Syntheses and initial evaluation of a series of indolo-fused heterocyclic inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2011.03.067

Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles

Full text of DOI:10.1016/j.bmcl.2011.03.067

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2925–2929
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and initial evaluation of a series of indolo-fused heterocyclic
inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus
Xiaofan Zheng a, , Thomas W. Hudyma a, Scott W. Martin a, Carl Bergstrom a, Min Ding a, Feng He a,
⇑
Jeffrey Romine a, Michael A. Poss a, John F. Kadow a, Chong-Hwan Chang c, John Wan c, Mark R. Witmer c,
Paul Morin c, Daniel M. Camac c, Steven Sheriff c, Brett R. Beno c, Karen L. Rigat b, Ying-Kai Wang b,
Robert Fridell b, Julie Lemm b, Dike Qiu b, Mengping Liu b, Stacey Voss b, Lenore Pelosi b, Susan B. Roberts b,
Min Gao b, Jay Knipe a, Robert G. Gentles a
a Discovery Chemistry and Candidate Optimization, Bristol Myers Squibb, Research and Development, 5 Research Parkway, Wallingford CT 06492, United States
b Virology Biology, Bristol Myers Squibb, Research and Development, 5 Research Parkway, Wallingford CT 06492, United States
c Bristol Myers Squibb, Research and Development, US Route 206 and Province Line Road, Lawrenceville, NJ 08540, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The
introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety
resulted in conformationally constrained heterocycles that possess multiple additional vectors for further
exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including:
13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC50 = 0.07
Received 11 January 2011
Revised 15 March 2011
Accepted 17 March 2011
Available online 23 March 2011
lM, %F = 18), are reported.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
NS5B
HCV
Indolo-fused heterocyclic inhibitors
Hepatitis C is a liver disease caused by infection with the
hepatitis C virus (HCV). It affects approximately 180 million of
the world’s population, with four million estimated to be infected
in the United States alone.1 Individuals exposed to the virus can
contract a persistent infection that leads to variable hepatic ne-
cro-inflammation. Those patients that proceed to develop high
inflammatory activity frequently deteriorate and ultimately pres-
ent with fibrosis and cirrhosis, the consequences of which include
liver failure, portal hypertension and hepatocellular carcinoma.
The current optimal therapy: extended treatment (48 weeks) with
pegylated interferon and ribavirin (Peg-IFN/RBV), is poorly effec-
tive in patients infected by genotype 1 HCV, with less than 50%
achieving a sustained viral response (SVR).2 This, coupled with
poor compliance due to a severe side effect profile has prompted
a number of pharmaceutical companies to attempt to develop
direct acting antiviral agents (DAAs) for the treatment of HCV.3
HCV RNA-dependent RNA polymerase (NS5B) has long been
considered an attractive target for therapeutic intervention. It is
essential for viral replication and several companies have identi-
fied active-site or allosteric inhibitors of the NS5B enzyme. Many
of these are being clinically evaluated.4 An early focus of our
research in this area was indole-based allosteric inhibitors5 of
the type shown in Figure 1.
In an attempt to further investigate these early indole leads we
evaluated a number of related indolo-fused heterocycles of the
types shown in Figure 2.6 We designed these structures to retain
known binding moieties, and to introduce bridging elements be-
tween the indole N1 and the 2-position of the pendant aryl group.
We envisioned that such functionality could induce a degree of
pre-organization through conformational constraint, as well as
provide a scaffold from which additional pendant functionality
could be evaluated. In the work presented here, we report on the
impact of the introduction of such novel bridging elements in the
context of both truncated 1 and extended 2 NS5B-inhibitor classes,
as well as discuss the mode of binding and pharmacokinetic (PK)
properties of select examples.
