Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047)

Article abstract of DOI:10.1021/jm801329v

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and Non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (β5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.

Full text of DOI:10.1021/jm801329v

3028
J. Med. Chem. 2009, 52, 3028–3038
Design and Synthesis of an Orally Bioavailable and Selective Peptide Epoxyketone Proteasome
Inhibitor (PR-047)
Han-Jie Zhou,* Monette A. Aujay, Mark K. Bennett, Maya Dajee, Susan D. Demo, Ying Fang, Mark N. Ho, Jing Jiang,
Christopher J. Kirk, Guy J. Laidig, Evan R. Lewis, Yan Lu, Tony Muchamuel, Francesco Parlati, Eileen Ring, Kevin D. Shenk,
Jamie Shields, Peter J. Shwonek, Timothy Stanton, Congcong M. Sun, Catherine Sylvain, Tina M. Woo, and Jinfu Yang
Proteolix, Inc., 333 Allerton AVenue, South San Francisco, California 94080
ReceiVed October 20, 2008
Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma
and Non-Hodgkin’s lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant
diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry
effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for
improved dosing flexibility and patient convenience over intravenously administered agents. The lead
compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L
activity of both the constitutive proteasome (ꢀ5) and immunoproteasome (LMP7) and demonstrated an
absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral
administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor
response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse
syngeneic models. The favorable pharmacologic profile supports its further development for the treatment
of malignant diseases.
Introduction
bond hydrolysis and activation of nucleophilic water by the
R-amino group to hydrolyze the resulting ester.
The proteasome is a multicatalytic protease complex that is
responsible for the ubiquitin-dependent turnover of cellular
proteins.1-3 Proteasome substrates include misfolded or mis-
assembled proteins as well as short-lived components of
signaling cascades that regulate cell proliferation and survival
pathways. Inhibition of the proteasome leads to an accumulation
of substrate proteins and results in cell death.4 The proteasome
consists of a 20S proteolytic core and two 19S regulatory caps
that assemble with the core at either end to form a 26S
complex.5,6 Two distinct forms of the 26S proteasome, the
constitutive proteasome and the immunoproteasome, have been
identified. The majority of cell types express the constitutive
form of the proteasome, while cells of the immune system
express the immunoproteasome. Nonimmune cells can also
express immunoproteasomes following exposure to inflamma-
tory cytokines such as interferon γ (IFN-γ). The 20S core of
the constitutive proteasome has three distinct catalytic activities:
chymotrypsin-like (CT-La), trypsin-like (T-L), and caspase-like
(C-L), which are encoded by the ꢀ5, ꢀ2, and ꢀ1 subunits,
respectively. Of these, the CT-L activity is thought to be the
rate-limiting step of proteolysis in vitro and in vivo, while
inhibition of multiple sites might be required to fully suppress
proteasome-mediated protein turnover.7,8 The immunoprotea-
some retains the structural subunits of the constitutive protea-
some but incorporates the catalytic subunits LMP7, MECL1,
and LMP2 in place of ꢀ5, ꢀ2, and ꢀ1, respectively.9-12 Each
proteasome active site utilizes the nucleophilic γ-hydroxyl group
of an amino-terminal threonine (Thr) residue to initiate amide
Clinical validation of the proteasome as a therapeutic target
in oncology has been provided by bortezomib 1 (Figure 1), a
dipeptide boronic acid,4,13 which is approved for the treatment
of patients with multiple myeloma14-17 and mantle cell
lymphoma.18,19 Although the clinical success of bortezomib is
encouraging, a significant fraction of patients relapse or are
refractory to treatment.14-19 Additionally, dose-limiting toxici-
ties (DLT), including a painful peripheral neuropathy and
thrombocytopenia, have been reported.16,20,21 It is unclear
whether these toxicities can be attributed to off-target effects
because bortezomib inhibits other enzymes such as serine
proteases, albeit with lower potencies.13,22,23 Recently, we
reported the discovery of carfilzomib 2 (also called PR-171),22,23
a structural analogue of the microbial natural product epoxo-
micin 3 that was initially identified for its antitumor activity
and subsequently shown to be a potent inhibitor of the
proteasome.24-28 Carfilzomib selectively inhibits the CT-L
activity of the 20S proteasome and displays equivalent potency
against ꢀ5 and LMP7 with minimal cross reactivity to other
protease classes. Preclinical studies and phase I clinical studies
demonstrated that consecutive day dosing schedules with
carfilzomib are both well tolerated and promote antitumor
activity in hematologic malignancies, including patients previ-
ously treated with bortezomib.22,23,29-33 Carfilzomib is currently
being evaluated in phase I and phase II clinical trials in multiple
myeloma, non-Hodgkin’s lymphoma, and solid tumors.
Clinical responses to proteasome inhibitor therapy require
frequent dosing (e.g., twice per week) and prolonged treatment.
Both bortezomib and carfilzomib are administered intravenously
(iv) on biweekly or more frequent dosing schedules with
treatment that can extend for over 6 months.23 Therefore, the
development of orally bioavailable proteasome inhibitors that
would allow for dosing flexibility and improve patient conven-
ience is warranted. We describe here the results of systematic
* To whom correspondence should be addressed. Phone: (650)-504-8346.
Fax: 650-872-1875. E-mail: hzhou@proteolix.com.
a Abbreviations: CT-L, chymotrypsin-like; T-L, trypsin-like; C-L, caspase-
like; MDR, multidrug resistance transporters; SGF, simulated gastric fluids;
SIF, simulated intestinal fluids; iv, intravenous administration; po, oral
administration; PK, pharmacokinetics; PD, pharmacodynamics; DLT, dose
limiting toxicities; MTD, maximum tolerated dose.
10.1021/jm801329v CCC: $40.75
2009 American Chemical Society
Published on Web 04/06/2009

Peptide Epoxyketone Proteasome Inhibitor (PR-047)
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3029
Figure 1. Structures of bortezomib, carfilzomib, and epoxomicin.
Table 1. Carfilzomib and Its Truncated Analogues
mice PO PD
(% CT-L activity @ 40 mg/kg)
MES cell viability (
stability
(% parent @ 15 min)
liver microsome
stability (Re)
MDRa IC50 (nM)
20S CT-La
IC50 (nM)
Molt-4 CT-La
IC50 (nM)
compd
liver
blood
adrenal
MDR-
MDR+
SGF
SIF
mouse human
2
4
5
5.7 ( 5.3 (139) 5.1 ( 2.7 (78)
109
NDb
22
77
83
18 ( 16 (46) 413 ( 237 (46)
∼15
NDb
∼90
<5
0.98
NDb
0.95
0.93
NDb
NDb
>1000
>1000
NDb
37
NDb
82
>1000
>1000
NDb
∼45
57 ( 42 (5)
45 ( 5 (4)
35 ( 10 (5)
308 ( 134 (5)
a All experiments to determine IC50 values were run with at least duplicates at each compound dilution; IC50 values were averaged when determined in
two or more independent experiments; the individual IC50 values listed in parentheses (n ) 2) or ( standard deviation denoted and n listed in parentheses
(n g 3). b Not determined.
SAR studies to develop orally bioavailable epoxyketone-based
proteasome inhibitors that maintain the potency, selectivity, and
antitumor activity of carfilzomib.
Biological Assays
Inhibition of the CT-L activity was tested in cell free systems
using purified human 20S proteasomes (Boston Biochem Inc.,
Cambridge, MA) and in cellular lysates prepared from Molt-4
(human leukemia) cells treated with inhibitors for 1 h. In both
assays, 50% inhibitory concentrations (IC50) were determined
and comparison of the IC50 values using purified enzyme to
intact cell exposure served as an assessment of the cell
permeability of compounds.22 Inactivation rates (kinact/Ki) were
determined for a subset of analogues using purified human 26S
proteasome. Selectivity for proteasomal subunits was measured
using an active site binding assay in lysates from inhibitor treated
Molt-4 cells.34 The sensitivity of compounds to multidrug
resistance transporters (MDR) was evaluated by comparing cell
viability on a pair of MES (uterine sarcoma) tumor cell lines:
the parental line (MDR-) and a doxorubicin resistant subline
known to express MDR (MDR+).35 Compound stability was
evaluated in simulated gastric and intestinal fluids (SGF and
SIF), and the percentage of parent remaining was determined
after 15 min of incubation. Metabolic stability was assessed in
liver microsomes from mouse, rat, dog, and human, and the
extraction ratio (Re) was calculated based on half-life to facilitate
cross-species comparisons.36 Bioactivity was determined by
pharmacodynamic (PD) measurements of residual CT-L activity
in blood and tissues 1 h after oral (po) administration. Absolute
bioavailability (F) was determined by pharmacokinetic (PK)
assessment of areas under the plasma concentration versus time
curves following iv and po administrations. Antitumor efficacy
was assessed in immunocompromised mice bearing established
human tumor xenografts and in normal mice bearing syngeneic
tumor cells (see Supporting Information for more details on
biological assays).
Figure 2. Structures of analogues 4 and 5.
activity 1 h postdose). A subset of compounds was subsequently
chosen for further evaluation in other aforementioned assays.
