Discovery of narlaprevir (SCH 900518): A potent, second generation HCV NS3 serine protease inhibitor

Article abstract of DOI:10.1021/ml9000276

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (～10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.

Full text of DOI:10.1021/ml9000276

pubs.acs.org/acsmedchemlett
Discovery of Narlaprevir (SCH 900518): A Potent, Second
Generation HCV NS3 Serine Protease Inhibitor†
Ashok Arasappan,* Frank Bennett,* Stephane L. Bogen, Srikanth Venkatraman,
Melissa Blackman, Kevin X. Chen, Siska Hendrata, Yuhua Huang, Regina M. Huelgas,
Latha Nair, Angela I. Padilla, Weidong Pan, Russell Pike, Patrick Pinto, Sumei Ruan,
Mousumi Sannigrahi, Francisco Velazquez, Bancha Vibulbhan, Wanli Wu, Weiying Yang,
Anil K. Saksena, Viyyoor Girijavallabhan, Neng-Yang Shih, Jianshe Kong, Tao Meng, Yan Jin,
Jesse Wong, Paul McNamara, Andrew Prongay, Vincent Madison, John J. Piwinski,
Kuo-Chi Cheng, Richard Morrison, Bruce Malcolm, Xiao Tong, Robert Ralston, and
F. George Njoroge
Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033
ABSTRACT Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease
inhibitor discovered in our laboratories, is currently undergoing phase III clinical
trials. Detailed investigations toward a second generation protease inhibitor
culminated in the discovery of narlaprevir (SCH 900518), 37, with improved
potency (∼10-fold over 1), pharmacokinetic profile and physicochemical char-
acteristics, currently in phase II human trials. Exploration of synthetic sequence for
preparation of 37 resulted in a route that required no silica gel purification for the
entire synthesis.
KEYWORDS Hepatitis C virus NS3 serine protease inhibitor, R-ketoamide,
narlaprevir, SCH 900518
epatitis C virus (HCV) infection is a global health crisis
leading to liver cirrhosis, hepatocellular carcinoma
and liver failure in humans.1 An estimated 3% of the
and telaprevir (VX950),12,13 from the slow-binding reversi-
ble R-ketoamide class, in phase III human evaluation. Inhi-
bitors in phase II studies, from the structurally distinct
noncovalent macrocyclic class, include ITMN-191,14 TMC-
435350,15 and MK-7009 (P2-P4 macrocycle).16 Other NS3
protease inhibitors currently in clinical evaluation (structure
not yet disclosed) include BI-201335, ABT-450, PHX-1766,
ACH-1625 and VX-813.8
Inhibitor 1 exhibited Ki* = 14 nM in the enzyme binding
assay,17 EC90 = 350 nM in the cell-based replicon assay,18
and acceptable pharmacokinetic profile in rats and dogs
(Figure 1). In our efforts to discover a second generation HCV
protease inhibitor, we focused mainly on improving the in
vitro potency and preclinical pharmacokinetic profile of the
inhibitor, specifically exposure in monkeys. Furthermore, to
alleviate possible synthetic/purification issues that could
arise during development, it was required to identify a
molecule that existed as a single isomer, unlike 1, which
was a mixture of diastereomers at P1.
H
human population worldwide is chronically infected with
HCV.2 Currently the only available treatment regimens are
subcutaneous R-interferon or long-acting pegylated-inter-
feron, alone or in combination with oral antiviral agent
ribavirin.3 The approved therapy is still far from ideal for
the hard to treat genotype-1 patients4 and is frequently
accompanied by adverse side effects. There is an unmet
medical need to discover new, more effective and tolerable
regimens for the treatment of HCV infection. Advances in the
understanding of molecular pathways of HCV replication
have resulted in several small molecule direct-acting anti-
virals entering clinical trials in the past few years.
Since identification of this virus, the NS3 serine protease
contained within the N-terminal region of the NS3 protein
has been studied extensively.5 This chymotrypsin-like serine
protease plays a pivotal role in viral replication and, there-
fore, is an attractive target for HCV antiviral therapeutics.6,7
Intense efforts were focused in the past decade to discover
novel small molecule agents that inhibit NS3 serine pro-
tease.8 Proof of concept studies in humans with BILN 2061, a
noncovalent P1-P3 macrocyclic inhibitor, validated this
hypothesis.9 Since then, several NS3 protease inhibitors
have progressed to human clinical trials. Currently the most
advanced among those are boceprevir (SCH 503034), 1,10,11
Based on the X-ray crystal structure of 1 bound to the NS3
protease, exploration of the P4 area seemed attractive, since
additional interaction with the enzyme could potentially
Received Date: December 22, 2009
Accepted Date: January 24, 2010
Published on Web Date: February 15, 2010
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Scheme 1a
Figure 1. Profile of 1, and SAR toward new P4 moiety.
improve potency. Previously, we explored introduction of
carbamate, keto, ester, imide or sulfonamide capped P4 moi-
eties onto the core of 1.8,19-21 While we were able to improve
the replicon potency and rat pharmacokinetic properties in
most cases, monkey exposure turned out to be a challenge.
