1,5-Benzodiazepine inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2009.03.035

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydr

Full text of DOI:10.1016/j.bmcl.2009.03.035

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2492–2496
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,5-Benzodiazepine inhibitors of HCV NS5B polymerase
David McGowan, Origène Nyanguile, Maxwell D. Cummings, Sandrine Vendeville, Koen Vandyck,
Walter Van den Broeck, Carlo W. Boutton, Hendrik De Bondt, Ludo Quirynen, Katie Amssoms,
Jean-François Bonfanti, Stefaan Last, Klara Rombauts, Abdellah Tahri, Lili Hu, Frédéric Delouvroy,
Katrien Vermeiren, Geneviève Vandercruyssen, Liesbet Van der Helm, Erna Cleiren, Wendy Mostmans,
Pedro Lory, Geert Pille, Kristof Van Emelen, Gregory Fanning, Frederik Pauwels, Tse-I Lin,
*
Kenneth Simmen, Pierre Raboisson
Tibotec BVBA, Gen. De Wittelaan L11 B3, B-2800 Mechelen, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase
inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-
10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and
(R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-
2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC50 = 400 nM and
Received 20 January 2009
Revised 11 March 2009
Accepted 12 March 2009
Available online 14 March 2009
270 nM, respectively) and selective (CC50 > 20
ther lead-optimization efforts.
l
M) inhibitors of HCV replication. These data warrant fur-
Keywords:
Hepatitis C virus
HCV
Ó 2009 Elsevier Ltd. All rights reserved.
Polymerase
Benzodiazepine
Approximately 170 million people (3% of the world population)
are chronically infected by hepatitis C virus (HCV), a major cause of
acute hepatitis and chronic liver disease.1 Amongst all individuals
contracting HCV, only 20% of them spontaneously clear the virus
from their body, while the majority develops a chronic infection.1
Untreated HCV infection can ultimately progress to cirrhosis, hepa-
tocellular carcinoma, and liver failure.2 As a result, HCV infection is
the major indication for liver transplants in western countries.2
Limited therapeutic options are currently available for patients in-
fected with HCV: the current standard of care therapy consists of
inhibitors of NS5B were initiated and have been subsequently val-
idated in the clinic. These NS5B inhibitors can be divided into two
distinct classes: nucleoside inhibitors (NIs) and non-nucleoside
inhibitors (NNIs). NNIs can be further subdivided into at least four
distinct families based on their respective binding pockets (NNI
sites 1–4, Fig. 1).5
O
NH
pegylated interferon-a combined with the broad spectrum antivi-
N
O
ral drug ribavirin.3 This treatment is often poorly tolerated due to
side effects and is characterized by limited efficacy, especially
when administered to patients infected with genotypes 1a and
1b, those most common in the European Union and the United
States.4 In this context, novel anti-HCV therapies directly targeting
the virus life cycle will hopefully address this unmet medical need,
by improving the efficacy and limiting the emergence of adverse
events.
HO
N
N
N
H
O
COOH
O
S
1
2
(NNI1)
(NNI2)
O
O
S
HN
OH
N
N
O
N
H
The RNA-dependent RNA polymerase (RdRp) NS5B is responsi-
ble for replication of the HCV genome. Intense drug discovery ef-
forts directed toward the identification of potent and selective
F
HO
O
O
N
S
O
O
3
4
(NNI3)
(NNI4)
* Corresponding author. Tel.: +32 15 44 42 62; fax: +32 15 40 12 57.
E-mail addresses: praboiss@its.jnj.com, PierreRaboisson@aol.com (P. Raboisson).
Figure 1. Representative examples of non-nucleoside HCV NS5B inhibitors and
their respective binding pockets (NNI sites 1–4).5
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.035

D. McGowan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2492–2496
2493
to explore the structure–activity relationship (SAR) studies for this
series of 1,5-BZDs at positions R1, R2, and R3.
H
N
H
N
Condensation of aryldiamines 7a–i with dimedone 8 in toluene
at reflux afforded intermediates 9a–i as major regioisomers, which
were then condensed with aldehydes R2CHO to form the 1,5-BZD
10a–x in 12–65% yield. Intermediates 10a–x were subsequently
acylated using either an anhydride or an acyl chloride in the pres-
ence of diisopropylethylamine (DIPEA) to give final products 11a–
c, 11e–g, and 11i (Table 1). Noteworthy, when R1 was a hydroxyl
group, more than one acyl group (R3CO–) was introduced on 1,5-
BZD 10. These undesired acyl functions were readily cleaved in ba-
sic conditions using a dilute solution of lithium hydroxide to afford
the final products 11d, 11h, and 11j–zo (Tables 1–3, 50–95% yield),
each comprising a polysubstituted three-ring system (rings A, B
and C, Scheme 1).