Results and Discussions
Carfilzomib Truncations. The starting point of our chemistry
effort was carfilzomib, a tetrapeptide epoxyketone. Initially, we
found that carfilzomib and its tetrapeptide derivatives failed to
exhibit significant oral bioavailability as measured by inhibition
of proteasome activity in blood or tissue following po admin-
istration to mice (Table 1). The instability of carfilzomib in SGF,
SIF, and liver microsomes may each contribute to the lack of
systemic exposure. These results are consistent with reports that
di- and tripeptides can cross intestinal epithelial barriers, while
tetrapeptides are generally not orally bioavailable.37,38 To
determine if shortening the peptide portion of carfilzomib would
maintain potency and improve bioavailability, truncated di- and
tripeptidyl analogues of carfilzomib were synthesized (Figure
2). The dipeptide epoxyketone 4 had weak potency against the
CT-L activity of the 20S proteasome (>1000 nM). In contrast,
the tripeptide epoxyketone 5 exhibited strong CT-L inhibitory
activity, and importantly, induced proteasome inhibition in blood
and liver following po administration to mice. Notably, com-
pound 5 was stable in SGF and partially stable in SIF but
unstable in liver microsomal assays across multiple species,
perhaps contributing to the limited PD response in peripheral
tissues such as adrenal after po dosing. On the basis of these
initial results, further chemistry efforts were focused on trip-
eptide epoxyketone analogues.
All compounds were tested against purified human 20S
proteasomes and Molt-4 cells for inhibition of the proteasome
CT-L activity. Compounds with IC50 less than 100 nM and
solubility g1.0 mg/mL in a vehicle of 10% (v/v) EtOH and
10% (v/v) PS80 in citrate buffer (pH 3.5) were then orally
administered to Balb/c mice (40, 20, 10, or 5 mg/kg) for initial
assessment of bioavailability by blood and tissue PD (CT-L
N-Cap Screening on Tripeptide Ketoepoxide. The chem-
istry strategy began with screening N-terminus capping groups
with a backbone of Leu-Phe-Leu-ketoepoxide. The N-caps
included a variety of amides (primarily five- or six-membered
aromatic or nonaromatic heterocycles) and ureas. Amides were
synthesized by deprotection of a shared Boc-Leu-Phe-Leu-
ketoepoxide intermediate and then coupling with various

3030 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Zhou et al.
Table 2. Screening N-Caps with Backbone of N-cap-Leu-Phe-Leu-Ketoepoxide
MES cell viability ( MDRa IC50 (nM)
compd
N-cap
20S CT-La IC50 (nM)
Molt-4 CT-La IC50 (nM)
MDR-
MDR+
5
6
morpholine-CH2
3-furan
57 ( 42 (5)
34
45 ( 5 (4)
116
35 ( 10 (5)
54
308 ( 134 (5)
125
7
8
2-thiophene
5-oxazole
10 ( 5 (3)
27
40
84
34
159
120
505
9
5-isoxazole
3-isoxazole
(5-Me)-3-isoxazole
(5-iPr)-3-Isoxazole
(5-MeOCH2)-3-isoxazole
3-pyrazole
5.8 (3.7, 7.9)
7.4 (3.7, 11)
1.8 ( 0.9 (5)
6.3
1.2 (1.1, 1.3)
36 ( 2 (3)
107 (103, 110)
76 ( 32 (3)
122 (77, 167)
38 (25, 51)
20 (16, 23)
12 (9, 14)
752
65 ( 28 (3)
50 ( 17 (3)
17 ( 12 (5)
17 (15, 18)
8.1 ( 4.3 (3)
188 (135, 242)
271 (222, 320)
290
161
245
55
50 (38, 63)
>1000
154
180
47
58 (56, 60)
73 (55, 92)
28 (22, 34)
63 (55, 71)
23 (12, 35)
109 (83, 134)
136 (129, 142)
137
67
103
33
18 ( 2 (3)
>1000
69
130
182
82 (76, 88)
123 (86, 159)
47 (32, 64)
30 (24, 37)
28 (26, 30)
>1000
>1000
205
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2-imidazole
(N-Me)-3-Pyrazole
(N-Me)-2-imidazole
(5-Me)-3-pyrazole
4-pyridine
4-pyridazine
2-pyrazine
2-(R)-Tetrahydrofuran
2-(S)-tetrahydrofuran
(5-Me)-3-isoxazole-NH
164
>1000
>1000
>1000
>1000
110
159
>1000
34
42 (31, 52)
13
a All experiments to determine IC50 values were run with at least duplicates at each compound dilution; IC50 values were averaged when determined in
two or more independent experiments; the individual IC50 values listed in parentheses (n ) 2) or ( standard deviation denoted and n listed in parentheses
(n g 3).
carboxylic acids under a coupling condition of HOBt/HBTU/
DIEA. Ureas were prepared from the deprotected backbone and
isocyanates. More than 100 N-cap variants were evaluated for
potency against CT-L inhibition in purified human 20S protea-
some and Molt-4 cells as well as MDR resistance in the paired
MES-SA cell lines. Results from representative compounds are
outlined in Table 2. Notably, five-membered heterocycles devoid
of an NH group such as furan 6, thiophene 7, oxazole 8, and
isoxazoles (9 and 10) were potent in both cell free and in Molt-4
cell proteasome CT-L activity assays and were relatively
insensitive to the MDR transporter. Methylation at the 5-position
of 3-isoxazole 11 substantially improved its potency against the
proteasome without affecting MDR sensitivity. Other substit-
uents such as isopropyl 12 and methoxymethyl 13 at the same
position were well tolerated but not significantly better than
methyl group. In contrast, five-membered heterocycles contain-
ing an NH group such as pyrazole 14 and imidazole 15 showed
similar potency but increased MDR sensitivity. Methylation on
the nitrogen of the capping groups (16, 17) but not other
positions (e.g., 18) made the analogues less MDR sensitive.
N-Containing six-membered heterocycles such as 4-pyridine 19
and 4-pyridazine 20 displayed adequate potency against the
proteasome but were MDR sensitive despite the absence of
hydrogen attached to the nitrogen. Compound 21 was a very
poor proteasome inhibitor even though it shares the same N-cap
(2-pyrazine), P1, and P2 with bortezomib. Nonaromatic het-
erocycles such as tetrahydrofurans (22, 23) were well tolerated
as N-cap. Urea-linked N-caps (e.g., 24) were also potent but
increased sensitivity of the compounds to MDR activity. While
a number of N-cap variants exhibit favorable potency, cell
permeability, and MDR insensitivity, none were soluble enough
to be formulated for oral administration (i.e., g1.0 mg/mL in
the aforementioned EtOH/PS80/citrate vehicle; see solubility
data of representative analogues listed in Table S1 of the
Supporting Information). Therefore, two approaches were
chosen to increase solubility of compound 11, the compound
displaying the most favorable combination of potency and MDR
insensitivity: (a) introduction of solubility-enhancing substituents
at the 5-position of 3-isoxazole, and (b) modification of the
peptide backbone of 5-Me-3-isoxazole-Leu-Phe-Leu-ketoep-
oxide.
A set of solubilizing groups were attached to the 5-position
of 3-isoxazole through a methenyl group, and the resulting
potent analogues are listed in Table 3. Introduction of triazole
25, imidazole 26, N-Me-piperazine 27, and morpholine 28
resulted in solubility sufficient for oral administration (i.e., g1.0
mg/mL). Among them, compound 28 was able to promote
significant proteasome inhibition in peripheral tissues such as
adrenal after po dosing. However, compounds (25, 26, and 27)
did not display oral bioactivity, perhaps due in part to their high
MDR sensitivity.
