Potent bisimidazole-based HCV NS5A inhibitors bearing annulated tricyclic motifs

Article abstract of DOI:10.1016/j.bmcl.2014.10.056

This Letter describes the synthesis and biological evaluation of a number of functionalized bisimidazoles bearing annulated tricyclic motifs as potent inhibitors of HCV NS5A protein. Compound 4h, which contains a substituted tricyclic 6-6-6 xanthene, demonstrated broad genotypic spectrum, compelling potency, and good oral bioavailability with dose-dependent drug exposure level in multiple animal species.

Full text of DOI:10.1016/j.bmcl.2014.10.056

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent bisimidazole-based HCV NS5A inhibitors bearing annulated
tricyclic motifs
⇑
⇑
Min Zhong , Eric Peng, Ningwu Huang, Qi Huang, Anja Huq, Meiyen Lau, Richard Colonno, Leping Li
Presidio Pharmaceuticals, Inc., 1700 Owens Street, Suite 585, San Francisco, CA 94158, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the synthesis and biological evaluation of a number of functionalized bisimidazoles
bearing annulated tricyclic motifs as potent inhibitors of HCV NS5A protein. Compound 4h, which
contains a substituted tricyclic 6-6-6 xanthene, demonstrated broad genotypic spectrum, compelling
potency, and good oral bioavailability with dose-dependent drug exposure level in multiple animal
species.
Received 12 August 2014
Revised 11 October 2014
Accepted 17 October 2014
Available online xxxx
Ó 2014 Published by Elsevier Ltd.
Keywords:
HCV
NS5A inhibitor
Annulated tricyclic
Bisimidazole
It has been estimated that more than 170 million people
worldwide have been chronically infected by hepatitis C virus
(HCV), which is one of the major pathogens causing chronic
hepatitis, liver cirrhosis, hepatocellular carcinoma, and the related
liver transplant and death.1–4 Due to the deficiency of public
awareness and the lack of diagnostic tools in the past, the number
of HCV infected population has been growing.
Unlike other epidemic liver viruses, such as hepatitis A virus
(HAV) and hepatitis B virus (HBV), there is no vaccine available
to prevent HCV infection and transmission.5 In the past, standard
of care (SOC) therapy for treating chronic HCV infection has been
continually evolving from pegylated interferon alpha (IFN-a) in
combination with ribavirin (RBV)6 to all oral regimens, such as
the recently FDA approved cocktail treatment of HCV NS5B
nucleotide inhibitor sofosbuvir with RBV.7 All-oral regimens con-
sisting of multiple direct acting agents (DAAs) targeting a variety
of HCV targets have become main stream for HCV drug discovery.8
The HCV NS5A protein has been considered as a key therapeutic
target,9,10 along with HCV NS3 protease and HCV NS5B polymer-
ase,8 for developing novel and potent oral HCV DAAs. In fact, a
number of HCV NS5A inhibitors, which can rapidly reduce viral
RNA levels up to 99.99% following a single dose, currently serve
as the core component of several highly effective regimens in
human clinical trials.11
Figure 1. Structure of daclatasvir (BMS-790052) and general structure of novel
functionalized bisimidazole derivatives bearing annulated tricyclic motifs (2–5).
Inspired by the reported structure of the fist-in-class novel
potent HCV NS5A inhibitor daclatasvir (BMS-790052) (Fig. 1),12 we
incorporated a bioisostere strategy13,14 into this structure by
replacing one of the phenyls with structurally constrained aliphatic
ring systems, which led to the discovery of several series of novel
potent HCV NS5A inhibitors.13a To extend such a plausible strategy,
we began an exploratory effort on potential biphenyl replace-
ments.13b Herein, we report the discovery of a number of bisimidaz-
ole-based HCV NS5A inhibitors bearing annulated tricyclic motifs.
⇑
Corresponding authors. Tel.: +1 (415) 655 7560; fax: +1 (415) 986 2864.
E-mail addresses: min.zhong@acmeca.com (M. Zhong), lli@presidiopharma.com
(L. Li).
http://dx.doi.org/10.1016/j.bmcl.2014.10.056
0960-894X/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Zhong, M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.056

2
M. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Scheme 1. Reagents and conditions: (a) ClCH2COCl, AlCl3, DCM, ꢀ20 °C to rt, overnight; (b) N-Boc-
L-Pro-OH, DIPEA, MeCN, rt; (c) NH4OAc, DIPEA, o-xylene, 145 °C, sealed
tube; (d) 4.0 N HCl in dioxane, rt; (e) N-Moc- -Val, HATU, DIPEA, DMF, rt; (f) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, N2, 90 °C, overnight; (g) (S)-tert-butyl 2-(5-
L
iodo-1H-imidazol-2-yl)pyrrolidine-1-carboxylate, NaHCO3, Pd(dppf)Cl2, DME/H2O = 4:1 (v/v), N2, 80 °C, overnight.
