Pyrazolo[1,5-a]pyrimidine-based inhibitors of HCV polymerase

Article abstract of DOI:10.1016/j.bmcl.2009.09.087

The present paper describes a novel series of HCV RNA polymerase inhibitors based on a pyrazolo[1,5-a]pyrimidine scaffold bearing hydrophobic groups and an acidic functionality. Several compounds were optimized to low nanomolar potencies in a biochemical RdRp assay. SAR trends clearly reveal a stringent preference for a cyclohexyl group as one of the hydrophobes, and improved activities for carboxylic acid derivatives.

Full text of DOI:10.1016/j.bmcl.2009.09.087

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6331–6336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolo[1,5-a]pyrimidine-based inhibitors of HCV polymerase
a
a
a
a
Janeta Popovici-Muller a, , Gerald W. Shipps Jr. , Kristin E. Rosner , Yongqi Deng , Tong Wang ,
Patrick J. Curran a, Meredith A. Brown a, M. Arshad Siddiqui a, Alan B. Cooper b, José Duca b,
Michael Cable b, Viyyoor Girijavallabhan b
*
a Schering-Plough Research Institute Cambridge, 320 Bent Street, Cambridge, MA 02141, United States
b Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Revised 18 September 2009
Accepted 22 September 2009
Available online 25 September 2009
The present paper describes a novel series of HCV RNA polymerase inhibitors based on a pyrazolo[1,5-
a]pyrimidine scaffold bearing hydrophobic groups and an acidic functionality. Several compounds were
optimized to low nanomolar potencies in a biochemical RdRp assay. SAR trends clearly reveal a stringent
preference for a cyclohexyl group as one of the hydrophobes, and improved activities for carboxylic acid
derivatives.
Ó 2009 Elsevier Ltd. All rights reserved.
Keyword:
HCV polymerase inhibitors
The hepatitis C virus (HCV) was identified in 19891 and has
been recognized as a major human pathogen associated with
chronic hepatitis leading to cirrhosis and, in some cases, to hepato-
cellular carcinoma.2 It is estimated that globally over 170 million
people are chronically infected with HCV, and no vaccine is cur-
rently available to prevent hepatitis C.3 The current standard ther-
apy is pegylated interferon (IFN) in combination with Ribavirin,
which has yielded modest sustained viral response (SVR) rates
(40–50%) particularly in genotype 1-infected patients, the majority
of the hepatitis C population in the US, Europe and Japan. Addition-
ally this therapy is often associated with side effects, thus treat-
ment of the chronic HCV infection represents an unmet medical
need.4
HCV is a single stranded RNA virus in the Flaviviridae family. Its
genome encodes for a polyprotein consisting of both structural
core and envelope proteins, as well as non-structural (NS) pro-
teins.5 Among the NS proteins the NS5B RNA dependent RNA poly-
merase (RdRp) is essential for viral replication, and represents an
ideal target for the development of small molecule anti-HCV com-
pounds.6 Inhibition of NS5B can be achieved through binding at the
active site, or at one of the several allosteric sites, and several
nucleoside and non-nucleoside NS5B inhibitors have been de-
scribed in the literature.7
itors 29 and 310 published concurrent to our synthetic efforts
shared common pharmacophore features: heterocyclic hydropho-
bic compounds that contained an acidic functionality and hydro-
phobe positioning adjacent to one another, preserving cyclohexyl
as one of the key hydrophobes. Our comparison of 1 with 2 and
3 resulted in the design of a new ‘6,7-pyrazolo[1,5-a]pyrimidine’
scaffold 4 where by shifting the C-5 hydrophobe over to C-6 (and
replacing it with p-benzyloxy phenyl), and by substituting the C-
7 aromatic ring with a cyclohexyl moiety and adjusting the carbox-
ylic acid position to C-3, compound 4 was obtained with improved
biochemical potency. A related approach has recently been pub-
lished by our group.11 Herein, we would like to report the synthesis
and optimization of novel HCV polymerase (HCV pol) inhibitors
based on the ‘6,7-pyrazolo[1,5-a]pyrimidine’ scaffold.
Two synthetic routes were developed to probe various sites of
the scaffold and enable efficient analog synthesis. Scheme 1 de-
scribes a general sequence that allows simultaneous exploration
of the distal hydrophobe at C-6, modification of the cyclohexyl
group at C-7 and exploration of the carboxylic acid and derivatives
at C-3 from a common intermediate 9. Thus, starting from methyl
2-(4-(benzyloxy)phenyl)acetate or methyl 2-(4-iodophenyl)ace-
tate, alkylation with carbonyl chlorides gave b-keto ester 6, which
in turn was saponified and decarboxylated to afford ketone 7. Upon
Our initial optimization efforts revealed compound 1 with a
‘5,7-pyrazolo[1,5-a]pyrimidine’ scaffold8a (Fig. 1) with low micro-
molar biochemical activity. Compound 1 was obtained as a result
of scaffold rigidification aimed at improving activity of a previously
explored pyrazole chemotype.8b Literature HCV polymerase inhib-
treatment with methoxy bis(dimethylamino)methane 7 was
converted to the corresponding enamino-ketone 8, which upon
cyclization with 5-amino-1H-pyrazole-4-carbonitrile or ethyl
5-amino-1H-pyrazole-4-carboxylate in acetic acid afforded inter-
mediate 9. Using intermediate 9 with substituent R1 as benzyloxy,
simple treatment with boron trichloride converted R1 to hydroxy
group, which can be manipulated to afford other suitably substi-
tuted benzyloxy or phenoxy analogs via simple transformations,12
* Corresponding author.
E-mail address: janeta.popovici-muller@spcorp.com (J. Popovici-Muller).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.087

