The discovery of a pan-genotypic, primer grip inhibitor of HCV NS5B polymerase

Article abstract of DOI:10.1039/c6md00636a

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a preclinical candidate.

Full text of DOI:10.1039/c6md00636a

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RESEARCH ARTICLE
The discovery of a pan-genotypic, primer grip
inhibitor of HCV NS5B polymerase†‡
Cite this: DOI: 10.1039/c6md00636a
*
Kyle J. Eastman, Kyle Parcella, Kap-Sun Yeung, Katharine A. Grant-Young,
Juliang Zhu, Tao Wang, Zhongxing Zhang, Zhiwei Yin, Brett R. Beno, Steven Sheriff,
Kevin Kish, Jeffrey Tredup, Adam G. Jardel, Vivek Halan, Kaushik Ghosh,
Dawn Parker, Kathy Mosure, Hua Fang, Ying-Kai Wang, Julie Lemm, Xiaoliang Zhuo,
Umesh Hanumegowda, Karen Rigat, Maria Donoso, Maria Tuttle, Tatyana Zvyaga,
Zuzana Haarhoff, Nicholas A. Meanwell, Matthew G. Soars,
Susan B. Roberts and John F. Kadow
Received 15th November 2016,
Accepted 20th January 2017
The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B
polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavail-
ability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered
and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a pre-
clinical candidate.
DOI: 10.1039/c6md00636a
rsc.li/medchemcomm
up to 1 trillion viral particles a day, 10- to 100-fold higher
than HIV-1, with a median rate of mutation recently esti-
Introduction
Hepatitis is a condition characterized by inflammation of the
liver. Viral infection is the most predominant cause of hepati-
tis, and hepatitis C virus (HCV) is currently the leading cause
of liver cirrhosis and liver cancer across the globe.1 HCV is a
blood-borne virus that specifically targets the liver. Of those
who become infected with HCV, nearly 20% will clear the dis-
ease without medical intervention while the remaining 80%
face chronic infection, which persists for a lifetime without
treatment with antiviral therapy. Progression of the disease
may ultimately lead to cirrhosis, hepatocellular carcinoma or
both.1 Furthermore, estimates have suggested that more than
15 000 HCV-related deaths occur annually in the US, and up-
wards of 1 000 000 deaths per year globally, with an inordinate
percentage of deaths occurring in underdeveloped nations.
HCV has a wide genetic diversity with seven major geno-
types (GT) designated 1–7, of which the most recently identi-
fied is GT-7,2 with each comprised of subtypes. The predomi-
nant GT varies from country to country with GT-1a
predominating in the in the U.S. and Western Europe while
GT-1b predominates in Japan and China. The HCV NS5B
polymerase is error-prone due to the lack of an effective proof
reading function. The high replication rate, estimated to be
mated at 2.5 × 10−5 or 1 nucleotide change for every 10 ge-
nomes replicated, anticipated the need for combination ther-
apy.3,4 Combination therapy, in which two or three direct-
acting antiviral agents (DAAs) with complementary mecha-
nisms of action are co-administered in order to minimize the
emergence of resistance, has become the therapeutic
standard.1
Until 2014, the standard of care (SOC) for the treatment of
HCV relied upon once-weekly injections of the immune mod-
ulator pegylated interferon-α (PEG-IFN) in combination with
twice-daily dosing of the nucleoside analogue ribavirin (RBV).
This regimen was only effective in curing GT-1 infections ap-
proximately 45% of the time after 48 weeks of therapy and
was associated with a high incidence of side effects that in-
cluded severe flu-like symptoms, depression, insomnia, ar-
thritis and gastrointestinal complications. The addition of ei-
ther of the NS3 protease inhibitors telaprevir or boceprevir
(Fig. 1) to PEG-IFN/RBV therapy improved the sustained viro-
logical response (SVR) in GT-1 patients to nearly 70% under
the idealized conditions of clinical trials, although additional
treatment-specific side effects including anemia, neutrope-
nia, and severe rash were often observed. The 2014 approval
of the dual combination of the NS5A inhibitor daclatasvir5
and the NS3 protease inhibitor asunaprevir6 in Japan repre-
sented the first all oral, interferon-free regimen for the treat-
ment of GT-1b patients. Harvoni®, the combination of the
nucleoside analogue sofosbuvir and the NS5A inhibitor
ledipasvir, was approved in the USA in 2014 but is not
Bristol-Myers Squibb Pharmaceutical Research and Development, Department of
Discovery Chemistry and Molecular Technologies, 5 Research Parkway,
Wallingford, Connecticut, USA. E-mail: kyle.eastman@bms.com
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c6md00636a
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Fig. 1 Former and currently marketed HCV drugs.