P3 Modification. A set of 30 natural and non-natural amino
acids were tested at the P3 position with either 5-methyl or
5-morpholinomethyl 3-isoxazole as the N-cap. Representative
analogues with potent in vitro activity are also listed in Table
3. P3 substituents containing ether and heterocyclic amino acids
had improved solubility relative to leucine (Table S1 of the
Supporting Information). Analogues with 5-methyl-3-isoxazole
as N-cap (11, 29, 31, 33, 35, 37, and 39) demonstrated reduced
MDR sensitivity and equivalent or greater oral bioactivity as
compared to their morpholinomethyl counterparts (28, 30, 32,
34, 36, 38, and 40, respectively). When comparing across P3
analogues, those with low stability in SIF, such as 4-thiazoly-
lalanine (31, 32) and 2-pyridylalanine (35, 36) showed the least
oral bioactivity. Conversely, P3 analogues with methylserine
(29, 30) and homomethylserine (33, 34) were highly stable in
SIF and demonstrated increased oral bioactivity. Most of these
analogues, however, were rapidly degraded in liver microsomes
across multiple species (Re > 0.85, data not shown). Among
the P3 substitutions, compound 29 displayed the best in vitro
and in vivo properties; therefore, 5-methyl-3-isoxazole and

Peptide Epoxyketone Proteasome Inhibitor (PR-047)
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3031
Table 3. Solubility-Enhancing Substituents on 5-Position of 3-Isoxazole-Leu-Phe-Leu-Ketoepoxide and P3 Modification with
5-Me-3-Isoxazole-P3-Phe-Leu-Ketoepoxide
MES cell viability ( MDRa
IC50 (nM)
mice PO PD
(% CT-L activity @
40 mg/kg)
SIF
stability
(% parent @
15 min)
20S CT-La Molt-4 CT-La
IC50 (nM) IC50 (nM)
compd
R
P3
adrenalb
MDR-
35
52
189
28 (22, 34)
22 ( 8 (3)
40 (31, 48)
13
MDR+
>1000
>1000
>1000
47 (32, 64)
70 ( 20 (3)
75 (53, 96)
147
25
26
27
11
28
29
30
31
32
33
34
35
36
37
38
39
40
1-(1,2,4)-triazole-CH2 Leu
1-imidazole-CH2
N-Me-piperazine-CH2 Leu
13 62
105
110
101
NDc
29
NDe
NDe
40
60
60
90
80
20
15
80
90
20
20
90
70
90
60
Leu
15
59
94
124
Me
Leu
Leu
1.8 ( 0.9 (5)
17 ( 12 (5)
morpholine-CH2
Me
morpholine-CH2
Me
morpholine-CH2
Me
morpholine-CH2
Me
morpholine-CH2
Me
morpholine-CH2
Me
4.1
11 ( 3 (3)
Ser(OMe)
Ser(OMe)
9.3 ( 5.9 (3)
5.7
36 ( 14 (3)
26 (15, 37)
9
57
(4-Thiazolyl)-ala 6.7 (5.1, 8.4)
(4-Thiazolyl)-ala 6.9 (6.1, 7.6)
56 (46, 66)
59
63@10d
93
15
77
7.9
164
24
841
286
1145
hSer(OMe)
hSer(OMe)
(2-Py)-ala
(2-Py)-ala
(3-Py)-ala
(3-Py)-ala
(4-Py)-ala
(4-Py)-ala
26 (19, 33)
69 (58, 80)
47
81
23
92
70
90
28
96
19 (17, 20)
13
5.2
1.6
6.4 ( 3.6 (3)
2.8
5.1
42
78
31
27
30
8.6
14
45
219
11
421
39
543
17
8.6
8.7
>1000
271
morpholine-CH2
>1000
a All experiments to determine IC50 values were run with at least duplicates at each compound dilution; IC50 values were averaged when determined in
two or more independent experiments; the individual IC50 values listed in parentheses (n ) 2) or ( standard deviation denoted and n listed in parentheses
(n g 3). b Adrenal selected as the peripheral tissue and inhibition in blood always equivalent or better than adrenal. c Not determined due to its poor
solubility. d Dosed at 10 mg/kg. e Not determined.
Table 4. P2 Modification with 5-Me-3-Isoxazole-Ser(OMe)-P2-Leu-Ketoepoxide
MES cell viability (
liver microsome
stability (Re)
mice PO PD
(% CT-L activity @
20 mg/kg),
MDRa IC50 (nM)
SIF
stability
20S CT-La Molt-4 CT-La
compd
P2
IC50 (nM)
IC50 (nM)
adrenal
MDR-
MDR+
(% parent @ 15 min) mouse human
29
41
42
43
44
45
46
47
48
49
Phe
cyhxy-ala
3-thienyl-ala
4-thiazolyl-ala 16 ( 3 (3)
Val
Leu
Ala
Abu
CN-Ala
Ser(OMe)
9.3 ( 5.9 (3)
36 ( 14 (3)
33
56
117
39
12
37
8
90
40
9
40 (31, 48) 75 (53, 96)
90
75
90
100
95
90
100
100
100
70
0.96
0.96
0.95
0.86
0.79
0.45
0.48
0.47
0.28
0.67
0.91
0.94
0.87
0.85
0.63
0.83
0.28
NDb
NDb
0.54
13
78
210
25
353
181
9.2
46
25 ( 23 (3)
147
41
57 ( 28 (3)
248
91
17 ( 2 (5) 367 ( 124 (5)
44
20
57
45
636
366
19 ( 5 (6)
54
40
28 ( 5 (5) 276 ( 57 (5)
92
29
468
941
11 ( 4 (10)
48 ( 26 (3)
22 ( 2 (4) 145 ( 52 (4)
a All experiments to determine IC50 values were run with at least duplicates at each compound dilution; IC50 values were averaged when determined in
two or more independent experiments; the individual IC50 values listed in parentheses (n ) 2) or ( standard deviation denoted and n listed in parentheses
(n g 3). b Not determined.
methylserine were selected as N-cap and P3, respectively, for
further optimization.
the exception of analogue 47, these analogues demonstrated
greater oral bioactivity. In addition, compound 49 displayed
better solubility (Table S1 of the Supporting Information). On
the basis of these results, 5-methyl-3-isoxazole was kept as
N-cap and methylserine was chosen as both P2 and P3 for
further optimization.
P1 Modification and N-Cap Rescreening. Carfilzomib is
>100 fold selective for the CT-L activity versus either the T-L
and C-L activities and mediates an equivalent inhibition of ꢀ5
and LMP7. We utilized an ELISA-based active site binding
assay with intact cells to profile the active site selectivity of
selected analogues.34 Most of the tripeptidyl analogues with
leucine as P1 (50, 49, 53, 55, and 57) displayed greater potency
for ꢀ5 than LMP7, making these compounds selective for the
P2 Modification. A similar set of 30 natural and non-natural
amino acids were chosen for P2 modification, and representative
potent analogues are outlined in Table 4. Although most of these
compounds were stable in SGF and SIF, they differed in
sensitivity to first pass metabolic pathways. Bulky amino acids
at P2 such as phenylalanine (29), cyclohexylalanine (41),
3-thienylalanine (42), 4-thiazolylalanine (43), and even valine
(44) and leucine (45) resulted in compounds that were unstable
in liver microsomal assays. On the other hand, compounds with
small amino acids at P2 such as alanine (46), 2-aminobutyric
acid (Abu) (47), cyanoalanine (48), and methylserine (49) were
more resistant to microsomal degradation. Consequently, with

3032 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Zhou et al.
Table 5. Subunit Profile Comparison of Leu and Phe as P1 with
Methylserine as Both P2 and P3 and Variant N-Caps
was calculated by comparing dose response curves for protea-
some inhibition in blood following po and iv administrations.
Oral bioactivity measured by PK and PD was found to be
comparable, which reconfirmed the rationale that measurement
of PD (CT-L activity 1 h postdose) following po administration
can be used as a primary screening assay to evaluate bioactivity
and bioavailability (F) of this series of peptide epoxyketone
analogues.
ELISA-based active
b
Because peptide epoxyketone proteasome inhibitors are
irreversible, their absolute potency is most accurately described
by their inactivation kinetics (kinact/Ki). The kinact/Ki for a subset
of key compounds (Table S3 of the Supporting Information)
demonstrated that rapid inactivation of the target was well
correlated with the relative potency reflected by IC50 values
measured at 1 h with purified enzyme and on cells. Furthermore,
the IC50 values of the all set of epoxyketone analogues for CT-L
inhibition in Molt-4 cells are also directly proportional to cellular
cytotoxicity (data not shown). Thus, the determination of IC50
values at a fixed time point (1 h) provides an efficient screening
strategy for defining the relative potency of analogues. Further-
more, the kinetics of proteasome inhibition in animals following
po administration of 58 demonstrated rapid absorption, tissue
distribution, and inactivation of the proteasome (Figure 4).
Within 15 min of dosing, proteasome inhibition in excess of
80% was achieved in blood and all tissues examined except
the brain. This rapid onset of proteasome inhibition is compa-
rable to that seen with iv administration of carfilzomib.22,23
Similar to carfilzomib, proteasome activity recovered through
new proteasome synthesis in all tissues, with the exception of
blood, within 24-72 h.
site IC50 (nM)
compd
N-cap
Ra
ꢀ5
LMP7
50
51
49
52
53
54
55
56
57
58
2
3-isoxazole
3-isoxazole
i-Bu
CH2Ph 44
i-Bu
48 ( 31 (3)
607 ( 273 (3)
93
5-Me-3-isoxazole
5-Me-3-isoxazole
5-EtO-3-isoxazole
5-EtO-3-isoxazole
17 ( 10 (5)
238 ( 114 (5)
21 (15, 27)
41 (40, 42)
5.0 (2.7, 7.3)
22 (17, 28)
36 (36, 37)
784
CH2Ph 11 (8.2, 13)
i-Bu
CH2Ph 3.7 (1.9, 5.4)
23 (20, 25)
3-MeO-5-isoxazole CH2Ph 14 (14, 14)
9.3 (5.4, 13)
3-MeO-5-isoxazole i-Bu
2-Me-5-thiazole
2-Me-5-thiazole
-
-
i-Bu
CH2Ph 36
-
-
73
82
5.2 ( 2.5 (27) 14 ( 9 (27)
8.7 ( 4.6 (6) 8.1 ( 5.4 (6)
1
a Leu: Rdi-Bu; Phe: RdCH2Ph. b All experiments to determine IC50
values were run with at least duplicates at each compound dilution; IC50
values were averaged when determined in two or more independent
experiments; the individual IC50 values listed in parentheses (n ) 2) or (
standard deviation denoted and n listed in parentheses (n g 3).
constitutive proteasome (Table 5). Previous reports have
indicated that tetrapeptide-based proteasome inhibitors with
phenylalanine as P1 may enhance inhibition of LMP7 (the CT-L
activity of the immunoproteasome).39,40 Therefore, we inves-
tigated the impact of leucine versus phenylalanine substitutions
at P1 in a series of compounds. Tripeptide analogues with
phenylalanine as P1 (51, 52, 54, 56, and 58) demonstrated an
equivalent potency for ꢀ5 and LMP7 (Table 5). Similar to
carfilzomib, these analogues also displayed selectivity for the
CT-L subunits over T-L and C-L activities of the proteasome
(data not shown). Furthermore, these P1 phenylalanine analogues
exhibited similar in vitro potency, MDR sensitivity, and stability
as compared to their P1 leucine counterparts (Table 6). Most
importantly, the P1 phenylalanine and leucine analogues
displayed similar oral bioactivity. Two analogues, 5-EtO-3-
isoxazole (54) and 2-Me-5-thiazole (58) (PR-047), from an
N-cap rescreening within the Ser(OMe)-Ser(OMe)-Phe-ketoe-
poxide backbone series, were selected for further in vivo
efficacy, PK, and PD testing based on their oral bioactivity.
