Identification of a potent and rapidly reversible inhibitor of the 20S-proteasome

Article abstract of DOI:10.1016/j.bmcl.2004.06.084

Synthesis and in vitro characterization of a novel, lactam boronic acid based, selective, and rapidly reversible inhibitor of the 20S-proteasome is presented. Synthesis and in vitro characterization of novel, lactam boronic acid based, selective, and rapi

Full text of DOI:10.1016/j.bmcl.2004.06.084

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4701–4704
Identification of a potent and rapidly reversible inhibitor
of the 20S-proteasome
Ashok V. Purandare,* Honghe Wan, Naomi Laing, Khalid Benbatoul,
Wayne Vaccaro and Michael A. Poss
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
Received 20 May 2004; revised 25 June 2004; accepted 26 June 2004
Available online 19 July 2004
Abstract—Synthesis and in vitro characterization of novel, lactam boronic acid based, selective, and rapidly reversible inhibitor 14
of the 20S-proteasome is presented.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The proteasome, also referred to as the multicatalytic
proteinase complex, is an unusually high molecular
weight complex (about 700kDa, 26S) that is found in
both the cytoplasm and the nucleus of a wide variety
of eukaryotic cell types.1 The proteasome is comprised
of the 20S central catalytic core, and two 19S regulatory
caps.2 The 19S regulatory caps are found at each end of
the 20S cylinder-shaped complex, and regulate the entry
of substrates into the central catalytic core. The 19S caps
play a role in the recognition of substrates that have
been targeted for degradation by the addition of multi-
ple molecules of the 8.5kDa polypeptide ubiquitin.
The 19S caps facilitate the removal of the ubiquitin mole-
cules from the substrate, and promote the unfolding of
the substrate protein as it enters the central catalytic
core. The catalytic core (20S) has a cylindrical shape
with four stacked rings, each ring having seven subunits
(a1-7b-1-7b1-7a1-7). The active site is an N-terminal
threonine residue, which is deeply buried inside. The
closest family member of this class of enzymes is the
bacterial penicillin acylase. Early studies on the protea-
some led to the delineation of three major different pro-
teolytic activities (similar in specificity to chymotrypsin,
trypsin, and peptidylglutamyl peptidase), each associ-
ated with a distinct component of the complex.
such as cell division, antigen processing, and the degra-
dation of short-lived regulatory proteins such as onco-
proteins, transcription factors, and cyclins. Since the
proteasome plays a key role in the orderly degradation
of cyclins during the progression of the cell cycle, it
plays a role in cell division. Additional studies demon-
strated that disruption in any one of 12 out of 13 genes
encoding yeast proteasome subunits results in an arrest
in cellular proliferation, or an inability to degrade pro-
teins, also suggesting a role for the proteasome in cell
growth. Therefore, inhibition of the proteasome may
be useful in the treatment of diseases resulting from
aberrant cell division.3 Further, the clinical proof of
principal to demonstrate that proteasome inhibition
may have utility in treating cancer was provided by
the dipeptide boronic acid (1) (PS-341, bortezomib, Vel-
cadeꢁ).4 In addition, several other classes of proteasome
inhibitors have been reported.2,5 Herein we report the
identification of a potent and rapidly reversible, inhibi-
tor of the proteasome.
Focused screening of the companyÕs compound collec-
tion identified 2 as a potent, lactam based boronate pro-
teasome inhibitor for the chypotrypsin-like activity.6
It is now well established that the proteasome is a major
extralysosomal proteolytic system involved in the pro-
teolytic pathways essential for diverse cellular functions
N
O
H
N
O
N
H
B
O
N
O
Keywords: : Proteasome inhibitor; Reversible.
*
Corresponding author. Tel.: +1-609-2524320; fax: +1-609-
2527446; e-mail: ashok.purandare@bms.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.084

