Discovery of 4H-pyrazolo[1,5-a]pyrimidin-7-ones as potent inhibitors of hepatitis C virus polymerase

Article abstract of DOI:10.1016/j.bmcl.2009.07.124

A series of 4H-pyrazolo[1,5-a]pyrimidin-7-one derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase. A number of these compounds exhibited potent activity in enzymatic assay. The synthesis and stru

Full text of DOI:10.1016/j.bmcl.2009.07.124

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5363–5367
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4H-pyrazolo[1,5-a]pyrimidin-7-ones as potent inhibitors
of hepatitis C virus polymerase
a
a
a
a
Yongqi Deng a, , Gerald W. Shipps Jr. , Tong Wang , Janeta Popovici-Muller , Kristin E. Rosner ,
*
M. Arshad Siddiqui a, Jose Duca b, Alan B. Cooper b, Michael Cable 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:
A series of 4H-pyrazolo[1,5-a]pyrimidin-7-one derivatives was synthesized and evaluated for inhibitory
activity against HCV NS5B RNA-dependent RNA polymerase. A number of these compounds exhibited
potent activity in enzymatic assay. The synthesis and structure–activity relationship are also described.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 June 2009
Revised 24 July 2009
Accepted 28 July 2009
Available online 6 August 2009
Keywords:
Hepatitis C virus
Polymerase
Pyrazolopyrimidin-ones
Anti-viral agents
Hepatitis C virus (HCV) was identified in 1989 as the pathogen
responsible for non-A and non-B hepatitis.1 It has been estimated
that 1–3% of the world’s population is infected and is a leading
cause of liver transplantation in the United States.2 Because the
virus often results in fibrosis and cirrhosis of the liver over a time
course that can last decades, it is projected that the societal costs of
this disease will increase over the next 20 years.3 Current treat-
thiophene or phenylalanine scaffolds. Given the availability of a
crystal structure for an inhibitor bound at the finger-loop site,
molecular modeling was employed as part of consideration of
alternative scaffolds and to rationalize observed SAR.
The improvement of intrinsic affinity through conformational
constraints is well-documented,9 and this prompted us to design
a new inhibitor series with a more rigid skeleton. Several scaffolds
were selected based on conformational analysis to see whether the
hydrophobic groups and carboxylic acid could be projected into a
similar orientation as in the aminothiazole series. One such scaf-
fold was 4H-pyrazolo[1,5-a]pyrimidin-7-one (PPO), which pos-
sesses good pharmacokinetic properties and has been widely
used in drug design (Fig. 1).10
ment options are limited, with interferon a-2b/ribavirin combina-
tions having the most widespread application. Unfortunately this
combination does not always produce sustained responses across
all viral genotypes, particularly genotype 1. Therefore, the develop-
ment of new inhibitors targeting HCV becomes urgent.
The HCV NS5B RNA-dependent RNA polymerase is a central en-
zyme in the replication of the virus and has been vigorously pur-
sued by several groups.4 Several nucleoside and non-nucleoside
NS5B inhibitors have been previously described.5,6 Recently we
described the discovery and initial optimization of aminothiazole
5-carboxylic acid derivatives 1 as HCV polymerase inhibitors.7 Sig-
nificant enhancement of the potency of this series proved to be
challenging. It was observed that the structure–activity relation-
ship (SAR), specifically the requirement for cyclohexyl, was
reminiscent of a series of inhibitors which bind to the ‘finger-loop’
region of NS5B.8 This binding site is distinct from the previously-
identified allosteric pocket that is occupied by compounds of either
Compound 2, the PPO analog of 1, was docked into the structure
of NS5B using an induced-fit docking procedure11 at the finger-
loop binding site. The three ‘anchors’ of compound 2 in the NS5B
finger-loop binding site—the carboxylate and neighboring phenyl
BnO
O
O
N
H
N
BnO
5
6
N
S
OH
O
N
2
N
OH
O
1
2
* Corresponding author. Tel.: +1 617 499 3516; fax: +1 617 499 3500.
E-mail address: yongqi.deng@spcorp.com (Y. Deng).
Figure 1. Design of pyrazolo[1,5-a]pyrimidin-7-one acid derivative 2 from amino-
thiazole 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.124

