Discovery of conformationally constrained tetracyclic compounds as potent hepatitis C virus NS5B RNA polymerase inhibitors

Article abstract of DOI:10.1021/jm0610245

We report a new series of hepatitis C virus NS5B RNA polymerase inhibitors containing a conformationally constrained tetracyclic scaffold. SAR studies led to the identification of 6,7-dihydro-5H-benzo[5,6][1,4]diazepino[7,1-a]indoles (19 and 20) bearing a basic pendent group with high biochemical and cellular potencies. These compounds displayed a very small shift in cellular potency when the replicon assay was performed in the presence of human serum albumin.

Full text of DOI:10.1021/jm0610245

6950
J. Med. Chem. 2006, 49, 6950-6953
Discovery of Conformationally Constrained
Tetracyclic Compounds as Potent Hepatitis C
Virus NS5B RNA Polymerase Inhibitors
Kazutaka Ikegashira,*,† Takahiro Oka,† Shintaro Hirashima,†
Satoru Noji,† Hiroshi Yamanaka,† Yoshinori Hara,†
Tsuyoshi Adachi,‡ Jun-Ichiro Tsuruha,‡ Satoki Doi,‡
Yasunori Hase,† Toru Noguchi,† Izuru Ando,† Naoki Ogura,†
Satoru Ikeda,† and Hiromasa Hashimoto*,†
Central Pharmaceutical Research Institute, Japan Tobacco Inc.,
1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan, and
Pharmaceutical Frontier Research Laboratories,
Japan Tobacco Inc., 1-13-2 Fukuura, Kanazawa-ku, Yokohama,
Kanagawa 236-0004, Japan
Figure 1.
Table 1. NS5B Enzyme Assay IC50 for Compounds 1-4 (Formation of
Tetracyclic Compounds)
ReceiVed August 25, 2006
Abstract: We report a new series of hepatitis C virus NS5B RNA
polymerase inhibitors containing a conformationally constrained
tetracyclic scaffold. SAR studies led to the identification of 6,7-dihydro-
5H-benzo[5,6][1,4]diazepino[7,1-a]indoles (19 and 20) bearing a basic
pendent group with high biochemical and cellular potencies. These
compounds displayed a very small shift in cellular potency when the
replicon assay was performed in the presence of human serum albumin.
More than 170 million people worldwide are infected with
hepatitis C virus (HCV),1 and a considerable portion of those
chronically infected are likely to develop cirrhosis of the liver
and hepatocellular carcinoma. However, the current standard
treatment for HCV, a combination therapy of pegylated
interferon-R with ribavirin, has limited efficacy especially
against the genotype 1 virus and is associated with severe
adverse events.2 Therefore, development of an improved anti-
HCV agent remains as an unmet medical need.
A viral protein, HCV NS5B RNA polymerase, is the key
enzyme involved in the replication of the HCV gene and has
been one of the main targets for drug development.3 Several
classes of potent NS5B inhibitors have been reported in the past
couple of years.4-7 Recently, antiviral effects in HCV infected
patients were shown by three inhibitors: nucleoside NS5B
inhibitors NM2838 and R-1626,9 and a non-nucleoside inhibitor
HCV-796.10
We previously found and reported benzimidazole 5-carboxylic
acid derivatives as inhibitors of the NS5B polymerase, which
led to the identification of I (JTK-109, Figure 1).7,11 In the study,
we investigated the effect of a substituent at a position ortho to
the benzimidazole on the central phenyl (C2-ring). We also
identified a fluorine atom as the most effective substituent for
enzyme inhibition and were able to establish that the biochemical
potency was influenced not by electronic effects of the sub-
stituent but by the the steric effect of the substituent.12 We
presumed that the fluorine atom at the ortho position to the
benzimidazole on the C2-ring might make the dihedral angle
between the benzimidazole ring and the C2-ring optimal for
binding to NS5B protein. We hypothesized that fixing the
dihedral angle between the 6,5-bicyclic core and the C2-ring
by bridging them with another ring would increase the potency
a Values are the mean of three independent experiments. Standard
deviations are within 30% of the mean.