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J. Popovici-Muller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6331–6336
O
CO2H
O
3
2
N
N
N
N
HO
O
N1
HO
4N
O
N
5
7
3
2
Cl
1
OPh
IC50 = 1.6 µM
IC50 ~ 0.53 µM
IC50 = 6 µM
O
N
HO
3
6
N
7
O
N
1
4
µ
IC50 ~ 0.59
M
Figure 1. Design of the ‘6,7-pyrazolo[1,5-a]pyrimidine’ scaffold 4.
O
R2
O
R2
O
O
O
O
R2
COOMe
(a)
COOMe
(b)
(a)
N
N
N
Br
(c)
(b)
12
10
11
R1
5
R1
R1
6
R1
R1 = OBn, I
COOEt
7
8
N
(c)
6
N
N
Br
Explore acid -> amides
7
R
3 = COOEt -> COOH,
carboxylic acid derivatives
R3 = CN -> tetrazole
R3
13
N
3
Explore C-6
hydrophobe
6
N
R2
(d)
N
7
R1
Scheme 2. Synthesis of advanced intermediate 13. Reagents and conditions: (a)
Bredereck’s reagent (t-butoxybis(dimethylamino) methane), 60 °C, 93%; (b) bro-
mine, DCM, triethylamine, 0 °C to rt; (c) 5-amino-1H-pyrazole-4-carboxylate,
ethanol, 30% hydrogen bromide in acetic acid, reflux, 27% (steps (b) and (c)).
9
Cyclohexyl
replacement
Distal hydrophobe
exploration
Scheme 1. Synthesis of advanced intermediate 9. Reagents and conditions: (a)
diisopropylamine, THF, n-butyllithium, methyl 2-(4-(benzyloxy)phenyl)acetate or
methyl 2-(4-iodophenyl)acetate, R2-carbonyl chloride, À78 °C to rt; ꢀ80%; (b)
DMSO, NaCl, water, 150 °C, 96%; (c) methoxy bis(dimethylamino)methane, toluene,
70 °C, quant.; (d) 5-amino-1H-pyrazole-4-carbonitrile or ethyl 5-amino-1H-pyra-
zole-4-carboxylate, acetic acid, reflux, 55–62%.
formed using radiolabeled GTP, a poly C/oligo G template/primer
and the
D-21 construct of NS5B according to a modified literature
procedure.7e,14
The SAR development began with an investigation of the acidic
functionality at C-3 and it’s replacement with carboxylic acid
derivatives and isosteres.15 For this study we maintained the
hydrophobes at C-6 and C-7 fixed as 4-benzyloxy-phenyl and
cyclohexyl, and the results are depicted in Table 1. Comparing to
the parent compound 4 reduction of the acid group to the primary
alcohol (14a) decreased the activity dramatically, while replace-
ment of the acid group with the tetrazole isostere gave 14b which
was equipotent to 4. The methylated tetrazole derivative 14c and
the triazole 14d showed a significant potency loss, however the
hydroxamic acid analog 14e retained some potency and had only
a threefold loss compared to parent compound 4. Conversion of 4
to the primary amide 14f yielded an inactive compound, however
the tetrazole carboxamide 14g displayed similar biochemical po-
tency to 4 and 14b. The most potent compound in this subset
while with R1 as iodo, directly attached distal hydrophobes can be
introduced via Suzuki coupling.13 The versatility of intermediate 9
was further utilized to prepare a variety of carboxylic acid deriva-
tives and tetrazoles at C-3 simply by the use of pyrazole nitrile or
carboxylate in the cyclization step (d). With R3 as ethyl carboxylate
simple hydrolysis using aqueous lithium hydroxide in tetrahydro-
furan (THF) afforded the carboxylic acid which was further con-
verted to
a variety of derivatives using standard organic
transformations, while with R3 as nitrile, treatment with triethyl-
amine hydrochloride and sodium azide in toluene/dimethylform-
amide (DMF) generated the corresponding tetrazole (e.g., 9,
R3 = tetrazole).
An alternative synthetic route was then developed to rapidly
access analogs with fixed cyclohexyl moiety at C-7 while varying
the C-6 hydrophobe and exploring carboxylic acid derivatives at
C-3 from common intermediate 13. This approach is depicted in
Scheme 2 and started from the commercially available 1-cyclo-
hexylethanone 10. Treatment of 10 with Bredereck’s reagent affor-
ded enamino-ketone 11, which was then brominated to generate
compound 12. Finally, cyclization of 12 with ethyl 5-amino-1H-
pyrazole-4-carboxylate in ethanol with 30% hydrogen bromide in
acetic acid gave intermediate 13 which was subsequently used in
structure–activity relationships (SAR) development.
was obtained by converting compound 4 to the L-tryptophan car-
boxamide 14h which was active at 90 nM in the biochemical assay,
and other more potent amino acid carboxamides were revealed in
a subsequent investigation and their SAR will be discussed later on
in the manuscript. Finally, a small set of acyl sulfonamides was
synthesized (compounds 14i–14j), but no significant potency gain
was achieved with these analogs.
Having discovered that carboxylic acid replacement at C-3 with
tetrazole is tolerated, and considering the tetrazoles’ enhanced
physicochemical and pharmacological properties compared to car-
boxylic acids,16 we next focused our attention to the study of cyclo-
hexyl modification and replacement at C-7, holding the tetrazole
moiety fixed at C-3. (Table 2) Briefly, replacement of cyclohexyl
To measure the efficacy of these compounds a scintillation
proximity assay (SPA)—based RNA polymerase assay was per-

