8S2) C, H, N.
1
Ac-Glu(OBn)-Ala-LeuVSMe (22j). H NMR (400 MHz): δ
8.15 (d, J ) 7.2 Hz, 1H), 8.09 (d, J ) 8.0 Hz, 1H), 7.96 (d, J )
8.4 Hz, 1H), 7.37 (m, 5H), 6.68 (dd, J ) 15.2, 4.4 Hz, 1H), 6.61
(d, J ) 15.2 Hz, 1H), 5.08 (s, 2H), 4.53 (m, 1H), 4.24 (m, 2H),
2.99 (s, 3H), 2.40 (broad t, J ) 7.6 Hz, 2H), 1.7-2.0 (m, 2H),
1.84 (s, 3H), 1.59 (m, 1H), 1.38 (m, 2H), 1.24 (d, J ) 7.2 Hz, 3H),
0.85 (d, J ) 6.8 Hz, 3H), 0.80 (d, J ) 6.4 Hz, 3H). MS m/z: 524
(MH)+. Anal. (C25H37N3O7S) C, H, N.
1
1
Ac-Glu(OtBu)-Ala-LeuVSMe (22a). H NMR (300 MHz): δ
Compound 22k. H NMR (400 MHz): δ 8.43 (d, J ) 6.4 Hz,
8.12 (d, J ) 7 Hz, 1H), 8.09 (d, J ) 8 Hz, 1H), 7.98(d, J ) 9 Hz,
1H), 6.66 (m, 2H), 4.55 (m, 1H), 4.23 (m, 2H), 3.00 (s, 3H), 2.22
(t, J ) 7 Hz, 2H), 1.85 (s, 3H), 1.5-1.9 (m, 3H), 1.38 (s, 9H),
1.1-1.5 (m, 2H), 1.24 (d, J ) 7 Hz, 3H), 0.85 (m, 6H). MS m/z:
490 (MH)+. Anal. (C22H39N3O7S) C, H, N.
1H), 8.12 (d, J ) 8.8 Hz, 1H), 8.04 (d, J ) 8.4 Hz, 1H), 7.51 (d,
J ) 8.0 Hz, 1H), 7.35 (m, 5H), 6.62 (s, 2H), 5.01 (ABq, 2H), 4.53
(m, 1H), 4.29 (q, J ) 7.6 Hz, 1H), 4.13 (m, 1H), 4.08 (d, J ) 8.8
Hz, 1H), 2.92 (s, 3H), 2.65 (d, J ) 7.2 Hz, 2H), 1.64 (m, 1H),
1.3-1.6 (m, 2H), 1.39 (s, 9H), 1.26 (d, J ) 6.8 Hz, 3H), 0.87 (m,
15H). MS m/z: 679 (M - H)-. Anal. (C33H52N4O9S‚0.5 H2O) C,
H, N.
Benzoyl-Glu(OtBu)-Ala-LeuVSMe (22b). 1H NMR (400
MHz): δ 8.49 (d, J ) 7.6 Hz, 1H), 8.15 (d, J ) 7.2 Hz, 1H), 7.94
(d, J ) 8.8 Hz, 1H), 7.87 (d, J ) 8.0 Hz, 2H), 7.53 (m, 1H), 7.45
(m, 2H), 6.64 (m, 2H), 4.53 (m, 1H), 4.38 (m, 1H), 4.25 (m, 1H),
2.99 (s, 3H), 2.32 (broad t, J ) 8.0 Hz, 2H), 1.8-2.1 (m, 2H),
1.59 (m, 1H), 1.34 (s, 9H), 1.3-1.5 (m, 2H), 1.24 (d, J ) 7.2 Hz,
3H), 0.83 (m, 6H). MS m/z: 552 (MH)+. Anal. (C27H41N3O7S) C,
H, N.
Compound 22l. 1H NMR (400 MHz): δ 8.04 (m, 3H), 7.50 (d,
J ) 7.6 Hz, 1H), 7.35 (m, 5H), 6.68 (dd, J ) 15.2, 8.0 Hz, 1H),
6.62 (d, J ) 15.2 Hz, 1H), 5.02 (m, 2H), 4.54 (m, 1H), 4.28 (m,
1H), 3.97 (m, 1H), 2.99 (s, 3H), 2.10 (m, 2H), 1.87 (m, 1H), 1.5-
1.8 (m, 2H), 1.38 (m, 2H), 1.34 (s, 9H), 1.25 (m, 9H), 0.88 (d, J
) 6.0 Hz, 3H), 0.82 (d, J ) 6.0 Hz, 3H). MS m/z: 667 (MH)+.
Anal. (C32H50N4O9S‚0.5 H2O) C, H, N.
