α-Ketoamides, α-ketoesters and α-diketones as HCV NS3 protease inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00074-3

Peptide-based α-ketoamides, α-ketoesters and α-diketones were designed, synthesized and evaluated against HCV NS3 protease. α-Ketoamides have the highest affinity among the three classes, with 8 being the most potent inhibitor with an IC₅₀ of 3

Full text of DOI:10.1016/S0960-894X(00)00074-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 711±713
ꢀ-Ketoamides, ꢀ-Ketoesters and ꢀ-Diketones as HCV NS3
Protease Inhibitors
Wei Han,* Zilun Hu, Xiangjun Jiang and Carl P. Decicco
Department of Chemical and Physical Sciences, DuPont Pharmaceuticals Company, Experimental Station, PO Box 80500,
Wilmington, DE 19880, USA
Received 14 December 1999; accepted 24 January 2000
AbstractÐPeptide-based a-ketoamides, a-ketoesters and a-diketones were designed, synthesized and evaluated against HCV NS3
protease. a-Ketoamides have the highest anity among the three classes, with 8 being the most potent inhibitor with an IC50 of 340
nM. # 2000 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.
The hepatitis C virus (HCV), ®rst identi®ed in 1989,1 is
the principal etiologic agent of both parenterally trans-
mitted and sporadic non-A non-B hepatitis.2 More than
170 million people worldwide are infected with HCV
according to the World Health Organization. Current
therapies for HCV infection include treatment with
interferon-a and its combination with ribavirin.3,4 These
therapies have limited ecacy and are accompanied by
frequent side e€ects.3,4 Hence, a new treatment of the
disease is of great interest. One of the most intensively
studied and best understood targets for HCV antiviral
therapy is the serine protease of the NS3 protein.4,5
In this communication, we wish to report a class of
peptide-based 1,2-dicarbonyl derivatives as HCV NS3
protease inhibitors.
P1 mercaptomethyl group in 1 with the less reactive allyl
and ethyl groups in our new targets.8,9
Hexapeptide 1 was synthesized early on in our organi-
zation and found to inhibit HCV NS3 protease with an
IC50 of 2.5 mM.6 In an attempt to enhance the inhibitory
potency, we chose to investigate the e€ect of serine
traps. 1,2-Dicarbonyl derivatives, such as a-ketoamides,
a-ketoesters, and a-diketones, are known serine traps
and have been used as inhibitors of related serine
proteases.7 Therefore, we designed target 2 by incor-
porating a-ketoamides, a-ketoesters, and a-diketones
into the peptide backbone of 1. Based on the observa-
tion that HCV NS3 protease has a shallow and hydro-
phobic S1 pocket in the crystal structure5 and on
preliminary data showing the e€ectiveness of the allyl
and ethyl P1 groups in a related series,6 we replaced the
Scheme 1 outlines the synthesis of a-ketoamide 8. dl-
Allylglycine 3 was converted to aldehyde 4 via a Wen-
reib amide intermediate. Cyanohydrin formation,
hydrolysis and Boc protection provided a-hydroxy-
carboxylic acid 5. Treatment of 5 with allylamine
yielded the corresponding a-hydroxyamide 6. After
removal of Boc group, the resulting amine was reacted
with pentapeptide 910 to give a-hydroxyamide 7. Finally,
*Corresponding author. Tel.: +1-302-695-1984; fax: +1-302-695-
1173; e-mail: wei.han@dupontpharma.com
0960-894X/00/$ - see front matter
# 2000 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00074-3

712
W. Han et al. / Bioorg. Med. Chem. Lett. 10 (2000) 711±713
Dess±Martin oxidation11 followed by acidic deprotec-
tion of the t-butyl groups completed the synthesis of a-
ketoamide 8. The a-ketoester 20 was synthesized fol-
lowing a similar sequence.
and 83% ee.12 The enantiomeric excess of 11 was
improved to 95% by a single recrystalization from
EtOAc/hexane. Reductive cleavage of the Cbz group
followed by coupling with pentapeptide 9 provided the
a-hydroxyester 12. Ester 12 was then saponi®ed with
LiOH and coupled with allylamine to give the a-hydro-
xyamide 13. Dess±Martin oxidation and deprotection
gave the ®nal product 14.