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Y. Deng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5363–5367
able to form a hydrogen bond interaction with the side chain of
His-428. The unsubstituted N-4 position projects out of the binding
pocket.
The IC50 value of 2 against HCV polymerase in the in vitro enzy-
matic assay was 1.4 lM, a fivefold improvement over 1. Hereafter,
we report the synthesis and optimization of a series of HCV poly-
merase inhibitors based on the 4H-pyrazolo[1,5-a]pyrimidin-7-
one scaffold.
Two synthetic routes were developed in order to produce deriv-
atives at C-5 and C-6 in a parallel manner. Scheme 1 depicts the
general synthetic route which was used to make analogs at C-6.
Reaction of the substituted acetophenone with diethyl carbonate
in the presence of sodium hydride generated the corresponding
b-ketone ester 3, which then alkylated with alkyl bromide to pro-
duce
a-substituted ketone ester 4. The substituted ketone ester
was then condensed with ethyl 5-amino-pyrazole-3-carboxylate
in the presence of catalytic amount of p-toluene sulfonic acid in
chlorobenzene to generate 6-substituted PPO ester 5. Subsequent
hydrolysis using aqueous lithium hydroxide in tetrahydrofuran
(THF) produced the desired PPO carboxylic acid 6 in quantitative
yield. Although analogs with various substitutions at C-5 can be
made from this route, various ketones of interest are limited and
this route is not optimal for large set of compounds. An alternative
synthetic route was then developed to access analogs with various
substitutions at C-5 and is highlighted in Scheme 2. In this syn-
Figure 2. Overlay of the docked orientation of 2 (magenta) with the finger-loop
inhibitor from the docking template structure (green; PDB 2brk). In addition to the
hydrogen bond donors Arg-503 and His-428, residues are labeled where the
sidechain orientation differs from the template resulting from the IFD procedure.
The figure was produced using PyMOL (Warren L. DeLano The PyMOL Molecular
Graphics System; DeLano Scientific: Palo Alto, CA, USA).
and cyclohexyl groups—overlay closely with their counterparts in
the model template as well as other reported indoles and benzim-
idazoles12 despite differences in scaffold and substitution (Fig. 2).
To our surprise, the exocyclic oxygen atom of the PPO core was
thetic route, the key intermediate
a-substituted b-ketone ester 7
was generated in one step by reacting aryl substituted ethyl ace-
O
O
O
O
O
a
b
OEt
OEt
R
BnO
BnO
BnO
3
4
BnO
BnO
H
N
H
N
d
c
CO2H
CO2Et
N
N
N
N
R
R
O
O
5
6
Scheme 1. Reagents and conditions: (a) diethyl carbonate, NaH, toluene, reflux, 16 h, 60–70%; (b) ArBr, KtBuO, EtOH; (c) 5-amino-2H-pyrazole-3-carboxylic acid ethyl ester,
p-toluene sulfonic acid, chlorobenzene, 12 h, 110 °C, 30–70%; d: LiOH, THF, rt, 14 h, 95%.
O
O
H
N
Ar
CO2Et
OEt
a
b c
Ar
OEt
CO2H
Ar
+
N
N
O
O
7
8
H
N
Ar
O
d
N
O
HN
R
N
9
Scheme 2. Reagents and conditions: (a) NaH, DME, reflux; (b) 5-amino-2H-pyrazole-3-carboxylic acid ethyl ester, p-toluene sulfonic acid, chlorobenzene, 12 h, 110 °C, 30–
70%; (c) LiOH, THF, rt, 14 h, 95%; (d) HATU, DIEA, rt, 2 h, 90%.

