Potent aza-peptide derived inhibitors of HCV NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2009.06.060

Chronic hepatitis C infection is the primary cause for cirrhosis of the liver and hepatocellular carcinoma leading to liver failure and transplantation. The etiological agent hepatitis C virus produces a single positive strand RNA that is processed furthe

Full text of DOI:10.1016/j.bmcl.2009.06.060

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4760–4763
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent aza-peptide derived inhibitors of HCV NS3 protease
*
Srikanth Venkatraman , Wanli Wu, Neng-Yang Shih, F. George Njoroge
Schering Plough Research Institute, K15-MS 3545, 2015, Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Chronic hepatitis C infection is the primary cause for cirrhosis of the liver and hepatocellular carcinoma
leading to liver failure and transplantation. The etiological agent hepatitis C virus produces a single posi-
tive strand RNA that is processed further with the help of NS3 serine protease to produce mature virus.
Inhibition of this protease can potentially be used to develop drugs for HCV infections. Boceprevir is a
ketoamide derived novel inhibitor of HCV NS3 protease that has been progressed to clinical trials and
proven to be efficacious in humans. Herein, we report our efforts in identifying an aza-peptide derivative
as a potential second generation compound, that lacks electrophilic ketoamide group and are potent in
enzyme and replicon assay.
Received 9 April 2009
Revised 8 June 2009
Accepted 15 June 2009
Available online 17 June 2009
Keywords:
Hydrazides
HCV
Ó 2009 Published by Elsevier Ltd.
NS3 Protease
Hepatitis C virus is the primary etiological agent responsible for
non-A, non B infections of the liver.1 The prognosis for patients in-
fected with hepatitis C is poor with a majority of these infections
turning chronic progressing to cirrhosis of the liver and hepatocel-
lular carcinoma. Pegylated alpha-interferon in combination with
ribivirin is the primary standard of care which is effective in
ꢀ50% of genotype-1 infected patients.2 Patients infected with
genotype-2 respond better to Pegylated alpha-interferon with
>80% of patients demonstrating sustained reduction in viral RNA.2
Lack of effective methods to treat genotype-1 infections and pa-
tients relapsing from interferon therapy necessitate discovery of
new drugs. Significant efforts are now directed towards develop-
ment of therapies that target key enzymes vital for HCV replication
and maturation.3
Hepatitis C virus is a positive strand virus that encodes a single
polyprotein of ꢀ3000 amino acids. This polyprotein which contains
all the structural and functional proteins is post translationally
modified by HCV NS3 protease.4 This enzyme with the assistance
of cofactor NS4B catalyzes the cleavage of NS2–NS3, NS3–NS4A,
NS4A–NS5B junctions to form functional proteins. HCV NS3, a ser-
ine protease has a shallow active site on the surface of the enzyme
catalyzes the cleavage of cysteine–serine or a cysteine–threonine
bond. Inhibition of this vital enzyme has been extensively investi-
gated in an effort to develop potential drugs for the treatment of
HCV infections. Boceprevir 15 and VX-950 are two ketoamide de-
rived inhibitors that have been advanced to Phase-III clinical trails
and demonstrated excellent efficacy in humans. In addition, mac-
rocyclic inhibitors ITMN-191, MK7006 and TMC435350 have also
been progressed to clinical studies and have demonstrated excel-
lent viral load reduction in patients. These compounds are cur-
rently being evaluated at various phases of clinical development.
Boceprevir 1 (Fig. 1), is a ketoamide containing inhibitor that
readily epimerizes in human serum under physiological conditions
at the P1 center. The (S)-diastereomer at this center is the more po-
tent isomer whereas the (R)-isomer is less active. Several attempts
to avoid this epimerization by introduction of quaternary amino
acids have resulted in significant loss in potency. A recent publica-
tion from a group at Boehringer Ingelhiem demonstrated that no-
vel hydrazide derived inhibitor
2
spanning from P6 to P10
demonstrated excellent enzyme binding6 (Fig. 2). However these
compounds were peptidic and no cellular activity in the replicon
based assay was reported.