against NS5B (Figure 1). Herein, we report the discovery of a
new class of tetracyclic compounds as potent NS5B inhibi-
tors.13,14 This initial work led to the identification of 20 with a
cellular potency of 84 nM in the presence of human serum
albumin. Our assay employed a C-terminally truncated genotype
1b NS5B544 enzyme (BK strain) and Huh-5-2 replicon cells as
previously reported.7
We first designed and synthesized tetracyclic 3 as a confor-
mationally constrained analogue of 1,11 in which the 6,5-bicyclic
core is linked to the C2-ring through a newly formed seven-
membered ring, and the benzimidazole ring was replaced with
an indole to allow for the ring fusion. The unfused tricyclic
N-substituted indole analogue 2 was also tested for compari-
son.15,16 As expected, 3 exhibited a 7-fold increased biochemical
potency (IC50 ) 17 nM) compared to 2 and was 82-fold more
potent than the benzimidazole 1 (Table 1). The added confor-
mational constraint translates to approximately a 10-fold gain
in potency. Furthermore, 4, lacking the benzyloxy substituent,
showed a comparable potency (IC50 ) 26 nM), leading one to
believe that this additional appendage is unnecessary for the
SAR.
* To whom correspondence should be addressed. For K.I.: phone, +81-
72-681-9905; fax, +81-72-681-9725; e-mail, kazutaka.ikegashira@ims.jti.co.jp.
For H.H.: phone +81-72-681-9905; fax, +81-72-681-9725; e-mail,
hiromasa.hashimoto@ims.jti.co.jp.
† Central Pharmaceutical Research Institute.
‡ Pharmaceutical Frontier Research Laboratories.
10.1021/jm0610245 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/02/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 6951
Table 3. SAR at the N5 Atom of Compound 10
Table 2. Evaluation of the A-Ring Size and X Atom Type
a Values are the mean of three independent experiments. Standard
deviations are within 30% of the mean. b The angles were measured in the
most stable conformation with a random conformational search.11 c n ) 1.
d n ) 2.
a Values are the mean of three independent experiments. Standard
deviations are within 30% of the mean. b Assays run with 4% human serum
albumin. EC50 values are the average of two experiments.
Since bridging the 6,5-bicyclic core and the C2-ring led to a
potent tetracyclic scaffold, we studied the effects of the size of
the A-ring and of C or O in position X (five- to eight-membered
rings, 4-9 in Table 2). Regardless of the atom type (X ) C,
O), the optimal A-ring size was found to be a seven-membered
ring. The more planar molecules (five- or six-membered rings)
and the more twisted compound (eight-membered ring) were
less potent. Oxygen as atom X (4, IC50 ) 26 nM) seems to be
better than a CH2 (7, IC50 ) 77 nM). Next, we fixed the A-ring
size to a seven-membered ring and examined other heteroatoms,
such as N and S, at X. Compound 10 (X ) NH, IC50 ) 21 nM)
was as potent as 4, whereas sulfur, as atom X (11, IC50 ) 38
nM), does not appear to be as potent as oxygen and NH. These
compounds, except 5, efficiently blocked the replication of HCV
RNA in the replicon cells at micromolar to submicromolar
concentrations. Compound 10 showed the best EC50 value (0.57
µM) in this series. To clarify the relationship between the NS5B
inhibitory potency and the dihedral angle, we calculated the most
stable conformation of the compounds with a random confor-
mational search.17 As shown in Table 2, the NS5B inhibitory
potency roughly correlates with the measured dihedral angle in
the calculated most stable conformation, suggesting that the
measured angle reflects the actual angle in the bioactive
conformation. The optimal angle seems to be approximately
46° (4 and 10). Although we presume the dihedral angle is the
most important feature, electronic effects also appear to
contribute, as seen by the preference of heteroatoms over a
methylene linkage.