The novel core heterocycles evaluated in this study were pre-
pared as outlined in Schemes 1–3. In the case of hydrocarbon-
bridged analogs such as 3 and 4, two complementary approaches
were developed. The first utilized a key ring-closing metathesis
(RCM) reaction7 for construction of the indolo-fused heterocycle,
as depicted in Scheme 1. Methyl 2-bromo-3-cyclohexylindole-6-
carboxylate8 (11) was coupled with 2-vinyl phenyl boronic acid
using standard Suzuki conditions. Subsequent alkylation of the
indole nitrogen with 3-bromoprop-1-ene afforded the cyclization
⇑
Corresponding author. Tel.: +1 203 6776795.
E-mail address: xiaofan.zheng@bms.com (X. Zheng).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.067

2926
X. Zheng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2925–2929
Figure 1. Early examples of truncated (1) and extended (2) indole-based allosteric
HCV NS5B inhibitors.
precursor 12. RCM using the Grubb’s 2nd generation catalyst7
followed by catalytic hydrogenation and subsequent hydrolysis,
provided the indolobenzazepine 3 in a concise fashion. This acid
was coupled with the cinnamate side chain shown in Figure 2
and subjected to a further saponification to access the extended
indolobenzazepine 4. Alternatively, 3 could also be synthesized
from indole 14 using a Heck reaction9 as the key cyclization step
(Scheme 1). Alkylation of the indole 14 with 1-bromo-2-(3-bromo-
propyl)benzene afforded the aryl bromide 15. This was treated
under standard Heck conditions to generate the desired indo-
lobenzazepine 3. A potential advantage that we anticipated for this
route was that it would allow the introduction of more highly func-
tionalized bridging elements that might be excluded when using
the RCM approach.
Figure 2. Novel truncated (1) and extended (2) indolo-fused heterocyclic NS5B
inhibitors.
Suzuki coupling of 11 with the pinacol ester of 2-hydroxy-
phenyl boronic acid provided the hydroxyphenyl indole derivative
16 in good yield (92%). Alkylation of this intermediate with 1,2-
dibromoethane provided access to the indolobenzoxazepine ring
system in a single step. Subsequent base hydrolysis afforded the
target analog 5. This, in turn, could be coupled with (E)-4-(3-meth-
oxy-3-oxoprop-1-enyl)phenyl-1-aminocyclopentane carboxylate
using standard coupling conditions, and the resultant product
saponified to provide the extended indolobenzoxazepine target 6.
For the synthesis of indolobenzodiazepine analogs 7 and 8, a re-
lated methodology was utilized, as described in the bottom of
The indolobenzoxazepine derivatives 5 and 6 were also pre-
pared from methyl 2-bromo-3-cyclohexylindole-6-carboxylate
(11), as shown in Scheme 2.
Scheme 1. Reagent and conditions: (a) 2-vinylphenylboronic acid, Pd(Ph3P)4, Na2CO3(1 N), LiCl, toluene, EtOH; (b) NaH, allyl bromide, DMF, rt; (c) RCM, Grubb’s 2nd
generation catalyst; (d) H2, Pd/C; (e) NaOH, MeOH, THF, H2O, 90 °C, microwave, 10 min; (f) 1-bromo-2-(3-bromopropyl)benzene, NaH, THF, rt; (g) Pd(Ph3P)4, KOAc, DMA,
150 °C, microwave, 30 min; (h) NaOH, MeOH, THF, H2O, 90 °C, microwave, 10 min; (i) (E)-4-(3-methoxy-3-oxoprop-1-enyl)phenyl 1-aminocyclopentanecarboxylate, TBTU,
diisopropylethyl amine, DMF, rt; (j) NaOH, MeOH, THF, H2O, 90 °C, microwave, 10 min.

X. Zheng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2925–2929
2927
Scheme 2. Reagents and conditions: (a) 2-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)phenol, Pd(Ph3P)4, Na2CO3(1 N), LiCl, toluene, EtOH; (b) NaH, THF, dibromoethane;
(c) NaOH, MeOH, THF, H2O, 90 °C, microwave, 10 min. (d) (E)-4-(3-methoxy-3-oxoprop-1-enyl)phenyl 1-amino-cyclopentanecarboxylate, TBTU, DIPEA, DMF, rt; (e) tert-
butylbromo-acetate, NaH, THF; (f) 2-(tert-butoxycarbonyl-amino)phenylboronic acid, Pd(Ph3P)4, Na2CO3(1 N), LiCl, toluene, EtOH; (g) TFA, CH2Cl2.