optimized for the treatment of GT-2 and GT-3-infected pa-
tients. More recently, the approval of Eplcusa®, a combina-
tion of sofosbuvir and velpatasvir, provides an interferon-free
treatment regimen for all HCV GTs.
cellent pharmacokinetic (PK) properties in normal healthy
volunteers. However, a notable drawback of this early family
of compounds was the lack of pan-genotype coverage, which
focused further optimization of this chemotype on broaden-
ing the GT coverage.
Of the non-structural (NS) genes comprising the HCV ge-
nome, Bristol-Myers Squibb has explored inhibitors of NS5A,5
the NS3 protease,6 NS5B site I7 and NS5B primer grip, also
known as site II.8 This communication details further efforts
toward the design of inhibitors of site II of the RNA-
dependent RNA polymerase NS5B. Essential for replication,
the NS5B polymerase has multiple allosteric sites, with site II
located approximately 10 Å from the active site and character-
ized as being both large and flexible in nature. The most
prominent site II inhibitor to date is HCV-796 which em-
bodies a benzofuran core and was progressed into phase II
clinical trials before being terminated due to liver toxicity.9,10
Influenced by the efficacy of HCV-796 and the promise of the
benzofuran chemotype, our own early studies merged this
chemotype with learnings from our anthranilic acid inhibitor
investigations11 to arrive at a family of benzofuran-based
compounds represented generically by 1 that embodies vari-
ably substituted phenyl substituents at C5 and no substitu-
tion at C6.8,12–14 A single representative from this chemotype
was advanced into clinical studies where it demonstrated ex-
Results and discussion
The single antiviral deficiency associated with inhibitors
based on 1 is the lack of inhibition of GT-2a virus. Address-
ing this gap in GT-2 coverage became the key objective for a
backup program which was part of a broader effort to iden-
tify a pan-GT-inhibiting drug combination. The inhibitory
profile targeted was one that encompassed the major GTs 1–
6 with extension to the pre-existing GT-1b C316N mutant,
with EC50 values of less than 20 nM in replicons. From the
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Fig. 2 Alternative cores evaluated.
Table 1 SARs associated with C6 azabenzofuran substitution
a
b
Cmpd
R
GT-1a EC50
GT-2a EC50
GT-1b C316N IC50
CC50
2
3
4
5
H
600 nM
112 nM
45 nM
22 nM
346 nM
57 nM
23 nM
18 nM
447 nM
61 nM
5 nM
13 μM
CH3CH2O
CH3CH2CH2
CH3CH2NH
>100 μM
>100 μM
>100 μM
17 nM
a
b
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details.
perspective of human PK properties, a low human dose pro-
jection to facilitate fixed-dose combinations with once daily
administration was sought.
With satisfactory replicon EC50 values of less than 20 nM
across GTs 1, 2 and the GT-1b C316N mutant and with a
>5000-fold in vitro therapeutic window, 5 was examined in a
rat PK study. The low peak (Cmax) to trough (C24) ratio,
resulting in a nearly flat PK curve, and the long half-life asso-
ciated with 5 were encouraging data toward the potential for
achieving sustained efficacious drug concentrations with a
low dose and an infrequent dosing schedule (Table 2).
With an aim towards maintaining a C5 aryl-substituted
fused 6/5 bicyclic core silhouette similar to 1,
a
pyrazolopyridine as well as the 4-, 6-, and 7-azabenzofuran
cores were evaluated from which the most promising with
respect to targeted inhibitory activity and PK properties was
the 7-azabenzofuran (Fig. 2). Early explorations of a series of
pyrazolopyridine analogues yielded compounds with EC50
values toward the C316N mutant replicon in excess of 500
nM and poor PK properties, while the 4- and
6-azabenzofuran analogues showed disappointing levels of
inhibitory activity toward GT-1a, 1b, 2a and GT-1b C316N
mutant replicons.