However, a cyclohexyl moiety at P4 with an appended tert-
butyl sulfone group provided encouraging results (Figure 1).
Thus, inhibitor 3 exhibited improved replicon potency (EC90
=
100 nM) and displayed good monkey plasma exposure
(AUC = 3.2 μM h, 3 mpk, po). SAR studies toward the
3
discovery of sulfone containing P4 cap is being communicated
separately. Improvement in potency and monkey exposure in
this series was accompanied with disproportionate loss in rat
PK. Herein, we describe our efforts in the P4 sulfone capped
series that culminated in the discovery of 37 (narlaprevir, SCH
900518), our second generation HCV NS3 serine protease
inhibitor currently in phase II studies.
a (a) BOM-Cl, KHMDS/THF, -78 °C; (b) (i) H2, 10% Pd/C, THF; (ii) MsCl,
Et3N, CH2Cl2; (c) NaStBu, EtOH, H2O, 100 °C; (d) (i) LiOH, THF, MeOH,
H2O, 80 °C; (ii) Oxone, MeOH, H2O; (e) DPPA, Et3N, toluene, 100 °C; (f)
(i) Et3N, CH2Cl2; (ii) D-M periodinane, CH2Cl2; (g) (i) DIPEA, CH2Cl2,
0°C; (ii) aq. LiOH, MeOH, THF; (h) (i) EDCI, HOOBt, NMM, CH2Cl2/DMF,
-20°C to 0 °C; (ii) D-M periodinane, CH2Cl2, 0°C to RT (HPLC separation
of P1 diastereomers).
Synthesis of the sulfone capped cyclohexyl P4 moiety
and further processing to target structures are shown in
Scheme 1. Alkylation of ester 4 with benzyloxymethyl
chloride afforded 5. Removal of the protecting group and
subsequent activation of the alcohol provided mesylate 6.
Displacement of the mesylate group with sodium tert-
butylthiolate resulted in thioether 7. Hydrolysis of the ester
moiety, followed by oxidation of the thioether, provided 8.
Curtius rearrangement of acid 8 resulted in isocyanate 9,
which was reacted with previously described intermediates
of type 10,11 followed by Dess-Martin oxidation to provide
target compounds of type 11 as a mixture of P1 diastereo-
mers. Alternatively, isocyanate 9 was reactedwith previously
described P3-P2 ester 12.11 Hydrolysis of the resultant ester
to acid 13, and subsequent coupling with P1-P0 intermedi-
ate of type 14, followed by Dess-Martin oxidation provided
the target compounds. HPLC separation of the mixture
afforded the required (S)-P1 diastereomer of type 15.
Synthesis of representative P1-P0 moiety is shown in
Scheme 2. Thus, reduction of L-norleucine 16 followed by
amino protection provided 17. Mild oxidation conditions
employing bleach/TEMPO resulted in aldehyde 18. Passerini
reaction of 18 with cyclopropylisonitrile 19 afforded the pro-
tected intermediate 20. Sequential deprotection unraveled
the required P1-P0 intermediate 21 in approximately 60%
overall yield.
Scheme 2a
a (a) (i) LAH, THF, reflux; (ii) Boc2O, THF (82%, 2 steps); (b) NaOCl,
TEMPO, LiBr, NaHCO3, EtOAc/H2O; (c) AcOH, EtOAc, 0 °C to RT; (d) (i)
LiOH, THF, H2O; (ii) 4 M HCl, dioxane (70-78%, 4 steps).
for SAR studies, we were challenged to develop a better
route that was amenable for scale-up. Lewis acid mediated
alkylation of 4 with thioether 30 provided 7 in one step.
Hydrolysis of the methyl ester to acid 31, followed by Oxone
oxidation, afforded sulfone 8 in 62% yield (3 steps). Treatment
of isocyanate 9 described above, with L-tert-leucine 32, in a
Our efforts toward an improved synthesis of target com-
pounds, specifically 37, are shown in Scheme 3. While the
synthetic sequence (Scheme 1) described earlier was useful
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Scheme 3a
Table 1. P1 SAR of Inhibitors of Type 11
a po (10 mpk). b iv/po (2/10 mpk). c iv/po (1/3 mpk). d po (3 mpk).
