Discovery of pentacyclic compounds as potent inhibitors of hepatitis C virus NS5B RNA polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.12.039

We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-d][2]benzazepines exposing basic groups. The compo

Full text of DOI:10.1016/j.bmcl.2008.12.039

Bioorganic & Medicinal Chemistry Letters 19 (2009) 633–638
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pentacyclic compounds as potent inhibitors of hepatitis C
virus NS5B RNA polymerase
*
Jörg Habermann , Elena Capitò, Maria del Rosario Rico Ferreira, Uwe Koch, Frank Narjes
Department of Medicinal Chemistry, IRBM ‘‘P. Angeletti” S.p.A., Merck Research Laboratories Rome, Via Pontina km 30,600, I-00040 Pomezia, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained
pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-
d][2]benzazepines exposing basic groups. The compounds displayed a high activity in the enzyme assay
and displayed good activity in the cell-based (replicon) assay in the presence of serum proteins.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 10 November 2008
Revised 5 December 2008
Accepted 7 December 2008
Available online 13 December 2008
Keywords:
HCV
HCV NS5B polymerase inhibitor
Pauson–Khand reaction
[1,3]-Dipolar cycloaddition
Hepatitis C is a viral infection of the liver and as such is a major
cause of acute hepatitis and chronic liver disease, including cirrho-
sis and liver cancer. Globally, an estimated 170 million persons are
chronically infected with HCV and 3–4 million persons are newly
infected each year. HCV is spread primarily by direct contact with
human blood.1 No vaccine is currently available to prevent hepati-
tis C. The front line therapy is a combination of pegylated inter-
largely invariable structural feature of these compounds is the
presence of a cyclohexyl ring and an aromatic ring attached to a
heterocyclic core.
In the course of our investigations X-ray crystallography and
computer modelling had indicated that the pendant N,N-dimethyl
acetamide chain in 1 is directed mostly towards the solvent-ex-
posed surface. Only to a minor extent this chain exhibits contacts
with the enzyme. Thus, we deemed it possible to use this part
mainly to modulate physico-chemical properties.8
Additionally, it is known that the enzyme contains a lipophilic
pocket in close proximity to the aryl ring of the inhibitor (e.g., 3,
Fig. 2). Studies by a group from Japan Tobacco had indicated that
this area might be reached by extending from N5 of compound 3.9
After our initial work in the tetracyclic indole series10 which
also indicated the beneficial effect of an exposed basic functional-
ity we considered to reduce the rotatable bond count by locking up
the side-chain in an extra ring system. In addition, we felt that the
presence of an anilinic nitrogen atom in compound 3 constituted a
potential liability from a drug safety point of view.11
Hence, we decided to move from a benzodiazepine scaffold to a
benzazepine scaffold. Since our previous studies had shown that
monoamines were tolerated the compounds 4–7 were designed
to generically probe the concept of the fifth anellated ring. Only
for 4 an activity in the cell-based assay13 was observed which
was in the range of previous tetracyclic compounds (Table 1).
In general, the intrinsic potency was largely maintained in most
of the new pentacycles with the exception of the cis-configured
pentacycle 6, which showed a nearly 5-fold loss compared with
3. The trans-anellated compound 7 was about 3.5-fold more active
in terms of intrinsic potency and cell-based potency under high
feron-a dosed in combination with the nucleoside analogue
ribavirin. However, this treatment is often poorly tolerated and
of limited success rate. Consequently, HCV continues to be a signif-
icant world health burden and remains the leading cause of liver
transplantation in the United States.2 Thus there is a need for im-
proved therapies.
The virally encoded non-structural proteins of the hepatitis C
virus are attractive targets for antiviral drug therapy. As such, the
hepatitis C virus NS5B polymerase is a key enzyme necessary for
the replication of the HCV gene in infected cells and has recently
been the focus of many research groups.3
Several classes of inhibitors have been reported for this enzyme
acting at the active site or one of the allosteric sites of the enzyme.