Cbz-Glu(OtBu)-Ala-LeuVSMe (22c). 1H NMR (400 MHz): δ
8.09 (d, J ) 7.6 Hz, 1H), 8.01 (d, J ) 8.8 Hz, 1H), 7.48 (d, J )
8.0 Hz, 1H), 7.33 (m, 5H), 6.65 (m, 2H), 5.01 (ABq, 2H), 4.54
(m, 1H), 4.24 (m, 1H), 4.00 (m, 1H), 2.99 (s, 3H), 2.23 (broad t,
J ) 8.0 Hz, 2H), 1.84 (m, 1H), 1.5-1.8 (m, 2H), 1.38 (s, 9H),
1.23 (d, J ) 7.0 Hz, 3H), 1.1-1.5 (m, 2H), 0.84 (m, 6H). MS m/z:
604 (M + Na)+. Anal. (C28H43N3O8S) C, H, N.
pTosyl-NeopentylAsn-Ala-LeuVSMe (22m). 1H NMR (400
MHz): δ 8.43 (d, J ) 6.4 Hz, 1H), 8.17 (d, J ) 8.8 Hz, 1H), 8.12
(t, J ) 6.0 Hz, 1H), 8.04 (d, J ) 8.4 Hz, 1H), 7.65 (d, J ) 8.6 Hz,
2H), 7.35 (d, J ) 8.6 Hz, 2H), 6.59 (dd, J ) 15.2, 4.0 Hz, 1H),
6.48 (d, J ) 15.2 Hz, 1H), 4.48 (m, 1H), 4.00 (m, 1H), 3.52 (m,
1H), 2.88 (s, 3H), 2.6-2.9 (m, 3H), 2.44 (m, 1H), 2.37 (s, 3H),
1.57 (m, 2H), 1.38 (m, 1H), 1.06 (d, J ) 7.6 Hz, 3H), 0.87 (d, J )
6.8 Hz, 3H), 0.82 (d, J ) 7.2 Hz, 3H), 0.79 (s, 9H). MS m/z: 601
(MH)+. Anal. (C27H44N4O7S2) C, H, N.
Cbz-Glu(OtBu)-Gly-LeuVSMe (22d). 1H NMR (400 MHz): δ
8.25 (broad t, J ) 5.6 Hz, 1H), 7.85 (d, J ) 8.0 Hz, 1H), 7.60 (d,
J ) 5.6 Hz, 1H), 7.32 (m, 5H), 6.69 (m, 2H), 5.02 (ABq, 2H),
4.57 (m, 1H), 3.99 (m, 1H), 3.73 (m, 2H), 2.98 (s, 3H), 2.25 (broad
t, J ) 7.2 Hz, 2H), 1.88 (m, 1H), 1.74 (m, 1H), 1.59 (m, 1H), 1.38
(s, 9H), 1.3-1.5 (m, 2H), 0.86 (m, 6H). MS m/z: 566 (M - H)-.
Anal. (C27H41N3O8S) C, H, N.
pTosyl-NeopentylAsn-Aib-LeuVSMe (22o). 1H NMR (400
MHz): δ 8.24 (s, 1H), 8.12 (m, 2H), 7.96 (d, J ) 8.4 Hz, 1H),
7.68 (d, J ) 8.4 Hz, 2H), 7.31 (d, J ) 8.4 Hz, 2H), 6.62 (d, J )
15.2 Hz, 1H), 6.55 (dd, J ) 15.2, 3.0 Hz, 1H), 4.41 (m, 1H), 4.02
(m, 1H), 2.86 (s, 3H), 2.6-2.9 (m, 3H), 2.46 (m, obscured by
DMSO, 1H), 2.35 (s, 3H), 1.56 (m, 2H), 1.32 (m, 1H), 1.09 (s,
3H), 0.95 (s, 3H), 0.85 (d, J ) 6.4 Hz, 3H), 0.78 (s, 12H). MS
m/z: 613 (M - H)-. Anal. (C28H46N4O7S2) C, H, N.
1,3,4-Ketooxadiazole Syntheses. {1-[(R/S)-Hydroxy-(5-phenyl-
[1,3,4]oxadiazol-2-yl)-(S)-methyl]-3-methylbutyl}-carbamic Acid
tert-Butyl Ester (24). A solution of 2-phenyl-[1,3,4]oxadiazole
(2.34 g, 16.0 mmol) in anhydrous THF (100 mL) was cooled with
a dry ice bath and stirred under N2. A solution of 1.6 M
n-butyllithium in hexanes (10.0 mL, 16.0 mmol) was added. After
Cbz-Glu(OtBu)-Ser-LeuVSMe (22e). 1H NMR (400 MHz): δ
8.04 (d, J ) 8.0 Hz, 1H), 7.94 (d, J ) 7.2 Hz, 1H), 7.52 (d, J )
6.8 Hz, 1H), 7.32 (m, 5H), 6.69 (broad s, 2H), 5.02 (m, 3H), 4.57
(m, 1H), 4.27 (m, 1H), 4.04 (m, 1H), 3.59 (broad s, 2H), 2.96 (s,
3H), 2.23 (broad t, J ) 7.2 Hz, 2H), 1.84 (m, 1H), 1.5-1.8 (m,
2H), 1.38 (s, 9H), 1.2-1.5 (m, 2H), 0.84 (m, 6H). MS m/z: 596
(M - H)-. Anal. (C28H43N3O9S) C, H, N.