Vehicle: po, 0.4% HPMC or 20% HPBCD; iv, 40% HPBCD.
Synthesis of P0 allyl amide containing inhibitor corres-
ponding to 3 afforded 38. While 38 lost replicon potency
compared to 3, there was a measurable improvement in rat
a (a) (i) LDA, TMSCl, -78 °C to -10 °C; (ii) ZnBr2, CH2Cl2; (b) LiOH, THF,
MeOH, H2O, 80 °C; (c) Oxone, MeOH, H2O (62%, 3 steps); (d) DPPA,
Et3N, toluene, 110 °C; (e) L-tert-Leu (32), Et3N, toluene, H2O (85%, 2
steps); (f) EDCI, HOBt, NMM, MeCN, 0 °C to RT; (g) aq LiOH, THF, MeOH,
0 °C to RT (86%, 2 steps); (h) EDCI, HOBt, DIPEA, MeCN, 0 °C to RT; (i)
bleach, TEMPO, KBr, NaHCO3, EtOAc, H2O (93%, 2 steps).
oral exposure (AUC = 2.8 μM h). Inhibitor 39 containing P1
3
cyclopropyl alanine moiety exhibited good replicon potency
(EC90 = 140 nM) andgoodratandmonkeyPK(AUC = 1.7 and
7.1 μM h, respectively). Inhibitors 40 and 42 showed similar
3
profile; good potency and rat PK, with suboptimal monkey AUC
and selectivity. Inhibitor 41 containing norleucine P1 moiety
demonstrated good overall profile, with EC90 = 180 nM, good
selectivity (HNE/HCV = 1350), and oral exposure in rodent
(AUC = 5.1 μM h) and large animal (AUC = 5.5 μM h). Based
biphasic medium gave 33 which crystallized from ethyl acetate.
Acid 33 was coupled with dimethylcyclopropylproline methyl
ester 34, resulting in the P4-P2 methyl ester 35. Hydrolysis of
35 afforded the fully assembled P4-P2 acid 13, after crystal-
lization, in 86% yield. The two advanced intermediates, P4-P2
acid 13 and P1-P0 amine hydrochloride 21, were coupled
employing essentially conditions described above. After a brief
study, we identified appropriate conditions for the final oxida-
tion. Thus, intermediate 36 was oxidized using bleach/TEMPO
under buffered conditions. The product obtained after workup
was crystallized from acetone-water mixture to provide 37 in
high yield. It is noteworthy to mention that the entire sequence
for the synthesis of 37 was carried out without the need for silica
gel purification, while maintaining the stereochemical integrity
at the P1 center.
Target compounds synthesized in these studies were
tested for HCV NS3 serine protease inhibition using the
continuous spectrophotometric assay,17 and inhibition of
HCV replication in vitro using the subgenomic replicon
assay.18 All compounds were tested for inhibition of human
neutrophil elastase (HNE) as a measure of selectivity. Selec-
ted compounds were then subjected to in vivo studies.
As described above, inhibitor 3, with tert-butyl sulfone
capped cyclohexyl moiety at P4 exhibited improved replicon
potency and monkey oral exposure compared to 1. However,
this series of inhibitors containing P0 primary amide residue
displayed poor rat PK profile. During our studies that led
to the discovery of 1, it was observed that P0 secondary amides,
specifically allyl amides, provided inhibitors with good
rat PK profile. In order to improve rat plasma exposure of P4
sulfone capped inhibitors, we then set out to introduce allyl
residue at the P0 position. We also knew from our previous
studies that P1 cyclobutyl alanine moiety, in general, was not
tolerated with allyl (or small alkyl) residue at P0 position. Hence,
introduction of P0 allyl amide was carried out with various P1
surrogates to identify the appropriate P1-P0 combination
(Tabl e 1 ).
3
3
on the potency, selectivity and pharmacokinetic profile, includ-
ing higher rat oral bioavailability, we decided to continue further
studies with norleucine moiety at P1.