The synthesis of ketoamides with an ethyl P1 group is
outlined in Scheme 2. Starting from methyl 2-pent-
enoate (10) Sharpless asymmetric aminohydroxylation
catalyzed with K2OsO2(OH)4 and (DHQ)2PHAL pro-
vided a-hydroxy-b-amino compound 11 in 78% yield
The synthesis of the a-diketone 19 is described in
Scheme 3. Key transformations include intermolecular
pinacol coupling under McMurry conditions13 to intro-
duce diol 17 and Dess±Martin oxidation to give the
diketone moiety in 19.
The HCV protease (NS3) inhibitory data is summarized
in Table 1.14 The a-ketoamides prepared in this study are
active inhibitors, with the best compound (8) having an
IC50 of 0.34 mM. Ketoamides of allylamine and ethyl-
amine gave comparable potency (entries 1 and 2). As
predicted from modeling, the analogue with an ethyl
group in P1 is also active. The IC50's for ethyl and allyl
P1 analogues are comparable (entries 1 and 3). However,
since 8 is a 1:1 mixture of P1 Ca isomers and only the l
isomer is expected to be potent, the allyl P1 may be
slightly better than the ethyl group. Replacing the a-
ketoamide with an a-ketoester or a-diketone reduced the
potency by an order of magnitude (entries 4±6). Blocking
of the N terminus and the side chains of Asp and Glu
with t-butyl groups also slightly reduced the potency in
the a-ketoamide series (entries 7 and 8). Interestingly, this
variation resulted in total loss of the inhibitory activity in
a-ketoester and a-diketone series (entries 9 and 10).
In summary, a series of a-ketoamides, a-ketoesters and
a-diketones were designed, synthesized, and found to be
HCV NS3 protease inhibitors. Among them, a-keto-
amides provided the most potent HCV NS3 protease
inhibitors.
Scheme 1. (a) (Boc)2O, NaOH, THF/H2O (95%); (b) MeONHMe
HCl, BOP, TEA, CH2Cl2 (90%); (c) LAH, THF (91%); (d)
Me2C(OH)CN, TEA, CH2Cl2 (81%); (e) HCl, H2O/dioxane; (f)
(Boc)2O, Na2CO3, H2O (73% for two steps); (g) allylamine, BOP,
DIEA, DMF (82%); (h) 4 N HCl in dioxane; (i) Boc-Asp(t-Bu)-Glu(t-
Bu)-Val-Val-Pro-OH (9), BOP, DIEA, DMF (70% for two steps); (j)
Dess±Martin oxidation (70%); (k) TFA, CH2Cl2 (95%).
Scheme 2. (a) (DHQ)2PHAL, K2[OsO2(OH)4], CbzNClNa, H2O/i-
PrOH (78%); (b) H2, Pd/C, MeOH (95%); (c) 9, BOP, DIEA, DMF
(82%); (d) LiOH, THF/H2O (95%); (e) allylamine, BOP, DIEA,
DMF (83%); (f) Dess±Martin oxidation (71%); (g) TFA, CH2Cl2
(95%).
Scheme 3. (a) VCl3(THF)3, Zn, CH2Cl2 (59%); (b) 4 N HCl in diox-
ane; (c) 9, BOP, DIEA, DMF (67% for two steps); (d) Dess±Martin
oxidation (47%); (e) TFA, CH2Cl2 (90%).

W. Han et al. / Bioorg. Med. Chem. Lett. 10 (2000) 711±713
713
Table 1.
Entry
1
throughout the course of the study and for reading this
manuscript.
Structure
IC50 (mM)
0.34
References and Notes
1. Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Brad-
ley, D. W.; Houghton, M. Science 1989, 244, 359.
2. Houghton, M. In Fields Virology, 3rd ed.; Raven: New
York, 1996; pp 1035±1058.
2
0.35
3. (a) Hoofnagle, J. H.; Di Bisceglie, A. M. N. Engl. J. Med.
1997, 336, 347. (b) Reichard, O.; Schvarcz, R.; Weiland, O.
Therapy of Hepatitis C: Alpha Interferon and Ribavirin; 1997;
Hepatol 26 (Suppl 1) 108S.