O
O
Cl
Cl
Cl
O
N
N
O
O
5
6
NS5B IC50 = 1.2 µM
Rep EC50 > 32 µM
NS5B IC50 = 0.9 µM
Rep EC50 = 2.3 µM
Figure 2. Previously reported 1,5-BZD HCV NS5B inhibitors.6
We have recently reported the discovery of the novel 1,5-ben-
zodiazepine (1,5-BZD) HCV NS5B inhibitor 5 (Fig. 2), initially iden-
tified by high-throughput screening of
a library of small
As shown in Table 1, preliminary SAR analysis both on ring A
and on substituent R2 was performed on a limited set of deriva-
tives. Amongst the small substituents R1 (compounds 11a–d), the
hydroxyl group was found to be of special interest, leading to a
31-fold improved activity (compare 5 to 11d), as reported by us
earlier.6 Importantly, this activity was found to be highly stereode-
pendent, since the (R)-enantiomer of 11d, separated from (S)-11d
by supercritical fluid chromatography (SFC), was found to be
molecules.6 Herein we report our early lead optimization effort,
using parallel synthesis, which led to the identification of sub-
micromolar inhibitors of subgenomic replicons.
The 3-step parallel synthesis procedure8 used for the synthesis
of the target compounds 11a–zo (Tables 1–3) is readily amenable
Table 1
SAR analysis on A ring and R4 substituents
178-fold more potent than (S)-11d (IC50 = 0.081
14.4 M, respectively).
lM, and
l
R1
H
With these encouraging results in hand, we decided to explore
the effect of incorporation of nitrogen atoms in the A ring at posi-
tions X and Y. Although a nitrogen at position Y was found to be
deleterious for activity (compare 11e with 5), its incorporation at
position X (pyridine 11f), or in both positions X and Y (pyrimidine
11g), led to compounds with over 10-fold improved potency com-
pared to 5.8 Surprisingly, and in contrast to the unsubstituted
N
C
X
A
Y
O
B
N
O
Cl
R4
pyrimidine 11g (IC50 = 0.51
lM), the corresponding 6-hydroxypy-
Compd #
X
Y
R1
R4
NS5B
HUH7-Rep
rimidine 11h proved to be completely inactive (IC50 > 32
lM).
IC50
(l
M)
EC50 (lM)
To help us understand our initial findings, and to guide our
56
11a
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
N
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3.1
>32
15.4
14.6
7.8
3
medicinal chemistry effort, the crystal structure of (R)-11d bound
CN
Me
Br
OH
OH
OH
H
6.97
4.78
6.12
0.1
to HCV NS5B (NS5BD
C21, J4, genotype 1b)5 was determined. Sim-
11b
11c
11d
(S)-11d
(R)-11d
11e
ilar to the results of our previous work with a related compound of
this chemotype,6 (R)-11d was found to bind at the NNI3 binding
site, adjacent to the polymerase active site (Fig. 3).7 One face of
the bound benzodiazepine fused ring system is solvent-exposed,
while the dichlorophenyl ring is buried in a deep and largely
hydrophobic pocket of NS5B. The hydroxyl group participates in
an extensive network of intra- and intermolecular hydrogen bonds,
partially mediated by bound water molecules and involving Ser367
and Tyr415 (Fig. 3). To our satisfaction, when the hydroxyl was
introduced in the benzyloxy derivative 11i, a very potent inhibitor
11j was obtained (IC50 = 40 nM).