A critical discovery toward achieving GT-2a inhibitory ac-
tivity in both the benzofuran and the 7-azabenzofuran series
was the introduction of substitution at C6 of the core hetero-
cycle. In the absence of C6 substitution GT-2a replicon inhib-
itory activity was well beyond the targeted upper end EC50
value of 20 nM (compound 2, Table 1). Introduction of a C6
ethyl ether (3) improved GT-2a inhibitory activity presumably
through increased van der Waals contact with the protein via
partially filling a narrow pocket abutting glutamine 414. Fur-
ther improvements to GT-2a replicon inhibition were
achieved with the incorporation of a propyl (4) or an N-ethyl
(5) substituent, resulting in EC50 values of 23 nM and 18 nM,
respectively. The near 3-fold improvement in GT-2a inhibi-
tion for both 4 and 5 may, in part, be rationalized by
hydrogen-bonding to glutamine 414, a weak hydrogen-bond
with one of the polarized benzylic protons of 4 and a stronger
hydrogen-bond with the NH of 5 (Fig. 3).
Fig. 3 Co-crystal structure of 5 bound to NS5B GT-2a L30S protein.
Compound 5 is depicted in ball and stick representation with orange
carbon atoms. Protein is shown with a green backbone cartoon and
specific residues are displayed in stick representation with either green
or cyan (Q414) carbon atoms. Hydrogen-bonds are denoted as black
dotted lines. The omitted Fo–Fc electron density is contoured at 3
rmsd in magenta mesh. Image generated with the PyMOL molecular
graphics system (v. 1.8, Schrödinger, LLC).15
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An in vitro metabolism study in an Aroclor-induced rat
liver S9 (ref. 16) assay was the next step in the progression of
5. In addition to the observation of amide demethylation,
amine demethylation, and oxidation of the tBu amide moiety,
12% of the material was converted to a glutathione (GSH) ad-
duct. This observation heightened concern for the production
of metabolism-induced reactive intermediates in vivo that
precipitated a systematic evaluation of differential functional-
ity on the western portion of the 7-azabenzofuran core. At this
juncture of the program, the eastern portion of the
chemotype had been subjected to extensive optimization with
the C2 pF-phenyl and C3 methyl amide determined to be op-
timal. This led to a focus on derivatization of the acid 6, a
versatile synthetic intermediate that was prepared by a se-
quence beginning with 2-chloro-6-methoxypyridine (7). Site-
good yield and was followed by a Sonogashira coupling with
the fluoro-substituted phenyl acetylene 11, which proceeded
with concomitant cyclization to afford the 7-azabenzofuran
12. C3 iodination with NIS in the presence of BF3IJOEt)2
furnished 13 in acceptable yield. Carbonylative coupling at
C3 of 13 in the presence of MeNH2 resulted in the formation
of the C3 methyl amide 14 in 67% yield. Finally, a selective
Suzuki coupling at C5 of 14 produced 6. For the purposes of
illustrating the preparation of 5, HATU-mediated coupling be-
tween 6 and tBu amine gave 15 which was subsequently
substituted at C6 under standard Buchwald coupling condi-
tions to give 5 in 43% yield. Similarly, Pd-mediated coupling
between 15 and a suitable coupling partners such as potas-
sium trifluoroIJtrifluoroethyl) borate furnished C6 carbon ana-
logues such as 16 in excellent yield.
selective bromination of 7 afforded the bromopyridine 8 in
68% yield which was deprotected using BBr3 at 75 °C in DCE
to effect clean conversion to the hydroxypyridine 9.
N-Iodosuccinimide-mediated iodination of 9 afforded 10 in
With the observation of in vitro oxidation of the tBu amide
and GSH conjugation, the identification of a suitable combi-
nation of amide and C6 substitution that maintained all of
the positive aspects of 5 while improving metabolic stability
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Table 2 Rat PK data for 5
Cmpda Cmax (μM) IV/PO IV clearance (mL min−1 kg−1
)
t1/2 (h) IV/PO PO (μM) 24 h liver PO (μM) 24 h plasma PO AUC (μM h) % oral F
5
1.7/0.6
12
14/29
1.6
0.56
18
64
a
IV dose = 2 mg kg−1; PO dose = 6 mg kg−1
.