4. (a) Bartenschlager, R. Antiviral Chem. Chemother. 1997, 8,
281. (b) Hijikata, M.; Mizushima, H.; Tanji, Y.; Komoda, Y.;
Hirowatari, Y.; Akagi, T.; Kato, N.; Kimura, K.; Shimo-
tohno, K. Proc. Natl. Acad. USA 1993, 90, 10773. (c) Grakoui,
A.; McCourt, D. W.; Wychowski, C.; Feinstone, S. M.; Rice,
C. M. Proc. Natl. Acad. USA 1993, 90, 10583
3
4
0.42
4.34
5. For X-ray structure of NS3 protease, see: (a) Kim, J. L;
Morgenstern, K. A.; Lin, C.; Fox, T.; Dwyer, M. D.; Landro,
J. A.; Chambers, S. P.; Markland, W.; Lepre, C. A.; O'Malley,
E. T.; Harbeson, S. L.; Rice, C. M.; Murcko, M. A.; Caron, P.
R.; Thomson, J. A. Cell 1996, 87, 343. (c) Love, R. A.; Parge,
H. E.; Wickersham, J. A.; Hostomsky, Z.; Habuka, N.; Moo-
maw, E. W.; Adachi, T.; Hostomska, Z. Cell 1996, 87, 331.
6. Unpublished data from Dr. Charles A. Kettner at DuPont
Pharmaceuticals Company.
7. Edwards, P. D.; Bernstein, P. R. Med. Res. Rev. 1994, 14, 127.
8. While our investigation was in progress, researchers at
Boehringer Ingelheim reported one example of a-ketoamide as
HCV protease inhibitor (Ac-DDIVP-NVA-CONHBn, IC50=
2.0 mM). Llinas-Brunet, M.; Bailey, M.; Deziel, R.; Fazal, G.;
Gorys, V.; Goulet, S.; Halmos, T.; Maurice, R.; Poirier, M.;
Poupart, M.; Rancourt, J.; Thibeault, D.; Wernic. D.;
Lamarre. D. Bioorg. Med. Chem. Lett. 1998, 8, 2719.
9. For literature reports on HCV protease inhibitors, see: (a)
Llinas-Brunet, M.; Bailey, M.; Fazal, G.; Goulet, S.; Halmos,
T.; Laplante, S.; Maurice, R.; Poirier, M.; Poupart, M.; Thi-
beault, D.; Wernic. D.; Lamarre. D. Bioorg. Med. Chem. Lett.
1998, 8, 1713. (b) Ingallinella, P.; Altamura, S.; Bianchi, E.;
Taliani, M.; Ingenito, R.; Cortese, R.; Francesco, R.; Stein-
kuhler, C.; Pessi, A. Biochemistry 1998, 37, 8906. (c) Chu, M.;
Mierzwa, R.; He, L.; King, A.; Patel, M.; Pichardo, J.; Hart,
A.; Butkiewicz, N.; Puar, M. S. Bioorg. Med. Chem. Lett.
1999, 9, 1949. (d) Marchetti, A.; Ontoria, J. M.; Metassa, V.
G. Synlett 1999, 1000.
5
6
7
4.23
4.80
0.57
8
9
0.84
>3 0
10
>3 0
10. The pentapeptide 9 was synthesized using a peptide syn-
thesizer under standard conditions.
11. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
12. (a) Tao, B.; Schlinglo€, G.; Sharpless, K. B. Tetrahedron
Lett. 1998, 39, 2507. (b) Li, G.; Bang, H.-T.; Sharpless, K. B.
Angew. Chem., Int. Ed. Engl. 1996, 35, 451.
13. Konradi, A. W.; Kemp, S. J.; Pedersen, S. F. J. Am. Chem.
Soc. 1994, 116, 1316.
14. The inhibition of HCV protease activity was determined
using a modi®cation of the reported method (Taliani et al.
Anal. Biochem. 1996, 240, 60). The assay measures the increase
in ¯uoresence on cleavage by the catalytic domain of type 1A
HCV protease of a self quenching depsi-peptide substrate.
Compounds were serially diluted in bu€er, then preincubated
with 4 nM enzyme for 30 min at room temperature in white
96 well plates. After addition of substrate, ¯uoresence was
measured at 5 min intervals for 40 min. (Ex=360 nm/Em=
530 nm).
Acknowledgements
We would like to thank Dr. Bruce D. Korant and Dr.
Marina G. Bukhtiyarova for providing HCV protease,
Dr. James L. Meek and Ms. Lorraine Gorey-Feret for
determination of IC50 values, and Dr. Nilsa Graciani
for providing pentapeptide 9. We are also grateful to
Dr. Charles A. Kettner for the helpful discussions