From our experience in developing ketoamide derived clinical
candidate 1, we discovered appropriate modifications of P2, P3
and P4 moieties allowed truncation of inhibitors spanning from
P6 to P0 achieving compounds with lower molecular weights, desir-
1
able binding and pharmacokinetic profile. In this present work we
envisioned that introduction of hydrazide analogs in our class of
inhibitors may potentially provide compounds with better binding
O
H
N
NH2
Ki* =14 nM
N
H
O
O
HN
N
HNE/HCV =2200
O
Replicon EC90 = 0.35 µM
O
1, Boceprivir
* Corresponding author. Tel.: +1 908 740 3758; fax: +1 908 740 7152.
E-mail address: Srikanth.Venkatraman@spcorp.com (S. Venkatraman).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.06.060

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4760–4763
4761
C6H5
O
BocHN
COOH
R3
a
CH3
COOH
O
H
COOCH3
N
N
COOCH3
.HCl
N
N
N
BocHN
O
O
H
N
H
H
O
H
N
O
N
8
R3
CH3
N
N
O
9
H
H
CH3
O
O
2
b
c
COOH
COOCH3
N
Ki = 0.078 µM
H
H
N
N
R4
O
Figure 2.
O
R3
10
than 2 and compounds with desirable cellular activity. In addition
these modifications would depeptidize our compounds and pro-
vide inhibitors that are free of labile stereocenters which epimer-
ized under physiological conditions. Pursuant to our
identification of 1 we had also discovered that introduction of no-
vel depeptidized P3 caps allowed improvement in binding and cel-
lular activity of 1.7 In this Letter we disclose our efforts to identify
novel hydrazide derived inhibitors with excellent binding and cel-
lular activity.
O
H
N
N
R1
N
H
H
N
O
R2
N
R4
O
O
R3
11
Synthesis of these hydrazide inhibitors is outlined in Schemes 1
and 2.
Scheme 2. Reagents and conditions: (a) HATU, NMM, CH2Cl2/DMF; (b) (i) 4 M HCl/
dioxane; (ii) R4NCO, NMM; (c) (i) aq LiOH, THF/MeOH; (ii) 7, HATU, NMM.
Condensation of Boc-carbazate 3 with desired aldehyde re-
sulted in hydrazone of type 4 which were reduced to alkylated
hydrazides using NaBH4 and toluenesulfonic acid. The resulting
alkylated hydrazides were acylated with acid chlorides or isocya-
nates to yield desired amides or ureas of type 6. These acylated
derivatives were deprotected to form hydrazide salts of type 7 that
were further used for syntheses of inhibitors.
evaluated various novel P01 groups derived from amides, carba-
mates and ureas (Table 1).
Introduction of benzamide at P01 resulted in compound 12 that
demonstrated a Ki = 52.0
moiety with acetamide group resulted in compound 13 with
Ki = 22 M, a marginal improvement in potency compared to 12.
However, introduction of cyclopropyl amide resulted in compound
14 with improved binding Ki = 3.0 M and EC90 = 15.0 M. Simi-
larly, incorporation of a phenethyl amide at P01 resulted in com-
pound 15 with much improved binding Ki = 0.72 and
EC90 = 3.5 M. This was our first compound in this series to dem-
onstrate a binding less than 1 M. We next explored the incorpo-
lM (Table 1). Replacement of benzamide
l
Coupling of dimethylcyclopropanated proline derivative 88 with
P3 amino acid using HATU coupling agent9 resulted in dipeptides of
type 9. The Boc group of 9 was deprotected using 4 M HCl in diox-
ane and capped with appropriate P3-capping group using corre-
sponding isocyanate to yield compounds of type 10. The methyl
ester 10 was hydrolyzed with aq lithium hydroxide and the result-
ing acid was coupled with hydrazide salt of type 7 to obtain aza-
peptide inhibitors of type 11.
Synthesized inhibitors of type 11 were evaluated for their abil-
ity to inhibit HCV NS3 protease in a continuous assay as previously
described10 to obtain a Ki. Compounds with good binding were fur-
ther evaluated in a replicon based cellular assay to determine EC90.