Table 3 Optimization of C6 azabenzofuran substitution
a
b
Cmpd
R
GT-1a EC50
GT-2a EC50
GT-1b C316N IC50
CC50
5
16
17
18
19
20
21
22
23
24
25
CH3CH2NH
CF3CH2CH2
CF3CH2NH
CH3CH2CH2NH
CF3CH2CH2NH
CH3CF2CH2NH
CH3CH2NCH3
CH3
18 nM
14 nM
13 nM
29 nM
13 nM
55 nM
77 nM
337 nM
109 nM
142 nM
145 nM
13 nM
6 nM
5 nM
16 nM
10 nM
10 nM
35 nM
20 nM
96 nM
31 nM
362 nM
64 nM
66 nM
187 nM
>100 μM
>100 μM
>100 μM
>100 μM
>100 μM
>100 μM
92 μM
15 nM
13 nM
39 nM
31 nM
139 nM
43 nM
54 nM
359 nM
77 μM
i-Pr
n-Butyl
HOIJCH2)3CH2
>100 μM
>100 μM
>100 μM
a
b
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details.
in vitro and reducing the extent of bioactivation became the
objective. Attention was first directed toward optimizing C6,
with a particular focus toward nitrogen- and carbon-linked
substituents. With a reliable route to attain significant quan-
tities of 6, SAR evaluation on the western portion of the
7-azabenzofuran core could be rapidly evaluated through am-
ide coupling followed by Pd-mediated derivatization at C6.
Table 3 summarizes the key SARs associated with a select set
of C6 substitutions that were evaluated.
Compared to the ethyl amine, a small improvement in GT-
2a inhibition was observed in association with the slight in-
crease size and lipophilicity associated with terminal fluori-
nation, as exemplified by 17, or with the installation of the
trifluoropropyl substituent in 16. Attempts to shorten the C6
substituent resulted in a significant loss in replicon inhibi-
tory activity (22). Extending the C6 N-substituents (18)
resulted in slightly decreased inhibitory activity toward GT-1a
and GT-1b C316N while the terminally fluorinated 19
maintained the activity of 5. Lengthening the chain in the
carbon series (24) or adding terminal polarity (25) had detri-
mental effects, significantly decreasing antiviral activity in all
3 assays. Branching the C6 chain with fluoro (20) or alkyl (23)
substituents or alkylating the nitrogen atom (21) all proved
Fig. 4 Co-crystal structure of 5 bound to NS5B GT-1b WT protein.
Compound 5 is depicted in ball and stick representation with orange
carbon atoms. Protein is shown with a green backbone cartoon and
specific residues are displayed in stick representation with either green
or cyan (M414) carbon atoms. Hydrogen-bonds are denoted as black
dotted lines. The surface of the binding site in the vicinity of C6 of the
7-azabenzofuran is shown in light gray. The omitted Fo–Fc electron
density is contoured at 2 rmsd in magenta mesh. Image generated with
the PyMOL molecular graphics system (v. 1.8, Schrödinger, LLC).15
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Table 4 Antiviral activity and liver microsomal half-life data for variably substituted C6 aryl amides 26–36
Liver microsomal half-lifec (min)
b
GT-1a
EC50 (nM)
GT-2a EC50
(nM)
GT-1b
CC50
Cmpd
R
C316N IC50a (nM)
(μM)
Human
60
Rat
16
Dog
19
Cyno
8
26
6
10
18
>100
27
17
12
18
>100
120
71
37
84
28
29
30
12
25
4
6
17
32
5
13
120
100
50
120
46
120
11
120
22
20
4
>100
2.2
18
96
1.2
31
32
35
46
16
8
43
50
9.5
4.2
64
99
112
20
13
22
>120
112
17
33
34
11
10
8
5
5
4
9
>100
>100
>100
44
96
>120
>120
1.7
7.9
36
13
15
11
>120
18
106
46
35
32
87
305
13.5
>120
>120
>120
39
a
b
c
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details. Human, rat, dog and cynomolgus monkey liver microsome
half-life: see ESI for experimental details.
detrimental to activity. Taken together, these data are consis-
tent with a narrow and shallow hydrophobic pocket (Fig. 4)
that appears to be optimally filled with either C6
trifluoroethyl amine (17) or C6 trifluoropropyl (16)
substitution.
