SAR and pharmacokinetic studies on phenethylamide inhibitors of the hepatitis C virus NS3/NS4A serine protease

Article abstract of DOI:10.1016/j.bmcl.2004.05.093

SAR on the phenethylamide 1 (K_i 1.2μM) in the P2- and the P′-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.

Full text of DOI:10.1016/j.bmcl.2004.05.093

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4575–4579
SAR and pharmacokinetic studies on phenethylamide inhibitors
of the hepatitis C virus NS3/NS4A serine protease
Savina Malancona,a Stefania Colarusso,a Jesus M. Ontoria,a Antonella Marchetti,a
Marco Poma,a Ian Stansfield,a Ralph Laufer,b Annalise Di Marco,b Marina Taliani,b
Maria Verdirame,b Odalys Gonzalez-Paz,a Victor G. Matassaa, and Frank Narjesa,*
aDepartment of Chemistry, Istituto di Ricerche di Biologia Molecolare, Merck Research Laboratories,
Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy
bDepartment of Pharmacology, Istituto di Ricerche di Biologia Molecolare, Merck Research Laboratories,
Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy
Received 9 February 2004; revised 21 May 2004; accepted 27 May 2004
Available online 19 July 2004
Abstract—SAR on the phenethylamide 1 (Ki 1.2 lM) in the P2- and the P0-position led to potent inhibitors, one of which showed
good exposure and low clearance when administered intramuscularly to rat.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infections constitute a global
health problem, which affects more than 170 million
individuals. Most of these individuals, if untreated, will
develop chronic liver disease, which can develop into
liver cirrhosis and hepatocellular carcinoma.1 In con-
trast to hepatitis B virus infections, no vaccine or gen-
eral therapy is available. The limited efficacy of the
current therapy, a-interferon in combination with riba-
virin,2 has stimulated intense research programs to find
a broadly effective antiviral therapy.3
Another approach, based on product inhibition led
to noncovalent macrocyclic inhibitors,7 one of
which showed a substantial antiviral effect in clinical
trials.8
We recently disclosed a novel class of noncovalent
inhibitors of the NS3/4A protease, which contain a C-
terminal phenethylamide moiety.9 The initial poly-
anionic hexapeptide lead could be truncated to tripep-
tides like 1 (Fig. 1), which contain only one carboxylic
acid. Amide 1 was shown to be a competitive, reversible
inhibitor of the NS3/4A protease. The compound has
comparable activity on the protease domain and on the
full-length NS3 protein/NS4A complex, comprising
protease-helicase/NTPase (Fig. 1).
The nonstructural (NS) protein 3 of the viral genome
harbors a serine protease, which forms a heterodimer
with the NS4A protein. This chymotrypsin-like NS3/4A
protease is essential for viral replication,4 and has been
studied intensively in the search for new effective
antiviral agents. Several classes of inhibitors of NS3
have been described in the literature, the majority de-
rived from the substrates of NS3.5 The classical serine
trap approach yielded small peptidic or peptidomimetic
inhibitors with good potency,5;6 but it is doubtful if such
electrophilic molecules would ever become drugs.
CHF2
O
O
Cl
H
N
H
N
O
N
H
N
H
O
O
CO2H
1
Protease
domain
Full-length
complex
* Corresponding author. Tel.: +39-06-910-93-625; fax: +39-06-910-93-
654; e-mail: frank_narjes@merck.com
Ki (µM)
2.1
1.2
Present address: Graffinity Pharmaceuticals AG, Im Neuenheimer
Feld 518, 69120 Heidelberg, Germany.
Figure 1.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.093