A few compounds have shown antiviral effects in clinical studies
(e.g., nucleoside prodrugs NM283, R-1626 and R-7128 and non-
nucleoside inhibitors HCV-796, BILB-1941 and VCH-759).4,5 We
and others have previously reported benzimidazole, indole and thi-
enopyrrole carboxylic acid derivatives as inhibitors of hepatitis C
NS5B polymerase (Fig. 1).6 These classes of compounds show selec-
tivity for thumb domain pocket I of the enzyme.7 A common and
* Corresponding author. Tel.: +39 06 91093851; fax: +39 06 91093225.
E-mail address: jorg_habermann@merck.com (J. Habermann).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.039

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J. Habermann et al. / Bioorg. Med. Chem. Lett. 19 (2009) 633–638
Table 1
serum conditions than its cis-anellated analogue 6. Since the bind-
ing of lead compounds and drugs to serum proteins is a ubiquitous
problem modulating the availability to the target our attention
turned to the serum shift.14 We noticed that the trans-configured
compound 7 did not exhibit any notable shift between low and
high serum conditions which made this compound an interesting
starting point for further evaluation. The high serum conditions
may be considered a physiologically more relevant system.
Additionally, ease of synthetic accessibility and the possibility
to reach out to the hydrophobic patch led to the choice of scaffolds
6 and 7 as starting points for further evaluation (Table 2).
We were able to gain potency by exchanging the N-methyl sub-
stituent of 7 with a N,N-dimethylaminoethyl side-chain. This
change from a monoamine system to the diamino system repre-
sented by 8a significantly improved both intrinsic and cell-based
activity with respect to 2 and 3.
A minor improvement in intrinsic activity was observed when
increasing the lipophilic character of the pendant side-chain by
replacing the N,N-dimethylamino ethyl group with a piperidin-1-
yl ethyl moiety (8c vs 8a). However, this gain was achieved at
the cost of cell-based potency in 50% NHS. We hypothesised that
this is most likely due to diminished cell-penetration of 8c since
the human plasma protein binding is virtually identical for both
compounds (91.5% vs 92%). We have not been able to establish a
clear correlation between this empirical finding and calculated
physico-chemical properties (cLogD, pKa, PSA) but in most cases
a higher cLogD (e.g., 8c: cLogD7.4 = 5.76 vs 8a: cLogD7.4 = 4.5) cor-
related with a higher serum shift.15 Elongation of the pendant
side-chain (8d, 8e) was permitted in terms of intrinsic potency,
but did not lead to a substantial gain in cell-based activity. Various
amidic substitutions (8f–k) were tolerated in terms of activity in
the biochemical assay but led in most cases to poorer cell-based
activity when compared with the basic amine substituents.
Thus, our attention turned to the modification of the benzaze-
pine aryl moiety. In related compounds of earlier studies, the intro-
duction of a para-methoxy substituent had proven beneficial, so
we attempted the same modification in the new trans-series. The
resulting compound 8b showed improved intrinsic activity paired
with a low shift (2.5-fold) for the cell-based assay between 10% FCS
and 50% NHS.
Monoamine pentacyclic series—variation of the scaffold12
Compound
NS5B
IC50
(nM)
Replicon
10% FCS
EC50 (nM)
Replicon
50% NHS
EC50 (nM)
Approx.
serum
shift
N
H
H
HOOC
N
30
130
890
7
4
5
6
N
H
H
HOOC
N
20
140
40
1200
3800
2540
700
3
3
1
H
N
H
HOOC
N
770
H
N
H
For comparison the cis-derivatives 9 were prepared. While in
general showing appreciable intrinsic activity, the cell-based activ-
ity was insufficient since no substantial gain over previously de-
scribed compounds was observed. For example, with compound
9a we noticed that while the intrinsic activity on the isolated
NS5B enzyme remained nearly invaried a nearly 8-fold loss in
cell-based activity against its trans-analogue 8a could be observed.
Due to the interesting activity level reached with the racemic
compound 8b we decided to separate it into the two enantiomers
10 and 11. We found that the activity largely resides only in one of
the two enantiomers, while the second is nearly inactive. The large
HOOC
N
830
7
difference in activity between the two enantiomers 10 and 11
prompted us to investigate this behaviour in detail.