J. Popovici-Muller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6331–6336
6333
Table 1
Table 2
Carboxylic acid and isosteres (C-3)
Modification of cyclohexyl group (C-7)
R
N
N
N
N
3
HN
6
N
N
N
7
1
N
N
BnO
7
R
BnO
Compd
R
HCV NS5B
0.59
D-21 IC50 (lM)
Compd
R
HCV NS5B
0.45
D-21 IC50 (lM)
O
4
OH
OH
14b
14a
14b
12.5
0.45
15a
15b
15c
na
12
3.4
N
N
N
HN
N
N
N
N
O
S
14c
14d
50
N
N
HN
O
5.7
15d
15e
15f
na
S
O O
OH
14e
14f
14g
1.9
na
N
H
12.5
8.6
na
CH3
O
NH2
N
O HN
N
0.67
N
N
H
15g
15h
HO
O
6
O
NH
14h
14i
0.09
N
H
15i
4.6
O
O
O
S
O
1.6
na = not active.
N
H
O
the finger-loop binding site. The three ‘anchors’ of compound 16f
in the NS5B finger-loop binding site–the tetrazole as a carboxylate
mimic, and neighboring phenyl and cyclohexyl groups–overlay
closely with their counterparts in the model template as well as
other reported indoles and benzimidazoles18 despite differences
in scaffold and substitution (Fig. 2). As a result, compound 16f
hydrogen bonds (H-bonds) to Arg-503 with the tetrazole and
makes a salt bridge interaction with Lys-491 through its benzoate
moiety.
Shortening the linkage to the proximal ring by one carbon as
shown in the phenoxy analogs 16g and 16h afforded equipotent
compounds to 14b, irrespective of the nature of the distal ring
(phenyl vs cyclohexyl), while adding meta substituents to the distal
ring (16i and 16j) resulted in analogs in the same potency range as
14b. Both benzyloxy and phenoxy linkages were slightly improved
compared to the biaryl linkage (16k and 16l) which afforded low
micromolar analogs, and by completely removing the distal ring
and adding fluorine atoms on the proximal hydrophobe, compound
16m was obtained and found to be only slightly less potent than
the parent compound 14b.
14j
0.76
S
N
H
O
na = not active.
group with phenyl (15a) or other smaller acyclic groups (15e, 15f,
15h, 15i), or modification of cyclohexyl to a pyran (15b) or thiopy-
ran ring (15c) resulted in completely inactive or less potent com-
pounds with micromolar biochemical activities. Therefore, we
decided to maintain the cyclohexyl group fixed at C-7 for subse-
quent analogs.
Continuing the SAR development of the pyrazolo[1,5-a]pyrimi-
dine scaffold, substitution patterns around the distal hydrophobe
and linkage to the proximal hydrophobe at C-6 were also investi-
gated, and the results are summarized in Table 3. Starting from
the parent benzyloxy compound 14b, replacement of phenyl ring
with 4-pyridine (16a) or addition of meta or para substituents to
the ring (16b, 16c, 16d, 16e, 16f) afforded compounds of compara-
ble or slightly improved potency (twofold improvement for 16d
and 16e). The most active compound identified in the benzyloxy
sub-series was 16f, bearing a carboxylic acid group para on the dis-
tal ring, with 42 nM in the biochemical assay. The binding mode of
16f was investigated, and the compound was docked into the
structure of NS5B using an induced-fit docking procedure17 at
Replacement of the distal hydrophobe with a fluorine atom en-
abled us to reduce the molecular weight of the scaffold, while
focusing next on amide derivatives of the carboxylic acid at C-3
(Table 4), which were previously reported in the HCV literature19
to produce inhibitors with significantly improved biochemical