Benzenesulfonyl-Glu(OtBu)-Ser-LeuVSMe (22f). 1H NMR
(400 MHz): δ 8.02 (d, J ) 6.8 Hz, 3H), 7.74 (d, J ) 7.6 Hz, 2H),
7.54 (m, 3H), 6.66 (m, 2H), 5.03 (t, J ) 9.2 Hz, 1H), 4.55 (m,

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2965
1 h, MgBr2 diethyl etherate (4.13 g, 16.0 mmol) was added in one
portion. After another 1 h, a solution of freshly prepared Boc
leucinal (1.94 g, 8.0 mmol)38 was added as a solution in 20 mL of
THF. After an additional 1.5 h at low temperature, 1 M HCl was
added, and the cold bath was removed. The mixture was allowed
to warm and then was transferred to a sep funnel. Ether (100 mL)
was added. The organic phase was separated, and the aqueous phase
was extracted twice more with ether. The combined organic phase
was washed with water, aq NaHCO3, and brine and then dried over
MgSO4. Filtration and solvent evaporation gave an oily residue that
was flash chromatographed on silica gel, eluting with a 25-50%
anhyd MgSO4 followed by filtration and solvent evaporation gave
a sticky brown oil. Trituration with ether followed by filtration and
drying of the precipitate gave compound 28d (1.93 g, 37% yield)
as a crude mixture of diastereomers that was used directly in the
next step. Mp 109-112 °C. 1H NMR (400 MHz): δ 7.65-8.1 (m,
7H), 7.57 (m, 4H), 7.26 (m, 2H), 6.27, 6.06 (2d, exchange with
D2O, J ) 6.4 Hz, 1H), 4.84, 4.59 (2t, J ) 6.4 Hz, 1H), 3.9-4.3
(m, 3H), 2.9-3.4 (m, 2H), 3.11, 2.99 (2s, 3H), 2.71 (m, 2H), 2.3-
2.5 (m, 2H), 2.35, 2.31 (2s, 3H), 1.3-1.7 (m, 3H), 0.75-0.9 (m,
6H), 0.80, 0.77 (2s, 9H). MS m/z: 702 (MH)+.
pTs-NeopentylAsn-Ser(OMe)-LeuKODPh (29d). Compound
28d (2.20 g, 3.14 mmol) was dissolved in 50 mL of water-saturated
CH2Cl2 and was vigorously stirred. Dess-Martin periodinane (2.00
g, 4.71 mmol) was added in one portion. After 2 h, more Dess-
Martin periodinane (0.67 g, 1.57 mmol) was added. After 4 h, a
solution of 0.26 M Na2S2O3 in sat. NaHCO3 (300 mL) was added
with vigorous stirring. After 30 min, the mixture was transferred
to a sep funnel, and CH2Cl2 (50 mL) was added. The organic phase
was separated and washed twice with the previously described
quenching solution and then once with sat. NaHCO3, water, and
brine. Drying over anhydrous MgSO4 followed by filtration and
solvent evaporation afforded 1.93 g of a pale yellow solid. Flash
chromatography on silica gel with 2.5/5.0/92.5 MeOH/acetone/CH2-
Cl2 gave 29d (1.73 g, 79%) as a pale yellow solid; mp 161-163
1
EtOAc/hexane gradient, to give 24 (4.27 g, 70% yield). H NMR
(400 MHz): δ 7.94 (d, J ) 6.0 Hz, 2H), 7.58 (s, 3H), 6.75, 6.64
(2d, J ) 9.2, 8.8 Hz, 1H total), 6.30, 6.10 (2d, J ) 6.0 Hz, 1H
total), 4.85, 4.52 (2m, 1H total), 3.85 (m, 1H), 1.1-1.7 (m, 12H),
0.86 (m, 6H). MS m/z: 384 (M + Na)+.
