Synthesis and biological activity of macrocyclic inhibitors of hepatitis C virus (HCV) NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2005.07.033

The 17-membered phenylalanine-based macrocycle 6 was prepared starting from 3-iodo-phenylalanine. Macrocyclization of alkene phenyl iodide 5 was effected through a palladium-catalyzed Heck reaction. The macrocyclic α-ketoamides were active inhibitors of t

Full text of DOI:10.1016/j.bmcl.2005.07.033

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4475–4478
Synthesis and biological activity of macrocyclic inhibitors
of hepatitis C virus (HCV) NS3 protease
Kevin X. Chen,* F. George Njoroge, Andrew Prongay, John Pichardo,
Vincent Madison and Viyyoor Girijavallabhan
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-3-3545, Kenilworth, NJ 07033, USA
Received 13 May 2005; revised 7 July 2005; accepted 11 July 2005
Available online 19 August 2005
Abstract—The 17-membered phenylalanine-based macrocycle 6 was prepared starting from 3-iodo-phenylalanine. Macrocyclization
of alkene phenyl iodide 5 was effected through a palladium-catalyzed Heck reaction. The macrocyclic a-ketoamides were active
inhibitors of the HCV NS3 protease, with the C-terminal acids and amides being more potent than tert-butyl esters.
Ó 2005 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection is the principal cause
of chronic liver disease, leading to cirrhosis, hepatocellu-
lar carcinoma, or liver failure in humans.1 It has infected
more than 170 million people worldwide and has
emerged as a major global health problem. Currently
available therapy is a-interferon, either alone or in com-
bination with ribavirin. Even the latest pegylated
a-interferon-ribavirin combination therapy can generate
sustained virological response in only 50% of infected
patients.2 Existing therapies are also fraught with several
considerable side effects. The limited efficacy and ad-
verse side effects of the current therapies have clearly
raised the need to develop even more effective antiviral
agents, stimulating intensive research in finding potent
and orally bioavailable small molecule drug candidates.3
substrates. It is often difficult to extract desirable phar-
macokinetic properties from these peptidic compounds.7
Lot of effort has been made to design peptidomimetics
with less peptidic character. One of the strategies devel-
oped was macrocyclization of a peptide chain.8 Many
biologically active macrocyclic molecules, such as van-
comycin family of antibiotics9 and chloropeptins,10 have
been observed in nature. A number of potent and selec-
tive macrocyclic inhibitors with novel structures have
also been designed for HIV protease,11 ACE,12 matrix
metalloproteases (MMP),13 and TACE.14 Macrocycles
are designed to pre-organize the conformation for en-
zyme binding, reducing entropy cost in the process.15
They are potentially more stable to proteolytic degrada-
tion than their acyclic counterparts.16
The HCV viral RNA genome encodes a polyprotein that
consists of structural and nonstructural (NS) proteins.
The chymotrypsin-like serine protease, located at N-ter-
minal of NS3 nonstructural protein, is essential for viral
replication in vivo.4 It has been a valuable target for
which a number of inhibitors have been described in
the literature.5
Herein, we report a short and efficient synthesis of a ser-
ies of phenylalanine-based macrocyclic HCV inhibitors.
The 17-membered macrocycles were designed as a pepti-
domimetic of a P2–P3 dipeptide moiety. We presumed
that incorporation of a macrocyclic ring in these inhibi-
tors could enhance binding potency and improve phar-
macokinetic profile. The size of the macrocycle was
determined, based on the analysis of X-ray crystal struc-
ture of the enzyme binding site. Clearly, the major chal-
lenge in the synthesis of this type of compounds was
macrocyclization step. We envisaged that the alkyl aryl
linkage could be effected through a Heck reaction,17 that
is, palladium-catalyzed carbon–carbon bond formation
between an aryl iodide and terminal alkene (Scheme 1).
A large number of protease inhibitors have been devel-
oped in recent years for the treatment of a variety of
diseases, many of which are peptidic in nature.6 Their
designs are normally based on the cleavage of native
Keywords: Macrocycle; Protease; Inhibitor; Hepatitis C virus.
*
Synthesis ofthe macrocycle commenced with the coupling
of commercially available N-Boc-cyclohexylglycine 1 and
Corresponding author. Tel.: +1 908 740 7556; fax: +1 908 740
7152; e-mail: kevin.chen@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.033