Figure 2. X-ray crystal structure (2.2 Å resolution) of compound 12
bound to the thumb domain of HCV NS5B544 polymerase. Nitrogen,
oxygen, and sulfur atoms are shown in blue, red, and orange,
respectively. Hydrogen bonds between the amide oxygen and Arg503
are shown as white dashed lines.
phenyl ring in the complex structure is 47°, which is in good
agreement with the angle predicted by the above SAR and the
conformational calculation.
At this point, we were eager to examine the actual angle in
the enzyme-bound structure and attempted to obtain a NS5B
crystal complexed with this tetracyclic series in various condi-
tions. Although the trials with the compounds in Tables 2 and
3 were unsuccessful, we obtained an enzyme/inhibitor complex
of an amide derivative 12 (IC50 ) 46 nM), which has been
newly synthesized for the purpose of a SAR study in the
6-carboxylic acid moiety (the result is not shown in this report),
by a soaking method (Figure 2).18 Compound 12 binds to the
allosteric pocket of thumb domain, which was exposed by
displacing the tip of the finger loop on the thumb surface, just
as the indole compounds that were previously reported.19 The
observed dihedral angle between the indole ring and the C2-
Following the discovery of low molecular weight tetracyclic
compounds with inhibitory potency against NS5B in the 20 nM
range, improvement of the cellular potency was attempted
through the introduction of various substituents on the N5 atom
of the diazepine ring of 10 (Table 3). As a first attempt, a methyl
group was introduced (13) and this compound showed equiva-
lent biochemical and cellular potencies compared to 10. Second-
ary alkyl (iPr, 14) and aromatic groups (Bn 16 and phenethyl
17) were well tolerated, while an acyl group (Ac 15) led to
reduced the potency. Since various substituents on N5 seem to
be tolerated, it was hoped that introduction of a polar functional-
ity at this position could also lead to improved cellular activity.
A N,N-dimethylamide analogue (18) was synthesized, and it

6952 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Letters
Scheme 1a
Scheme 2a
a Reagents: (a) Pd(PPh3)4, Na2CO3, LiCl, DME-H2O; (b) BrCH2-
CH2OTHP, NaH, DMF; (c) 6 N HCl (aq), THF-MeOH; (d) DIPAD, PPh3,
THF; (e) BnBr, K2CO3, DMF; (f) 4 N NaOH (aq), THF-MeOH.
exhibited a 3-fold improvement in cellular potency (EC50
)
0.21 µM) compared to 10 with no change in biochemical
potency. Further improvement in cellular potency was achieved
by introduction of a substituent with an amino functionality such
as 2-dimethylaminoethyl (19), resulting in a 10-fold improve-
ment (EC50 ) 53 nM). It is believed that the high cellular
potency of this compound comes from the lower affinity for
serum protein (10% FCS in the assay).20 This rationale is also
substantiated by the fact that 19 did not decrease the cellular
potency greatly (EC50 ) 0.20 µM) in the replicon assay
containing additional human serum albumin (HSA), while 10
and 18 show >15-fold shift in potency (Table 3). Additionally,
given that HSA is the most abundant protein in human blood
plasma and produced in liver, which is the target organ of HCV
inhibitors, the reduced affinity to HSA is an attractive feature
of 19. Further improvements include changing the dimethyl-
amino to a piperidine, which led to a more potent compound
(20) with a 2-fold increase in biochemical potency (IC50 ) 9
nM) and a 16-fold increase in cellular potency (EC50 ) 35 nM)
compared to 10. Again, a very small shift in cellular potency
(2-fold) was observed in the presence of HSA (EC50 ) 84 nM).
Consequently, a >100-fold improvement in cellular activity in
the presence of HSA compared to 10 has been achieved. These
compounds showed very weak cytotoxicity (CC50 > 20 µM)
and good cell permeability using a Caco-2 cell membrane assay.