2-(S)-Amino-4-methyl-1-(5-phenyl-[1,3,4]oxadiazol-2-yl)-(R/
S)-pentan-1-ol Hydrochloride (25). Compound 24 (11.9 g, 32.9
mmol) was dissolved in 25 mL of CH2Cl2. A 4 M solution of HCl
in 1,4-dioxane (100 mL, 400 mmol) was added with stirring. After
2 h, ether (400 mL) was added, which caused an oil to form. The
supernatant was decanted, and the oil was triturated with more ether
to afford 25 as a pale yellow solid (9.73 g, >99% yield). 1H NMR
(400 MHz): δ 8.00 (m, 2H), 7.62 (m, 3H), 4.57 (m, 1H), 4.12
(broad s, 1H), 3.06 (m, 1H), 1.80 (m, 1H), 1.0-1.5 (m, 2H), 0.87
(m, 6H). MS m/z: 262 (MH)+.
1
°C. H NMR (400 MHz): δ 8.56 (d, J ) 6.4 Hz, 1H), 8.16 (d, J
) 7.6 Hz, 1H), 8.06 (d, J ) 8.0 Hz, 2H), 7.92 (d, J ) 8.8 Hz, 1H),
7.77 (t, J ) 6 Hz, 1H), 7.65 (m, 5H), 7.29 (d, J ) 8.0 Hz, 2H),
5.13 (m, 1H), 4.15 (m, 2H), 3.42 (m, 1H), 3.25 (m, 1H), 3.13 (s,
3H), 2.79 (m, 2H), 2.35 (s, 3H), 2.31 (m, 2H), 1.71 (m, 3H), 0.93
(broad s, 6H), 0.78 (s, 9H). MS m/z: 699.6 (MH)+. Anal.
(C34H46N6O8S) C, H, N.
(1-{1(R/S)-[Hydroxy-(5-phenyl-[1,3,4]oxadiazol-2-yl)-(S)-meth-
yl]-3-methyl-butylcarbamoyl}-2-(S)-methoxyethyl)-carbamic Acid
tert-Butyl Ester (26). N-R-Boc-O-methyl-L-serine (2.19 g, 10.0
mmol), HOBT (1.62 g, 12.0 mmol), EDC (2.01 g, 10.5 mmol),
and NMM (1.4 mL, 12.5 mmol) were stirred in anhyd CH2Cl2 (150
mL). After 5 min, compound 25 (2.61 g, 10.0 mmol) dissolved in
CH2Cl2 (50 mL), and more NMM (1.4 mL, 12.5 mmol) was added.
After 3 h, the reaction mixture was transferred to a sep funnel and
washed twice with 100 mL portions of 0.5 M HCl. The organic
phase was separated, washed once with water (50 mL) and twice
with saturated NaHCO3 (100 mL). The organic phase was dried
over MgSO4. Filtration and solvent evaporation gave a tan foam.
This was flash chromatographed on silica gel with 5% MeOH in
CH2Cl2 to give 26 as a brittle, pale yellow foam, (3.65 g, 79%), a
Other Compounds Prepared by This General Method. Ms-
1
NeopentylAsn-Ala-LeuKODPh (29a). H NMR (400 MHz): δ
8.52 (d, J ) 6.4 Hz, 1H), 8.26 (d, J ) 7.2 Hz, 1H), 8.07 (d, J )
7.2 Hz, 2H), 7.85 (broad t, J ) 6.0 Hz, 1H), 7.67 (m, 3H), 7.41 (d,
J ) 8.4 Hz, 1H), 5.16 (m, 1H), 4.29 (m, 1H), 4.14 (m, 1H), 2.75-
2.95 (m, 2H), 2.86 (s, 3H), 2.55 (m, obscured by DMSO, 2H),
1.6-1.9 (m, 3H), 1.20 (d, J ) 7.2 Hz, 3H), 0.95 (d, J ) 6.0 Hz,
6H), 0.82 (s, 9H). MS m/z: 615.5 (M + Na)+. Anal. (C27H40N6O7S)
C, H, N.
1
mixture of diastereomers. H NMR (400 MHz): δ 7.96 (m, 2H),
mTs-NeopentylAsn-Ala-LeuKODPh (29b). 1H NMR (400
MHz): δ 8.44 (d, J ) 6.4 Hz, 1H), 8.15 (d, J ) 7.6 Hz, 1H), 8.06
(d, J ) 7.6 Hz, 2H), 8.00 (d, J ) 9.2 Hz, 1H), 7.82 (t, J ) 6.0 Hz,
1H), 7.65 (m, 3H), 7.54 (m, 2H), 7.38 (m, 2H), 5.13 (m, 1H), 4.08
(m, 1H), 3.94 (m, 1H), 2.81 (m, 2H), 2.3-2.6 (m, overlaps with
DMSO, 2H), 2.34 (s, 3H), 1.70 (m, 3H), 1.00 (d, J ) 7.2 Hz, 3H),
0.93 (m, 6H), 0.80 (s, 9H). gCOSY shows coupling between δ 1.00
(Ala CH3) and δ 3.94(Ala CH) and between δ 2.81 (HNCH2t-Bu)
and δ 7.82 (HNCH2t-Bu). MS m/z: 691 (M + Na)+. Anal.