Antitumor Activity, Absolute Bioavailability, and PD
Kinetics. Carfilzomib has been shown to mediate antitumor
responses in multiple mouse models of cancer.22,23 We evaluated
the antitumor activity of the lead compounds (54, 58) along
with carfilzomib in immunocompromised mice bearing estab-
lished xenografts of the Non-Hodgkin’s lymphoma cell line RL
and in BALB/c mice bearing the mouse colorectal tumor cell
line CT-26. When each compound was administered on a
weekly QD×2 schedule (Figure 3), compound 58 (po admin-
istered) promoted an equivalent antitumor response to carfil-
zomib (iv administered) in both models. However, 54 (po
administered) was not able to achieve significant antitumor
response in either model (further statistical analysis of antitumor
responses are listed in Table S2 of the Supporting Information).
It is noteworthy that 58 was administered below its maximum
tolerated dose (MTD), while carfilzomib and 54 were delivered
at their respective MTDs.
Conclusions
Although a variety of peptide-based drugs have been com-
mercialized successfully, most of these therapeutics are admin-
istered by the parenteral route because of insufficient oral
bioavailability.38 Furthermore, epoxides are typically chemically
reactive and unstable in low pH environments,41 presenting an
additional challenge to the identification of orally active peptidyl
epoxyketone proteasome inhibitors. Through a systematic SAR
optimization and in vivo PD screening effort, we improved
solubility, metabolic stability (gastric fluid, intestinal fluid, liver
microsomes, and hepatocytes), and sensitivity to the multidrug
resistance protein 1 (MDR1) and, most importantly, oral
bioavailability in this series of compounds. These efforts
culminated in the identification of 58, a compound that promoted
antitumor activity in multiple animal models by oral administra-
tion at doses below the MTD. Taken together, the data presented
here demonstrate that 58 is an orally bioavailable and selective
proteasome inhibitor with favorable pharmacologic properties
and support the further development of this molecule for the
treatment of malignant diseases.
Synthesis
The generalized route for the synthesis of peptide epoxyke-
tone, exemplified by synthesis of 58, is illustrated in Schemes
1 and 2).40,42,43 Cbz-protected phenylalanine 59 was converted
into the Weinreb amide 60, followed by treatment with Grignard
reagent to yield enone 61. The enone was then reduced with
sodium boronate and cerium(III) chloride into alcohols 62a
(2S,3R) and 62b (2S,3S) in a ratio of 6/1. The mixed allylic
alcohols were treated with tert-butylhydroperoxide in the
presence of vanadyl acetylacetonate as catalyst to achieve allylic
epoxides 63a (1R,2S,3S) and 63b (1S,2R,3S) in a similar ratio.
These allylic epoxides are unstable and were oxidized directly
Compound 58 displayed a moderate absolute oral bioavail-
ability (F) across multiple species by plasma PK measurement
and blood PD measurement (Table 7). In mice, PD bioactivity

Peptide Epoxyketone Proteasome Inhibitor (PR-047)
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3033
Table 6. P1 and N-Cap Modification with Methylserine as Both P2 and P3
MES cell viability ( MDRb
liver microsome
stability (Re)
mice PO PD
(% CT-L activity @
IC50 (nM)
SIF
20S CT-Lb Molt-4 CT-Lb
10 mg/kg),
adrenal
stability
compd
N-cap
Ra
IC50 (nM)
IC50 (nM)
MDR-
MDR+
(% parent @ 15 min) mouse human
50
51
49
52
53
54
55
56
57
58
3-isoxazole
3-isoxazole
5-Me-3-isoxazole i-Bu
5-Me-3-isoxazole CH2Ph 24 ( 6 (3)
5-EtO-3-isoxazole i-Bu 3.1 ( 0.3 (3)
5-EtO-3-isoxazole CH2Ph 1.4
i-Bu
CH2Ph 73
43 ( 8 (3)
168
47
48 ( 26 (3)
35 (24, 45)
35
3.0
32
10
164
66
14
34
21
43
4
37
43
260
756
145 ( 52 (4)
155 (139, 171)
90
85
70
90
70
85
35
100
80
90
0.39
0.49
0.67
0.59
0.69
0.77
0.37
0.54
0.21
0.51
0.16
0.42
0.54
0.59
0.69
0.77
0.40
0.64
0.41
0.60
11 ( 4 (10)
22 ( 2 (4)
17 (12, 21)
5.4 (4.2, 6.5) 66 (39, 94)
4.3
12
3
54
82
269
1157
3-MeO-5-isoxazole i-Bu
3-MeO-5-isoxazole CH2Ph 50 (45, 56)
11 ( 6 (3)
21
19
45
6
12
38
2-Me-5-thiazole
2-Me-5-thiazole
i-Bu
74
CH2Ph 55 ( 19 (9)
25 ( 15 (4)
1322 ( 316 (4)
a Leu: Rdi-Bu; Phe: RdCH2Ph. b All experiments to determine IC50 values were run with at least duplicates at each compound dilution; IC50 values were
averaged when determined in two or more independent experiments; the individual IC50 values listed in parentheses (n ) 2) or ( standard deviation denoted
and n listed in parentheses (n g 3).
by deprotection of Boc group.44 Dipeptide BocNH-Ser(OMe)-
Ser(OMe)-OBn (68) was then prepared by coupling of protected
methylserines 65 and 67 under a condition of HOBt, HBTU,
and DIEA. Similarly, 2-Me-thiazol-5-yl-Ser(OMe)-Ser(OMe)-
OBn (71) was prepared from coupling of 2-methylthiazole-5-
carboxylic acid and intermediate 69. However, hydrogenation
of 71 using Pd/C to remove benzyl ester group failed to yield
acid 72, most likely due to poisoning of the catalyst by thiazole.
In contrast, hydrogenation under the same condition to remove
the benzyl ester group of dipeptide 68 yielded acid 73
quantitatively. Removal of Cbz group from 64a by hydrogena-
tion with Pd/C in trifluoroacetic acid (TFA) resulted in TFA
salt 74, which was then coupled with acid 73 into Boc-
Ser(OMe)-Ser(OMe)-Phe-ketopoxide (75). The latter was treated
with TFA to remove Boc group and coupled with 2-methylthi-
azole-5-carboxylic acid under a condition of HOBt, HBTU, and
DIEA to yield the target molecule 58.
Experimental Section
General Methods. Chemicals, reagents and solvents were
obtained from commercial sources and used as received. NMR
spectra were obtained on a Varian Mercury-300 spectrometer in
the indicated solvents. Analytical LCMS was run on Water 2996
eluting with a mixture of acetonitrile and water containing 0.1%
acetic acid. Mass spectra were obtained using Water EMD1000
mass spectrometer, and UV absorption was recorded at wavelength
of 214 nm. Preparative reverse phase HPLC was run using Water
2695 instrument with a YM 25 cm × 50 mm column eluting with
a mixture of acetonitrile and water containing 0.1% ammonium
acetate. Normal phase flash chromatography was performed on
silica gel 60.
Benzyl (S)-1-(N-Methoxy-N-methylcarbamoyl)-2-phenyleth-
ylcarbamate (60). A suspension of N,O-dimethylhydroxylamine
hydrochloride (41.0 g, 420 mmol) in DCM (700 mL) was stirred
vigorously for 0.5 h and then TEA (42.5 g, 420 mmol) was added
via an addition funnel. In a separate flask, to a 0 °C solution of
Cbz-phenylalanine (59) (125.0 g, 420 mmol) in DCM (700 mL)
was added isobutylchloroformate (57.3 g, 420 mmol) dropwise via
an addition funnel, and the resulting mixture was further cooled to
-20 °C and NMM (42.5 g, 420 mmol) was added via an addition
funnel at such a rate to maintain the temperature below -10 °C.
The freshly prepared dimethyl hydroxylamine solution was then
added via a wide bore Teflon canula at a rate to maintain the
temperature below -5 °C. The reaction mixture was warmed to
room temperature for 1.5 h followed by dilution with water (500
Figure 3. Antitumor activity of compounds 2, 54, and 58. BNX mice
bearing established human tumor xenografts derived from RL and
BALB/c mice challenged with the murine tumor cell line CT-26 were
treated twice weekly on days 1 and 2 (QD×2) with 5 mg/kg 2 (iv), 20
mg/kg 54 (po), or 30 mg/kg 58 (po). Arrow indicates the start of dose
period. Data are presented as mean tumor volume ( SEM (n ) 8-10/
group). Statistical comparisons between treatment groups and vehicle
controls were made by two-way ANOVA followed by Bonferroni post
hoc analysis (Table S2, Supporting Information). P values reflect the
statistical significance by the end of study.
Table 7. Oral Bioavailability (F) of Compound 58
mouse
F calculated by PK (Dose) 17 (40 mg/kg) 21 (40 mg/kg) 39 (19 mg/kg)
F calculated by PD
36a
rat
dog
-
-
a iv dose range: 1-5 mg/kg; po dose range: 5-20 mg/kg.
into epoxyketones with Dess-Martin reagent. The major isomer
64a (1R,3S) was isolated from others by flash chromatography.
