Discovery of GS-9451: An acid inhibitor of the hepatitis C virus NS3/4A protease

Article abstract of DOI:10.1016/j.bmcl.2012.01.017

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monke

Full text of DOI:10.1016/j.bmcl.2012.01.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2629–2634
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of GS-9451: An acid inhibitor of the hepatitis C virus NS3/4A protease
⇑
X. Christopher Sheng , Todd Appleby, Thomas Butler, Ruby Cai, Xiaowu Chen, Aesop Cho,
Michael O. Clarke, Jeromy Cottell, William E. Delaney IV, Edward Doerffler, John Link, Mingzhe Ji,
Rowchanak Pakdaman, Hyung-Jung Pyun, Qiaoyin Wu, Jie Xu, Choung U. Kim
Gilead Sciences, Inc., 333 Lakeside Dr, Foster City, CA 94404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solu-
bilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved
pharmacokinetic properties in rats, dogs and monkeys.
Received 2 November 2011
Revised 4 January 2012
Accepted 9 January 2012
Available online 1 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
HCV
GS-9451
NS3 Protease
Solubilizing group
Hepatitis C virus (HCV) has infected an estimated 3% of the
world’s population (170–200 million), making it a serious public
health concern.1 Since this disease was first reported in the
1980s,2 extensive research has led to the first generation treatment
increased interactions using a fused bicyclic P3 cap and polar
groups such as a weak amine attached to the P2 quinoline. In doing
so, we envisioned we could (1) pick up additional interactions with
the protease; (2) use polar groups to improve pharmaceutical
properties such as aqueous solubility and PK. Herein we describe
our efforts that culminated in the identification of a potent and
highly bioavailable HCV NS3 inhibitor.
The chemistry used for our P3 cap modification is shown in
Scheme 1. The bicyclic alcohol shown was converted to carbonate
2 in the presence of Et3N. Activation of commercially available
hydroxyl proline as the brosylate, followed by removal of the Boc
group provided amine 3. Coupling of 3 with (s)-tert-butyl glycine
in the presence of HATU and subsequent saponification, provided
the carboxylic acid dipeptide 4. HATU mediated coupling provided
tripeptide 5. This tripeptide served as a key intermediate for the
preparation of a number of analogs. For example, after the Boc
protecting group removal, the free amine was reacted with
cis-cyclopropylpentyl carbonate 2 to provide 6. Displacement of
the brosylate with the P2 quinoline and subsequent hydrolysis of
the methyl ester provided compound 7. Compounds 8 (cis-diflu-
of
a
-interferon (Peg-IntronÒ and PegasysÒ) in combination with
ribavirin. This therapy is only partially effective with ꢀ50% of geno-
type 1 HCV patients demonstrating sustained viral responses (SVR).
Recently, direct acting antivirals which inhibit the key enzymes in
the viral replication process are emerging as new treatments with
better overall response rates.3 One of these viral targets is the serine
protease NS3/4A. Efforts toward improved HCV therapy during the
past several years have led to the recent FDA approval of telaprevir
(VX-950)4 and boceprevir (SCH 503034)5, two
a-keto-amide HCV
NS3 protease inhibitors. However, some characteristics of these
drugs can be improved, such as inconvenient dosing frequency
(both are used three times a day).
The first carboxylic acid inhibitor of HCV NS3 protease, BILN-
2061 (Fig. 1, compound 1) was reported by Boehringer–Ingelheim
with clinical proof of concept.6 Through excellent design based on
an inhibitor bound to the protease, cyclization of P1 to P3 led to a
picomolar binding affinity and bioavailable NS3 protease inhibitor.7
It is well known that HCV protease has a featureless and rela-
tively solvent exposed active site.8 A potent inhibitor requires a
series of weak lipophilic and electrostatic interactions distributed
along its contact with the protease. As part of our ongoing antiviral
drug discovery program, we decided to explore the potential for
orocyclopropylpentyl) and
9 (trans-difluorocyclopropylpentyl)
were synthesized in a similar fashion from the corresponding
alcohols.
Compounds 7–9 were evaluated in a genotype 1b enzyme assay,
and cellular activity was determined using the genotype 1b repli-
con. Table 1 summarizes the biochemical and replicon activities
of these analogs. All three analogs are potent biochemical inhibitors
of the HCV NS3 protease with IC50’s in the single digit nanomolar
range. However, in the replicon cells, cis-cyclopropylpentyl analogs
⇑
Corresponding author. Tel.: +1 650 522 5902.
E-mail address: csheng@gilead.com (X.C. Sheng).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.017