(C33H44N6O7S) C, H, N.
7.82, 7.72 (2d, J ) 9.6, 8.4 Hz, 1H), 7.60 (m, 5H), 6.85, 6.73 (2d,
J ) 8.4, 7.6 Hz, 1H), 6.33, 6.28 (2d, J ) 6.0, 4.8 Hz, 1H), 4.90,
4.64 (2 broad t, J ) 4.4, 7.6 Hz, 1H), 4.15-4.35 (m, 1H), 4.09,
3.93 (2m, 1H), 3.10, 2.96 (2s, 3H), 1.3-1.7 (m, 3H), 1.35, 1.34
(2s, 9H), 0.88 (d, J ) 6.8 Hz, 3H), 0.83 (d, J ) 6.0 Hz, 3H). gCOSY
shows coupling between δ 6.33 (OH of diastereomer 1) and δ 4.64
(CHOH of diastereomer 1) peaks, and between δ 6.28 (OH of
diastereomer 2) and δ 4.90 (CHOH of diastereomer 2) peaks. MS
m/z: 485 (M + Na)+.
pTs-NeopentylAsn-Ala-LeuKODPh (29c). 1H NMR (400
MHz): δ 8.43 (d, J ) 6.0 Hz, 1H), 8.12 (d, J ) 6.8 Hz, 1H), 8.05
(d, J ) 7.6 Hz, 2H), 7.95 (d, J ) 8.8 Hz, 1H), 7.80 (broad t, J )
6.0 Hz, 1H), 7.62 (m, 5H), 7.28 (d, J ) 7.6 Hz, 2H), 5.11 (m, 1H),
4.02 (m, 1H), 3.92 (m, 1H), 2.80 (m, 2H), 2.51 (m, overlaps with
DMSO, 2H), 2.34 (s, 3H), 1.69 (m, 3H), 1.00 (d, J ) 7.2 Hz, 3H),
0.93 (broad s, 6H), 0.79 (s, 9H). MS m/z: 667 (M - H)-. Anal.
(C33H44N6O7S‚0.5 H2O) C, H, N.
2-(S)-Amino-N-{1(R/S)-[hydroxy-(5-phenyl-[1,3,4]oxadiazol-
2-yl)-(S)-methyl]-3-methylbutyl}-3-methoxy-propionamide Hy-
drochloride (27). Compound 26 (3.55 g, 7.68 mmol) was dissolved
in 4 M HCl (36 mL, 144 mmol) in 1,4-dioxane. After 2 h, the
volatiles were removed by rotary evaporation, and the residue was
triturated with ether (15 mL). Filtration and drying gave compound
27 as a beige solid (3.06 g, >99%), a mixture of diastereomers. 1H
NMR (400 MHz): δ 8.51 (d, J ) 8.8 Hz, 1H), 7.98 (m, 3H), 7.62
(m, 5H), 4.97, 4.66 (2d, J ) 2.0, 8.4 Hz, 1H), 4.31 (m, 1H), 3.95,
3.87 (2m, 1H), 3.2-3.8 (m, 2H), 3.12, 3.07 (2s, 3H), 1.66 (m, 1H),
1.51 (m, 2H), 0.90 (t, J ) 6.8 Hz, 6H). MS m/z: 363 (MH)+.
N4-(2,2-Dimethylpropyl)-N1-(1-{1(R/S)-[hydroxy-(5-phenyl-
[1,3,4]oxadiazol-2-yl)-(S)-methyl]-3(S)-methylbutylcarbamoyl}-
2-methoxyethyl)-2(S)-(toluene-4-sulfonylamino)-succinamide (28d).
Compound 21 (2.68 g, 7.52 mmol) and HOBT (3.00 g, 7.52 mmol)
were stirred in CH2Cl2 (130 mL). EDC (1.51 g, 7.90 mmol) and
N-methylmorpholine (1.65 g, 15.0 mmol) were added. After 8 min,
compound 27 (3.00 g, 7.52 mmol) and more NMM (1.65 g, 15.0
mmol) were added. After 2.5 h, the reaction mixture was transferred
to a sep funnel and washed thrice with 0.5 M HCl. The organic
phase was washed with water, sat. NaHCO3, and brine. Drying over
pTs-NeopentylAsn-Ala-AibKODPh (29e). 1H NMR (400
MHz): δ 8.78 (s, 1H), 8.12 (d, J ) 6.8 Hz, 1H), 8.04 (d, J ) 7.6
Hz, 2H), 7.90 (broad s, 1H), 7.84 (broad t, J ) 6.0 Hz, 1H), 7.62
(m, 5H), 7.27 (d, J ) 7.6 Hz, 2H), 4.01 (m, 1H), 3.67 (m, 1H),
2.82 (d, J ) 6.0 Hz, 2H), 2.3-2.6 (m, overlaps with DMSO, 2H),
2.32 (s, 3H), 1.54 (s, 3H), 1.43 (s, 3H), 0.80 (s, 12H). gCOSY
shows coupling between peaks at δ 0.90 (Ala CH3) and δ 3.67
(Ala CH), between δ 2.40 (CHCH2CO) and δ 4.01 (CHCH2CO),
and between δ 7.84 (t-BuCH2NH) and δ 2.82 (t-BuCH2NH). MS
m/z: 639 (M - H)-. HPLC homogeneity 98.6% (System A); 100%
(System B).