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A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4701–4704
The compound was a 1:1 diastereomeric mixture at the
quaternary center (indicated by * in the structure) at
the ring junction.
O
c,d
CbzHN
a, b
OH
O
CbzN
O
4
3
O
O
O
O
S
e
CbzHN
OCH3
O
N
HN
H
O
*
CbzHN
N
N
O
B(OH)2
OMe
6
O
5
2
O
f, g
HN
OCH3
SO2
N
At first, we set out to examine the stereospecificity of the
inhibition. The required lactam was synthesized as
shown in Scheme 1.
O
7
Scheme 1. Reagents and conditions: (a) paraformaldehyde, p-toluene-
sulfonic acid, toluene, 110ꢂC; (b) LiHMDS, allyl bromide, THF,
À78ꢂC; (c) MeOH; (d) O3, Me2S, MeOH, À20ꢂC; (e) L-cyclohexyl
alanine methyl ester, NaHB(OAc)3, ClCH2CH2Cl, 0–80ꢂC; (f) H2,
10%, Pd/C, MeOH; (g) m-CH3–PhSO2Cl, Et3N, DMAP (cat), CH2Cl2.
Intermediate 7 was resolved into two diastereomers (8
and 9) using reversed phase preparative HPLC.7 The
stereochemistry at the ring junction for these diastereo-
mers was assigned based on X-ray crystallographic
analysis.
intermediate step, thereby providing flexibility and
convenience.
O
Similarly, the corresponding R-valine boronate ester
was synthesized. These boronate esters were converted
into 20 and 21 using the methodology as described in
Scheme 3. As seen in Table 1, the isobutyl side chain
was preferred. The ethyl and isopropyl side chains were
of similar activity.
HN
SO2
OCH3
N
O
7
8
+
Next, we examined the effect of replacement of the sul-
fonamide and cyclohexylmethyl alanine. These analogs
O
HN
SO2
OCH3
N
O
c
O
O
a, b
9
B(O-iPr)3
B
10
11
The required enantiomerically pure, protected, boronic
acid component 13 was prepared using the modified lit-
erature procedure8 as shown in Scheme 2. In a similar
manner, the corresponding S-isomer was prepared from
(À)-pinanediol.
ClH H2N
O
O
Cl
O
O
d, e
B
B
13
12
Scheme 2. Reagents and conditions: (a) EtMgBr, THF, À78ꢂC; (b)
(+)-pinanediol; (c) CH2Cl2, LDA, ZnCl2, THF, À78ꢂC to rt; (d)
LiHMDS, THF, À78ꢂC to rt; (e) HCl, 1,4-dioxane.
8 and 9 were separately converted into the correspond-
ing (R)- and (S)-diastereomers (14 and 15; respectively)
(Scheme 3).
The diastereomer with R-stereochemistry at the ring
junction (14) (IC50 0.031lM) was 12-fold more active
than the corresponding S-isomer 15 (IC50 0.4lM),
clearly indicating the preferential binding of one of the
diastereomers.
a, b, c
O
O
S
8 and 9
O
H
HN
N
N
B(OH)2
We further embarked on exploration of the boronate
side chain. Since the methodology (shown in Scheme
2) was not suitable to explore substituted boronic acids,
we investigated an alternative modular approach as
shown in Scheme 4. In this modified approach, as dem-
onstrated for R-leucine boronate ester 19, the required
O
14 - R- isomer
15 - S- isomer
Scheme 3. Reagents and conditions: (a) LiOH, DME–H2O; (b) 8 or 9,
PyOP, CH2Cl2; (c) NaIO4, acetone–0.1M aq NaOAc.
substitution could be introduced at
a
common