4576
S. Malancona et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4575–4579
From SAR data, molecular modeling, and inhibition
data generated on mutagenized enzymes, it was con-
cluded that the phenethyl group is binding in the prime
site of the enzyme, with the strictly required phenyl
group occupying a unique position, where it stacks
against the aliphatic part of the side-chain of Lys136.9
F
F
F
F
a, b
R
OH
+
H
N
H2N
PGHN
ClH3N
R
O
O
13a PG = Cbz
13b PG = Boc
14
F
In this letter we report the further optimization of 1
towards more potent inhibitors and disclose first
encouraging PK data.
F
H
N
O
O
c - f
H
N
O
P3 P2
N
H
R
O
O
The synthesis of representative phenethylamines 4a,b, 8
and 12 is shown in Scheme 1. For the installation of the
ethylamine moiety we relied on the ortho-directing effect
of the fluorine group.10 For example, tert-butyl-3,5-di-
fluorobenzoate 2a was treated with LDA and the
resulting anion quenched with DMF. The formyl group
was then converted into the nitroethyl group via the
nitrostyrene to give 3a, whose reduction lead to the
phenethylamine 4a.
15-29
Scheme 2. (a) EDCI, HOBt, DCM, i-Pr2NEt; (b) 13a: H2, Pd/C
MeOH; HCl/Et2O; 13b: HCl/EtOAc; (c) P2-residue, EDCI, HOBt,
i-Pr2NEt; HCl/EtOAc (Boc) or 4-AMP, DCM; phosphate buffer,
pH 5.5 (Fmoc); (d) i-Boc-P3-residue, HATU, 2,6-lutidine, DMF;
(e) deprotection: TFA/DCM/H2O (1:1:0.1) or NaOH, MeOH; (f) RP-
HPLC.
Several groups had reported that substituted proline
derivatives in the P2-position can significantly enhance
potency in product inhibitor and serine-trap based
series.7;13 We tried this approach in our phenethylamide
series14a and the results are presented in Table 1.
Tripeptides 15–29 were assembled as previously re-
ported (Scheme 2).9 trans-4-Cyclohexyl proline was ob-
tained by hydrogenation of the commercially available
4-phenylproline.11 Acylsulfonamide 22 was obtained
from 18 and methyl sulfonamide using EDCI.
Exchanging leucine in 15 with trans-4-benzyloxyproline
gave equipotent 16a. Inverting the stereochemistry of
the 4-substituent to cis as in 16b, resulted in a 10-fold
loss of potency. Due to the potential metabolic insta-
bility of the benzyloxy residue, we investigated carbon-
linked 4-substituents and found that a 4-phenyl- or a 4-
cyclohexyl residue are equally well tolerated. The
resulting inhibitors 17 and 18 are not significantly more
potent compared to the leucine containing compound
15, but the cyclic P2-residue renders these structures less
peptidic and therefore should increase their hydrolytic
stability. As we have recently reported,14b a major gain
All inhibitors were then evaluated for inhibitory activity
on the NS3/NS4A full-length complex.12
Extensive SAR in the previous Glu-Leu-Cys series had
established the requirement of the 4-benzoic acid moiety
as the optimal residue. Improvement of potency in this
series could be achieved by incorporation of a chlorine
substituent.9 To evaluate if another halogen would fur-
ther enhance the activity, we synthesized tripeptide 15,
containing the 2,6-difluorobenzoic acid, which was in-
deed threefold more potent than 1 (Table 1).
F
F
F
O2N
H2N
d for 3a
e, f for 3b
a-c
F
R
F
R
F
R
t
t
3a, b
i
2a R = CO2 Bu
2b R = CN
4a R = CO2 B u (HCl salt)
4b R = tetrazole
Br
j
ClH3N
CO2Et
g, h
O2N
Br
BocHN
Br
F
F
F
F
5
6
7
8
O
F
F
F
F
OH
OHC
F
O
CO2Me
H2N
O
CO2Me
k, l
m
n, o
O
F
F
F
9
10
11
12
Scheme 1. Reagents and conditions: (a) LDA, THF, )78 ꢁC, 1 h; DMF, 2a 77%; 2b 60%; (b) MeNO2, KF, NMM, i-PrOH, 0 ꢁC; Ac2O, NaOAc,
60 ꢁC; (c) NaBH4, SiO2, i-PrOH, CHCl3, 3a 45% (two steps), 3b 40% (two steps); (d) H2, Pd/C; Et2O/HCl, 98%; (e) NaN3, NH4Cl, DMF, 42%; (f) H2,
Pd/C, 67%; (g) as in (a), 83%; (h) as in (b) and (c), 65%; (i) NiCl2, NaBH4, MeOH; Boc2O, NEt3, DCM, 57%; (j) Pd(OAc)2, P(o-tol)3, NaOAc,
CH2@CHCO2Et, DMF; EtOAc, HCl, 75%; (k) LDA, THF, DMF; ethylenglycol, PPTs, 85%; (l) BuLi, TMEDA; B(OMe)3; oxone, 63%; (m)
Me(Br)CHCO2Me, K2CO3, acetone; PPTs, H2O, acetone, reflux, 76%; (n) as in (b) and (c), 55%; (o) as in (f), 85%.