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K. X. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4475–4478
OH
I
OH
CO2H
O
H
N
I
a
b
BocHN
BocHN
OBn
BocHN CO2H
O
a
O
OMe
BocHN
+
N
H
OMe
HCl.H2N
O
O
8
7
2
1
3
OH
O
OH
O
I
O
H
H
c
BocHN
N
OtBu
BocHN
N
N
OH
c
b
H
O
O
O
OMe
HCl.H2N
N
H
10
O
9
4
OH
O
d
O
H
HCl.H2N
N
OtBu
N
I
H
O
d
O
O
N
N
H
OMe
OMe
H
N
N
11
H
H
O
O
O
O
Scheme 2. Reagents and conditions: (a) glycine benzyl ester, HOOBt,
EDC, 4-methylmorpholine, DMF/CH2Cl2, ꢀ20 °C, 75%; (b) H2, Pd–
C, EtOH, rt, 98%; (c) phenylglycine t-butyl ester hydrochloride,
HOOBt, EDC, 4-methylmorpholine, DMF/CH2Cl2, ꢀ20 °C, 81%; (d)
2 M HCl, dioxane/EtOAc (1:1), rt, quant.
6
5
Scheme 1. Reagents and conditions: (a) HOOBt, EDC, 4-
methylmorpholine, DMF/CH2Cl2, ꢀ20 °C, 84%; (b) 4 M HCl, diox-
ane, rt; (c) oct-7-enoic acid, HOOBt, EDC, 4-methylmorpholine,
DMF/CH2Cl2, ꢀ20 °C, 68% (two steps); (d) Pd(PPh3)4, Et3N, CH3CN/
DMF, 80 °C, 37%.
assemble target molecules (Scheme 3). Thus, methyl es-
ter 6 was hydrolyzed to macrocyclic acid 12, which was
coupled to amine 11 under standard conditions. The
resulting a-hydroxyamide was then oxidized using
Dess–Martin periodinane19 to afford a-ketoamide 13.
This compound was an inseparable diastereomeric
mixture of (R)- and (S)-isomers at P1 norvaline a-center.
The corresponding C-terminal carboxylic acid 14, also
(3-iodo)phenylalanine methyl ester 2 (Scheme 1) using
3-hydroxy-1,2,3-benzotriazin-4(3H)-one
(HOOBt),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
hydrochloride, and 4-methylmorpholine (NMM) at
ꢀ20 °C to give dipeptide 3. The Boc-protecting group
was removed and the resulting amine hydrochloride 4
was reacted with oct-7-enoic acid under similar coupling
conditions (HOOBt, EDC, and NMM) to provide 5, the
precursor to the macrocycle. The key macrocyclization
step came up next, wherein the terminal alkene and phenyl
iodide in 5 were cyclized to a 17-membered macrocycle
through a Heck reaction.17 The reaction was performed
in a sealed tube at 80 °C in anhydrous acetonitrile–di-
methylformamide media in the presence of 10% palladi-
um tetrakistriphenylphosphine and 10 equiv of
triethylamine. The desired macrocycle 6 was obtained in
37% isolated yield. The product was a mixture of cis-
and trans-isomers at the newly formed double bond.
O
a
O
N
N
OH
OMe
H
H
N
N
H
H
O
O
O
O
12
6
O
O
b, c
O
H
H
N
N
H
N
OR
N
N
The right-hand tripeptide fragment of the molecule was
prepared in a series of peptide couplings and protecting
group manipulations (Scheme 2). First, norvaline
hydroxyacid18 7 was coupled to glycine benzyl ester un-
der standard conditions (HOOBt, EDC, and NMM) to
give compound 8, which was hydrogenated to give acid
9. Coupling of 9 to commercially available phenylgly-
cine tert-butyl ester provided tripeptide 10. The Boc-
protecting group was then selectively removed in the
presence of tert-butyl ester using 2 M hydrogen chloride
in 1:1 dioxane/ethyl acetate to give the desired amine 11
as a hydrochloride salt.
H
H
O
O
O
O
P3
P2
P1
P1'
P2'
13 R = OtBu Ki* = 1.2 µM
14 R = OH
Ki* = 0.084 µM
15 R = NMe2 Ki* = 0.12 µM
d
e
Scheme 3. Reagents and conditions: (a) LiOH, MeOH/THF/H2O, rt,
quant.; (b) 11, HOOBt, EDCI, 4-methylmorpholine, DMF/CH2Cl2,
ꢀ20 °C, 77%; (c) Dess–Martin periodinane, CH2Cl2, rt, 55%; (d)
trifluoroacetic acid, CH2Cl2, quant.; (e) Me2NHÆHCl, HOOBt, EDC,
4-methylmorpholine, DMF/CH2Cl2, ꢀ20 °C, 65%.
With both the left-hand macrocycle and the right-hand
a-hydroxyamide fragment in hand, the stage was set to

