A new structural class of selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome

Article abstract of DOI:10.1016/S0960-894X(01)00205-0

We describe the identification and in vitro characterization of a series of 2-aminobenzylstatine derivatives that inhibit non-covalently the chymotrypsin-like activity of the 20S proteasome. Our initial SAR data demonstrate that the 2-aminobenzylstatine core structure can effectively serve as the basis for designing potent, selective and non-covalent inhibitors of the chymotrypsin-like activity of the 20S proteasome.

Full text of DOI:10.1016/S0960-894X(01)00205-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1317–1319
A New Structural Class of Selective and Non-covalent Inhibitors
of the Chymotrypsin-like Activity of the 20S Proteasome
C. Garcıa-Echeverrıa,a,* P. Imbach,a,* D. France,a P. Furst,a M. Lang,a
M. Noorani,a D. Scholz,b J. Zimmermanna and P. Fureta,*
aOncology Research, Novartis Pharma Inc., CH-4002 Basel, Switzerland and Summit, NJ-07901, USA
bNovartis Research Institute, A-1235 Vienna, Austria
Received 22 December 2000; accepted 19 March 2001
Abstract—We describe the identification and in vitro characterization of a series of 2-aminobenzylstatine derivatives that inhibit
non-covalently the chymotrypsin-like activity of the 20S proteasome. Our initial SAR data demonstrate that the 2-aminobenzyl-
statine core structure can effectively serve as the basis for designing potent, selective and non-covalent inhibitors of the chymo-
trypsin-like activity of the 20S proteasome. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
that inhibit non-covalently the chymotrypsin-like activity
of the 20S proteasome. This new structural class shows
good selectivity over the trypsin-like and post-glutamyl-
peptide hydrolytic activities of the 20S proteasome.
The 20S proteasome, which is the catalytic core of the
26S proteasome,1 is a threonine protease that exhibits at
least three distinct peptidase activities: chymotrypsin-
like, trypsin-like, and post-glutamyl-peptide hydrolytic
activities.2 Because of the role of this enzyme in the non-
lysosomal ATP-dependent degradation of proteins
involved in critical intracellular regulatory cascades,3
inhibitors of the 20S proteasome are being explored for
use as potential anti-inflammatory agents and for the
treatment of cancer and auto-immune diseases.4 Our
specific target in the search of novel cytotoxic and anti-
proliferative agents is the chymotrypsin-like activity of
the 20S proteasome. Modulation of this enzymatic
activity by b-subunit-specific proteasome inhibitors may
convey an anti-tumor effect by induction of cell cycle
arrest and apoptosis in tumor cells.4c,d,5 Early inhibitors
of the chymotrypsin-like activity of the 20S proteasome
exert their inhibitory activity via adduct formation with
the N-terminal threonine on the active site of the b-
subunits.6 The inherent drawbacks of these classical
protease inhibitors (e.g., non-target specific, too reactive
or unstable) prompted us to the search for alternative
subunit-specific proteasome inhibitors.
Synthesis and Biological Assays
The synthesis of compounds 1–4 has been reported
previously7 and compounds 6–12 were prepared
according to known methods.7ꢀ9 The general route for
the syntheses of 2-heterosubstituted derivatives of 4-
amino-3-hydroxy-5-phenylpentanoic acid is illustrated
for compound 12 in Scheme 1. Compound 6, synthe-
sized according to the general route (Scheme 1), was the
starting point for the synthesis of compounds 7–12.
After removal of the tert-butoxycarbonyl protecting
group, the amino terminus was acylated with various
carbonic acid esters or carboxylic acids. The ability of
these compounds to inhibit the 20S proteasome was
determined in vitro using purified human placenta 20S
proteasome10 and the following fluorogenic peptides as
substrates: Suc-Leu-Leu-Val-Tyr-AMC (substrate for
chymotrypsin-like assay), Boc-Leu-Arg-Arg-AMC
(substrate for trypsin-like assay) and Z-Leu-Leu-Glu-
AMC (substrate for post-glutamyl-peptide hydrolytic-
like assay). Fluorescence excitation/emission wave-
lengths were 355 nm/460 nm for 7-amido-4-methyl-
coumarin (AMC). The rates of hydrolysis were mon-
itored by the fluorescence increase and the initial linear
portions of curves were used to calculate the IC50 values
(Tables 1 and 2).
We describe herein the identification and in vitro char-
acterization of a series of 2-aminobenzylstatine derivatives
*Corresponding authors. Fax: +41-61-696-6246; e-mail: carlos.garcia-
echeverria@pharma.novartis.com; patricia.imbach@pharma.novartis.
com; pascal.furet@pharma.novartis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00205-0