2630
X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2629–2634
P2 Quinoline
solubilizing groups
S
S
N
Cl
NH
NH
O
N
O
O
N
O
N
X
P2
O
O
H
N
H
N
N
N
H
N
H
N
OH
O
O
OH
O
O
O
O
O
O
1
P3
P1
IC50 = 1 nM
EC50 = 1 nM
P3 Cap
lipophilic interaction
Figure 1. Carboxylic acid HCV NS3 protease inhibitors.
O
O
O
a
O
O
N
N
N
OH
O
O
O
O
O
O
O
O
2
Br
Br
O
OH
H
N
Br
O
O
S
HO
O
O
O
S
O
SO2Cl
O
b
c
d
e
OMe
O
N
OH
N
O
OMe
H
Boc
O
N
N
O
O
H
O
4
3
Br
Br
O
H2N
O
OMe
HCl
S
O
O
S
O
O
O
g
h
O
O
H
N
O
N
OMe
H
N
N
O
f
H
N
BocHN
OMe
O
O
O
O
5
6
S
N
Cl
NH
S
Cl
O
N
O
NH
O
N
N
OH
O
H
N
i
j
N
O
H
OH
O
N
O
O
7
Scheme 1. Preparation of compound 7. (a) alcohol, CH2Cl2, Et3N, 75%; (b) DABCO, PhMe; (c) 4 N HCl, CH2Cl2, 94% for two steps (d) HATU, Huniq’s base, DMF; (e) LiOH, THF–
MeOH–H2O, 82% for two steps; (f) HATU, Huniq’s base, DMF, 90%; (g) 4 N HCl, CH2Cl2, 90%; (h) 2, satd NaHCO3, EtOAc, 92%; (i) Cs2CO3, NMP, 60 °C, 75%; (j) LiOH, THF–MeOH–
H2O, 85%.
(compound 7 and 8) were much more active than the trans analog
(compound 9). To further differentiate the two cis analogs, we
tested their thermodynamic solubility and Caco-2 permeability.
Compound 7 showed very promising solubility, especially at
neutral pH as well as good forward permeability. Given the
excellent potency and favorable aqueous solubility profile, the

X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2629–2634
2631
Table 1
SAR of P3 pharmacophore
S
Cl
NH
O
N
O
N
O
H
N
N
H
N
O
OH
O
R
O
O
Caco-2 A À B/B À A (Â10À6 cm/s)
b
Compound
R
IC50a/EC50 (nM)
Sol (lM) pH7.3/2.2
7
1.4/9.0
72/BLQ
57/20
7.3/25
8
2.6/5.0
4.5/18
F
F
F
F
9
5.0/115
9/<6
6.8/49
a
IC50 determined by enzymatic assay using an HCV genotype 1b NS3/4A protein.
EC50 determined by cell based assay using Huh-luc cells harboring subgenomic HCV genotype 1b replicon.
b
Cl
OMe
Cl
a
O
NH2
O
O
NH2
O
MeO
OMe
Br
MeO
11
10
b
S
NH
S
OMe
Cl
N
HO2C
NH
O
N
N
c
MeO
d
OH
12
Br
S
N
OMe
Cl
NH
O
O
N
O
MeO
O
O
S
O
O
f
12
e
O
H
N
H
N
N
N
H
N
H
N
OMe
OMe
O
O
O
O
O
O
O
13
6
S
N
S
N
Cl
Cl
NH
O
NH
O
O
N
O
N
O
O
N
O
amine
g
O
H
N
H
N
N
N
h
H
N
H
OMe
O
OH
O
N
O
O
O
O
O
O
15
14
Scheme 2. Preparation of modified quinoline derivatives. (a) 48% HBr, reflux, 61%; (b) Cs2CO3, DMF, 65 °C, 67%; (c) POCl3, Py, À40 to 0 °C, 85%; (d) NaH, PhMe, reflux, 86%; (e)
Cs2CO3, NMP, 65 °C, 60%; (f) 1.4 N HCl, HOAc, 60°; (g) amine, CH2Cl2, NaBH(OAc)3; (h) LiOH, THF–MeOH–H2O, 71% over three steps.