S. Malancona et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4575–4579
4577
Table 1. SAR on inhibitor 1
F
F
O
O
O
R1
H
N
O
P3
P2
N
H
O
R2
CO2H
No.
P2
P3
R1, R2
Ki (lM)
1
Leu
Leu
Val
Val
Val
Val
Val
Val
Cl, H
F, F
F, F
F, F
F, F
F, F
1.2
15
16a
16b
17
18
0.40
0.55
4.5
trans-4-BnO-Pro
cis-4-BnO-Pro
trans-4-Phe-Pro
trans-4-Cy-Pro
0.45
0.35
N
O
19
20
t-Bu-gly
F, F
0.03
0.02
MeO
MeO
N
Ph
N
O
N
Val
F, F
N
NH
S
in potency can be achieved by incorporation of a large
(2-aryl-7-methoxy)-4-quinolyl fragment, discovered by
the Boehringer group.7 Compounds 1914b and 20 are
about 20-fold more active than 15–18, but their mole-
cular weight is now well above 850. For this reason we
chose 17 and 18 for the investigation of the SAR in the
P0-site.
two fluorines, and this is demonstrated with tripeptides
29a,b, both of which achieve potencies similar to tetra-
zole 23.
Having increased potency about 10-fold, we investigated
the pharmacokinetic behavior of our molecules.
Some selected compounds were dosed intravenously to
rats to examine their pharmacokinetic parameters
(Table 3). Their behaviour was mostly characterized by
high clearance and a short half-life, with compound 16a
exhibiting the highest plasma clearance. Exchanging the
benzyloxy substituent on proline to phenyl (17) or cy-
clohexyl (18) improved the clearance, although half-life
for the latter compound is short. Exchanging the acid in
18 for acid mimetics was beneficial in the case of tetra-
zole 23, which exhibited low clearance and reasonable
half-life and AUC, whereas acylsulfonamide 22 was
comparable to parent acid 18.
An acid is required for activity, as inactive methyl ester
21 shows (Table 2), but it can be successfully converted
to known isosteres like an acylsulfonamide (22, Ki
0.45 lM) or a tetrazole (23, Ki 0.1 lM). Interestingly,
this compound is threefold more active than the parent
acid 18.
Previous SAR in the series related to 1 had shown that
elongation of the acid led to an increase in potency only
in the meta-position.15 In the P2-proline series, the meta-
cinnamic acid 24 (Ki 1.3 lM) is fourfold less active than
18, but also here incorporation of a fluorine increases
potency (25, Ki 0.35 lM). The position of the halogen is
not relevant for the activity, as equipotent 26 shows
(Table 2).
Acylsulfonamide 22 was also dosed intramuscularly to
rat and showed a remarkable profile (Fig. 2). Very good
exposure with an estimated bioavailability of 85% was
observed over an extended period of time.
Since numerous attempts to increase activity by
increasing the size of the aromatic ring were not suc-
cessful in the Glu-Leu-Cys series,9 we investigated the
possibility of appending hydrophobic residues in the
context of a structure similar to 24. Indeed, we had seen
earlier that substituted phenyl acetic acid derivatives
give compounds with good affinity for NS3 (e.g. 27a and
27b). Further investigation of this series showed that the
phenyl ring does not contribute significantly to potency,
as the ethyl derivative 28, (Ki 0.5 lM) illustrates. The
major gain in potency is thus due to the presence of the
In summary we have optimized the potency of the
noncovalent tripeptide phenethylamide series of NS3/4A
inhibitors. Although a substantial gain in potency can
be achieved with large lipophilic substituents in P2, as
compound 20 (Ki 0.02 lM) demonstrates, this results in
compounds with high molecular weight (933 Daltons),
which is above the optimal range for most orally active
drugs. Small changes in the phenethylamide moiety by
incorporation of two fluorines and modifications on the
acidic group led to tripeptides 23 and 29b with Ki’s of

4578
S. Malancona et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4575–4579
Table 2.
10000
F
F
O
O
O
1000
100
10
H
N
N
H
O
N
N
H
R2
O
R1
i.m.
i.v.
No.
R1
R2
Ki (lM)
F
F
F
21
Cy
Nab
F
F
F
CO2Me
H
1
0.00
1.00
2.00
3.00
Time (h)
4.00
5.00
6.00
22
23
Cy
Cy
0.45
0.10
N
CH3
S
Figure 2. Profile of compound 22 after iv (2 mg/kg) and im dosage
(10 mg/kg, solution in 80% DMSO and 20% water) to rat.
O
O
O
N
Acknowledgements
NH
N
N
The authors thank Mirko Brunetti, Gabriella Biasiol,
Mauro Cerretani, Sergio Serafini, Sergio Altamura and
CO2H
24
25
26
Ph
Ph
Ph
1.30
0.35
0.40
€
Christian Steinkuhler for providing the assay data, the
CO2H
analytical chemistry group for quantitative analysis of
the plasma samples, Silvia Pesci for NMR spectra and
Michael Rowley for valuable discussions. This work was
supported in part by a grant from the MIUR.
F
F
CO2H
CO2H
References and notes
27a
27b
Ph
Ph
1.0a
0.65a
X
X = O
X = NH
Ph
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No.
t1=2 (min)
Clp (mL/
min/kg)
Vdss (L/kg)
AUC
(lM · h)
16a
17
48
72
15
19
74
222
36
44
46
9
4.5
2.4
0.4
1.3
18
0.45
0.8
2.7
0.9
22b
23
0.15
12.4
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€
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15. For example: i-BocGluLeuCysNH(CH2)2 Ph(4-CHCH-
CO2H), Ki 5 lM; i-BocGluLeuCysNH(CH2)2 Ph(3-CH@
CH–CO2H), Ki 1.5 lM; i-BocValLeu-difluoroAbuNH
(CH2)2Ph(2-CH@CHCO2H), Ki >30 lM.
ꢀ
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