Based on our experience from ketoamide derived inhibitors we
decided to evaluate inhibitors derived from dimethylcylclopropa-
nated proline as P2, and glutarimide derived P3 capping. We first
l
l
lM
l
l
ration of carbamates and ureas at this position. Synthesis of the
tert-butyl carbamate analog resulted in compound 16 with
Ki = 3.1
lM and EC90 = 24.0
lM whereas introduction of tert-butyl
urea resulted in compound 17 with Ki = 1.6
lM. Since the urea ana-
logs demonstrated partially better binding than carbamates, we
further explored SAR of the ureas analogs by synthesizing cyclo-
hexyl, cyclopropyl and phenyl urea derivatives. The cyclohexyl
urea analog 18 demonstrated a Ki = 1.3
lM with an EC90 = 16 lM
whereas the cyclopropyl analog demonstrated a binding activity
Ki = 0.75
Ki = 0.95
l
l
M and EC90 = 6.0
M a similar activity as cyclopropyl derivative 19 but
M).
lM. The phenyl urea analog 20 had a
H
demonstrated a better cellular activity (EC90 = 2.4
l
H
a
b
N
N
A close analysis of Table 1 showed that the phenethyl amide
derivative 15 and phenyl urea analog 20 were the more active
R2
O
Boc
Boc
NH2
Boc
N
3
4
compounds with enzyme binding less than 1
prove binding in this series we decided to evaluate the SAR by
incorporating -methylbenzyl urea present in 2 (Table 2). This
lM. To further im-
H
H
N
d
a
c
N
R2
Boc
N
R1
N
H
5
was also observed as the desirable group in identification of 2. Con-
comitant to this modification we evaluated the effect of modifica-
R2
6
tion of the P1, P3 and P3-capping groups. Introduction of
methylbenzyl urea had a profound effect on the binding activity
of these inhibitors. -methylbenzyl urea analog with bicyclic imide
cap and P1 cyclopropylmethyl group resulted in compound 21 with
Ki = 0.25 M and EC90 = 1.1 M. Replacement of bicyclic P3 cap
with dimethylcyclohexylimide cap resulted in compound 22 with
Ki = 0.20 M similar in activity to compound 21 and five fold
improvement in activity compared to phenylurea 20.
a-
O
a
ClH3N
N
R1
R2
l
l
7
Scheme 1. Reagents and conditions: (a) R2CHO, toluene, TsOHꢁH2O, reflux; (b)
TsOHꢁH2O, NaBH4, CH2Cl2; (c) R1COCl, Et3N, CH2Cl2 ꢂ78 °C; (d) 4 M HCl/dioxane.
l

4762
S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4760–4763
Table 1
Table 2
CH3
O
O
H
H
N
O
N
N
N
R1
N
N
N
H
N
H
H
H
H
N
O
O
R4
R2
N
N
N
O
O
O
O
R3
O
Entry
R2
R3
R4
Ki (
l
M)
EC90
1.1
(lM)
Entry
R1
Ki (
l
M)
EC90
NA
(lM)
O
12
52.0
22.0
3.0
21
0.25
0.2
N
O
O
13
14
15
16
17
18
19
20
NA
CH3
22
23
24
25
26
NA
0.7
NA
0.3
0.25
N
O
15.0
3.5
O O
S
0.11
0.23
N
0.72
3.1
O O
S
24.0
8.0
N
O
N
1.6
N
H
O
0.064
0.032
N
N
1.3
16.0
6.0
O
N
H
O
.
0.75
0.95
N
H
O
2.4
N
H
In conclusion we have identified a series of novel aza-peptide
derived series of HCV NS3 protease inhibitors that demonstrated
excellent binding and cellular activity. This provides a possible
avenue for depetidization of peptidic analogs of our first genera-
We next evaluated the effect of modification of the P3 group and
its capping. Incorporation of methylsulfonamide derived cap with
n-propyl P1 residue resulted in compound 23 with Ki = 0.11
tion compound 1. By truncation of P6—P0 derived inhibitors we
1
lM
have identified a series of novel imide P3-capped aza-peptide ana-
logs that demonstrated excellent inhibition of HCV NS3 protease.