1
Compound 29f. H NMR (400 MHz): δ 8.45 (d, J ) 6.0 Hz,
1H), 8.18 (d, J ) 7.6 Hz, 1H), 7.80 (broad t, J ) 5.2 Hz, 1H), 7.36

2966 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Rydzewski et al.
(d, J ) 9.2 Hz, 1H), 5.08 (m, 1H), 4.27 (m, 1H), 4.03 (m, 1H),
3.29 (m, obscured by HOD, 1H), 2.85 (d, J ) 6.0 Hz, 2H), 2.60
(s, 6H), 2.4-2.7 (m, overlaps with DMSO, 2H), 1.74 (m, 1H), 1.62
(m, 2H), 1.32 (dd, J ) 6.8, 1.2 Hz, 6H), 1.17 (d, J ) 7.2 Hz, 3H),
0.90 (m, 6H), 0.81 (s, 9H). MS m/z: 587 (M - H)-. Anal. Calcd
for C25H45N7O7S: C, 51.09; H, 7.72; N, 16.68. Found: C, 51.10;
H, 7.47; N, 16.11.
least-squares regression. For reversible, competitive inhibitors, the
apparent inhibition constant, Kiapp, is related to the true thermo-
dynamic binding constant, Ki, by the following relationship: Ki )
Kiapp/(1 + [Substrate]/Km). Because, except for trypsin-like pro-
teasome activity, substrates are supplied in the assays at their Km,
Ki ) Kiapp/2.
Assaying the Chymotryptic Activity of the 20S Proteasome.
Human 20S proteasome was incubated at a final concentration of
2 nM with test compound present at variable concentrations. The
reaction buffer consisted of 50 mM Tris (pH 7.4), 50 mM NaCl,
0.5 mM EDTA, 0.05% Tween-20, 0.035% SDS, and 5% DMSO.
After 60 min of enzyme and inhibitor incubation, the reactions were
initiated with the addition of the substrate, Suc-Leu-Leu-Val-Tyr-
AMC (Bachem Bioscience Inc., King of Prussia, PA). The final
concentration of substrate in the reaction was 50 µM.
Assaying the Tryptic Activity of the 20S Proteasome. Human
20S proteasome was incubated at a final concentration of 20 nM
with test compound present at variable concentrations. The reaction
buffer consisted of 50 mM Tris (pH 7.4), 50 mM NaCl, 0.5 mM
EDTA, 0.05% Tween-20, 0.035% SDS, and 5% DMSO. After 60
min of enzyme and inhibitor incubation, the reactions were initiated
with the addition of the substrate, Boc-Leu-Arg-Arg-AMC (Bachem
Bioscience Inc., King of Prussia, PA). The final concentration of
substrate in the reaction was 85 µM.
Assaying the Post Glutamyl-Peptide Hydrolyzing Activity of
the 20S Proteasome. Human 20S proteasome was incubated at a
final concentration of 10 nM with the test compound present at
variable concentrations. The reaction buffer consisted of 50 mM
Tris (pH 7.4), 50 mM NaCl, 0.5 mM EDTA, 0.05% Tween-20,
0.035% SDS, and 5% DMSO. After 60 min of enzyme and inhibitor
incubation, the reactions were initiated with the addition of the
substrate, Z-Leu-Leu-Glu-AMC (California Peptide Research, Inc.,
Napa, CA). The final concentration of the substrate in the reaction
was 80 µM.
Determination of the Reversibility of 20S Proteasome Inhibi-
tion. The reversibility of 20S proteasome inhibition by 29c was
carried out by preforming the enzyme-inhibitor complex during a
60 min incubation phase. Compound 29c was supplied at 150 nM
during the incubation phase along with the 150 nM active sites of
the 20S proteasome. Following incubation, the reaction was diluted
50-fold into an assay buffer containing 50 µM substrate such that
the final concentration of 29c and enzyme was 3.0 nM. The recovery
of chymotryptic activity of the 20S proteasome was monitored as
described above as a function of time. The kinetic parameters for
the first order recovery of enzyme activity were obtained by
standard methods. Control reactions were performed in parallel in
which the inhibitor was omitted from the initial incubation phase
of the experiment to obtain the uninhibited rate. Additionally,
control assays were performed to demonstrate that the enzyme was
fully inhibited during the incubation phase.