While exploring allyl residue as an appropriate P0 substi-
tuent to improve rat pharmacokinetic profile of the inhibi-
tors, we were cognizant of the fact that a terminal double
bond may potentially be a toxicological liability. In order to
assess this potential liability, an analogue (structure not
shown) containing an allyl residue at P0 was subjected to
rat in vivo study. Results indicated that minor amounts of
hydroxy-glutathione (GSH) adduct were formed. This obser-
vation raised the possibility of in vivo formation of a reactive
epoxide from the terminal olefin, which would be highly
undesirable and would have toxicological concerns. Thus,
our next task was to find a suitable replacement for the allyl
moiety. Furthermore, inhibitors described above were all
diastereomeric mixtures at P1, similar to 1. A key require-
ment that we had set forth was identification and develop-
ment of an appropriate inhibitor as a single isomer. Hence
we conducted further SAR studies on the P0 region using
norleucine P1 moiety as a single isomer (S-stereochemistry).
Results of the P0 SAR studies are shown in Table 2.
In our efforts to replace the allyl moiety, we introduced a
methyl group at P0 resulting in compound 43, with desirable
enzyme (Ki* = 4 nM) and replicon potency (EC90 = 55 nM),
and very good selectivity (HNE/HCV = 1500). While the rat
AUC was reasonable (2.2 μM h) for 43, monkey AUC was
3
rather low (0.4 μM h). Extending the chain to P0 ethyl moiety
3
provided 44 with slight loss in potency and selectivity, albeit
moderate improvement in rat AUC. n-Propyl, 45, and
cyclopropylmethyl analogue, 46, exhibited similar potency
(EC90 = 100 nM). Introduction of cyclopropyl moiety at P0
afforded inhibitor 37, with significantly improved overall
profile. Thus, 37 showed excellent potency (EC90 = 40 nM),
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Table 2. P0 SAR of Inhibitors of Type 15
Table 3
a iv/po (2 or 4/10 mpk). b po (10 mpk). c iv/po (1/3 mpk). d po (3 mpk).
e P3 = β-MeChg. Vehicle: po, 0.4% HPMC or 20% HPBCD; iv, 40% HPBCD.
exception of 66 (EC90 = 50 nM). Unfortunately, 66 displayed
no plasma exposure on oral dosing in rats.
From these explorations, it was clear that the cyclopropyl
moiety was the best P0 residue (37), providing a superior
overall profile compared to 1. Results from Table 1 demon-
strated that P1 variations could have a beneficial influence
on the potency profile of the inhibitor. Having identified the
appropriate P0 moiety, we then investigated the P1 area
in order to find the most suitable combination (Table 3).
Inhibitor 67, with norvaline P1, exhibited similar potency
and PK profile as 37. Unfortunately, the selectivity (HNE/
HCV) was poor. Introduction of trifluoronorvaline P1 resi-
due, expected to improve the selectivity based on our
previous studies, resulted in inhibitor 68. A measurable
enhancement in selectivity was observed for 68, albeit with
less than acceptable rat exposure. While P1 cyclopro-
pylalanine containing inhibitor 69 was equipotent to 37,
rat exposure and selectivity were not optimal. Incor-
poration of homocyclopropylalanine P1 residue, expected
to enhance selectivity, resulted in inhibitor 70 with EC90
=
30 nM, good selectivity (HNE/HCV=820) and monkey expo-
sure similar to 37.
Thus, our efforts led to the identification of two promising
compounds, 70 and 37, with desirable characteristics. While
both the inhibitors were essentially equipotent, 37 demon-
strated a better overall pharmacokinetic profile. When dosed
a po (10 mpk). b iv/po (4/10 mpk). c po (3 mpk). d iv/po (1/3 mpk).
Vehicle: po, 0.4% HPMC or 20% HPBCD; iv, 40% HPBCD.
∼10-fold improvement over compound 1. Moreover, rat
and monkey exposure were also superior to 1, with AUC =
6.5 and 1.1 μM h, respectively. Increasing the P0 ring size to
3
orally in rats, 37 displayed very good AUC of 6.5 μM h, and
3
cyclobutyl (47) and cyclopentyl moiety (48) resulted in
gradual loss in enzyme potency. Similarly, substitution of
the cyclopropyl ring proved to be detrimental (49, Ki* =
300 nM). While ether containing P0, 50, exhibited good
replicon potency (EC90 = 80 nM), the sulfone containing
inhibitor, 51, showed no improvement in potency com-
pared to 1. Aromatic and heteroaromatic substitutions at
P0 position (inhibitors 52-61) displayed good replicon
potency, except 53 and 57. Thiazole containing inhibitor
56 exhibited excellent potency (Ki* = 2.7 nM, EC90 = 40
nM, ∼10-fold improvement over 1) and selectivity (HNE/
HCV = 1200). While monkey exposure for 56 was signifi-
cantlyimproved(AUC=1.9 μM h), ratPK(AUC = 1.2 μM h)
46% bioavailability. 37 was well distributed in the target
organ; rat liver concentration at 8 h post dosing (C8h) was
750 ng/g. In a similar rat in vivo study, 70 exhibited lower
AUC and oral bioavailability (2.1 μM h, and 21%), and liver
3
C8h was only 90 ng/g. Dog oral exposure for 37
was moderate (AUC = 0.9 μM h, F = 29%). The corres-
3
ponding data for 70 were slightly lower, AUC = 0.5 μM h and
3
F = 16%. More importantly, in monkeys inhibitor 37
displayed an AUC of 1.1 μM h, with 46% oral bioavailability,
3
a significant improvement over 1. This improvement, while
not apparent based on the structural features of 37, could
have possibly occurred through some form of transport
mechanism that was specific to monkeys. Based on the
aforementioned pharmacokinetic profile in three species,
inhibitor 37 was considered for further evaluation.