With maintenance or improvement in antiviral activity ac-
companying two structural permutations at C6 (16 and 17),
focus was directed at the amide substitution. Successful de-
ployment of a cyclopropylpyrimidine amide in the context of
efforts toward 1, which offered favorable metabolic stability,
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warranted inquiry into similarly-substituted amides in the
7-azabenzofuran context. The data presented in Table 4 dem-
onstrates that there are minimal differences between
cyclopropyl-phenyl (26), -pyridine (27), and -pyrimidine (28)
amides with respect to activity toward the GT-1a and GT-2a
replicons and the GT-1b C316N mutant. While consistent for
this SAR comparison, it should be noted that the cyclopropyl
moiety does not universally impart an activity advantage
across all of the western aryls evaluated, vide infra. Clearer
differences between the amide substitution pattern emerge
when evaluating liver microsomal (LM) half-life data, with 26
demonstrating unsatisfactory half-life values in rat, dog and
cynomolgus monkey LMs and 27 similarly falling short of a
desired half-life in dog. Most differentiated was the cyclo-
propylpyrimidine 28 with a t1/2 of >120 minutes across all
four species of LMs evaluated. It should be mentioned at this
point that a five membered heterocycle appended to the
cyclopropyl amide that was evaluated early in the program,
oxadiazole 29, offered borderline antiviral activity that taken
together with relatively low LM half-life data contributed to
this chemotype not being pursued at this point of the study.
The oxadiazole moiety would, however, reemerge later in the
program as possessing a unique balance of PK and antiviral
activity (vide infra).
In an attempt to replicate the PK observed with tBu amide
5 and avoid the metabolic liability associated with this func-
tionality, several tBu isosteres were evaluated. In the context
of the C6 trifluoroethylamine substituent, acceptable antiviral
activity was observed with the tBu amide 17, the
bicyclo[1.1.1]pentane12,17,18 33 and the ipso-substituted
methyl cyclobutane 34, while the ipso-substituted methyl
cyclopropane 35 fell just outside of the targeted activity value
in the GT-1a replicon (Table 4). The tBu amide 17 exhibited
poor stability in human and cynomolgus monkey LMs while
the t1/2 for the ipso-substituted methyl cyclobutane amide 34
was short in all species LMs. The bicyclo[1.1.1]pentane-based
amide 33 exhibited very good stability in human, rat and dog
LMs, and while the half-life in cynomolgus monkey LMs was
36 minutes, the overall profile was considered acceptable for
further progression, thus positioning the bicyclo[1.1.1]pentane
as a tBu isostere suitable for further evaluation.
As a result of the SAR evaluation and optimization of C6
substitution and the western amide functionality, the cyclo-
propylpyrimidine amides 28 and 36 and the bicyclo[1.1.1]
pentane amides 33 and 37 were identified as the leading can-
didates for further study. All four compounds expressed repli-
con EC50 values of less than 15 nM toward all GTs tested.
Both of the C6 trifluoroethylamine analogues 28 and 33
demonstrated time-dependent inhibition (TDI) of cytochrome
P450 (CYP) 3A4 when evaluated in human LMs. Detection of
5% GSH adducts upon evaluation of 36 in an in vitro Aroclor-
induced rat liver S9 assay halted progression of this com-
pound. Combining the bicyclo[1.1.1]pentane and the C6
trifluoropropane substituents in 37 resulted in a compound
with undetectable bioactivation, no CYP liability and an ex-
cellent balance of pan genotype activity (Table 5) and good
in vitro metabolic stability.
With the identification of a cyclopropylpyrimidine moiety
imparting a good balance of antiviral activity and metabolic
stability, efforts were undertaken to identify additional
binding interactions through alternative functionalization of
the benzylic cyclopropyl element. Table 4 shows that the
cyclobutane 30 achieved a 3-fold improvement in GT-1a and
GT-1b C316N mutant inhibitory activity compared to 28;
however, a short half-life in rat and cynomolgus monkey LMs
coupled with the reduced in vitro therapeutic index due to
a CC50 value of 2.2 μM eliminated 30 from consideration.