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X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2629–2634
Table 2
IC50/EC50 measurements of modified quinoline analogs
S
Cl
NH
R
N
O
N
O
H
N
N
H
N
O
OH
O
O
O
Compound
R
IC50/EC50 (nM)
3.2/2.0
Sol (
l
M) pH7.3/2.2
Caco-2 A À B/B À A (Â10À6 cm/s)
O
O
N
N
15
38/790
0.8/8.2
O
O
16
4.2/3.2
22/1900
6.6/10
O
O
N
17
18
2.9/3.8
25/7.5
59/>100
80/>100
6.1/30
6.7/20
O
N
Table 4
PK parameters of compound 15 (GS-9451)
Rat
Dog
Cyno Monkey
Cl (L/h/kg)
Vdss (L/kg)
iv t1/2 (h)
4.4
2.3
0.62
62
1409
576
1.1%
>395
2.6%
>395
0.26
1.15
4.2
0.29
0.76
3.9
F (%)
143
49
Oral AUC (nM h LÀ1
Oral Cmax (nM)
)
21760
3937
2.6%
>395
13121
3467
1.6%
79
Unbound free fraction
MS stability t (min)
Human free fraction
Human MS stability t (min)
½
½
Dose, iv 1.0 mg/kg; po 4.0 mg/kg.
quinoline 12 following literature reported procedures.7 Displace-
ment of the brosylate 6 with quinoline 12 went smoothly to pro-
vide 13. The conversion of the acetal group to aldehyde was
accomplished with aqueous HCl in acetic acid to yield the key alde-
hyde intermediate 14. Our synthesis of different analogs relies on
reductive amination with a variety of amines using this aldehyde.
As shown in Scheme 2, reductive amination with morpholine pro-
ceeded smoothly, and hydrolysis of the methyl ester afforded com-
Figure 2. The refined 1.85 Å resolution 2Fo–Fc electron density map superimposed
on the X-ray crystal structure of compound 15 bound to HCV protease.
Table 3
Dog PK parameters
pound 15. In
a
similar manner dimethylmorpholine,
Compound iv t1/2
Oral AUC
(nM h LÀ1
Cl (L/h/
kg)
Vdss (L/
kg)
F
(%)
methoxypyrrolidine and pyperidine, provided compounds 16, 17
and 18 in high yield.
(h)
)
15
16
17
18
4.2
5.8
2.1
3.0
21760
38287
8331
0.26
0.03
0.22
0.13
1.15
0.21
0.66
0.39
142
24
45
All compounds of this series showed excellent antiviral activity
in the cell-based HCV replicon assay (Table 2). In particular, the
simple morpholine analog, compound 15, was four-fold more ac-
tive than the methoxy analog 7. Compared to 7, compound 15
19740
32
Dose, iv 1.0 mg/kg; po 4.0 mg/kg.
showed dramatically increased solubility at low pH (790 lM vs
BLQ) obviously a result of the charged morpholine under acidic
conditions.
cis-cyclopropylpentyl carbamate functionality (P3 cap for com-
pound 7) was selected in our next round of SAR study which ex-
plores modified P2 qinoline analogs.
The synthesis of modified quinoline analogs is depicted in
Scheme 2. Commercially available aniline 10 was treated with
aqueous HBr to remove the methyl group. Alkylation with 2-bro-
mo-1,1-dimethoxyethan provided 11, which was converted to
In order to confirm the binding mode for this class of inhibitors,
we crystallized the HCV NS3/4A protease in the presence of com-
pound 15 (Fig. 2). The X-ray crystal structure reveals that the cis-
cyclopropyl bicycle stabilizes the required axial orientation of the
cyclopentyl group. Addition of the bicyclic P3 cap also results in in-
creased van der Waals contacts with the enzyme. As predicted
from earlier modeling studies, the morpholine group is solvent