N
NMe2
O
5
N
N
N
8
HOOC
HOOC
HOOC
N
N
N
1
2
3
IC50 430 nM
EC50 (10% FCS) 1540 nM
EC50 (50% NHS) 9850 nM
IC50 27 nM
EC50 (10% FCS) 140 nM
EC50 (50% NHS) 850 nM
IC50 28 nM
EC50 (10% FCS) 130 nM
EC50 (50% NHS) 360 nM
Figure 1. Examples of HCV NS5B inhibitors from previous work.

J. Habermann et al. / Bioorg. Med. Chem. Lett. 19 (2009) 633–638
635
Table 2
SAR in the pentacyclic series
H
H
R1
R1
N
N
H
H
HOOC
HOOC
N
N
R2
R2
8a-k
10, 11
9a-b
Compound
R1
R2
NS5B IC50 (nM)
Replicon 10% FCS EC50 (nM)
Replicon 50% NHS EC50 (nM)
8a
9a
8b
9b
8c
8d
8e
8f
8g
8h
8i
8k
10*
11*
CH2CH2NMe2
CH2CH2NMe2
CH2CH2NMe2
CH2CH2NMe2
CH2CH2pip
H
H
OMe
OMe
H
H
H
H
H
20
30
8
32
260
25
150
40
58
53
630
78
170
32
98
620
63
270
220
80
110
480
96
210
110
225
>1000
49
21
11
24
11
24
17
31
23
21
312
4
CH2CH2CH2NMe2
CH2CH2CH2pip
C(@O)NHCH2CH2NMe2
CH2C(@O)NMe2
C(@O)CH2NMe2
C(@O)CH2NMe2
C(@O)OCH2CH2NMe2
CH2CH2NMe2
H
OMe
OMe
OMe
OMe
180
>1000
29
CH2CH2NMe2
*
Separated enantiomers of 8b.
Figure 2. Hydrophobic patch (yellow) in proximity to allosteric site 1.
Figure 3. Modelling study explaining the difference in activity of the two trans-
enantiomers 10 (red) and 11 (green).
A modelling study16 (Fig. 3) indicated that binding to the en-
zyme is compatible only with 11 (3aS,14bR, shown in green). The
minimum conformation of the inactive enantiomer 10 (3aR,14bS,
shown in red) does not fit into the pocket as the pendant side-
chain clashes with the backbone of the enzyme.
fore, hydrogenation of the corresponding oxime 17 over platinum
was carried out.19 Reductive amination and basic hydrolysis com-
pleted the synthesis of 4.
The relative stereochemical outcome of the ring-closure and the
oxime reduction was confirmed by the observation of nOe’s in the
proton NMR.
The compounds evaluated in this study were prepared as de-
scribed in Schemes 1–3.17
Scheme 2 exemplifies the synthesis of cis-configured com-
pounds of type 6. Depending on the desired substitution pattern,
the sequence started with a Suzuki reaction of bromoindole 12
with a 2-formyl benzene boronic acid or an aryl boronic acid bear-
ing a protected benzylic alcohol as the precursor for the 2-formyl
group. This reaction was followed by N-allylation.
When a protected benzyl alcohol had been used as the precur-
sor, the alcohol was liberated and subjected to Swern oxidation.
Intermediate 21 was thus set up for a [1,3]-dipolar cycloaddition.
The cycloaddition was carried out with glycine or N-substituted
The pentacyclic indolobenzazepine 4 could be prepared by
applying the Pauson–Khand reaction18 to the vinyl/propargyl-
substituted aryl indole 14 which in turn had been obtained by Su-
zuki reaction of bromoindole 12 with 2-vinylbenzene boronic acid
followed by N-alkylation of 13 with propargylbromide (Scheme 1).