Neither the oxetane 31 nor the gem-dimethyl substituted
homologue 32 offered an advantage in GT-1a replicon or
GT-1b C316N mutant inhibition, thus establishing the
cyclopropyl as the preferred substituent for pairing with the
pyrimidine.
The rat PK profile of 37 (Table 5) was promising, demon-
strating lower clearance and a PO half-life similar to 5 as well
as a flat oral PK curve that resulted in very similar Cmax and
24 h liver levels that were sustained for an extended period,
declining by an additional 10% at 48 h. When evaluated in
the presence of 40% human serum (HS), the EC50 value in
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Table 5 Rat PK comparison of 37 and 5 and full GT activity for 37
IV
PO
Cmax
(μM)
IV/PO
clearance
(mL
PO (μM)
24 h
liver
(μM)
48 h
liver
PO (μM)
24 h
plasma
t1/2 (h) IV/
PO
PO AUC
(μM h)
Cmpda
min−1 kg−1)
% oral F
5
37
1.7/0.6
3.2/0.9
12
7.3
14/29
14/20
1.6
0.9
na
0.82
0.56
0.16
18
10
64
44
EC50 (nM)
GT: 1a
EC50 (nM)
1b C316Nb
9.6
CC50
>100 μM
1aFBS
16
1aHS
158
c
cmpd
1b
4
2a
8
2b
3a
4
4a
2
5a
6a
10
37
9.6
3
0.2
a
b
c
IV dose = 2 mg kg−1; PO dose = 6 mg kg−1
.
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details.
the GT-1a replicon increased ten-fold to 158 nM and target
exposure in vivo is typically set as a multiple of this value.
The HS-adjusted EC50 in the human dose prediction gave an
estimated 150 mg QD for 37, twice that of our previous clini-
cal candidate.8 Consequently, further modification of amide
substitution was initiated in an effort to improve upon the
HS shift and thus lower the human dose prediction, based
on the anticipation of final deployment within a fixed dose
combination.
The direction of the next phase of the program emerged
from an analysis of the shift in antiviral activity in the pres-
ence of HS for a series of analogues. These data indicated
that aliphatic or phenyl (26, Table 6) substituents experi-
enced a higher shift in the presence of HS while hetero aro-
matic substituents were more generally associated with lower
HS shifts. While the antiviral potency of both the pyridine 27
and the pyrimidine 28 shifted by 3.6-fold in the presence of
HS, the pyridine series was dropped from consideration due
Table 6 FBS and HS activity data imparted by western aromatic substituents
EC50 (nM)
Cmpd
R
GT-1a (FBS)
8
GT-1a (HS)
102
HS shift
12.8
26
27
28
29
22
31
56
80
112
226
3.6
3.6
4
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to its less than optimal metabolic stability in LMs from sev-
eral species. Despite a good balance of antiviral activity and
metabolic stability, challenges with bioactivation, human
liver microsome (HLM) CYP TDI, as well as universally poor
rat PK properties in the 7-azabenzofuran series rendered
pyrimidines as suboptimal. 1,2,4-oxadiazole 29 showed a sim-
ilarly low HS shift as the pyridine and pyrimidine and optimi-
zation of compounds incorporating this ring system were
pursued in an effort to identify compounds with the targeted
antiviral profile, a favorable HS shift, and maintenance of all
other beneficial aspects of 37.
Data for the three oxadiazoles 38–40 evaluated in the con-
text of the more active gem-dimethyl benzylic substitution are
compiled in Table 7. Both the methylated 1,2,4-oxadiazole 38
and its topological isomer 40 emerged as possessing excellent
antiviral activity. However, LM stability for both of these com-
pounds was marginal while the 2-methyl-1,3,4-oxadiazole 39
failed to meet targeted antiviral activity. In an effort to block
suspected oxidative metabolism on the C3 methyl of 40, the
trifluoromethylated 1,2,4-oxadiazole 41 was prepared. Unfor-
tunately, fluorination proved detrimental to antiviral activity.
A key observation in this series was that removal of substitu-
tion from the 1,2,4-oxadiazole offered the best balance of prop-
erties, as exemplified by the C6 trifluoroethylamine analogue
42 and the C6 trifluoropropyl analogue 43, both of which
exhibited EC50 values of less than 10 nM across all 3 assays, a
HS shift of only 5-fold and targeted in vitro metabolic stability.