K. X. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4475–4478
4477
as a diastereomeric mixture, was obtained from com-
pound 13 by treatment with trifluoroacetic acid. The
dimethyl amide 15 was prepared via coupling of 14 with
dimethylamine.
as a cis/trans mixture, unsaturation in the macrocycle
did not seem to affect the inhibition activity of these
compounds.
The X-ray crystal structure of inhibitor 18 bound to
HCV NS3 protease was obtained (Fig. 1, PDB ID code
2A4Q). The diastereomer with (S)-norvaline at P1 was
observed in the X-ray structure as it was more potent
than (R)-isomer. A reversible covalent bond was formed
between enzyme active site serine (Ser139) hydroxyl and
the ketone carbonyl of the inhibitor. The macrocycle
encircled the methyl group of alanine 156, which estab-
lished a ꢀdonut-shapedꢁ conformation that provided ex-
tra interaction between the inhibitor and the protease.
The n-propyl side chain of P1 norvaline fit very well into
the S1 pocket. In addition, the benzene ring of P20 phe-
nylglycine reached the other side of the lysine136 side
chain such that it and the P1 propyl group formed a
ꢀC-shapedꢁ clamp around the lysine residue. The com-
pound also entered into multiple hydrogen bonding with
the protease through its amide chain.
To evaluate the effect of the double-bond on the potency
of inhibitors, saturated analogs of compounds 13–15
were also synthesized, as outlined in Scheme 4. Thus,
macrocycle 6 was hydrogenated to give the saturated
macrocycle, which was hydrolyzed to its corresponding
carboxylic acid 16. The coupling between 16 and amine
11 provided an a-hydroxyamide, which on Dess–Martin
periodinane oxidation afforded the desired a-ketoamide
17. The tert-butyl ester was treated with trifluoroacetic
acid to give acid 18. The dimethyl amide analog 19
was prepared from 18 in a similar manner as the prepa-
ration of 15.
The six macrocyclic compounds 13–15 and 17–19 were
tested as hepatitis C virus NS3 serine protease inhibitors
in a continuous assay.20 The two tert-butyl esters were
modestly active toward the enzyme (13: Kꢁi ¼ 1.2 lM;
17: Kꢁi ¼ 2.3 lM). The carboxylic acids (14, 18), howev-
er, were potent inhibitors of HCV NS3 protease, with Kiꢁ
values of 0.084 and 0.066 lM, respectively. The dimeth-
yl amides (15, 19) were also very active HCV inhibitors
with Kꢁi at 0.12 and 0.11 lM, respectively. The tert-butyl
esters (13/17) were more than ten times less potent than
their corresponding acids (14/18) and amides (15/19).
Presumably, the bulky tert-butyl group must have
undergone unfavorable steric interactions with the en-
zyme surface. The smaller acid and amide groups were
more tolerated. The potency was very close for any pair
of inhibitors (13/17, 14/18, and 15/19) with or without
the presence of a double bond in the macrocyclic ring.
Although the double bond in compounds 13–15 existed
In summary, a short and concise synthesis of 17-mem-
bered macrocycles 12 and 16 has been carried out. The
key macrocyclization step was achieved through a Heck
reaction between the aryl iodide and terminal alkene in
the presence of a palladium catalyst. Macrocyclic inter-
mediates were coupled to a tripeptide right-hand frag-
ment to provide a-ketoamides 13–15 and 17–19 after
oxidation. The C-terminal carboxylic acids and amides
14–15 and 18–19 were potent HCV protease inhibitors
with Kꢁi values between 0.066 and 0.12 lM, while
the bulky tert-butyl esters 13 and 17 were less active.
X-ray structure of compound 18 bound to protease
revealed that the macrocycle ring adopted a ꢀdonut-
shapedꢁ conformation around the methyl group of
Ala156. These macrocyclic inhibitors were at least a
few fold more potent than similar noncyclic analogs.
The investigation of pharmacokinetic properties is
underway.
O
a, b
O
N
N
H
OH
OMe
H
N
N
H
H
O
O
O
O
6
16
O
O
O
His57
c, d
Ser139
H
H
N
H
N
N
R
N
N
H
H
O
O
O
O
Ala156
17 R = OtBu Ki* = 2.3 µM
18 R = OH Ki* = 0.066 µM
19 R = NMe2 Ki* = 0.11µM
e
f
S1
Lys136
Scheme 4. Reagents and conditions: (a) H2, Pd–C, EtOH, rt, quant.;
(b) LiOH, MeOH/THF/H2O, rt, quant.; (c) 11, HOOBt, EDC, 4-
methylmorpholine, DMF/CH2Cl2, ꢀ20 °C, 73%; (d) Dess–Martin
periodinane, CH2Cl2, rt, 46%; (e) trifluoroacetic acid, CH2Cl2, quant.;
(f) Me2NHÆHCl, HOOBt, EDC, 4-methylmorpholine, DMF/CH2Cl2,
ꢀ20 °C, 71%.
Figure 1. X-ray structure of compound 18 bound to HCV NS3
protease.

4478
K. X. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4475–4478
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