SAR investigation of P01 identified a phenethyl amide analog 15
and phenylurea derivative 20 which demonstrated submicro-mo-
lar enzyme binding activity and moderate cellular activities. Fur-
ther modification of P01 P3 and P3-capping residue resulted in
and EC90 = 0.7 M; a 10-fold improvement in binding compared
l
to 20. This was the first compound with sub micro-molar cellular
activity in this series of inhibitors. Introduction of pyridylsulfona-
mide derived P3 cap with P3 cyclohexyl glycine and P1 propyl res-
idue resulted in compound 24 with Ki = 0.23 lM a two fold loss in
binding compared to methylsulfonamide derivative 23.
Compound containing, dimethylglutarimide P3-cap, P1 propyl
-methylbenzyl urea resulted in inhibitor 25 with much
improved binding (Ki = 0.064 M) and EC90 = 0.3 M. This was an
identification of potent analogs such as 25 (Ki = 0.064
EC90 = 0.30 M) and 26 (Ki = 0.032 M; EC90 = 0.25 M) that con-
tained imide P3 capping and
-methylbenzyl urea P01 and a propyl
lM;
l
l
l
group, P01
a
a
l
l
P1 groups. These inhibitors are similar in potency to our first gen-
eration clinical candidate Sch 503034, but desirably lack the elec-
trophilic trap and epimerizable ketoamide center. In addition the
urea carbonyl moiety is non-electrophilic and serine does not make
a covalent bond with the inhibitor.
aza-peptide analog with an EC90 comparable to our first generation
clinical candidate 1. Analog 25 was evaluated for its selectivity
against human neutrophil elastase a structural homologous en-
zyme to HCV NS3 protease. The selectivity was determined to be
Ki(HNE)/Ki(HCV) = 960. Incorporation of bicyclic imide derived P3
cap with P1 propyl group and P0
a
-methylbenzyl urea resulted in
1
References and notes
compound 26 with a binding of Ki = 0.032 M and EC90 = 0.25 M.
l
l
This compound was more potent in binding and replicon cellular
activity than the dimethylcyclohexylimide analog 25. It was also
slightly more active than our first generation compound 1.
1. (a) World health organization Fact sheet number 164, October 2000.; (b)
Wasley, A.; Alter, M. J. Semin. Liver Dis. 2000, 20, 1; (c) Brown, R. S., Jr.; Gaglio, P.
J. Liver Transpl. 2003, 9, S10–S13.

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4760–4763
4763
2. (a) McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.; Shiffman, M. L.; Lee, W. M.;
Rustgi, V. K.; Goodman, Z. D.; Ling, M.-H.; Cort, S.; Albrecht, J. K. N. Eng. J. Med.
1998, 339, 1485; (b) Davis, G. L.; Esteban-Mur, R.; Rustgi, V.; Hoefs, J.; Gordon,
S. C.; Trepo, C.; Shiffman, M. L.; Zeuzem, S.; Craxi, A.; Ling, M.-H.; Albrecht, J. N.
Eng. J. Med. 1998, 339, 1493; (c) Zeuzem, S.; Feinman, S. V.; Rasenack, J.;
Heathcote, E. J.; Lai, M.-Y.; Gane, E.; O’Grady, J.; Reichen, J.; Diago, M.; Lin, A.;
Hoffman, J.; Brunda, M. J. N. Eng. J. Med. 2000, 343, 1666; (d) Heathcote, E. J.;
Shiffman, M. L.; Cooksley, W. G. E.; Dusheiko, G. M.; Lee, S. S.; Balart, L.;
Reindollar, R.; Reddy, R. K.; Wright, T. L.; Lin, A.; Hoffman, J.; De Pamphilis, J. N.
Eng. J. Med. 2000, 343, 1673; (e) Manns, M. P.; McHutchison, J. G.; Gordon, S. C.;
Rustgi, V. K.; Shiffman, M.; Reindollar, R.; Goodman, Z. D.; Koury, K.; Ling, M.-
H.; Albrecht, J. K.International Hepatitis Interventional Therapy Group Lancet
2001, 358, 958.