The rat PK results for both 42 and 43 are compared to 37
in Table 8. Trifluoroethylamine 42 demonstrated a slightly
lower Cmax, higher IV clearance, and a shorter half-life than
37, while drug levels in plasma and liver 24 hours post-dose
were low and oral bioavailability was modest at 31%. With a
slightly lower IV and comparable oral Cmax, 43 marked an im-
provement in clearance and exhibited a similar IV half-life to
37. The oral half-life was comparably short; however, when
viewed in context of the high liver concentration of 1.5 μM at
24 hours, nearly matching the benchmark oral liver concen-
tration of 37, 43 was well positioned for further evaluation.
When assayed in Aroclor-treated rat liver S9 cells, 43 suffered
no observable bioactivation. Additionally, 43 exhibited a
dose-proportional increase of exposure across the dosing
range of 5, 15 and 30 mg kg−1 in rats while 37 did not.
Table 7 Antiviral activity and liver microsomal half-life data for a series of substituted oxadiazole derivatives
GT-1b
Liver microsomal half-lifec (min)
GT-1a
EC50
GT-2a
EC50
C316N
IC50
a
b
CC50
Cmpd
R
x
(nM)
(nM)
(nM)
(μM)
Human
13
Rat
25
Dog
7
Cyno
5
38
NH
5
3
15
8.8
39
40
41
42
43
NH
NH
NH
NH
CH2
21
4
49
2
27
17
99
9
5.0
na
na
na
na
14.7
>100
5.2
108
na
59
3
13
39
9
53
3
na
na
na
>120
>120
>120
>120
>120
>120
>120
67
9
4
6
>100
a
b
c
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details. Human, rat, dog and cynomolgus monkey liver microsome
half-life: see ESI for experimental details.
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Table 8 Rat PK comparison of 42 and 43 with 37
cmpda
Cmax (μM) IV/PO
IV clearance (mL min−1 kg−1
)
t1/2 (h) IV/PO
PO (μM) 24 h plasma
PO AUC (μM h)
% oral F
37
42
43
3.2/0.9
2.5/0.6
1.7/1.1
7.3
9.3
5.7
14/20
5.0/5.4
12/6.7
0.91
0.38
1.5
0.16
0.039
0.15
44
31
58
a
IV dose = 2 mg kg−1; PO dose = 6 mg kg−1
.
Table 9 Full GT activity data and three species PK for 43
EC50 (nM)
EC50 (nM)
1a (FBS)
11
Cmpd
GT: 1a
9
1b
3
2a
4
2b
3
3a
3
4a
2
5a
6a
6
1b C316Na
6
CC50
>100 μM
1a (HS)
52
b
43
0.3
IV clearance
(mL min−1
t1/2 (h)
IV/PO
PO AUC
(μM h)
Species
kg−1
)
Vss (L kg−1
)
% oral F
Ratc
5.7
1.2
5.8
5.6
3.3
2.8
12/6.7
34/35
6.1/6.4
13
25
8.3
58
85
61
Dogd
Cynod
a
b
c
d
Enzyme data (polyC:pGpG). GT-1b LUC CC50: see ESI for experimental details. IV dose = 2 mg kg−1; PO dose 6 mg kg−1
.
IV dose = 1 mg
kg−1; PO dose 3 mg kg−1
.
Table 9 summarizes the measured antiviral activity data as
well as the rat, dog and cynomolgus monkey PK for 43. The
predicted human dose calculated based on allometric scaling
of rat, dog and monkey PK using the HS-adjusted EC50 value
of 52 nM was 35 mg QD, less than half of that for the previ-
ous clinical compound.8 Clean in both Ames and genotoxicity
assays, 43 was progressed into investigational new drug (IND)
toxicology studies where its progress was halted do to highly
unexpected microcrystallization in multiple tissues at ele-
vated doses in both rats and dogs.
the western amide and C6 resulted in many compounds
meeting antiviral activity requirements. The 1,1,1-
bicyclopentane amide derivative 37 emerged as a promising
lead in all respects. However, stimulated by a desire to
minimize the predicted human dose, further efforts identi-
fied a 1,2,4-oxadiazole heterocycle as a promising western
amide appendage that balanced HS binding while
maintaining benchmark levels of antiviral activity and PK
properties. Ultimately, the effort was rewarded with the dis-
covery of 43 which met all program objectives and was
progressed into IND toxicology studies. While an unusual
finding of microcrystallization in many tissues at elevated
doses during the toxicological studies halted progression of
43, these data informed a parallel backup effort that fo-
cused on improved physicochemical properties. The nature
of the structural changes implemented to successfully im-
part further improvements in PK and solubility will be
disclosed in due course.