Table 1
Scheme 1 describes a general approach to 2–5. Friedel–Crafts
Representative SAR of functionalized bisimidazole derivatives bearing annulated
tricyclic motifs (2-5)
reaction of commercially available 6 with chloroacetylchloride in
the presence of AlCl3 gave 7, which reacted with N-Boc- -proline
L
to generate ester 8. Imidazole formation of 8 with NH4OAc afforded
bisimidazole 9. Subsequently, acid-prompted Boc-deprotection of
9 yielded amine 10, which coupled with N-Moc-L-valine to gener-
ate 2–5. Alternatively, 9 could be prepared using readily available
triflate or bromide 11 as the starting material through a Suzuki
coupling of the corresponding borate 12 with (S)-tert-butyl 2-(5-
iodo-1H-imidazol-2-yl)-pyrrolidine-1-carboxylate.15
Entry
Compd
Bridge
HCV gt-1a
EC50 (nM)a
HCV gt-1b
EC50 (nM)a
For the initial evaluation of those functionalized bisimidazoles
bearing annulated tricyclic motifs, both HCV genotype (gt) 1a
and 1b replicons were used as the primary cell-based assays. As
shown in Table 1, when the symmetric 4,40-biphenyl residue in
daclatasvir (1, entry 1) was replaced with a 2,6-biphenylenyl moi-
ety, the resulting compound (2) has much weaker potency in the
gt-1a and gt-1b replicons (entry 2). Among pseudo-symmetric
2,7-, 2,8-, and 3,7-substitution patterns of dibenzo[b,d]furan
(entries 3–5), the 3,7-dibenzo[b,d]furyl containing analog 3b (entry
4) demonstrated comparable potency to daclatasvir, while the
other two compounds (3a and 3c) had significant loss of potency
for gt-1a. Substitution of the central five-member furan ring in
dibenzo[b,d]furan by a six-member tetrahydro-2H-pyran residue
led to symmetric 2,6- and 2,7-9H-xanthenyl analogs (4a–b, entries
6 and 7). Compound 4b exhibited better gt-1b potency while
retaining similar gt-1a potency relative to daclatasvir (1); however,
4a showed significantly reduced potency. Further expansion of
central ring size to a seven-member ring resulted in compounds
with much weaker potency (5a–c, entries 8–10) relative to 3b.
To better understand the SAR of the central five member ring in
3b, a set of tricyclic 6-5-6 compounds with the –O– atom being
replaced with various substituents was prepared. For initial geno-
typic spectrum assessment, an HCV gt-3a replicon was employed.
As outlined in Table 2, when the –O– atom in 3b was substituted
by a –CH2–, the resulting compound (3d) showed comparable gt-1a
and gt-1b potency while losing some of the gt-3a potency (entry 3).
Reduction of gt-1a potency was observed when the size of –X– was
increased (3d vs. 3e, entries 3 and 4). When the oxidation-labile –
CH2– in 3d was replaced with an oxidation-inert –C(O)– (3f, entry
5), no impact on the gt-1a and gt-1b potency was noted; however,
an oxidation-inert –S(O)2– (3g, entry 6) diminished the gt-1a
potency. Moreover, –NH– (3h, entry 7) and –N(Me)– (3i, entry 8)
were not tolerated. It is worth noting that no compound bearing such
a tricyclic 6-5-6 motif was identified to have superior antiviral profile
to daclatasvir (1) with our limited effort on this series of compounds.
Meanwhile, SAR on the –CH2– in potent 4b (entry 1) bearing
a symmetric tricyclic 6-6-6 motif was established (Table 3).
0.14
0.073
1
2
1
2
(0.050 0.013)b
(0.009 0.004)b
3.0
9.0
0.4
1.8
0.55
3
4
5
3a
3b
3c
0.170
0.041
0.033
6
7
8
9
4a
4b
5a
5b
>100
0.098
4.8
>3.7
0.013
0.069
0.055
7.4
10
5c
>100
>3.7
a
Both HCV gt-1a_LucNeo and 1b_LucNeo Luciferase replicon stable cell lines
were generated in Huh-7 Naïve cells.
b
Reported data, see Ref. 12a.