3. (a) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.;
Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A.-M.;
Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagacé, L.; LaPlante, S. R.; Narjes, H.;
Poupart, M.-A.; Rancourt, J.; Sentjens, R. E.; George, S. T.; Simoneau, B.;
Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C.-L.; Llinàs-
Brunet, M. Nature 2003, 186; (b) Yip, Y.; Victor, F.; Lamar, J.; Johnson, R.; Wang,
Q. M.; Barket, D.; Glass, J.; Jin, L.; Liu, L.; Venable, D.; Wakulchik, M.; Xie, C.;
Heinz, B.; Villarreal, E.; Colacino, J.; Yumibe, N.; Tebbe, M.; Munroe, J.; Chen, S.-
H. Bioorg. Med. Chem. Lett. 2004, 14, 251; (c) Dymock, B. W. Emerging Drugs
2001, 6, 13; (d) De Francesco, R.; Migliaccio, G. Nature 2005, 436, 953; (e) Perni,
R. B.; Almquist, S. J.; Byrn, R. A.; Chandorkar, G.; Chaturvedi, P. R.; Courtney, L.
F.; Decker, C. J.; Dinehart, K.; Gates, C. A.; Harbeson, S. L.; Heiser, A.; Kalkeri, G.;
Kolaczkowski, E.; Lin, K.; Luong, Y.-P.; Rao, B. G.; Taylor, W. P.; Thomson, J. A.;
Tung, R. D.; Wei, Y.; Kwong, A. D.; Lin, C. Antimicrob. Agents Chemother. 2006, 50,
899; (f) Lin, T.-I.; Lenz, O.; Fanning, G.; Verbinnen, T.; Delouvroy, F.; Scholliers,
A.; Vermeiren, K.; Rosenquist, A.; Edlund, M.; Samuelsson, B.; Vrang, L.; de
Kock, H.; Wigerinck, P.; Raboisson, P.; Simmen, K. Antimicrob. Agents Chemother.
2009, 53, 1377; (g) Rajagopalan, R.; Misialek, S.; Stevens, S. K.; Myszka, D. G.;
Brandhuber, B. J.; Ballard, J. A.; Andrews, S. W.; Seiwert, S. D.; Kossen, K.
Biochemistry 2009, 48, 2559.
4. (a) Bartenschlager, R. J. Viral Hep. 1999, 6, 165; (b) Bartenschlager, R.; Ahlborn-
Laake, L.; Mous, J.; Jacobsen, H. J. Virol. 1993, 67, 3835; (c) Reed, K. E.; Rice, C. M.
In Hepatitis C Virus; Reesink, H. W., Ed.; Molecular Characterization of Hepatitic
C Virus; Karger: Basel, Switzerland, 1998; pp 1–37; (d) Lindenbach, B. D.; Rice,
C. M. Nature 2005, 436, 933.
5. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu,
Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.;
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Yang, W.;
Kong, J.; Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agarwal,
S.; Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.;
Prongay, A.; Madision; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, T. Y.; Brisson, J.-
M.; Prelusky, D.; Kormacher, W.; White, R.; Bogonowich-Knipp, S.; Pavlovsky,
A.; Prudence, B.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.;
Njoroge, F. G. J. Med. Chem. 2006, 49, 6074.
6. Bailey, M. D.; Halmos, T.; Goudreau, N.; Lescop, E.; Llinas-Brunet, M. J. Med.
Chem. 2004, 47, 3788.
7. Chen, K. X.; Nair, L.; Vibulbhan, B.; Yang, W.; Arasappan, A.; Bogen, S. L.;
Venkatraman, S.; Bennett, F.; Pan, W. B.; Melissa, L.; Padilla, A. I.; Prongay, A.;
Cheng, K.-C.; Tong, X.; Shih, N.-Y.; Njoroge, G. J. Med. Chem. 2009, 52, 1370.
8. (a) Mamai, A.; Zhang, R.; Natarajan, A.; Madalengoitia, J. S. J. Org. Chem. 2001,
66, 455; (b) Zhang, R.; Madalengoitia, J. S. J. Org. Chem. 1999, 64, 330.
9. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
10. (a) Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge, F. G.; Windsor, W. T.;
Malcolm, B. A. Anal. Biochem. 1999, 270, 268; b Morrison, J. F.; Walsh, C. T. The
behavior and significance of slow binding enzyme inhibitors. In Advances in
enzymology, Meister A. Ed.; Wiley: 1988; Vol. 61, p 201.