1
Compound 29g. H NMR (400 MHz): δ 8.36 (d, J ) 6.0 Hz,
1H), 8.08 (d, J ) 7.6 Hz, 1H), 7.93 (d, J ) 8.4 Hz, 1H), 7.82 (d,
J ) 7.2 Hz, 2H), 7.76 (broad t, J ) 5.6 Hz, 1H), 7.60 (d, J ) 7.2
Hz, 2H), 7.28 (d, J ) 8.0 Hz, 2H), 6.83 (d, J ) 8.0 Hz, 2H), 5.10
(m, 1H), 4.04 (m, 1H), 3.94 (m, 1H), 3.02 (s, 6H), 2.79 (m, 2H),
2.3-2.55 (m, obscured by DMSO, 2H), 2.33 (s, 3H), 1.5-1.9 (m,
3H), 0.99 (d, J ) 7.2 Hz, 3H), 0.92 (m, 6H), 0.78 (s, 9H). MS m/z:
710.5 (M - H)-. Anal. (C35H49N7O7S‚0.5 H2O) C, H, N.
1
Compound 29h. H NMR (400 MHz): δ 8.49 (d, J ) 6.8 Hz,
1H), 8.14 (d, J ) 8.0 Hz, 1H), 7.92 (d, J ) 9.2 Hz, 1H), 7.83 (d,
J ) 7.6 Hz, 2H), 7.75 (broad t, J ) 6.0 Hz, 1H), 7.61 (d, J ) 7.6
Hz, 2H), 7.28 (d, J ) 7.6 Hz, 2H), 6.83 (d, J ) 7.6 Hz, 2H), 5.11
(m, 1H), 4.16 (m, 2H), 3.40 (m, 1H), 3.31 (s, 3H), 3.24 (m, 1H),
3.03 (s, 6H), 2.78 (d, J ) 6.0 Hz, 2H), 2.25-2.55 (m, obscured by
DMSO, 2H), 2.32 (s, 3H), 1.6 - 1.8 (m, 3H), 0.90 (m, 6H), 0.78
(s, 9H). MS m/z: 741 (M - H)-. Anal. (C36H51N7O8S‚0.5 H2O)
C, H, N.
1
Compound 29i. H NMR (400 MHz): δ 8.53 (d, J ) 6.4 Hz,
1H), 8.21 (d, J ) 7.2 Hz, 1H), 8.07 (dd, J ) 7.2, 1.2 Hz, 2H), 8.02
(d, J ) 7.6, 1.2 Hz, 1H), 7.62 (m, 5H), 7.27 (m, 5H), 7.18 (d, J )
7.2 Hz, 2H), 5.13 (m, 1H), 4.34 (m, 1H), 4.31 (s, 2H), 4.10 (m,
1H), 3.38 (m, 2H), 3.31 (s, 3H), 3.25 (d, J ) 6.0 Hz, 2H), 2.32 (s,
3H), 1.69 (m, 2H), 1.55 (m, 1H), 0.89 (m, 6H). MS m/z: 692
(MH)+. Anal. Calcd for C35H41N5O8S‚H2O: C, 59.22; H, 6.11; N,
9.87. Found: C, 59.05; H, 5.59; N, 9.83.
1
Compound 29j. H NMR (400 MHz): δ 8.47 (d, J ) 6.4 Hz,
1H), 8.13 (d, J ) 7.6 Hz, 1H), 8.06 (d, J ) 6.4 Hz, 2H), 7.98 (d,
J ) 8.8 Hz, 1H), 7.62 (m, 5H), 7.24 (d, J ) 8.0 Hz, 2H), 7.11 (m,
4H), 5.12 (m, 1H), 4.30 (s, 2H), 4.16 (m, 1H), 4.03 (m, 1H), 3.41
(m, 2H), 2.33 (s, 3H), 2.14 (s, 3H), 1.70 (m, 2H), 1.53 (m, 1H),
1.02 (d, J ) 7.2 Hz, 3H), 0.91 (m, 6H). MS m/z: 699 (M + Na)+.
Anal. (C35H41N5O7S‚0.5 H2O) C, H, N.
1
Compound 29k. H NMR (400 MHz): δ 8.45 (d, J ) 6.8 Hz,
1H), 8.14 (d, J ) 7.2 Hz, 1H), 8.04 (m, 3H), 7.61 (m, 5H), 7.26
(m, 5H), 7.13 (m, 1H), 5.13 (m, 1H), 4.32 (s, 2H), 4.16 (m, 1H),
4.02 (m, 1H), 3.39 (d, J ) 6.4 Hz, 2H), 2.31 (s, 3H), 1.70 (m, 2H),
1.53 (m, 1H), 1.05 (d, J ) 7.2 Hz, 3H), 0.90 (m, 6H). MS m/z:
718.3 (M + Na)+. Anal. (C34H38ClN5O7S‚0.5 H2O) C, H, N.