Replacement of the –CH2– with an –O– atom gave 4c (entry 2) with
much improved gt-3a potency while retaining excellent gt-1a and
gt-1b potency. As observed in the tricyclic 6-5-6 series, the –CH2–
can be readily exchanged with a –C(O)– (4d, entry 3) without hav-
ing any significant impact on the potency. Substitution at the Z
position by either tuning the electronic density of the phenyl ring
or twisting the conformation of the phenyl-imidazole moiety
resulted in 3–20 fold loss of potency for gt-1a (4d vs 4e–g, entries
3–6). Unlike the tricyclic 6-5-6 series, bulkier residues, such as
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M. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 2
Representative SAR of functionalized bisimidazole derivatives bearing a tricyclic 6-5-6 motif (3b and 3d–i)
Entry
1
Compd
X
HCV gt-1a
EC50 (nM)a
HCV gt-1b
EC50 (nM)a
HCV gt-3a
EC50 (nM)a
1
0.14
0.073
0.67
(0.050 0.013)b
0.40
(0.009 0.004)b
0.041
(0.146 0.034)b
2
3
3b
3d
–O–
–CH2–
1.3
3.8
0.25
0.042
(0.094)c
1.14
(0.013)c
0.037
d
4
5
3e
3f
–C(Me)2–
–C(O)–
—
(1.2)c
0.45
(0.014)c
0.045
—
(0.30)c
2.0
(0.018)c
0.13
6
7
8
3g
3h
3i
–S(O)2–
–NH–
–N(Me)–
—
—
—
16.5
7.70
0.18
0.17
a
b
c
HCV gt-1a_LucNeo, 1b_LucNeo, and 3a_(1b)_LucNeo Luciferase replicon stable cell lines were generated in Huh-7 Naïve cells.
Reported data, see Ref. 12a.
Reported data, see Ref. 10n.
Not determined.
d
gem-dimethyl (4h, entry 7) and cyclopropyl (4i, entry 8) can read-
ily interchange with the –CH2– in 4b of this tricyclic 6-6-6 series by
maintaining great gt-1a and gt-1b potency. Moreover, replacement
of the –O– with –N(Me)– in the central tricyclic 6-6-6 ring is not
tolerated (4d vs 4j, entries 3 and 9). As expected, the compound
bearing a tricyclic 6-6-6 phenazinyl (4k, entry 10) as the central
bridge exhibited strong gt-1a and gt-1b potency; however, it
processes an extremely poor solubility, which prevented it from
further consideration.
Following the discovery of this tricyclic 6-6-6 series of potent
HCV NS5A inhibitors, selected members of this series were further
evaluated for their pharmacokinetic (PK) profiles. As presented in
Table 4, both 4b and 4c showed reasonable iv PK profiles in
Sprague-Dawley (SD) rats; however, the highest concentration
in plasma (Cmax) achieved after oral administration was low
(entries 1 and 2). The exposure level of 4d in rats (entry 3)
through either iv or po administration was much lower than its
close analog 4b, which has an oxidation-labile –CH2– in its
structure. Compound 4h demonstrated reasonable Cmax, high
exposure level, good ratio of liver/plasma concentration, great
bioavailability, and dose-dependent increase of drug exposure
level in rats after oral administration (entries 4–7). For the
purpose of potential exploratory toxicity study, 4h was also orally
dosed with elevated dosing levels in beagle dogs (entries 8–10)
and Cynomolgus monkeys (entries 11–13), respectively. As
observed in rats, 4h also showed respectable PK profiles with
dose-dependent drug exposure level in these two animal species.
Based on the data, SD rat was selected as the species for non-GLP
exploratory toxicity study of 4h and the corresponding data will
be discussed in due course.
Furthermore, 4h was evaluated in a broader panel of gt-1a to -
6a replicon assays with daclatasvir (1) being tested side-by-side as
Table 3
Representative SAR of functionalized bisimidazole derivatives bearing a tricyclic 6-6-6 motif (4b–k)
Entry
Compd
X
Y
Z
HCV gt-1a
EC50 (nM)a
HCV gt-1b
EC50 (nM)a
HCV gt-3a
EC50 (nM)a
1
2
3
4
5
6
7
8
9
10
4b
4c
4d
4e
4f
4g
4h
4i
–CH2–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–H
–H
–H
–F
–Cl
–Me
–H
–H
–H
–H
0.098
0.044
0.173
0.60
2.5
3.4
0.073
0.065
11.1
0.013
0.023
0.028
0.030
0.029
0.055
0.011
0.020
0.028
0.037
1.3
0.06
1.4
–C(O)–
–C(O)–
–C(O)–
–C(O)–
–C(Me)2–
–C(CH2CH2)–
–C(O)–
–N@
b
—
—
—
0.29
—
—
4j
4k
–N(Me)–
–N@
0.088
—
a
HCV gt-1a_LucNeo, 1b_LucNeo, and 3a_(1b)_LucNeo Luciferase replicon stable cell lines were generated in Huh-7 Naïve cells.