The tetracyclic compounds tested in this study were prepared
as shown in Schemes 1-3. Compound 3 was synthesized from
2-bromoindole derivative 21 (Scheme 1).16 Suzuki coupling21
with phenylboronic acid 22 provided the 2-phenylindole deriva-
tive 23. N-Alkylation of 23 with NaH/Br(CH2)2OTHP followed
by unmasking of the OH protections followed by an intra-
molecular Mitsunobu reaction22 furnished tetracyclic 24. Alkyl-
ation of the phenol and subsequent hydrolysis of the ester
yielded 3.
The compounds in Table 2 were synthesized as shown
Scheme 2. Compounds 5 and 7 were synthesized from indole
2516 in three steps: N-alkylation to afford 27, intramolecular
Heck reaction,23 and subsequent hydrolysis of the ester.
Compounds 4, 6, 8-11 were synthesized from 21 through route
A or route B. Suzuki coupling of 21 or its pinacol borate with
32-35 provided 28a,b and 29a,b, respectively. Compounds
28a,b were converted to 30a,b in three steps, respectively:
unmasking of the THP ether, conversion of the OH to the
bromide or mesylate followed by intramolecular cyclization.
Selective O-alkylation of 29a and subsequent intramolecular
alkylation afforded the cyclized 30c. Acylation of aniline 29b
with chloroacetyl chloride followed by intramolecular alkylation
and borane reduction of the resulting lactam furnished 30d. The
esters on 30a-d were hydrolyzed to give 4, 6, 8-11.
a Reagents: (a) NaH, DMF; (b) Pd(PPh3)4, KOAc, DMA; (c) 4 N NaOH
(aq), THF-MeOH; (d) 32, Pd(PPh3)4, NaHCO3, DME-H2O; (e) pinacol
borane, Pd(OAc)2, PCy2‚biphenyl, Et3N, 1,4-dioxane; (f) 33, Pd(PPh3)4,
NaHCO3, DME-H2O; (g) 6 N HCl (aq), THF-MeOH; (h) CBr4, PPh3,
CHCl3; (i) MsCl, Et3N, CHCl3; (j) 34, Pd(PPh3)4, NaHCO3, DME-H2O;
(k) 35, Pd(PPh3)4, NaHCO3, DME-H2O; (l) Cl(CH2)nBr, K2CO3, acetone;
(m) CH2Br2, K2CO3, DMF; (n) chloroacetyl chloride, AcONa, AcOH, THF;
(o) BH3/THF, THF.
Scheme 3a
a Reagents: (a) HCHO (aq), NaBH(OAc)3, CHCl3; (b) Ac2O, pyridine;
(c) alkyl halide, K2CO3, DMF; (d) bromoacetic acid tert-butyl ester, K2CO3,
DMF; (e) TFA, CHCl3; (f) amine, WSC‚HCl, HOBt, DMF; (g) BH3/THF,
THF; (h) 4 N NaOH (aq), THF-MeOH.
Compounds 13-20 (Table 3) were synthesized from 30d
(Scheme 3). Reductive aminomethylation of the aniline NH of
30d and subsequent hydrolysis of the ester 31a gave 13.
N-Alkylation or N-acylation of 30d afforded the esters 31b,c.
Compound 31d was derived from 30d in four steps: N-
alkylation with bromoacetic acid tert-butyl ester, unmasking of
tert-butyl ester, coupling with the corresponding amine, and
borane reduction of the amide. The ester 31d was hydrolyzed
to afford 19 and 20.
In summary, we have described the discovery of a new class
of tetracyclic compounds as potent HCV NS5B inhibitors.