14.40
0.081
8.04
0.51
0.51
>32
0.90
0.04
0.16
0.10
0.12
14.46
2.42
>32
>32
>32
>32
2.3
1.5
9.9
7.1
6.3
11f
11g
CH
N
H
H
Cl
Cl
N
11h
111s
11j
11k
11l
N
N
OH
H
OH
OH
OH
OH
Cl
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
OBn
OBn
OPh
O(2-Br)Ph
O(2-Br-6-F)Ph
11m
In an effort to improve the potency of the 1,5-BZD series, we
analyzed the inhibitor-NS5B interactions of other chemotypes
known to bind in the NNI-3 site (Figs. 1 and 4).9 For example,
superimposition of the NS5B-13b complex with that of 126 shows
extensive overlap of the phenoxy moiety of 13b9 and the benzyl-
oxy group of 12 (Figs. 4 and 5).6 We were able to exploit this par-
ticular 3D relationship to advance our lead optimization, although
ultimately we found that the SAR of the acrylic acid series9 was
only partially transferable to the 1,5-BZDs. Although the introduc-
tion of the phenoxy moiety on the para position of the phenyl ring
of 56 conferred a ꢀ20-fold increase in potency when compared to
Table 2
SAR analysis on R2 susbstituent
OH
O
H
N
O
N
R2
Compd #
R2
NS5B IC50
(lM)
HUH7- Rep EC50 (lM)
the dichlorophenyl derivative 56 (IC50 = 3.1
lM and 0.16 lM,
11n
11o
11p
11q
11r
11s
11t
2-Furyl
2-Pyridyl
A/-Me-imidazol-2-yl
A/-Me-indol-3-yl
5-OBn-2-thiophenyl-
Cyclohexyl
21
43
respectively for 56 and 11k), 11k was found to be less potent than
the corresponding benzyloxy derivative 11j. Moreover, and in di-
rect contrast to the 170-fold NS5B IC50 difference observed for
acrylamide derivatives 13a and 13b,9 the introduction of a bromo
substituent at position 2 of the 1,5-BZD phenoxy ring gave a min-
>32
>32
13
0.18
22
>32
>32
>32
11
0.74
>32
H
>32
imal 1.6-fold improvement in potency (IC50 = 0.16 lM and 0.10 lM

2494
D. McGowan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2492–2496
Table 3
Table 3 (continued)
SAR analysis on R3 and R4 position
Compd#
R3
R5
F
NS5B IC50 (nM)
79
HUH7-Rep EC50/CC50
0.57/28
(lM)
OH
H
N
N
11zk
O
O
N
R5
O
N
R3
(R)-11zk
(S)-11zk
11zl
F
F
F
F
F
F
29
0.27/28
3.6/13
O
OBn
N
Compd#
R3
R5
NS5B IC50 (nM)
HUH7-Rep EC50/CC50 (lM)
27,000
120
50
O
11j
CH3
i-Pr
i-Pr
i-Pr
i-Pr
Cl
Cl
F
Br
CH3
50
1.5/15
1.5/17
1.3/14
2.4/12
1.6/15
11u
11v
11w
11x
154
123
87
1.8/45
N
N
O
N
124
N
11y
F
F
51
0.36/15
11zm
11zn
1.0/25
N
N
11za
109
2.3/20
21
2.4/>32
0.73/>32
NH
N
N
11zo
60
11zb
F
F
F
86
70
89
3.3/76
N
N
N
N
11zc
0.50/18
0.40/21
R1
R1
O
O
H
N
ii
NH2
NH2
(R)- 11zc
X
X
i
X
X
+
Y
7a-i
Y
NH2
O
8
9a-i
N
R1
R1
(S)- 11zc
F
F
>11,000
77
5.7/18
H
N
H
N
C
iii
A
B
O
Y
O
N
Y
R2
N
H
10a-x
R2
F
N
O
11zd
0.86/>32
R3
11a-zo
(Tables 1-3)
R2
X
Y
R1
X
Y
R1
7a,9a CH CH CN
10a CH CH CN
Cl
N
7b,9b CH CH CH3 10b CH CH CH3
11ze
11f
F
F
F
108
75
0.69/13
2.2/16
1.7/19
7c,9c CH CH Br
7d,9d CH CH OH
10c CH CH
Br
10d CH CH OH
2,4-diClPh
7e,9e CH CH
H
H
10e CH
N
CH
N
H
H
H
OH
H
7f,9f CH
N
CH
N
10f
10g
10h
N
N
N
7g,9g
7h,9h
7i,9i
N
N
N
H
O
H
N
N
OH
10i CH CH
4-(BnO)-2-ClPh
10j CH CH OH 4-(BnO)-2-ClPh
10k CH CH OH 4-(PhO)Ph
N
11zg
111
10l CH CH OH 2-Br-4-(PhO)Ph
10m CH CH OH 2-Br-6-F-4-(PhO)Ph
10n CH CH OH 2-furyl
S
10o CH CH OH 2-pyridyl
N
10p CH CH OH N-CH3-imidazol-2-yl
10q CH CH OH N-CH3-indol-3-yl
10r CH CH OH 5-BnO-thiazol-2-yl
10s CH CH OH cyclohexyl
11zh
11zi
11zj
F
F
F
39
0.76/29
0.97/>32
1.6/29
S
10t CH CH OH
H
N
10u CH CH OH 2-furyl
143
63
S
10v CH CH OH 4-(BnO)-2-FPh
10w CH CH OH 4-(BnO)-2-BrPh
10x CH CH OH 4-(BnO)-2-CH3Ph
N
Scheme 1. Reactions and conditions: (i) toluene reflux; (ii) R2CHO, 10% acetic acid
in ethanol 70 °C; (iii) acetic anhydride, reflux for 11a–t or (a) R3COCl, DIPEA, CH2Cl2
then for 11d, h, j–m, n–zo; (b) LiOH, MeOH/H2O/THF.