Not determined.
b
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4
M. Zhong et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 4
PK profiles of representative compounds
Entry
Compd
Species
iv
po
Dose
(mg/kg)
CL
(L/h/kg)
Vss
(L/kg)
t1/2
(h)
AUClast
(h * ng/mL)
Dose
(mg/kg)
Tmax
(h)
Cmax
(ng/mL)
AUClast
(h * ng/mL)
F
(%)
Liver concdc
(ng/mL)
Plasma concdd
(ng/mL)
1
2
3
4
5
6
7
8
4ba
4ca
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Dog
Dog
Monkey
Monkey
Monkey
1
1
1
1
0.95
0.67
3.30
1.07
1.32
1.18
3.57
1.80
1.33
2.53
0.92
1.41
1055
2570
626
5
5
5
5
5
75
250
5
75
250
5
3.33
1.33
0.50
3.00
0.50
4.00
3.33
1.00
4.00
5.00
3.00
3.00
4.00
380
339
96.5
874
761
12367
16150
2765
6395
12155
486
2091
1470
197
5250
4116
136667
187000
16500
83000
191500
3371
41.1
20.1
6.7
112
88.9
—
—
53.4
—
462
973
25
371
199
14
833
—
—
—
—
—
—
—
—
—
4da
4ha
4hb
4hb
4hb
4hb
4hb
4hb
4hb
4hb
4hb
926
2606
e
—
—
—
—
—
—
—
—
—
1
1
0.16
0.42
0.74
1.33
3.77
3.56
6175
2377
9
10
11
12
13
—
28.4
—
75
250
3595
8095
17800
46600
—
a
b
c
The compound was formulated with 10% NMP + 5% solutol + 85% saline (v/v) for both iv and po administration.
The compound was formulated with 5% DMSO + 95% saline for iv and 0.5% MC in saline for po administration, respectively.
Data represent an average of two data points collected at 4 h post a given po dose. The current liver homogenate concentration can be converted into the corresponding
liver tissue concentration by taking a dilution factor of 3 mL/g liver tissue.
d
Data represent an average of multiple data points collected at 4 h post a given po dose. The number of data points varies, depending on the number of animals used in a
given study.
e
Not determined.
Table 5
Comparison of the genotypic profile of 1 to 4h
Compd
HCV gt-1a
EC50 (nM)a
HCV gt-1b
EC50 (nM)a
HCV gt-2a
EC50 (nM)a
HCV gt-3a
EC50 (nM)a
HCV gt-4a
EC50 (nM)a
HCV gt-5a
EC50 (nM)a
HCV gt-6a
EC50 (nM)a
1
0.14
0.073
0.15
0.67
0.041
0.051
0.45
(0.050 0.013)b
0.073
(0.009 0.004)b
0.011
(0.071 0.017)b
0.058
(0.146 0.034)b
0.29
(0.012 0.004)b
0.025
(0.033 0.010)b
0.017
4h
0.062
a
HCV gt-1a_LucNeo, 1b_LucNeo, 2a_(1b)_LucNeo, 3a_(1b)_LucNeo, 5a_(1b)_LucNeo, and 6a_(1b)_LucNeo Luciferase replicon stable cell lines were generated in Huh-7
Naïve cells while gt-4a_(1b)_LucNeo Luciferase replicon stable cell lines were generated in Huh-7 Lunet cells. The variation of the data was typically less than one fold
observed in all of these seven replicons. Data represent an average of multiple data points (n P 3).
b
Reported data, see Ref. 12a.
M.; Lam, A. M.; Micolochick Steuer, H. M.; Niu, C.; Otto, M. J.; Furman, P. A. J.
Med. Chem. 2010, 53, 7202; (b) ‘‘FDA approves Sovaldi for chronic hepatitis C’’.
FDA News Release on Dec. 16, 2013.
a reference. The data suggested that 4h has a broad genotypic
spectrum comparable to daclatasvir (1) (Table 5).
In short, with the extension of the bioisostere strategy, we have
successfully discovered a number of potent bisimidazole-based
HCV NS5A inhibitors bearing annulated tricyclic motifs. Particu-
larly, 4h bearing a novel symmetric tricyclic 6-6-6 motif demon-
strated compelling potency, broad genotypic spectrum, and good
oral bioavailability with dose-dependent effect of drug exposure
level in multiple animal species.
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Acknowledgments
The authors thank Drs. Jason Zhang and Zhi-jie Ni and their
chemistry team at Acme Bioscience, Inc. and Dr. Dejian Xie and
his chemistry team at Shanghai ChemPartner for their excellent
chemistry CRO services, respectively.
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