Conformational constraint of benzimidazole and indole inhibitors

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 6953
(9) Roberts, S.; Cooksley, G.; Shaw, D.; Berns, H. K.; Brandl, M. T.;
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(12) The Boehringer Ingelheim group reported in their study of benz-
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benzimidazole ring sterically influences the conformation of the
cyclohexyl ring at the N1-position and that steric bulk at the ortho
position reduces the biochemical potency: (a) Lapante, S; Jakalian,
A.; Aubry, N.; Bousquet, Y.; Ferland, J.-M.; Gillard, J.; Lefebvre,
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(14) Two other groups have recently discovered very similar tetracyclic
inhibitors: (a) Hudyma, T. W.; Zheng, X.; He, F.; Ding, M.;
Bergstrom, C. P.; Hewawasam, P.; Martin, S. W.; Gentles, R. G.
Inhibitors of HCV replication. U.S. Patent Appl. 2006/0166964, 2006.
(b) Conte, I.; Ercolani, C.; Narjes, F.; Pompei, M.; Rowley, M.;
Stansfield, I. Tetracyclic indole derivatives as antiviral agents. Int.
Patent Appl. WO 2006/046030, 2006.
(15) Indole inhibitors have been recently reported by several groups. See
refs 5 and 16. Compound 2 was prepared according to the following
patent application: Oka, T.; Yata, S.; Ikegashira, K.; Noji, S.; Akaki,
T.; Hirashima, S.; Niwa, Y.; Ando, I.; Sato, T. Condensed ring
compound and use thereof as HCV polymerase inhibitor. Int. Patent
Appl. WO 2005/014543, 2005.
(16) Beaulieu, P. L.; Fazal, G.; Kukolj, G.; Jolicoeur, E.; Gillard, J.;
Poupart, M.-A.; Rancourt, J. Viral polymerase inhibitors. Int. Patent
Appl. WO 03/010140, 2003.
(17) The random conformational searches were performed with SYBYL
6.9.1 (Tripos Inc., 1669 S. Hanley Road, St. Louis, MO). Details
were described in the supporting information.
(18) The cocrystal structure coordinate has been deposited in the Protein
Data Bank (PDB code 2DXS). For details, see the Supporting
Information. The molecular image was generated with PyMOL
(PyMOL Molecular Graphics System; DeLano Scientific: San Carlos,
CA).
(19) (a) The Boehringer Ingelheim group disclosed an indole derivative’s
binding pocket: Coulombe, R.; Beaulieu, P. L.; Jolicoeur, E.; Kukolj,
G.; Laplante, S.; Poupart, M.-R. Hepatitis C virus NS5B polymerase
inhibitor binding pocket. Int. Patent. Appl. WO 2004/099241, 2004.
(b) The IRBM group reported in their study of enzyme-bound crystal
structure of an indole derivative that the observed dihedral angle is
51°. See ref 5.
through a seven-membered-ring bridge afforded a tetracyclic
scaffold with significantly enhanced enzyme inhibition poten-
cies. We also showed that incorporation of a basic group
significantly improve the cellular potency with a very small
potency shift in the replicon assay containing HSA (20, EC50
) 84 nM). These findings may offer new prospects for the
benzimidazole and indole class of inhibitors in the development
of new anti-HCV agents. Further SAR and optimization studies
including DMPK profiles of this series will be reported in due
course.
Acknowledgment. We thank Mr. Mitsumasa Takahashi and
Mr. Eita Nagao for analytical support. We are grateful to Mr.
Yasushi Niwa and Ms. Yukiyo Tomonaga for support in running
the biological assays and to Mr. Yukihiro Nomura for Caco-2
experiments. We also thank Dr. Ken Murase, Mr. Takuya Orita,
Mr. Akihiro Nomura, and Dr. Masafumi Kamada for assistance
with the synchrotron data collection, Prof. Soichi Wakatsuki
for the data collection at the Photon Factory, KEK, and finally
Dr. Jun-ichi Haruta for support.
Supporting Information Available: Synthetic procedures and
characterization data for 3-20, computational methods, X-ray
crystallographic data, and Caco-2 assay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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