1
Compound 29l. H NMR (400 MHz, CDCl3): δ 8.15 (d, J )
6.8 Hz, 2H), 7.71 (d, J ) 8.4 Hz, 2H), 7.59 (m, 1H), 7.54 (m, 2H),
7.27 (m, 3H), 7.13 (m, 4H), 7.07 (d, J ) 8 Hz, 1H), 5.54 (m, 1H),
5.13 (m, 1H), 4.50 (m, 1H), 4.33 (ABq, 2H), 3.77 (m, 2H), 3.44
(m, 1H), 2.44 (s, 3H), 2.36 (s, 3H), 1.6-2.0 (m, 3H), 1.37 (d, J )
7.2 Hz, 3H), 1.04 (d, J ) 6.4 Hz, 3H), 0.99 (d, J ) 6.0 Hz, 3H).
MS m/z: 699 (M + Na)+. Anal. (C35H41N5O7S) C, H, N.
Assays for Cathepsin B and Cathepsin S Inhibition. Inhibition
of cathepsin B and cathepsin S was determined using the methods
previously described.26
In Vitro 20S Proteasome Inhibition Assays. Human 20S
proteasome was obtained from Affiniti Research Products Ltd.,
Mamhead, Exeter, U.K. The three distinct proteolytic activities of
the 20S proteaseome were measured by monitoring the hydrolysis
of an optimized peptidal 7-amino-4-methyl coumarin substrate for
either the chymotryptic, tryptic or post glutamyl-peptide hydrolyzing
activity (PGPH Activity) of the enzyme. Hydrolysis of the given
substrate was measured using an fMax plate reader (Molecular
Devices, Sunnyvale, CA) at ambient temperature with a 355 ( 38
nm excitation filter and a 460 ( 25 nm emission filter. The velocity
of the enzyme-catalyzed reaction was obtained from the linear
portion of the progress curves (usually the first 10 min after the
addition of substrate). Apparent inhibition constants, Kiapps, were
calculated for reversible inhibitors from the reaction velocity data
generated at the various inhibitor concentrations using the software
package, BatchKi (Biokin Ltd., Pullman, WA). BatchKi provides
a parametric method for the determination of inhibitor potency using
a transformation of the tight binding inhibition model described
by Morrison39,40 and further refines the Kiapp values by nonlinear
Determination of Compound Solubility. Kinetic solubility was
determined in PBS at pH 7.4, containing 5% DMSO. The samples
were prepared by adding 10 µL of 10 mM DMSO stock to 190 µL
of PBS. After vortexing, the samples were centrifuged at 4300 rpm
for 10 min to precipitate any insoluble materials. The supernatant
was then analyzed by HPLC with UV detector and quantified
against a calibration standard.
Plasma Stability Assessment. Human plasma stability was
determined by incubating compound at 5 µg/mL in human plasma.
The samples were incubated at 37 °C for 120 min. The disappear-
ance of the parent compound was monitored by LC/MS/MS. The
percentage remaining of the parent compound was calculated on
the basis of the peak area of the parent compound at the end of
incubation to that obtained at 0 min.
Cell Growth Inhibition Assay. The human prostate tumor cell
line PC3 was obtained from the American Type Tissue Culture
Collection (ATCC). The effect of proteasome inhibitors on human
PC3 cell growth inhibition was monitored in an alamarBlue

Syntheses and Properties of Proteasome Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2967
fluorometric assay as described by de Fries and Mitsuhashi.36
Briefly, 5000 cells/well were plated in 96-well plates in complete
media. The compounds were assayed in triplicate wells at eight
concentrations from 0.005 to 20 µM, except for epoxomicin, which
was tested at six concentrations from 0.0036 to 360 nM. Each well
contained 0.2% DMSO. The cells were exposed to compounds for
two doubling times (44 h). At 44 h, the alamarBlue reagent was
added to cells, and the plates were incubated for 4 h at 37 °C. At
48 h, the fluorescence was determined using the plate reader, and
the IC50 values were calculated for each compound. The concentra-
tion required to inhibit cell growth by 50% (IC50) was determined
by two point linear interpolation.
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Acknowledgment. We express our gratitude to Mimi Li,
Jeremy Parr, and Joanne Fanucchi for their expert help in
compound purification, compound analysis, and manuscript
preparation, respectively.
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specificity using positional-scanning libraries of covalent inhibitors.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2967-2972.
Supporting Information Available: Elemental analyses for the
products synthesized. The material is available free of charge via
the Internet at http://pubs.acs.org.
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JM058289O