Benzyl ester-protected methylserine 67 was prepared from Boc-
protected methylserine 65 with benzyl chloroformate followed

3034 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Zhou et al.
Figure 4. Kinetics of proteasome inhibition following po administration of compound 58. BALB/c mice (n ) 3/time point) received a single po
administration of 30 mg/kg, and proteasome inhibition was measured at the indicated time points using LLVY-AMC substrate. Data are presented
as mean residual activity ((SEM) relative to vehicle treated controls.
a
Scheme 1. Generalized Route to Synthesize Cbz-Phe Epoxyketone
a Reagents and conditions: (i) i-BuOCOCl, NMM, N,O-dimethylhydroxylamine hydrochloride, TEA, DCM, -20 °C; (ii) isopropenylmagnesium bromide,
THF, 0 °C; (iii) NaBH4, CeCl3 ·7H2O, MeOH, THF, 0°C-rt; (iv) vanadyl acetylacetonate, t-BuO2H, DCM, 0 °C; (v) Dess-Martin periodinane, DCM, 0
°C-rt.
mL). The layers were separated, and the aqueous layer was extracted
with DCM (2 × 150 mL). The combined organic layers were
washed with 1 N HCl (4 × 500 mL), water (1 × 150 mL), aqueous
NaHCO3 solution (4 × 150 mL), brine (1 × 250 mL), and dried
over Na2SO4. The Na2SO4 was removed by filtration, and the
volatiles were removed under reduced pressure to give Weinreb
amide 60 (166 g). LRMS (M + H+) m/z: calcd 343.16; found
343.16. 1H NMR (300.05 MHz, CDCl3): δ 2.91 (dd, J ) 7.2, 13.8
Hz, 1H, CHCH2Ph), 3.08 (dd, J ) 5.8, 13.8 Hz, 1H, CHCH2Ph),
3.17 (s, 3H, NCH3), 3.68 (s, 3H, OCH3), 5.00-5.11 (m, 3H,
OCH2Ph + NHCH), 5.45 (d, J ) 8.8 Hz, 1H, NHCH), 7.12-7.38
(m, 10H, 2C6H5).
Benzyl (S)-4-Methyl-3-oxo-1-phenylpent-4-en-2-ylcarbamate
(61). To a 0 °C solution of the isopropenyl magnesium bromide
(2 L, 1000 mmol, 0.5 M solution in THF) was added a solution of
amide 60 (116.0 g, 484 mmol) in dry THF (1 L) dropwise via an
addition funnel under an atmosphere of argon. The reaction was
stirred at 0 °C for 6 h and then slowly poured into a beaker
containing 1.25 L of saturated NH4Cl solution and approximately
1.25 L of ice. The pH of the solution was adjusted to 1.5 using
concentrated HCl when all the ice melted, and the resulting mixture
was extracted with EtOAc (2 × 2 L). The combined organic layers
were washed with aqueous NaHCO3 solution (1 × 1 L), brine
(1 × 1 L), and dried over MgSO4. The MgSO4 was removed by
filtration, and the volatiles were removed under reduced pressure.
Purification of residue by flash chromatography gave enone 61
(103.3 g, 76% over the first two steps) as oil. LRMS (M + H+)
m/z: calcd 324.15; found 324.15. 1H NMR (300.05 MHz, CDCl3):
δ 1.86 (s, 3H, CCH3), 2.97 (dd, J ) 6.0, 13.8 Hz, 1H, CHCH2Ph),
3.15 (dd, J ) 6.3, 13.8 Hz, 1H, CHCH2Ph), 5.09 (dd, J ) 12.4,
18.2 Hz, 2H, OCH2Ph), 5.35 (dd, J) 6.0, 14.03 Hz, 1H, NHCH),
5.58 (d, J ) 8.0 Hz, 1H, NHCH), 5.89 (br s, 1H, CdCH2), 6.04
(br s, 1H, CdCCH2), 7.01-7.39 (m, 10H, 2C6H5).
Benzyl (2S,3R)-3-Hydroxy-4-methyl-1-phenylpent-4-en-2-yl-
carbamates (62a, 62b). To a 0 °C solution of compound 61 (103.3
g, 320 mmol) in MeOH (1.3 L) and THF (1.3 L) was added
CeCl3 ·7H2O (172.6 g, 463 mmol) under an atmosphere of argon.
Then NaBH4 (17.5 g, 463 mmol) was added once it became
homogeneous. The reaction mixture was stirred at the same
temperature for 6 h and quenched with glacial acetic acid (230 mL).
The resulting mixture was stirred until homogeneous. The volatiles
were removed under reduced pressure, and the remaining oil was
diluted with water (1 L) and extracted with EtOAc (3 × 500 mL).
The combined organic layers were washed with water (2 × 500

Peptide Epoxyketone Proteasome Inhibitor (PR-047)
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3035
Scheme 2. Generalized Route to Synthesize Tripeptide Epoxyketone 58a
a Reagents and conditions: (vi) BnOCOCl, TEA, DMAP, DCM, 0 °C; (vii) TFA, DCM, rt; (viii) 65, HOBt, HBTU, DIEA, THF, 0 °C; (ix) TFA, DCM,
0 °C; (x) HOBt, HBTU, DIEA, THF, 0 °C; (xi) THF, Pd/C, H2, rt; (xii) THF, Pd/C, H2, rt; (xiii). TFA, Pd/C, H2, rt; (xiv) HOBt, HBTU, DIEA, THF, 0 °C;
(xv) TFA, DCM, 0 °C; (xvi) 70, HOBt, HBTU, DIEA, THF, 0 °C.
mL), brine (2 × 500 mL), and dried over MgSO4. The MgSO4
was removed by filtration, and the volatiles were removed under
reduced pressure to give a mixture of compounds (62a, 62b) (102.5
g, 98%, ratio 6:1 based on LCMS analysis) as oil. LRMS (M +
H+) m/z: calcd 326.17; found 326.17. 1H NMR (300.05 MHz,
CDCl3): δ 1.68 (s, 3H, CCH3, minor isomer), 1.82 (s, 3H, CCH3,
major isomer), 2.02 (br s, 1H, OH), 2.91 (dd, J ) 3.3, 14.2 Hz,
2H, CHCH2Ph), 3.97-4.20 (br s, 2H, NHCH + CHOH), 4.90-5.07
(m, 5H, NHCH + OCH2Ph + CdCCH2), 7.15-7.33 (m, 10H,
2C6H5).
L). The combined organic layers were washed with water (3 × 1
L), brine (1 × 1 L), and dried over MgSO4. The MgSO4 was
removed by filtration, and the volatiles were removed under reduced
pressure to give crude residue as oil. Purification by flash chro-
matography with ethyl acetate/hexane gave epoxyketone (64a) (38.7
g, 27% yield of the two-step). LRMS (M + H+) m/z: calcd 340.15;
found 340.15. 1H NMR (300.05 MHz, CDCl3): δ 1.52 (s, 3H, CH3),
2.78 (dd, J ) 7.7, 14.0 Hz, 1H, CHCH2Ph), 2.93 (d, J ) 5.0 Hz,
1H, OCH2), 3.14 (dd, J ) 5.1, 13.9 Hz, 1H, CHCH2Ph), 3.30 (d,
J ) 5.0 Hz, 1H, OCH2), 4.66 (ddd, J ) 5.0, 8.0, 12.4 Hz, 1H,
NHCH), 5.03 (dd, J ) 12.4, 14.9 Hz, 2H, OCH2Ph), 5.24 (d, J )
8.3 Hz, 1H, NHCH), 7.14-7.38 (m, 10H, 2C6H5).
tert-Butyl (S)-1-((Benzyloxy)carbonyl)-2-methoxyethylcar-
bamate (66). To a solution of Boc-methylserine 65 (43.8 g, 200
mmol) in DCM (400 mL) were added TEA (26.5 g, 260 mmol)
and DMAP (2.4 g, 20 mmol). The resulting solution was cooled to
-5 °C, and benzyl chloroformate (41.0 g, 240 mmol) was then
slowly added via an addition funnel under an atmosphere of argon.
The reaction was kept at the same temperature for 3 h and then
diluted with brine (100 mL). The layers were separated, and the
aqueous layer was extracted with DCM (2 × 200 mL). The organic
layers were combined and dried over Na2SO4. The Na2SO4 was
removed by filtration, and the volatiles were removed under reduced
pressure. The resulting residue was purified by flash chromatog-
raphy using a mixture of hexane and ethyl acetate to provide
intermediate 66 as white solid (54 g, 88% yield). LRMS (M +
H+) m/z: calcd 310.16; found 310.16. 1H NMR (300.05 MHz,
CDCl3): δ 1.44 (s, 9H, C(CH3)3), 3.29 (s, 3H, OCH3), 3.60 (dd, J
) 3.2, 9.3 Hz, 1H, CH2OCH3), 3.81 (dd, J ) 3.1, 9.3 Hz, 1H,
CH2OCH3), 4.45 (m, 1H, CHNH), 5.14 (d, J ) 12.3 Hz, 1H,
Benzyl (S)-1-((R)-2-Methyloxiran-2-yl)-1-oxo-3-phenylpropan-
2-ylcarbamate (64a). To a 0 °C solution of alcohols (62a, 62b)
(102.5 g, 300 mmol) in dry DCM (300 mL) was added VO(acac)2
(2.9 g, 10.7 mmol) under an atmosphere of argon. Then t-BuO2H
(105.5 mL, 580 mmol, 5.5 M solution in decane) was added
dropwise via an addition funnel. The reaction was warmed to room
temperature for 6 h and became light yellow. The resulting mixture
was filtered through celite, and the layers were separated. The aque-
ous layer was extracted with DCM (2 × 100 mL). The combined
organic layers were washed with aqueous sodium bisulfite solution
(2 × 200 mL), brine (2 × 200 mL), and dried over Na2SO4. The
Na2SO4 was removed by filtration, giving a DCM solution of
intermediates (63a, 63b), which were used in the next step. LRMS
(M + H+) m/z: calcd 342.16; found 342.16.