It was observed that the ring-closure reaction proceeded best
using an excess of dicobalt octacarbonyl without the application
of external carbon monoxide pressure. Transformation of the ke-
tone 16 (obtained by hydrogenation of intermediate 15) into the
amine could not be achieved by direct reductive amination. There-

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J. Habermann et al. / Bioorg. Med. Chem. Lett. 19 (2009) 633–638
H
H
MeOOC
MeOOC
MeOOC
N
N
N
(a)
(b)
Br
12
13
14
O
O
H
H
(c)
(d)
(e)
MeOOC
MeOOC
N
N
15
16
NOH
NH2
N
H
N
H
N
H
(f)
(g), (h)
H
H
H
MeOOC
MeOOC
HOOC
N
17
18
4
Scheme 1. Pauson–Khand reaction to form cis-pentacycle 4. Reagents and conditions: (a) 2-vinylbenzene boronic acid, PdCl2(PPh3)2, 2 M Na2CO3, dioxane, 110 °C, 83%; (b)
NaH, propargylbromide, DMF, 84%; (c) Co2(CO)8, 4 Å MS, toluene, 110 °C, 36%; (d) Pd/C, H2, EtOAc/iPrOH, 54%; (e) NH2OH HCl, NaOAc, H2O/MeOH/EtOAc, 60 °C, 83%; (f) PtO2,
H2 (50 psi), HOAc, quant; (g) HCHO, NaCNBH3, DCM, HOAc, 91%; (h) KOH, THF/MeOH/H2O, 45 °C, 37%.
OTIPS
OTIPS
H
H
MeOOC
MeOOC
MeOOC
N
N
N
(a)
(b)
Br
12
19
a
20
H
N
O
N
H
H
N
(c), (d)
(e), (f)
HOOC
MeOOC
21
6
Scheme 2. [1,3]-Dipolar cycloaddition for cis-pentacycles. Reagents and conditions: (a) (4-methoxy-2-{[(triisopropylsilyl)oxy]methyl}phenyl) boronic acid, PdCl2(PPh3)2, 2 M
Na2CO3, dioxane, 110 °C, 81%; (b) NaH, allylbromide, DMF, 84%; (c) TBAF, THF, 88%; (d) (COCl)2, DMSO, DCM, À70 °C, then NEt3, 0 °C, 88%; (e) sarcosine, toluene/DMF, 110 °C,
61%; (f) KOH, MeOH/dioxane/H2O, 59%.
derivatives of glycine (e.g., sarcosine to directly introduce
R1 = methyl) setting up a cis-pentacyclic system.20 Finally, basic
hydrolysis of the ester moiety gave the free acid 6.
For the preparation of trans-pentacyclic systems a similar ap-
proach to the one described before was taken (Scheme 3). Starting
from intermediate 19b (prepared in analogy to 19a) the indole
nitrogen was propargylated to give 22. The benzylic alcohol was
liberated and oxidised to give the aldehyde 23.
[1,3]-Dipolar ring-closure with glycine resulted in the forma-
tion of an intermediate 2,5-dihydro-1H-pyrrole system 24 which
is prone to oxidation and thus needed to be reacted onward
quickly. Hydrogenation over palladium led to the trans-pentacyclic
system 25.
Since some formation of the corresponding pyrrole 30 during
the hydrogenation process was observed, it could be speculated
that the reaction proceeds via an intermediary (and not observed)
2,3-dihydro-1H-pyrrole system 28 (Scheme 4). Reductive amina-
tion of 25 with various aldehydes or acylation worked smoothly.
Using N-Boc-amino acetaldehyde and subsequent cleavage of
the protecting group gave 26. After reductive amination and
hydrolysis of the methyl ester, the racemate 8b was obtained
and separated by SFC21 into the single enantiomers 10 and 11.
The peak separation was complete and gave pure products of
99% enantiomeric excess.