3
3
was similar to that of 1. Amino acid derivatives as P0 surro-
gates (inhibitors 62-66) resulted in loss in potency, with the
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the potency. Optimization of the synthetic sequence resulted
in a route that did not require any silica gel purification for
the entire synthesis, with 37 being obtained in a crystalline
form, thus solving a key developmental requirement. Having
achieved all the criteria set forth for a suitable second
generation HCV NS3/4A serine protease inhibitor, 37 was
advanced for further toxicological studies and was found to
be safe.23 Inhibitor 37 is currently undergoing phase II
clinical studies in humans.
SUPPORTING INFORMATION AVAILABLE Experimental
details of synthetic procedures and characterization data. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Figure 2. NS3 protease bound X-ray structure and profile of 37.
Preclinical resistance studies indicated that 37 was cross-
resistant to mutations raised against 1, in both enzymatic and
replicon assays. However, 37 retained more activity against
many of the mutants due to its higher intrinsic potency
compared to 1.22 Inhibitor 37 displayed no significant issues
in the in vitro CYP or hERG assay. Unlike an early compound
containing P0 allyl moiety, no glutathione or acyl glucuronide
conjugates were observed with 37 in rats, dogs and monkeys,
thus indicating no reactive metabolite issue. Plasma protein
binding data (in vitro, hu) were essentially similar for both 1 and
37. Thus, based on all the preclinical investigations, and having
achieved improvements in potency (∼10-fold over 1), phar-
macokinetic profile (improved monkey and rat exposure),
and physicochemical characteristics (crystalline, single isomer),
inhibitor 37 was advanced as development candidate.
Accession Codes: †Coordinates for 37 have been deposited
with the RCSB Protein Data Bank under the code 3LON.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Phone: 908-740-2607. Fax: 908-740-7152. E-mail:
ashok.arasappan@spcorp.com.
ACKNOWLEDGMENT We thank Structural Chemistry Depart-
ment at Schering-Plough Research Institute for NMR and MS
analysis for all new compounds.
REFERENCES
Binding of 37 to the active site was confirmed by X-ray
crystal structure of the protease bound complex (Figure 2).
As disclosed previously, the core (P3-P0) region of the
inhibitor 37 was bound to the protease via a series of
hydrogen bonding and hydrophobic interactions.11 The
newly introduced P4 cyclohexyl moiety was positioned
appropriately for additional hydrophobic contact with the
enzyme S4 pocket. Moreover, one of the P4 sulfone oxygen
atoms was in close proximity for hydrogen bonding inter-
action with Cys-159. These two additional favorable inter-
actions with the enzyme resulted in improved potency.
In conclusion, our efforts toward identification of a suita-
ble second generation HCV NS3 protease inhibitor were
initiated based on the P4 sulfone capped inhibitor, 3, ex-
hibiting good replicon potency, good monkey exposure but
low rat PK. Introduction of P0 allyl amide moiety, with
concomitant P1 modifications, resulted in inhibitor 41, with
enhanced rat exposure, while retaining monkey PK. Since
replacement of allyl moiety was necessary to alleviate any
potential toxicological issues, concerted efforts were direc-
ted at identification of a suitable nonolefin containing P0
residue. These investigations were carried out on P1 norleu-
cine containing inhibitor, as a single isomer. P0 cyclopropyl
amide turned out to be the optimal group giving rise to
compound 37, with ∼10-fold improvement in replicon
potency over 1. Furthermore, 37 displayed very good rat
exposure, and significantly improved monkey exposure in
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target organ (rat liver C8h/EC90 ∼25). X-ray crystal structure
of the inhibitor (37)-protease complex revealed the impor-
tance of P4 sulfone capped cyclohexyl moiety in improving
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