O

D. McGowan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2492–2496
2495
Figure 3. X-ray crystal structure of (R)-11d bound to HCV NS5B polymerase.7 NS5B
from monomer A of this structure is shown as white ribbon and white color-by-
atom stick, and (R)-11d shown as green color-by-atom stick. Upper: the refined
Figure 5. Superposition of the crystal structures of the NS5B complexes of 13b9
(upper panel; purple color-by-atom) and 35 (lower; purple color-by-atom) onto 12
(green color-by-atom). Superposition was based on all common C of the two
a
2Fo À Fc electron density map of bound (R)-11d is contoured at 1r. Lower: selected
protein structures. Selected residues of NS5B from the complex with 12 are also
shown. The cyan sphere in the lower panel depicts a bound water molecule from
the crystal structure of 35 displaced by the phenyl ring of NS5b-bound 12.
residues of the binding site showing a bound water from the A (cyan sphere) and B
(magenta sphere) monomers of the asymmetric unit of this crystal structure, as
well as one from another as yet unpublished structure from this series of inhibitors
(green sphere).
Attempts to replace the benzyloxyphenyl moiety of 11j with a
cyclohexyl (11s) or with a mono- (11n and 11o) or di-heteroaro-
matic ring systems (11p–r) resulted in a loss in potency (Table
2). Moreover, the removal of the benzyloxyphenyl moiety led to
O
O
HO
H
N
N
H
a completely inactive compound 11t (IC50 > 32 lM). Although the
importance of the R5 substituent was shown in our earlier commu-
nication,6 replacement of the chloro (11u) with a fluoro (11v), bro-
mo (11x) or methyl (11w) was found to be well tolerated, causing
only minor differences on both replicon and enzymatic activity
(Table 3). Since the fluoro atom was the smallest substituent re-
quired to maintain potency and was readily obtained from the
common intermediate 10o, we decided to pursue further SAR anal-
ysis around the R3 substituent on a series of 4-benzyloxy-2-fluoro-
phenyl derivatives (11y–zo, Table 3). X-ray structure analysis of
11d bound to NS5B suggested that the acetyl moiety at position
10 (R3 = CH3) could be replaced by a larger substituent. Given that
this position is close to the protein surface, we envisaged that the
introduction of other R3-acyl groups, readily introduced at the end
of the synthesis, would lead to compounds with improved potency.
Indeed, compared to the isopropyl analog (11v), the 2-pyridylcar-
bonyl moiety at position 10 (compound 11y) was found to increase
both the enzymatic and replicon activities by ꢀ3-fold. Interest-
ingly, the 3- and 4-pyridyl derivatives (11za and 11zb, respec-
O
N
R'
O
O
O
Cl
R
13a9: R = H, R' = H; IC50 = 17 µM*
13b9: R = Br, R' = H; IC50 = 0.10 µM*
13c9: R = Br, R' = F; IC50 = 0.03 µM*
126: IC50 = 1.0 µM
Figure 4. Structure of previously reported 1,5-BZD 126 and acrylic acid derivatives
*
13a–c9 and reported IC50
.
for 11k and 11l, respectively). Finally, while in the acrylamide ser-
ies the addition of an extra fluoro at position 6 of the phenoxy 13b
led to a further ꢀ3-fold improvement (IC50 = 0.10
respectively for 13b and 13c), similar modification of 11l did not
further improve the potency (11m-IC50 = 0.12 M).