Scheme 3. Reagents and conditions: (a) 2-(tert-butoxycarbonyl-amino)phenylboronic acid, Pd(Ph3P)4, Na2CO3 (1 N), LiCl, toluene, EtOH; (b) NaH, allyl bromide, DMF, rt; (c)
RCM, Grubb’s 2nd generation catalyst; (d) TFA, CH2Cl2; (e) H2, Pd/C; (f) NaOH, MeOH, THF, H2O, 90 °C, microwave, 10 min; (g) (E)-4-(3-methoxy-3-oxoprop-1-enyl)phenyl 1-
aminocyclopentanecarboxylate, TBTU, diisopropylethyl amine, DMF, rt.
Scheme 2. Intermediate 11 was first alkylated using t-butyl 2-bro-
moacetate to provide the bromoindole derivative 17. A subsequent
Suzuki reaction allowed the introduction of a pendant N-protected
2-aminoaryl group. Removal of the Boc protecting moiety led to
spontaneous ring closure and the generation of the indo-
lobenzodiazepine 18. Hydrolysis of the ester functionality in this
derivative resulted in the concomitant cleavage of the diazepinone
ring system. However, this was easily reversed under acidic condi-
tions and target 7 could be isolated in reasonable yield. Coupling of
intermediate 7 with the aforementioned cinnamate and final
saponification also resulted in the opening of the lactam ring,
which fortunately, once again spontaneously closed on acidificat-
ion to provide the targeted extended indolobenzodiazepine 8.
For the syntheses of the indolobenzodiazonines 9 and 10, an
RCM reaction was again employed, as shown in Scheme 3. Starting
from intermediate 11, Suzuki coupling with an N-protected 2-ami-
nophenyl boronic acid provided intermediate 19 in satisfactory
yield.
Treatment with an excess of sodium hydride and allyl bromide
resulted in a double alkylation, and the intermediate 20 could be
obtained in good yield (77%). RCM of 20 subsequently yielded
the target heterocycle 21. After removal of the Boc moiety, similar
procedures to those outlined above for the preparation of 3, al-
lowed access to the desired indolobenzodiazonines, 9 and 10.
With these compounds in hand, we evaluated the initial propo-
sition that such bridged structures might demonstrate improved
potency with respect to their non-bridged congeners due to appro-
priate pre-organization and conformational constraint.9
The acyclic analogue 1 was used as a basis for comparison. The
minimized structure of 1 ( Fig. 3) displays a dihedral of 65.6°

2928
X. Zheng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2925–2929
Table 2
Rat PKa results observed with select examples of the indolo-fused tetracyclic NS5B
inhibitors shown in Figure 2
Compound
Fb (%)
AUC (
l
M⁄h)
Clearancec (mL/min/kg)
3
6
1.3
53
4
6
7
8
8
5
17.6
3
12.3
0.28
0.79
2.2
0.66
3.61
250
17
35
13
8.7
10
a
b
c
n = 3. Vehicle iv and po PEG400.
Oral bioavailability.
iv (dose 2 mg/kg).
1 (65o). Despite this, 9 displays potency similar to compounds with
more rigid seven-membered rings and smaller dihedral angles. In
these compounds, the IC50s depend more on the content of the
bridge than on the enforced dihedral angle. Taken collectively,
one interpretation of these results is that the ring systems evalu-
ated here do not lock the pendant aryl group in an optimal posi-
tion. Instead, the observed differences in potencies may be
attributed to direct interactions between the bridging elements
and the enzyme. This idea will be elaborated upon in future
publications.
Figure 3. Molecular mechanics (OPLS)10 minimized structures of the indolo-fused
heterocycles depicted in Figure 2. The dihedral angle between the phenyl and
indole ring systems for each compound is listed in Table 1.