Conclusion
With C6 unsubstituted benzofurans 1 as a starting point, a
promising new azabenzofuran core was discovered that
addressed the absence of HCV GT-2 antiviral activity
exhibited by 1 through the introduction of C6 functionality.
Systematic evaluation of combinations of substituents at
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Ethical statement
Rajamani, K. Kish, J. Tredup, H. E. Klei, Q. Gao, L. Mueller,
R. J. Colonno, D. M. Grasela, S. P. Adams, J. Loy, P. C.
Levesque, H. Sun, H. Shi, L. Sun, W. Warner, D. Li, J. Zhu,
N. A. Meanwell and F. McPhee, J. Med. Chem., 2014, 57,
1730–1752.
All these studies were approved by local institutional review
boards and conducted in accordance with Good Clinical Prac-
tice, as defined by the International Conference on Harmoni-
zation, in accordance with the ethical principles underlying
European Union Directive 2001/20/EC and the United States
Code of Federal Regulations, Title 21, Part 50, and in accor-
dance with the ethical principles that have their origin in the
Declaration of Helsinki. Informed written consent was
obtained from all subjects.
7 R. G. Gentles, M. Ding, J. A. Bender, C. P. Bergstrom, K.
Grant-Young, P. Hewawasam, T. Hudyma, S. Martin, A.
Nickel, A. Regueiro-Ren, Y. Tu, Z. Yang, K.-S. Yeung, X.
Zheng, S. Chao, J.-H. Sun, B. R. Beno, D. M. Camac, C.-H.
Chang, M. Gao, P. E. Morin, S. Sheriff, J. Tredup, J. Wan,
M. R. Witmer, D. Xie, U. Hanumegowda, J. Knipe, K.
Mosure, K. S. Santone, D. D. Parker, X. Zhuo, J. Lemm, M.
Liu, L. Pelosi, K. Rigat, S. Voss, Y. Wang, Y.-K. Wang, R. J.
Colonno, M. Gao, S. B. Roberts, Q. Gao, A. Ng, N. A.
Meanwell and J. F. Kadow, J. Med. Chem., 2014, 57,
1855–1879.
8 K.-S. Yeung, K. E. Parcella, B. R. Beno, J. A. Bender, K. Grant-
Young, K. Rigat, Y.-K. Wang, M. Liu, J. Lemm, K. Mosure, U.
Hanumegowda, X. Zhuo, D. Parker, M. Sinz, K. Santone, D.
Smith, J. Li, K. J. Fraunhoffer, A. Delmonte, E. Colston, C.
Pasquinelli, M. Gao, N. A. Meanwell, S. Roberts, J. Knipe and
J. F. Kadow, presented in part at the 250th ACS National
Meeting & Exposition, Boston, MA, United States, August 16–
20, 2015, 2015.
Acknowledgements
Use of the IMCA-CAT beamline 17-ID and 17-BM at the Ad-
vanced Photon Source was supported by the companies of the
Industrial Macromolecular Crystallography Association
through a contract with Hauptman-Woodward Medical Re-
search Institute. This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science
by Argonne National Laboratory under Contract No. DE-AC02-
06CH11357.
9 N. M. Kneteman, A. Y. M. Howe, T. Gao, J. Lewis, D. Pevear,
G. Lund, D. Douglas, D. F. Mercer, D. L. J. Tyrrell, F.
Immermann, I. Chaudhary, J. Speth, S. A. Villano, J.
O'Connell and M. Collett, Hepatology, 2009, 49, 745–752.
10 A. Feldstein, D. Kleiner, D. Kravetz and M. Buck, J. Clin.
Gastroenterol., 2009, 43, 374–381.
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