lM and 0.03 lM,
l

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D. McGowan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2492–2496
tively) were found to be 10-fold less potent against replicon, while
the enzymatic effect was less than twofold. None of the small sub-
stituents tested in the alpha position of the 2-pyridyl moiety were
found to positively impact the potency in either the biochemical or
replicon assays (compare 11zc, 11zd, and 11ze with 11y). The re-
sults for the pyridyl series prompted us to evaluate other small
heterocycles bearing either an oxygen (2-furyl, 11zf) or a nitrogen
atom (11zg–zo) at the alpha position.
oral administration. These preliminary results suggest to us that
further lead optimization efforts are warranted to optimize the po-
tency and selectivity index of this novel series of HCV NS5B
inhibitors.
References and notes
1. Shepard, C. W.; Alter, M. J. Lancet 2005, 5, 524.
Amongst the compounds synthesized, the dimethyloxazole
derivative 11zk was the most active of the series, exhibiting similar
enzymatic and replicon potencies when compared to the more ac-
tive pyridyl analogs 11y and 11zc. Separation of the two enantio-
mers of 11zc and 11zk led to two very potent inhibitors both in
the biochemical (IC50 (R)-11zc = 89 nM, IC50 (R)-11zk = 29 nM)
and the replicon assays (EC50 (R)-11zc = 400 nM, EC50 (R)-
11zk = 270 nM). Noteworthy, the selectivity index was found to
be over 50-fold for (R)-11zc and 100-fold for (R)-11zk. For these
two compounds the (S)-enantiomers were essentially inactive on
2. Willems, M.; Metselaar, H. J.; Tilanus, H. W.; Schalm, S. W.; De Man, R. A. Transpl.
Int. 2002, 15, 61.
3. (a) Strader, D. B.; Wright, T.; Thomas, D. L; Seeff L. B. Hepatology 2004, 39, 1147.;
(b) Dixit, N. M.; Layden-Almer, J. E.; Layden, T. J.; Perelson, A. S. Nature 2004, 432,
922; (c) Fried, M. W. et al N. Engl. J. Med. 2002, 347, 975.
4. Fried, M. W.; Peter, J.; Hoots, K.; Gaglio, P. J.; Talbut, D.; Davis, P. C.; Key, N. S.;
White, G. C.; Lindblad, L.; Rickles, F. R.; Abshire, T. C. Hepatology 2002, 36, 967.
5. Pauwels, F.; Mostmans, W.; Quirynen, L.; van der Helm, L.; Boutton, C. W.; Cleiren,
E.; Raboisson, P.; Surleraux, D.; Nyanguile, O.; Simmen, K. A. J. Virol. 2007, 81, 6909.
6. Nyanguile, O.; Pauwels, F.; Van den Broeck, W.; Boutton, C. W.; Quirynen, L.;
Ivens, T.; Van der Helm, L.; Vandercruyssen, G.; Mostmans, W.; Delouvroy, F.;
Dehertogh, P.; Cummings, M. D.; Bonfanti, J.-F.; Simmen, K. A.; Raboisson, P.
Antimicrob. Agents Chemother. 2008, 52, 4420.
7. Coordinates for structure have been deposited at Protein Data Bank
(www.rcsb.org) under file name 3GOL.
8. Cortés Cortés, E.; Valencia Cornejo, A. L.; Garcia-Mellado de Cortés, O. J.
Heterocycl. Chem. 2007, 44, 183.
9. Pfefferkorn, J. A.; Greene, M. L.; Nugent, R. A.; Gross, R. J.; Mitchell, M. A.; Finzel,
B. C.; Harris, M. S.; Wells, P. A.; Shelly, J. A.; Anstadt, R. A.; Kilkuskie, R. E.;
Schwende, F. J.; Koptab, L. A. Bioorg. Med. Chem. Lett. 2005, 15, 2481.
the enzyme (IC50 > 10 lM), while the replicon activity was found
to be more than one order of magnitude lower for the (S)- versus
the (R)-enantiomers. Moreover, based on the high permeability
coefficient Papp observed for both (R)-11zc and (R)-11zk on
CACO-2 cells (Papp (A–B) = 11.6 Â 10À6 and 10.9 Â 10À6 cm/s,
respectively), these compounds are predicted to be absorbed after