Table 1
Comparison of inhibitor potency with dihedral angle, as indicated in Figure 3
a
Compound
Dihedral angle (°)
IC50
(
lM)
Previous reports have noted that the introduction of an
(E)-3-(4-(1-aminocyclopentanecarboxamido)phenyl) acrylic acid
side chain can significantly increase the potency of indole-6-car-
boxylate NS5B inhibitors.12 As described above, this was explored
with analogs 4, 6, 8, and 10, as shown in Figure 2. In all cases, sig-
nificant increases in potency of 10- to 20-fold were observed rela-
tive to the 6-carboxylates. In the case of compound 8, the EC50 of
the extended indolobenzodiazepinone was 10 nM, which repre-
sented the optimal activity achieved with these early examples.
Subsequently, a co-crystal of this analog complexed to NS5B [1b
Bartenschlager construct]13 was obtained, and is shown in Figure 4.
From this structure it can be seen that compound 8 binds in an
area in the NS5B thumb domain.14 The key-cyclohexyl ring binds in
a small hydrophobic pocket that is known to interact with a resi-
due in the finger domain of the enzyme when NS5B adopts a trans-
criptionally active conformation. The amide N–H moiety in the
pendant side chain forms a hydrogen-bond with ARG 503, and
the terminal carboxylate interacts with ARG 498. The pendant phe-
nyl group appears to lie in a hydrophobic cleft and adopts a dihe-
dral angle relative to the indole ring of ꢀ46o, suggesting the
bioactive conformation to be similar to that observed in the mini-
mized structures reported above. Notable by their apparent ab-
sence are specific interactions between the bridging element and
the enzyme. However, in the structures that we have obtained to
7
3
5
1
9
43
45
49
66
71
0.07
0.84
0.69
0.12
0.60
a
Values are means of at lease two test occatsions run in duplicate, standard
deviations are <40% of mean.
between the pendant phenyl and the core indole ring, and has an
IC50 of 0.12
tam bridge, as in 7, the dihedral angle is 43° and a modest increase
in potency is observed to 0.07 M. However, locking in similar
dihedral angles, as in 3 and 5, causes a loss in potency (Table 1).
The cyclic analogs 3, 5, and 9 are approximately equipotent with
IC50s of 0.84, 0.69, and 0.60 lM, respectively. Compound 9 contains
a flexible nine-membered ring that adopts a preferred dihedral an-
gle of ꢀ71°; closer to the dihedral angle observed with compound
l
M.11 When constrained with a seven-membered lac-
l
date, the
D1 fingertip region of the enzyme, which normally binds
at this site, appears disordered, and no such contacts can be
assigned.
Finally, the PK profiles obtained in the rat for select examples of
the tetracyclic indoles described above are summarized in Table 2.
PK data were obtained for the truncated analogs 3 and 7, and for
the extended inhibitors 4, 6, 8, and 10. In the case of the former,
the more polar lactam 7 showed significantly increased exposure
and improved bioavailability relative to the propano-bridged
derivative 3. The extended inhibitors 4, 6, 8, and 10 all exhibited
poor PK profiles in rat. Of the compounds examined, compound 7
appeared to be the most promising from a PK perspective. Replace-
ment of the acid functionality in this molecule might be expected
to improve the exposure achievable with this series, and as we
have established that this vector can be exploited to improve the
potency, an obvious path for the further optimization of this class
of inhibitor is apparent. This will be commented upon in future
manuscripts.
Figure 4. Compound 8 bound to HCV genotype1b NS5B (Bartenschlagger).14 The
protein in the vicinity of the P495 site is represented by a surface (gray) and cartoon
(green). The side chains of Pro495, Arg498, and Arg503 and compound 8 are
represented by sticks (cyan and yellow carbon atoms, respectively). The initial
(prior to placing compound 8 in the model) 2Fo-Fc electron density is contoured at
1
r
(magenta). Figure drawn with PyMola.15

X. Zheng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2925–2929
2929
6. Hudyma, T.W.; Zheng, X.; He, F.; Ding, M.; Bergstrom, C.P.; Hewawasam, P.;
Martin, S.W.; Gentles, R.G. PCT Int. Appl. 2006, WO 2006020082.
7. Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: Weinheim;Germany, 2003;
(b) Furstner, A. Angew. Chem., Int. Ed. 2000, 39, 3012; Furstner, A. Alkene
Metathesis in Organic Synthesis; Springer: New York, 1998; (d) Ponzi, S.;
Habermann, J.; del Rosario Rico Ferreira, M.; Narjes, F. Synlett 2009, 9, 1395.
8. (a) (a) Beaulieu, P. L.; Fazal, G.; Kukolj, G.; Jolicoeur, E.; Gillard, j.; Poupart, M.-
A.; Rancourt, J. Int. Patent Appl. 2003, WO 03010140.; (b) Ikegashira, K.; Oka, T.;
Hirashima, S.; Noji, S.; Yamanaka, H.; Hara, Y.; Adachi, T.; Tsuruha, J.-I.; Doi, S.;
Hase, Y.; Noguchi, T.; Ando, I.; Ogura, N.; Ikeda, S.; Hashimoto, H. J. Med. Chem.
2006, 49, 6950.
9. Stansfield, I.; Ercolani, C.; Mackay, A.; Conte, I.; Pompei, M.; Koch, U.; Gennari,
N.; Giuliano, C.; Rowley, M.; Narjes, F. Bioorg. Med. Chem. Lett. 2009, 19, 627.
10. Kozikowski, A. P.; Ma, D. Tetrahedron Lett. 1991, 61, 6860.
11. (a) Wang, Y.-K.; Rigat, K. L.; Roberts, S. B.; Gao, M. Anal. Biochem. 2006, 359,
106; (b) Wang, Y.-K.; Rigat, K. L.; Sun, J.-H.; Gao, M.; Roberts, S. B. Arch. Biochem.
Biophys. 2008, 470, 146.
Acknowledgment
The authors would like to thank Dr. Louis Chupak for his help
and advice in reviewing this manuscript.
References and notes
1. (a) Choo, Q.-L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359; (b) Lavanchy, D. Liver Int. 2009, 29, 74. Suppl. 1.
2. Farnik, H.; Mihm, U.; Zeuzem, S. Liver Int. 2009, 29, 23.
3. McHutchison, J. G.; Manns, M. P.; Muir, A. J.; Terrault, N. A.; Jacobson, I. M.;
Afdhal, N. H.; Heathcote, E. J.; Zeuzem, S.; Reesink, H. W.; Garg, J.; Bsharat, M.;
George, S.; Kauffman, R. S.; Adda, N.; Di Bisceglie, A. M. N. Eng. J. Med. 2010, 362,
1292.
4. (a) Brown, N. A. Expert Opin. Investig. Drugs 2009, 18, 709; (b) Beaulieu, P. L.
Expert Opin. Ther. Patents 2009, 19, 145; (c) Lin, K.; Hazuda, D. J.; Otto, M. J.
Expert Opin. Investig. Drugs 2010, 19, 963.
12. Beaulieu, P. L.; Dansereau, N.; Duan, J.; Garneau, M.; Gillard, J.; McKercher, G.;
LaPlante, S.; Lagacee, L.; Thauvette, L.; Kukolj, G. Bioorg. Med. Chem. Lett 2010,
20, 1825.
5. Beaulieu, P. L.; Gillard, J.; Bykowski, D.; Brochu, C.; Dansereau, N.; Duceppe,
J.-S.; Hache, B.; Jakalian, A.; Lagace, L.; LaPlante, S.; McKercher, G.; Moreau,
E.; Perreault, S.; Stammers, T.; Thauvette, L.; Warrington, J.; Kukolj, G. Bioorg.
Med. Chem. Lett. 2006, 16, 4987.
13. Lohmann, V.; Körner, F.; Koch, J. O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110.
14. PDB 3Q0Z at 2.29 Å resolution with R-work = 0.200 and R-free = 0.224.
15. Accessible at www.pymol.org.