To a 5 °C solution of aforementioned intermediates (63a, 63b)
in DCM (2.5 L) was added Dess-Martin periodinane (316 g, 16.4
mmol) in DCM (1 L) under an atmosphere of argon. The reaction
was kept at room temperature overnight. The resulting mixture was
cooled in an ice-bath, diluted with aqueous NaHCO3 solution
(1 L), and EtOAc (2 L), and filtered through celite. The layers were
separated and the aqueous layer was extracted with EtOAc (2 × 1

3036 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Zhou et al.
CH2Ph), 5.27 (d, J ) 12.3 Hz, 1H, CH2Ph), 5.40 (br s, 1H, NHCH),
7.26 (m, 5H, C6H5).
removed under reduced pressure. The residual TFA salt 74 was
dried over high vacuum and used in the next step without further
purification. LRMS (M + H+) m/z: calcd 206.11; found 206.11.
To a -5 °C mixture of the resulting acid 73 and epoxyketone
TFA salt 74, HOBt (6.0 g, 39.2 mmol), and HBTU (14.8 g, 39.2
mmol) in THF (600 mL) was added DIEA (23 mL) slowly via an
addition funnel. The reaction was kept at the same temperature for
4 h, followed by dilution with EtOAc (200 mL) and brine (200
mL). The layers were separated, and the aqueous layer was extracted
with EtOAc (2 × 300 mL). The organic layers were combined and
dried over Na2SO4. The Na2SO4 was removed by filtration, and the
volatiles were removed under reduced pressure. The resulting
residue was purified by flash chromatography using a mixture of
hexane and ethyl acetate to provide tripeptide 75 as white solid
(9.2 g, 69% overall yield). LRMS (M + H+) m/z: calcd 508.58;
Benzyl (2S)-2-[(2S)-2-{[tert-Butoxy)carbonyl]amino}-3-meth-
oxypropanamido]-3-methoxypropanoate (68). To a 0 °C solution
of intermediate 66 in DCM (100 mL) was added TFA (100 mL)
slowly via an addition funnel. The reaction was kept at the same
temperature for 1 h, concentrated, and dried under high vacuum
overnight. The resulting residual TFA salt 67 was used in the next
step without further purification. LRMS (M + H+) m/z: calcd,
210.11; found 210.1.
To a -5 °C mixture of aforementioned TFA salt 67, Boc-
methylserine 65 (36.7 g, 167 mmol), HOBt (27.0 g, 200 mmol),
and HBTU (71.4 g, 200 mmol) in THF (600 mL) was added DIEA
(76.6 g, 600 mmol) slowly via an addition funnel. The reaction
was kept at the same temperature for 4 h, followed by dilution
with EtOAc (500 mL) and brine (300 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (2 ×
300 mL). The organic layers were combined and dried over Na2SO4.
The Na2SO4 was removed by filtration, and the volatiles were
removed under reduced pressure. The resulting residue was purified
by flash chromatography using a mixture of hexane and ethyl acetate
to provide dipeptide 68 as white solid (65 g, 95% yield). LRMS
(M + H+) m/z: calcd 411.21; found 411.21. 1H NMR (300.05 MHz,
CDCl3): δ 1.45 (s, 9H, C(CH3)3), 3.29 (s, 3H, OCH3), 3.34 (s, 3H,
OCH3), 3.44 (dd, J ) 6.9, 9.4 Hz, 1H, CH2OCH3), 3.61 (dd, J )
3.0, 9.4 Hz, 1H, CH2OCH3), 3.78 (dd, J ) 3.9, 9.4 Hz, 1H,
CH2OCH3), 3.84 (dd, J ) 3.0, 9.4 Hz, 1H, CH2OCH3), 4.27 (m,
1H, CHNH), 4.75 (dt, J ) 3.3, 6.3, 8.3 Hz, 1H, CHNH), 5.20 (q,
J ) 12.4, 28.6 Hz, 2H, CH2Ph), 5.42 (br s, 1H, NH), 7.26 (m, 5H,
C6H5).
Benzyl (2S)-3-Methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-
thiazol-5-yl)formamido]propanamido]propanoate (71). To a 0
°C solution of aforementioned intermediate 68 (18.0 g, 38 mmol)
in DCM (100 mL) was added TFA (100 mL) slowly via an addition
funnel. The reaction was kept at the same temperature for 2 h,
concentrated, and dried under high vacuum overnight. The resulting
residual TFA salt 69 was used in the next step without further
purification. LRMS (M + H+) m/z: calcd 311.15; found 311.15.
To a -5 °C mixture of TFA salt 69, 2-methylthiazole-5-
carboxylic acid (70) (5.8 g, 38 mmol), HOBt (6.8 g, 45 mmol),
and HBTU (16.0 g, 45 mmol) in THF (600 mL) was added DIEA
(10.0 g, 80 mmol) slowly. The reaction was kept at the same
temperature for 4 h and then diluted with EtOAc (200 mL) and
brine (200 mL). The layers were separated, and the aqueous layer
was extracted with EtOAc (2 × 300 mL). The organic layers were
combined and dried over Na2SO4. The Na2SO4 was removed by
filtration, and the volatiles were removed under reduced pressure.
The resulting residue was purified by flash chromatography using
a mixture of hexane and ethyl acetate to provide benzyl ester 71 as
white solid (14 g, 75% yield). LRMS (M + H+) m/z: calcd 436.15;
found 436.15. 1H NMR (300.05 MHz, CDCl3): δ 2.76 (s, 3H, CH3-
thiazole), 3.31 (s, 3H, OCH3), 3.34 (s, 3H, OCH3), 3.50 (dd, J )
8.3, 9.1 Hz, 1H, CH2OCH3), 3.63 (dd, J ) 3.3, 9.6 Hz, 1H,
CH2OCH3), 3.84 (dd, J ) 4.1, 6.1 Hz, 1H, CH2OCH3), 3.87 (dd, J
) 3.3, 5.5 Hz, 1H, CH2OCH3), 4.72 (m, 2H, 2CHNH), 5.20 (q, J
) 12.4, 29.2 Hz, 2H, CH2Ph), 7.06 (d, J ) 6.6 Hz, 1H, NH), 7.35
(m, 5H, C6H5), 7.44 (d, J ) 8.3 Hz, 1H, NH), 8.10 (s, 1H,
H-thiazole).
tert-Butyl N-[(1S)-2-Methoxy-1-{[(1S)-2-methoxy-1-{[(2S)-4-me-
thyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]carbamoyl}-
ethyl]carbamoyl}ethyl]carbamate (75). A solution of dipeptide 68
(13.4 g, 32.7 mmol) in THF (200 mL) was stirred with Pd/C (2.7
g) under atmosphere of hydrogen for 2 h at room temperature. The
Pd/C was then removed by filtration and washed with THF (100
mL). The volatiles were removed under reduced pressure. The
residual acid 73 was dried and used in the next step without further
purification. LRMS (M + H+) m/z: calcd 321.16; found 321.16.
A 0 °C solution of aforementioned epoxyketone 64a (8.0 g, 32.7
mmol) in TFA (80 mL) was stirred with Pd/C (2.0 g) under
atmosphere of hydrogen for two hours. The Pd/C was removed by
filtration and washed with DCM (50 mL). The volatiles were
1
found 508.58. H NMR (300.05 MHz, CDCl3): δ 1.44 (s, 9H,
C(CH3)3), 1.47 (s, 3H, CH3-oxirane), 2.86 (dd, J ) 7.5, 14.1 Hz,
1H, CH2Ph), 2.88 (d, J ) 4.5 Hz, 1H, CH2 of oxirane), 3.11 (dd,
J ) 5.4, 14.1 Hz, 1H, CH2Ph), 3.26 (d, J ) 6.5 Hz, 1H, CH2 of
oxirane), 3.30 (s, 6H, 2OCH3), 3.38 (dd, J ) 6.0, 9.0 Hz, 1H,
CH2OCH3), 3.46 (dd, J ) 7.2, 9.0 Hz, 1H, CH2OCH3), 3.73 (dd, J
) 3.9, 9.0 Hz, 1H, CH2OCH3), 3.78 (dd, J ) 3.3, 9.0 Hz, 1H,
CH2OCH3), 4.25 (m, 1H, CHNH), 4.43 (dq, J ) 3.3, 6.0, 7.2, 9.3
Hz, 1H, CHNH), 4.86 (dt, J ) 5.4, 7.5, 13.2 Hz, 1H, CHNH),
5.39 (d, J ) 6.0 Hz, 1H, NH), 7.04-7.31 (m, 7H, C6H5 + 2NH).