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J. Popovici-Muller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6331–6336
Table 3
Table 3 (continued)
Distal hydrophobe and linkage
Compd
R
HCV NS5B
0.2
D-21 IC50 (lM)
N
N
N
HN
O
N
16j
O
6
N
S
N
O
R
16k
16l
2.8
Compd
R
HCV NS5B
0.45
D-21 IC50 (lM)
O
O
14b
0.93
0.7
O
S
O
16a
16b
0.71
16m
3,4-DiF
N
O
0.6
OMe
O
16c
0.4
CF3
O
16d
16e
16f
16g
0.22
0.19
0.042
0.4
COOMe
Figure 2. Induced-fit docking of 16f into NS5B. The compound is shown is stick
representation. The H-bonds to Arg-503 and Lys-491 are indicated. The figure was
produced using PyMOL (Warren L. DeLano The PyMOL Molecular Graphics System;
DeLano Scientific: Palo Alto, CA, USA).
O
MeOOC
activity. For this exploration we used 4-fluorophenyl ring as the C-
6 hydrophobe and this gave the baseline carboxylic acid analog 17a
with an IC50 of 0.93 lM in the biochemical assay. Coupling this car-
boxylic acid with aliphatic amines generated the amides with com-
O
plete loss of activity (data not shown). However, by synthesizing
carboxamides of commercially available
aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
a
-amino acids via 2-(7-
hexa-
HOOC
O
fluorophosphate (HATU) mediated coupling, several analogs with
improved biochemical activity were obtained and the results are
depicted in Table 4. With
and 17c) the biochemical activity showed a 2–3-fold improvement
compared to the parent 17a. When switching to -phenylalanine
(17d) the potency dropped slightly to 2 M, but simple addition
of a para hydroxy group in -tyrosine 17e afforded a significant
100-fold potency improvement. The -tyrosine amide (17i) and
tryptophan analog (17f) were equipotent to 17e, however the cor-
responding -tyrosine and -tryptophan analogs (17g and 17h)
exhibited a 25–50-fold potency loss. The most potent compound
synthesized in this sub-series was 5-hydroxy -tryptophan deriva-
L-leucine and L-lysine side chains (17b
L
l
L
L
L-
O
16h
16i
0.4
0.4
D
D
L
O
tive 17j, which had an IC50 of 11 nM in the biochemical assay. The
extra potency of compound 17j can be rationalized using modeling.
The docking pose of 17j presents a H-bond between the carboxyl-
ate and Arg-503 and a H-bond interaction between the 5-hydroxyl
MeO

J. Popovici-Muller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6331–6336
6335
Table 4
Carboxylic acid amides (C-3)
O
R
N
N
N
F
Compd
R
HCV NS5B
0.93
D-21 IC50 (lM)
17a
OH
H
N
O
17b
17c
17d
17e
17f
17g
17h
17i
0.29
0.48
2.0
OH
O
OH
HN
NH2
Figure 3. Docking model of compound 17j in NS5B. The compound is shown in
stick representation and the H-bonds to Arg-503 and Ser-431 are indicated with
dotted lines.
O
H
N
OH
O
O
O
Acknowledgments
H
N
OH
OH
0.02
0.02
0.96
0.45
0.024
0.011
The authors would like to acknowledge Drs. B. A. Malcolm, B. M.
Baroudy, and Charles Lesburg for insightful discussions.
OH
O
References and notes
NH
HN
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N
OH
OH
OH
O
NH
HN
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H
N
NH2
OH
OH
NH
O
HN
17j
OH
group and Ser-431 (Fig. 3). The H-bond to Ser-431 can only be opti-
mally reached with a 5-hydroxy -tryptophan group.
L
Some of the sub-micromolar inhibitors generated during this
optimization study (4, 14b, 17a) were subsequently tested in a
HCV cell-based replicon assay of RNA replication.20 However; these
inhibitors displayed only modest potencies in this assay, poten-
tially due to the higher molecular weight of the compounds.
In summary a novel series of HCV RNA polymerase inhibitors
based on a ‘6,7-pyrazolo[1,5-a]pyrimidine’ scaffold has been de-
scribed. Several compounds were optimized to low nanomolar
potencies in a biochemical RdRp assay. This series contributes to
further insights into the field of HCV pol inhibition.
9. Hashimoto, H.; Mizutani, K.; Yoshida, A. WO 03/000254.