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C. Garcıa-Echeverrıa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1317–1319
1318
Results and Discussion
unchanged. To this end, a series of derivatives were
prepared to examine the effects of N-terminal substitu-
tion on potency.14 Representative examples of the com-
pounds analyzed are shown in Table 2. A 27-fold
decrease in potency was observed when the benzyloxy-
carbonyl group was replaced by the less bulky tert-
butyloxycarbonyl group (6, Table 1). The possibility to
increase potency by establishing additional hydrophobic
or hydrogen-bonding interactions was explored by
increasing the size of the N-terminal group (7 and 12),
adding substituents to the aromatic ring (8 and 11) or
by incorporating hydrogen-bond donors (9 and 10).
Modifications on the N-terminal phenyl ring did not
affect substantially the inhibitory activities of the
To search for novel non-covalent structures inhibiting
specifically the chymotrypsin-like activity of the 20S
proteasome, we screened our in-house compound
archive. Among the compounds identified, compound 1
(Table 1) was selected for further profiling.11 This com-
pound, which was originally synthesised to target the
HIV-1 proteinase,7,12 inhibits the chymotrypsin-like
activity of the 20S proteasome with an IC50 value of
0.9 mM (compound 1, Table 1) and shows good selec-
tivity over the trypsin-like and post-glutamyl-peptide
hydrolytic activities (IC50>20 mM). Encouraged by
these results, a series of available 2-aminobenzylstatine
derivatives were tested in our in vitro assay to try to
identify key interactions of this new structural class of
inhibitors with the X and HC5 subunits of the 20S pro-
teasome.13 The SAR data obtained is shown in Table 1.
Replacement of tert-leucine by the less bulky valine (2)
resulted in a 3-fold decrease in binding affinity. A more
dramatic effect on potency was observed by introducing
variations at the C- and N-terminal groups. Methyla-
tion of the phenol group (3) or removal of the methoxy
group (4) in the C-terminal benzylamide ring resulted in
a substantial drop in binding activity (>20-fold). A
similar drop in activity was observed for a derivative
that lacks the bulky N-terminal benzyloxycarbonyl
group (5). On the basis of this limited SAR data, we
decided to focus our initial medicinal chemistry efforts
on the N-terminal part of the 2-aminobenzylstatine
template. In this modular approach, the N-terminal part
of the molecule was modified while the other remained
Table 1. Inhibition of the chymotrypsin-like activity of the 20S pro-
teasome by 2-aminobenzylstatine derivativesa
Compound
R1
R2
R3
R4
IC50 (mM)
1
2
3
4
5
CO2CH2Ph C(CH3)3
CO2CH2Ph CH(CH3)2
CO2CH2Ph CH(CH3)2 OCH3 OCH3
OH
OH
OCH3
OCH3
0.9
3.3
>20
72
CO2CH2Ph
H
C(CH3)3
C(CH3)3
OH
OH
H
OCH3
70
aThe IC50 value is the concentration of inhibitor at which the rate of
the chymotrypsin-like activity of the 20S proteasome catalyzed
hydrolysis of the substrate Suc-Leu-Leu-Val-Tyr-AMC is reduced at
50%.
Table 2. Inhibition of the chymotrypsin-like activity of the 20S pro-
teasome by 2-aminobenzylstatine derivativesa
Compound
R
IC50 (mM)
1
6
7
8
CO2CH2Ph
CO2C(CH3)3
0.9
27.0
2.0
1.1
0.6
0.6
0.5
0.1
CO2CH2(1-Naphthalene)
CO2CH2Ph[3,5-(CH3)2]
CO2CH2Ph(3-NH2)
C(O)CH2CH2Ph(3-NH2)
CO2CH2Ph(3-CH3)
C(O)CH2(1-Naphthalene)
9
10
11
12
Scheme 1. Synthesis of compound 12. Conditions: (a) (COCl)2,
DMSO, CH2Cl2, Et3N, ꢀ55 ꢁC ! rt; (b) Ph3P¼CHCO2Et, toluene,
80 ꢁꢁC; (c) MCPBA, CH2Cl2, rt; (d) 4-Methoxybenzylamine, EtOH,
aThe IC50 value is the concentration of inhibitor at which the rate of
the chymotrypsin-like activity of the 20S proteasome catalyzed
hydrolysis of the substrate Suc-Leu-Leu-Val-Tyr-AMC is reduced at
50%.
.
70 C; (e) 1 N LiOH, THF, rt; (f) HCl (S)-Val-(2-hydroxy-4-methoxy)-
benzylamine, TPTU, DIEA, DMF, rt; (g) 4 N HCl in dioxane, rt; (h)
Na-Boc-l-Tleu-OH, TPTU, DIEA, DMA, rt; (i) CF3CO2H, water, rt;
(j) 1-naphthylacetic acid, TPTU, DIEA, DMA, rt.