(2S)-3-Methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-
5-yl)formamido]propanamido]-N-[(2S)-1-[(2R)-2-methyloxiran-
2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide (58). To a 0 °C
solution of intermediate 75 (18 g, 35 mmol) in DCM (100 mL)
was added TFA (100 mL) slowly via an addition funnel. The
reaction was kept at the same temperature for 2 h and concentrated
under high vacuum. The resulting residual TFA salt 76 was dried
overnight and used in the next step without further purification.
LRMS (M + H+) m/z: calcd 408.21; found 408.21.
To a -5 °C mixture of aforementioned TFA salt 76, 2-me-
htylthiazole-5-carboxylic acid 70 (5.8 g, 38 mmol), HOBt (6.8 g,
45 mmol), and HBTU (16.0 g, 45 mmol) in THF (600 mL) was
added DIEA (10 g, 80 mmol) slowly via an addition funnel. The
reaction was kept at the same temperature for 4 h and then diluted
with EtOAc (200 mL) and brine (200 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (2 ×
300 mL). The organic layers were combined and dried over Na2SO4.
The Na2SO4 was removed by filtration, and the volatiles were
removed under reduced pressure. The resulting residue was purified
by HPLC using a mixture of acetonitrile and aqueous NH4Ac
solution (0.1%) as mobile phases, and the desired product 58 (12
g, 64% yield) was isolated as white solid. LRMS (M + H+) m/z:
1
calcd 533.20; found 533.20. H NMR (300.05 MHz, CDCl3): δ
1.50 (s, 3H, CH3-oxirane), 2.74 (s, 3H, CH3-thiazole), 2.88 (dd, J
) 7.2, 14.0 Hz, 1H, CH2Ph), 2.91 (d, J ) 5.0 Hz, 1H, CH2 of
oxirane), 3.14 (dd, J ) 5.0, 14.0 Hz, 1H, CH2Ph), 3.27 (d, J ) 5.0
Hz, 1H, CH2 of oxirane), 3.32 (s, 6H, 2OCH3), 3.40 (dd, J ) 5.8,
9.1 Hz, 1H, CH2OCH3), 3.55 (t, J ) 8.8 Hz, 1H, CH2OCH3), 3.80
(d, J ) 9.0 Hz, 1H, CH2OCH3), 3.81 (dd, J ) 2.1, 9.3 Hz, 1H,
CH2OCH3), 4.47 (dq, J ) 3.0, 5.8, 7.4, 10.5 Hz, 1H, CHNH), 4.68
(dq, J ) 4.4, 6.3, 8.3, 10.7 Hz, 1H, CHNH), 4.86 (dt, J ) 5.5, 7.7,
12.9 Hz, 1H, CHNH), 6.92 (d, J ) 6.1 Hz, 1H, NH), 7.08-7.31
(m, 7H, C6H5 + 2NH), 8.06 (s, 1H, H-thiazole). 13C NMR (75.46
MHz, CDCl3): δ 16.80, 19.90, 52.70, 52.74, 53.13, 59.32, 59.44,
59.52, 71.43, 71.71, 127.25, 128.68, 129.73, 133.41, 135.90, 143.88,
160.66, 169.60, 169.80, 170.95, 207.38. Elemental analysis
(C25H32N4O7S, 532.61), found (calcd), C: 56.47 (56.38); H: 6.06
(5.74); N: 10.55 (10.52).
1
Other analogues were prepared through similar procedures. H
NMR and 13C NMR spectra of representative compounds were listed
in Supporting Information. All of these compounds possess a purity
of not less than 95% (Table S4 of the Supporting information).
Supporting Information Available: Experimental details on
biological assays, DMPK assays, animal studies, spectral charac-
1
terization of compounds 5, 11, 28, 29, 49, and 54 (including H

Peptide Epoxyketone Proteasome Inhibitor (PR-047)
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3037
NMR, 13C NMR spectra), HPLC purity and solubility data, as well
as inactivation rates (Kinact/Ki) of compounds 5, 11, 28, 29, 46, 49,
54, and 58, and statistical analysis of antitumor response (2-way
ANOVA) of compounds 2, 54, and 58. This material is available
free of charge via the Internet at http://pubs.acs.org.
Schenkein, D.; Cabanillas, F.; Rodriguez, A. M. Phase II study of
proteasome inhibitor bortezomib in relapsed or refractory B-cell non-
Hodgkin’s lymphoma. J. Clin. Oncol. 2005, 23, 667–675.
(20) Richardson, P. G.; Briemberg, H.; Jagannath, S.; Wen, P. Y.; Barlogie,
B.; Berenson, J.; Singhal, S.; Siegel, D. S.; Irwin, D.; Schuster, M.;
Srkalovic, G.; Alexanian, R.; Rajkumar, S. V.; Limentani, S.; Alsina,
M.; Orlowski, R. Z.; Najarian, K.; Esseltine, D.; Anderson, K. C.;
Amato, A. A. Frequency, characteristics and reversibility of peripheral
neuropathy during treatment of advanced multiple myeloma with
bortezomib. J. Clin. Oncol. 2006, 24, 3113–3120.
(21) Lonial, S.; Waller, E. K.; Richardson, P. G.; Jagannath, S.; Orlowski,
R. Z.; Giver, C. R.; Jaye, D. L.; Francis, D.; Giusti, S.; Torre, C.;
Barlogie, B.; Berenson, J. R.; Singhal, S.; Schenkein, D. P.; Esseltine,
D. L.; Anderson, J.; Xiao, H.; Heffner, L. T.; Anderson, K. C. Risk
factors and kinetics of thrombocytopenia associated with bortezomib
for relapsed, refractory multiple myeloma. Blood 2005, 106, 3777–
3784.
(22) Demo, S. D.; Kirk, C. J.; Aujay, M. A.; Buchholz, T. J.; Dajee, M.;
Ho, M. N.; Jiang, J.; Laidig, G. J.; Lewis, E. R.; Parlati, F.; Shenk,
K. D.; Smyth, M. S.; Sun, C. M.; Vallone, M. K.; Woo, T. M.;
Molineaux, C. J.; Bennett, M. K. Antitumor activity of PR-171, a novel
irreversible inhibitor of the proteasome. Cancer Res. 2007, 67, 6383–
6391.
(23) Bennett, M. K.; Kirk, C. J. Development of proteasome inhibitors in
oncology and autoimmune diseases. Curr. Opin. Drug DiscoVery DeV.
2008, 11, 616–625.
(24) Groll, M.; Kim, K. B.; Kairies, N.; Huber, R.; Crews, C. M. Crystal
structure of epoxomicin: 20S proteasome reveals a molecular basis
for selectivity of ꢀ-epoxyketone proteasome inhibitors. J. Am. Chem.
Soc. 2000, 122, 1237–1238.
(25) Hanada, M.; Sugawara, K.; Kaneta, K.; Toda, S.; Nishiyama, Y.;
Tomita, K.; Yamamoto, H.; Konishi, M.; Oki, T. Epoxomicin, a new
antitumor agent of microbial origin. J. Antibiot. (Tokyo) 1992, 45,
1746–1752.
(26) Sin, N.; Kim, K. B.; Elofsson, M.; Meng, L.; Auth, H.; Kwok, B. H.;
Crews, C. W. Total synthesis of the potent proteasome inhibitor
epoxomicin: a useful tool for understanding proteasome biology.
Bioorg. Med. Chem. Lett. 1999, 9, 2283–2288.
References
(1) Ciechanover, A. Proteolysis: From the Lysosome to Ubiquitin and
the Proteasome. Nat. ReV. Mol. Cell Biol. 2005, 6, 79–87.
(2) Dalton, W. S. The proteasome. Semin. Oncol. 2004, 31, 3–9.
(3) Kisselev, A. F.; Goldberg, A. L. Proteasome inhibitors: from research
tools to drug candidates. Chem. Biol. 2001, 8, 739–758.
(4) Adams, J. The proteasome: a suitable antineoplastic target. Nat. ReV.
Cancer 2004, 4, 349–360.
(5) Goldberg, A. L. Functions of the proteasome: from protein degradation
and immune surveillance to cancer therapy. Biochem. Soc. Trans. 2007,
35, 12–17.
(6) Groll, M.; Ditzel, L.; Lo¨we, J.; Stock, D.; Bochtler, M.; Bartunik,
H. D.; Huber, R. Structure of 20S proteasome from yeast at 2.4 Å
resolution. Nature (London) 1997, 386, 463–471.
(7) Orlowski, M.; Wilk, S. Catalytic activities of the 20S proteasome, a
multicatalytic proteinase complex. Arch. Biochem. Biophys. 2000, 383,
1–16.
(8) Kisselev, A. F.; Callard, A.; Goldberg, A. L. Importance of the different
proteolytic sites of the proteasome and the efficacy of inhibitors varies
with the protein substrate. J. Biol. Chem. 2006, 281, 8582–8590.
(9) Groll, M.; Heinemyer, W.; Ja¨ger, S.; Ullrich, T.; Bochtler, M.; Wolf,
D. H.; Huber, R. The catalytic sites of 20S proteasomes and their role
in subunit maturation: a mutational and crystallographic study. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 10976–10983.
(10) Rock, K. L.; Gramm, C.; Rothstein, L.; Clark, K.; Stein, R.; Dick, L.;
Hwang, D.; Goldberg, A. L. Inhibitors of the proteasome block the
degradation of most cell proteins and the generation of peptides
presented on MHC class I molecules. Cell 1994, 78, 761–771.