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17. The X-ray structure of an indole-based inhibitor that binds to the finger-loop
region of NS5B (PDB access code: 2brk; Di Marco, S., Volpari, C., Tomei, L.,
Altamura, S., Harper, S., Narjes, F., Koch, U., Rowley, M., De Francesco, R.,
Migliaccio, G., Carfi, A., J. Biol. Chem. 2005, 280, 29765) was utilized as the frame
of reference. In preparation for docking, all ligand and water molecules were
deleted and hydrogen atoms were added and minimized using Macromodel as
implemented in Maestro 8.5 (Schrödinger, LLC, New York, NY, 2009). Docking of
16f to NS5B was performed using Glide XP (Halgren, T. A.; Murphy, R. B.;
Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. J. Med. Chem.
2004, 47, 1750; Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J.
J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.;
Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.) An inner box region (the
ligand midpoint remains within this box during docking) of 10 Å and outer box
region of 24 Å were used. The induced-fit docking protocol (Sherman, W.; Day,
T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. J. Med. Chem. 2006, 49, 534), IFD, was
used to perturb the initial NS5B model in the presence of the docked ligand 16f,
12. When R1 = OH, conversion to various substituted phenoxy analogs was done
via copper promoted arylation of phenol with aryl boronic acids following a
procedure by: Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39,
2937, while synthesis of other aryl substituted benzyloxy analogs was
accomplished either via alkylations with benzyl bromides and cesium
carbonate in DMF, or Mitsunobu reactions with ‘benzyl type’ alcohols,
triphenylphosphine and diisopropyl azodicarboxylate (DIAD).
13. Suzuki cross couplings were done using PdCl2(dppf), aryl boronates and
potassium phosphate in 1,4-dioxane. This procedure was also used to explore
aryl hydrophobes at C-6 as depicted in Scheme 2.
14. Briefly, 50
RNasIN, 0.25
MgCl2, 121 mM NaCl, 10 mM MgCl2, 2% DMSO, 0.05% glycerol, 100
and 0.05 M NS5B ( -21) were incubated at room temperature for 3 hours in
l
L reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT, 1 unit
g polyC/0.025 g oligoG12, 5 M GTP, 1
Ci/mL [3H]-GTP, 10 mM
g/mL BSA,
l
l
l
l
l
while allowing a flexible adaptation of the protein environment upon
compound binding. All the options used during IFD were standard, as
provided by the python interface in Maestro 8.5.
l
D
96-well plates with or without test compounds. Assay was terminated by the
addition of 0.5 mg streptavidin-coated SPA beads supplemented with 50 mM
EDTA, and the incorporation of labeled GTP was determined by a TopCount
Scintillation Counter. IC50 values were calculated from single experiments
using 11 serial twofold dilutions (0.05–50 lM), and data was considered
reliable only when the IC50 value of a positive internal control was within
standard deviation range.
18. (a) De Francesco, R.; Carfí, A. Adv. Drug Delivery Rev. 2007, 59, 1242; (b)
Beaulieu, P. L. Curr. Opin. Drug Discovery Dev. 2006, 9, 618.
19. Beaulieu, P. L.; Bös, M.; Bousquet, Y.; DeRoy, P.; Fazal, G.; Gauthier, J.; Gillard, J.;
Goulet, S.; McKercher, G.; Poupart, M.-A.; Valois, S.; Kukolj, G. Bioorg. Med.
Chem. Lett. 2004, 14, 967.
20. Lohmann, V.; Körner, F.; Koch, J. O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110.
15. Acid replacements on thumb domain inhibitors have been described in:
Stansfield, I.; Pompei, M.; Conte, I.; Ercolani, C.; Migliaccio, G.; Jairaj, M.;
Guiliano, C.; Rowley, M.; Narjes, F. Bioorg. Med. Chem. Lett. 2007, 17, 5143.