The pyrroles 30 while maintaining intrinsic activity in the low
nanomolar range lost significantly in cell-based potency, a fact

J. Habermann et al. / Bioorg. Med. Chem. Lett. 19 (2009) 633–638
637
OTIPS
OTIPS
H
MeOOC
MeOOC
N
N
(a)
(d)
(b), (c)
OMe
OMe
19b
22
O
NH
H
(e)
MeOOC
MeOOC
N
N
OMe
OMe
23
24
H
H
NH2
NH
H
N
H
(f), (g)
(h), (i), (k)
10 + 11
MeOOC
MeOOC
N
N
OMe
OMe
25
26
Scheme 3. Synthesis of trans-pentacycles. Reagents and conditions: (a) NaH, propargylbromide, DMF, 96%; (b) TBAF, THF, 64%; (c) (COCl)2, DMSO, DCM, À70 °C, then NEt3,
0 °C; (d) glycine, NEt3, DMF, 140 °C; (e) Pd/C, H2, EtOAc, 43% over three steps; (f) N-Boc-aminoacetaldehyde, MeOH, AcOH, NEt3, NaCNBH3; (g) TFA, DCM; (h) HCHO, MeOH,
AcOH, NaCNBH3; (i) KOH, MeOH/dioxane/H2O; (k) separation by SFC (Chiralpak AD-H, modifier 35% iPrOH + 0.4% DEA, Tcol 35 °C, pcol 100 bar), 12% over four steps.
H
R1
R1
N
N
H
MeOOC
MeOOC
N
N
Pd/C, H2
EtOAc
R2
R2
27
(24, when R1 = H, R2 = OMe)
28
H
R1
N
H
R1
N
MeOOC
MeOOC
N
N
R2
R2
29
(25, when R1 = H, R2 = OMe)
30
Scheme 4. Hypothesis regarding the formation of the trans-product during the hydrogenation.
which can be attributed to the poor solution stability of those
derivatives investigated.
identification of 11, which efficiently blocks subgenomic HCV RNA
replication in HUH7-cells at low nanomolar concentrations. This
compound is 7-fold more potent in the biochemical assay than
our starting point, the tetracyclic compound 3. In addition, the
pentacycle 11 displays a lower serum shift than 3.
In conclusion, we have identified a new class of allosteric inhibi-
tors of HCV NS5B polymerase based on a pentacyclic scaffold. Our
work led to the discovery of several new scaffolds 4–7.
While the substituent R2 on the aryl ring helped in gaining
intrinsic activity, the substituent R1 on the pyrrolidine ring in-
creased the cell-based potency as evidenced by the change in rep-
licon activity. Furthermore, the ring junction (cis/trans) plays an
important role in that the trans-pentacyclic derivatives are clearly
more potent.
Acknowledgements
The authors thank Renzo Bazzo and Silvia Pesci for analytical
support and structure confirmation by NMR, and Cecilia M. Palum-
bi for her assistance with the purification of some compounds and
for determining the enantiomeric excess. We also thank Nadia
Gennari, Monica Bisbocci and Sergio Altamura for running the bio-
logical assays.
Finally, we were able to show that in the trans-pentacyclic ser-
ies a clear preference for one of the two enantiomers exists. A mod-
elling study could rationally explain this behaviour. This led to the

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J. Habermann et al. / Bioorg. Med. Chem. Lett. 19 (2009) 633–638
template RNA using a modification of the procedure described in Ref. 7.
References and notes
Inhibition of the replication of subgenomic HCV RNA was measured in HUH-7
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determinations. Cell-based data was obtained in the presence of 10% fetal calf
serum (FCS) or 50% normal human serum (NHS).
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11. Although for the compounds in question this property has not been evaluated,
in some cases aromatic amines may constitute a potential risk for being
mutagenic and/or carcinogenic. For some studies see: (a) Franke, R.; Gruske, A.;
Giuliani, A.; Benigni, R. Carcinogenesis 2001, 22, 1561; (b) Turesky, R.J. Drug
Metab. Rev. 2002, 34, 625.
12. All compounds were profiled as racemic mixture if not denoted otherwise.
13. The compounds described were assessed for intrinsic activity (IC50) against the
21. Super-critical fluid chromatography (SFC) was performed using
system equipped with a Berger ALS 719.
a Berger
purified BK strain of NS5BDC21 enzyme in the presence of heterogenic