X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2629–2634
2633
exposed and is disordered in the structure as indicated by weak
electron density. The structure of compound 15 bound to NS3/4A
suggests that this class of inhibitors is capable of forming extensive
interactions with protein atoms in the active site and shallow sub-
strate binding groove of the protease. In addition, the structure
mean elimination half-life was around 12–14 h for all dose groups,
suitable for once daily dosing. For the 300 mg dose group, the
trough plasma concentration (C24 ꢀ400 nM) is 3 times the protein
adjusted EC50 (120 nM).
10000.0
1000.0
100.0
10.0
1.0
0.1
0
8
16
24
32
40
48
56
64
72
80
88
96
Time (hr)
Cohort 1 10 mg
Cohort 4 300 mg
Cohort 2 30 mg
Cohort 5 600 mg
Cohort 3 100 mg
1X Protein Adjusted EC50 (120 nM)
Cohort 7 1000 mg
5X Protein Adjusted EC50 (600 nM)
Cohort 6 300 mg Fed
confirms our initial hypothesis that we can introduce functional
groups which confer enhanced pharmaceutical and pharmacoki-
netic properties to the inhibitors without interfering with binding
to the protease.
When administered orally to patients infected with HCV geno-
type 1a, median HCV RNA declines were À0.88, À3.2 and À3.6 lo-
g10 IU/mL for three days at doses of 60, 200, and 400 mg,
respectively.9
We next evaluated the pharmacokinetic profile of compounds
15–18. In general, the compounds were characterized by low clear-
ance and good oral bioavailability (Table 3). Compound 15 exhib-
ited the highest oral bioavailability (142%) and volume of
distribution (1.15 L/kg).
Because of its excellent potency and bioavailability, compound
15 was selected as a clinical development candidate. Table 4 sum-
marizes pharmacokinetic parameters of 15 in rat, dog and cyno-
molgus monkey. Our aim was to determine if sufficient drug
exposure would permit once daily dosing. Excellent plasma levels
were observed after oral administration to dog and monkey. In rat,
15 was rapidly cleared (Cl 4.4 L/h/kg). The biliary excretion was
examined in bile-duct cannulated rats following intravenous
administration. Approximately 45 to 61% of the total dose was
recovered as the parent compound; with glucuronide conjugates
accounted for the remainder of total dose.
In summary, a new class of carboxylic acid HCV NS3 protease
inhibitors with excellent antiviral activity and bioavailability was
identified. Compound 15 (GS-9451) exhibited low toxicity as
determined by cell culture CC50 and high selectivity over mamma-
lian proteases. In a phase 1b study, GS-9451 showed a rapid onset
of viral load reduction, comparable to known inhibitors reported so
far. GS-9451 is currently in phase 2b clinical trial for the treatment
of hepatitis C infection.
Acknowledgments
The authors thank Huiling Yang, Margaret Robinson and Scott
Hluhanich for determining biological activities.
References and notes
Compound 15 (GS-9451) was further profiled against a panel of
mammalian proteases such as cathepsin D/L, human proteinase 3
and leukocyte elastase. GS-9451 showed selectivity of more than
50,000-fold selectivity over the human proteases assayed. In safety
pharmacology screens, GS-9451 was not an inhibitor of CYP-450
enzymes and did not block the hERG channel at concentrations
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up to 25
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