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C. Garcıa-Echeverrıa et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1317–1319
´
1319
compounds and only a major effect on potency was
observed for the inhibitors containing bulkier groups.
While the compound containing the carbamic acid
New Drugs 2000, 18, 109. (d) Murray, R. Z.; Norbury, C.
Anti-Cancer Drugs 2000, 11, 407. (e) Dou, Q. P.; Li, B. Drug
Resist. Updates 1999, 2, 215. (f) Elliott, P. J.; Adams, J. Curr.
Opin. Drug. Disc. Dev. 1999, 2, 484. (g) Kalogeris, T.; Gray,
L.; Laroux, F. S.; Cockrell, A.; Fuseler, J.; Conner, E. M.;
Brand, S.; Grisham, M. B. Expert Opin. Invest. Drugs 1999, 8,
1397. (h) Lee, D. H.; Goldberg, A. L. Trends Cell Biol. 1998,
8, 397.
5. A dipeptide boronic acid analogue (PS-341) is currently
under clinical evaluation in advanced cancer patients. For
additional data on this compound, see: Adams, J.; Palombella,
V. J.; Sausville, E. A.; Johnson, J.; Destree, A.; Lazarus, D. D.;
Maas, J.; Pien, C. S.; Prakash, S.; Elliott, P. J. Cancer Res.
1999, 59, 2615.
naphthalen-1-ylmethyl ester (7) showed
a 2-fold
decrease in binding affinity relative to our reference
compound (1), the 1-napthyl acetic acid synthon (12)
increased 9-fold the potency of our initial compound.
Compound 12 is not only a potent inhibitor of the chy-
motrypsin-like activity of the 20S proteasome but also
very selective for this catalytic site. This compound
shows at least 200-fold selectivity over the trypsin-like
and post-glutamyl-peptide hydrolytic activities.
6. To date, several classes of 20S proteasome inhibitors have
been described: peptide aldehydes, peptide a-keto aldehydes
(glyoxals), peptide a0,b0-epoxyketones, peptide vinyl sulfones,
peptide indanones, peptide boronic acids (e.g., PS-341, see ref
5), bifunctional inhibitors or natural products (e.g., lactacys-
tin, epoxomicin, TMC-95A-D or UCK 14A2); see also ref 4.
7. Billich, A.; Scholz, D.; Charpiot, B.; Gstach, H.; Lehr, P.;
Peichi, P.; Rosenwirth, B. Antiviral Chem. Chemother. 1995, 6,
327.
In summary, our initial SAR data demonstrate that the
2-aminobenzylstatine core structure can effectively serve
as the basis for designing potent, selective and non-
covalent inhibitors of the chymotrypsin-like activity of
the 20S proteasome. Further modular modifications of
this series hold the promise for future inhibitors with
increased in vitro activity.
8. Lehr, P.; Billich, A.; Charpiot, B.; Ettmayer, P.; Scholz, D.;
Rosenwirth, B.; Gstach, H. J. Med. Chem. 1996, 39, 2060.
9. Scholz, D.; Billich, A.; Charpiot, B.; Ettmayer, P.; Lehr, P.;
Rosenwirth, B.; Schreiner, E.; Gstach, H. J. Med. Chem. 1994,
37, 3079.
Acknowledgements
The authors thank D. Arz, R. Wille, V. von Arx, W.
Beck and E. Boss for technical assistance.
10. The human placenta 20S proteasome was obtained from
Diabetes Forschungsinstitut, Dusseldorf, Germany.
11. Compound 1 was identified during the high-throughput
screening of our entire in-house compound collection.
12. Other inhibitors of the HIV-1 protease have been descri-
bed as inhibitors of the 20S proteasome: (a) Andre, P.; Lot-
teau, V.; Zinkernagel, R.; Klenerman, P.; Groettrup, M. WO
9963998, 1999. (b) Andre, P.; Groettrup, M.; Klenerman, P.;
de Giuli, R.; Booth, B. L., Jr.; Cerundolo, V.; Bonneville, M.;
Jotereau, F.; Zinkernagel, R. M.; Lotteau, V. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 13120.
13. The active site of the chymotrypsin-type activity of the
human 20S proteasome is formed by the association of these
two b-subunits.
14. The N-terminal building blocks were determined by
molecular modeling on the basis of our binding model for this
structural class of compounds (Furet, P.; Imbach, P.; Furst,
P.; Lang, M.; Noorani, M.; Zimmermann, J.; Garcıa-
Echeverrıa, C. Bioorg. Med. Chem. Lett. 2001, 11, 1321).
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