A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4701–4704
4703
Table 3.
c
Cl
Cl
O
a, b
B
B(OMe)3
Shift in IC50 upon dilution (fold)
O
16
Compd #
20·
40·
17
d, e
14
21
16
2
35
5
ClH H2N
O
O
Cl
B
O
O
B
18
19
pounds (14 and 21) were analyzed for reversibility
using the following dilution method. Briefly, enzyme
was pre-incubated with a wide range of inhibitor con-
centrations for 1h, and then processed three different
ways: (1) Dilution of the enzyme–inhibitor mixture 20-
fold, and immediate addition of substrate, to follow
enzyme activity for 1h. (2) Dilution of the enzyme–
inhibitor mixture 40-fold prior to the addition of sub-
strate, to follow activity. (3) Confirmation of IC50 by
addition of small aliquots of substrate directly to the
enzyme–inhibitor mixture, to follow enzyme activity.
A rapidly reversible inhibitor can be detected with this
technique since the IC50 will shift approximately 20- to
40-fold after dilution due to the reduced free inhibitor
concentration. Alternatively, an irreversible inhibitor
would be expected to show little shift in the IC50 values
after dilution.
Scheme 4. Reagents and conditions: (a) CH2Cl2, n-BuLi, THF,
À100ꢂC; (b) (+)-pinanediol; (c) i-BuMgBr, ZnCl2, THF, À78ꢂC; (d)
LiHMDS, THF, À78ꢂC to rt; (e) HCl, 1,4-dioxane.
Table 1.
O
O
S
O
H
HN
N
N
B(OH)2
O
R
Compd #
R
Proteasome IC50 lM
14
20
21
–CH2CH3
–CH(CH3)2
–CH2–CH(CH3)2
0.031 ( 0.003)
0.028 ( 0.002)
0.008 ( 0.001)
The results from these experiments indicated that there
was a significant difference in the reversibility of com-
pounds 14 and 21 (Table 3).
aThe average of two independent IC50 determinations is reported, with
the range between the two values in parantheses.
The results for the dilution experiment with compound
14 are consistent with rapidly reversible inhibition, since
the loss in inhibitor potency was similar to the extent of
dilution of the enzyme–inhibitor mixture (i.e., 16-fold
loss in potency after a 20-fold dilution). In contrast,
the effects of dilution on the inhibitory potency of com-
pound 21 were significantly reduced. A 20-fold dilution
of enzyme–inhibitor produced only a 2-fold reduction in
potency, whereas a 40-fold dilution reduced proteasom-
al inhibition just 5-fold. These results suggested that
compound 21 was not a rapidly reversible inhibitor. Fol-
low-up experiments with compound 21 indicated that
there was a slow off-rate for dissociation of the com-
pound from the proteasome (>5h; data not shown).
These results indicate that varying the R group, which
is in the alpha position with respect to the boronate moi-
ety, can alter not only the potency of inhibition of the
proteasome, but also the reversible nature of the inhibi-
tion. Alteration of both the potency and reversibility of
a proteasome inhibitor, as described in this report,
would be predicted to significantly alter the therapeutic
properties of these compounds.
were synthesized from intermediate 5 using a sequence
analogous to Schemes 1 and 3.
SARfrom this exercise indicated that benzyloxy carbo-
nyl is tolerated in place of m-tolylsulfonamide at R1.
Interestingly, replacement with pyrazine amide, the side
chain from PS-341, resulted in a loss of activity (23).
Compound 24 having a phenyl group at R2 in place of
cyclohexyl was active. Replacement of cyclohexyl, with
a smaller group such as methyl (25), resulted in a loss
of activity (Table 2).
In order to further characterize the proteasomal inhibi-
tion by these boronates, two of the most potent com-
Table 2.
O
R1
HN
R2
O
H
N
N
B(OH)2
Compd R1
#
R2
Proteasome
IC50 lM
Compound 14 also displayed >100-fold selectivity
against chymotrypsin, trypsin, elastase, and several
coagulation factors (Xa, XIa, VIIa).
14
22
23
24
25
–SO2–(m-Me)Ph Cyclohexylmethyl 0.031 ( 0.003)
–CO–O–CH2Ph Cyclohexylmethyl 0.032 ( 0.0019)
–CO-pyrazine
Cyclohexylmethyl >10
In summary we have identified a potent, selective, and
rapidly reversible, lactam boronic acid based inhibitor
of the 20S-proteasome. Further characterization of this
inhibitor and its cellular effects will be reported
separately.9
–CO–O–CH2Ph –CH2–Ph
–CO–O–CH2Ph –Me
0.031 ( 0.004)
1.00 ( 0.17)
aThe average of two independent IC50 determinations is reported, with
the range between the two values in parantheses.

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A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4701–4704
proteasome enriched fraction was assayed at 50lg/mL
Acknowledgements
concentration in a buffer containing 150mM Tris–EDTA
(pH7.4). The pure 20S proteasomes were assayed at 3lg/
mL concentration in a buffer containing 25mM hepes,
0.5mM EDTA, 0.03% sodium dodecyl sulfate (pH7.6). The
activities of both preparations were monitored over time by
measuring the increase in fluorescent signal (excitation at
360nm; emission at 460nm) in a Cytofluor Series 4000
multiwell platereader (Applied Biosystems). Compounds
were evaluated for proteasome inhibition under steady
state conditions where substrate concentration is approx-
imately equal to the Km, and the amount of substrate
hydrolyzed during the reaction is less than 10% of the
total substrate in the reaction. Appropriately diluted
proteasome preparations were incubated with the inhibitor
for 30min at 37ꢂC prior to the initiation of the reaction
by the addition of substrate. The substrate concentration
for each assay was at, or below, the apparent Km
levels determined for each preparation (pure 20S protea-
some, 10lM; proteasome-enriched fraction, 50lM). The
reactions proceeded for 60min at 37ꢂC before being read
in the fluorescent platereader. Inhibition was quantitated
by determination of the IC50 values for the
given conditions. Two independent IC50 determinations is
done. Values are reported as IC50 with range between
the two values in paranthesis. There was less than 25%
difference between the two IC50 determinations for all
cases.
We would like to thank Dr. Charles Kettner and Ms.
Indavati DeLuca for their suggestions and help.
References and notes
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6. The proteolytic activity of the proteasome was determined
by utilizing synthetic peptide substrates specific for the
chymotrypsin-like activity of the proteasome. The activity
of the proteasome toward the cleavage of succinyl-Leu-
Leu-Val-Tyr-(7-amino-4-methyl coumarin) was measured
by the increased fluorescence of the coumarin moiety after
cleavage from the peptide. The sources of proteasomes were
the proteasome enriched fraction from HL60 cells described
above, and pure commercially available 20S human pro-
teasomes (Affiniti Research Products, Mamhead, UK). The
7. The diastereomers were separated using a chiral preparative
column (chiral OJ column, 50mm·500mm, flow rate
50mL/min), eluting with ethanol/heptane (5/95).
8. Dembitsky, V. M.; Srebnik, M. Tetrahedron 2003, 59,
579–593, and references cited therein.
9. Laing, N.; Purandare, A. V., to be communicated.