(11) Rock, K. L.; York, I. A.; Saric, T.; Goldberg, A. L. Protein degradation
and the generation of MHC class I presented peptides. AdV. Immunol.
2002, 80, 1–70.
(12) Rivett, A. J.; Hearn, A. R. Proteasome function in antigen presentation:
immunoproteasome complexes, peptide production, and interactions
with viral proteins. Curr. Protein Pept. Sci. 2004, 5, 153–161.
(13) Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.;
Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L.
Potent and selective inhibitors of the proteasome: dipeptidyl boronic
acids. Bioorg. Med. Chem. Lett. 1998, 8, 333–338.
(27) Meng, L.; Mohan, R.; Kwok, B.; Sin, N.; Crews, C. M. Eponemycin
exerts its antitumor effect through the inhibition of proteasome
function. Cancer Res. 1999, 59, 2798–2801.
(28) Meng, L.; Mohan, R.; Kwok, B.; Elofsson, M.; Sin, N.; Crews, C. M.
Epoxomicin, a potent and selective proteasome inhibitor, exhibits in
vivo anti-inflammatory activity. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 10403–10408.
(14) Orlowski, R. Z.; Stinchcombe, T. E.; Mitchell, B. S.; Shea, T. C.;
Baldwin, A. S.; Stahl, S.; Adams, J.; Esseltine, D. L.; Elliott, P. J.;
Pien, C. S.; Guerciolini, R.; Anderson, J. K.; Depcik-Smith, N. D.;
Bhagat, R.; Lehman, M. J.; Novick, S. C.; O’Connor, O. A.; Soignet,
S. L. Phase I trial of the proteasome inhibitor PS-341 in patients with
refractory hematologic malignancies. J. Clin. Oncol. 2002, 20, 4420–
4427.
(15) Richardson, P. G.; Barlogie, B.; Berenson, J.; Singhal, S.; Jagannath,
S.; Irwin, D.; Rajkumar, S. V.; Srkalovic, G.; Alsina, M.; Alexanian,
R.; Siegel, D.; Orlowski, R. Z.; Kuter, D.; Limentani, S. A.; Lee, S.;
Hideshima, T.; Esseltine, D. L.; Kauffman, M.; Adams, J.; Schenkein,
D. P.; Anderson, K. C. A phase 2 study of bortezomib in relapsed,
refractory myeloma. N. Engl. J. Med. 2003, 348, 2609–2617.
(16) Richardson, P. G.; Sonneveld, P.; Schuster, M. W.; Irwin, D.;
Stadtmauer, E. A.; Facon, T.; Harousseau, J. L.; Ben-Yehuda, D.;
Lonial, S.; Goldschmidt, H.; Reece, D.; San-Miguel, J. F.; Blade´, J.;
Boccadoro, M.; Cavenagh, J.; Dalton, W. S.; Boral, A. L.; Esseltine,
D. L.; Porter, J. B.; Schenkein, D.; Anderson, K. C. Bortezomib or
high-dose dexamethasone for relapsed multiple myeloma. N. Engl.
J. Med. 2005, 352, 2487–2498.
(17) Orlowski, R. Z.; Nagler, A.; Sonneveld, P.; Blade´, J.; Hajek, R.;
Spencer, A.; San Miguel, J.; Robak, T.; Dmoszynska, A.; Horvath,
N.; Spicka, I.; Sutherland, H. J.; Suvorov, A. N.; Zhuang, S. H.; Parekh,
T.; Xiu, L.; Yuan, Z.; Rackoff, W.; Harousseau, J. L. Randomized
phase III study of pegylated liposomal doxorubicin plus bortezomib
compared with bortezomib alone in relapsed or refractory multiple
myeloma: combination therapy improves time to progression. J. Clin.
Oncol. 2007, 25, 3892–3901.
(18) O’Connor, O. A.; Wright, J.; Moskowitz, C.; Muzzy, J.; MacGregor-
Cortelli, B.; Stubblefield, M.; Straus, D.; Portlock, C.; Hamlin, P.;
Choi, E.; Dumetrescu, O.; Esseltine, D.; Trehu, E.; Adams, J.;
Schenkein, D.; Zelenetz, A. D. Phase II clinical experience with the
novel proteasome inhibitor bortezomib in patients with indolent non-
Hodgkin’s lymphoma and mantle cell lymphoma. J. Clin. Oncol. 2005,
23, 676–684.
(29) Kuhn, D. J.; Chen, Q.; Voorhees, P. M.; Strader, J. S.; Shenk, K. D.;
Sun, C. M.; Demo, S. D.; Bennett, M. K.; van Leeuwen, F. W.;
Chanan-Khan, A. A.; Orlowski, R. Z. Potent activity of carfilzomib,
a novel, irreversible inhibitor of the ubiquitin-proteasome pathway,
against preclinical models of multiple myeloma. Blood 2007, 110,
3281–3290.
(30) O’Connor, O. A.; Orlowski, R. Z.; Alsina, M.; Stewart, K.; Trudel,
S.; Vallone, M. K.; Woo, T. M.; Urquilla, P. R.; Molineaux, C. J.;
Goy, A. Multicenter phase I studies to evaluate the safety, tolerability,
and clinical response to intensive dosing with the proteasome inhibitor
PR-171 in patients with relapsed or refractory hematological malignan-
cies [abstract]. Blood (ASH Annual Meeting Abstracts) 2006, 108,
2430.
(31) Stapnes, C.; Døskeland, A. P.; Hatfield, K.; Ersvaer, E.; Ryningen,
A.; Lorens, J. B.; Gjertsen, B. T.; Bruserud, O. The proteasome
inhibitors bortezomib and PR-171 have antiproliferative and proapo-
ptotic effects on primary human acute myeloid leukaemia cells. Br. J.
Hamaetol. 2007, 136, 814–828.
(32) Orlowski, R. Z.; Stewart, K.; Vallone, M.; Molineaux, C.; Kunkel,
L.; Gericitano, J.; O’Connor, O. A. Safety and antitumor efficacy of
the proteasome inhibitor carfilzomib (PR-171) dosed for five consecu-
tive days in hematologic malignancies: Phase I results. Blood 2007,
110, 409.
(33) Alsina, M.; Trudel, S.; Vallone, M.; Molineaux, C.; Kunkel, L.; Goy,
A. Phase I single agent antitumor activity of twice weekly consecutive
day dosing of the proteasome inhibitor carfilzomib (PR-171) in
hematologic malignancies. Blood 2007, 110, 411.
(34) Demo, S. D.; Kirk, C., J.; Parlati, F.; Aujay, M. A. et al. A proteasome
active site ELISA (PASE) for rapid quantitation of proteasome activity
in vitro and in vivo. Manuscript in preparation.
(35) Harker, W. G.; MacKintosh, F. R.; Sikic, B. I. Development and
characterization of a human sarcoma cell line, MES-SA, sensitive to
multiple drugs. Cancer Res. 1983, 43, 4943–4950.
(36) Obach, R. S. Predication of human clearance of twenty-nine drugs
from hepatic microsomal intrinsic clearance data: An examination of
in vitro half-life approach and nonspecific binding to microsomes. Drug
Metab. Dispos. 1999, 27, 1350–1359.
(19) Goy, A.; Younes, A.; McLaughlin, P.; Pro, B.; Romaguera, J. E.;
Hagemeister, F.; Fayad, L.; Dang, N. H.; Samaniego, F.; Wang, M.;
Broglio, K.; Samuels, B.; Gilles, F.; Sarris, A. H.; Hart, S.; Trehu, E.;

3038 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Zhou et al.
(37) Pauletti, G. M. Peptidomimetics designed for oral absorption. AdV.
Amino Acid Mimetics Peptidomimetics 1999, 2, 89–123.
(38) Hamman, J. H.; Enslin, G. M.; Kotze´, A. F. Oral delivery of peptide
drugs. Biodrugs 2005, 19, 165–177.
(39) Vinitsky, A.; Cardozo, C.; Sepp-Lorenzino, L.; Michaud, C.; Orlowski,
M. Inhibition of the proteolytic activity of the multicatalytic proteinase
complex (proteasome) by substrate-related peptidyl aldehydes. J. Biol.
Chem. 1994, 269, 29860–29866.
(40) Shenk, K. D.; Parlati, F.; Zhou, H. J.; Sylvain, C.; Smyth, M. S.;
Bennett, M. K.; Laidig, G. J. Preparation of peptide epoxyketones as
selective immunoproteasome inhibitors. U.S. Patent 2007293465 A1
20071220, 2007.
(41) Fretland, A. J.; Omiecinski, C. J. Epoxide hydrolases: biochemistry
and molecular biology. Chem.-Biol. Interact. 2000, 129, 41–59.
(42) Laidig, G. J.; Radel, P. A.; Smyth, M. S. Synthesis of amino acid
keto-epoxide. U.S. Patent 2005256324 A1 20051117, 2005.
(43) Zhou, H. J.; Sun, C. M.; Shenk, K. D.; Laidig, G. J. Compounds for
enzyme inhibition. U.S. Patent 2007105786 A1 20070510, 2007.
(44) Kim, S.; Lee, J. I.; Kim, Y. C. A simple and mild esterification method
for carboxylic acids using mixed carboxylic-carbonic anhydrides. J.
Org. Chem. 1985, 50, 560–565.
JM801329V