Y. Deng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5363–5367
5365
Table 1
tate with ethyl cyclohexanecarboxylate in the presence of sodium
Modification of cyclohexyl group
hydride in dimethoxyethane (DME). The subsequent transforma-
tion was then carried out under the same condition as that de-
scribed in Scheme 1 to give carboxylic acid 8 with various
functional groups at C-5 position. Compounds that were further
derivatized as amide 9 were prepared from acids generated from
these schemes and the corresponding amines via amide couplings
mediated by 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HATU) in dimethylformamide
(DMF) with N,N-diisopropylethylamine (DIEA).
BnO
H
N
O
N
N
OH
R
O
Compd
R
HCV NS5B
IC50 M)
D
-55
HCV NS5B
IC50 M)
D-21
(l
(l
To measure the efficacy of these compounds, a RNA polymerase
assay was performed utilizing scintillation proximity assay (SPA)
using radiolabeled GTP and a polyC/oligoG template/primer. Both
2
1.5 0.1
>50
1.0 0.2
>12.5
D
-55 and D-21 constructs of NS5B have been used according to a
modified literature procedure.13,14 Structure–activity relationship
studies were carried out using both assays. In general, the potency
6a
6b
of inhibitors using the
D-21 construct was higher than using D-55
construct, which may suggest a perturbation of the binding envi-
ronment. The rank order of IC50 values was similar between the
15 1.1
9.5 0.8
two assays for early lower potency compounds; however the
D-
21 assay proved to be more discerning for higher potency com-
pounds and ultimately this assay proved more informative.
The optimization began with an examination of substitution on
the 6-position of the PPO scaffold. Our initial study indicated that
an aryl group directly attached to C-6 was not tolerated as substi-
tution at C-6 with a phenyl group resulted in a loss of activity. Sub-
sequently, substitution at C-6 mainly focused on derivatives linked
through an aliphatic carbon. In general, larger hydrophobic groups
were preferred and resulted in compounds (6b and 6d–6g) with
moderate potency. This was consistent with the observation we
made in the aminothiazole series. Introduction of an oxygen atom
resulted compound 6c with similar potency. Although the phenyl
group was not tolerated when directly attached to the core, inser-
tion of a carbon spacer resulted in compounds with moderate po-
tency (6k–6m). The carbon spacer was also tolerated for the
cyclohexyl group, as compound 6j show comparable potency to
the parent compound 2 while compound 6n showed better activity
in both assays. However, due to the poor solubility of compound
6n and no significant improvement when compared to the parent
compound 2, we decided to maintain the cyclohexyl group at C-6
for subsequent analogs (Table 1).
Next, we investigated the substitutions on the C-5 position and
the SAR is summarized in Table 2. Replacing the benzoxy with a
methoxy group (compound 8a), a phenoxy group (compound
8b), a phenyl group (compound 8f) or a benzyl group (compound
8g) resulted in modest loss of potency compared to 2. However,
the distal phenoxy group substituted with either a methoxy or a
trifluoro group resulted in compounds 8c and 8d with similar po-
tency as 2. Further increasing the hydrophobicity at C-5 by a
substituted benzoxyl group in 2 led to small improvements in po-
tency in compounds 8h–8j. Interestingly, substitution at C-5 with
small aromatic groups resulted in compounds (8k and 8l) showing
6c
13 0.9
5.8 1.2
3.7 0.4
1.5 0.7
O
6d
6e
6f
>50
>12.5
15 1.0
10 0.4
6.7 0.8
3.5 0.2
6g
6h
6i
8.8 0.5
>12.5
2.8 0.3
10 1.2
O
6j
3.8 0.4
8.9 0.6
2.2 0.6
0.4 0.2
4.2 0.5
1.2 0.3
6k
6l
6m
6n
5.9 0.3
0.6 0.3
2.5 0.4
comparable potency in the
D-55 assay, and some improvement in
the -21 assay compared to compound 2. This SAR was distinct to
D
this series of compounds and differed from the aminothiazole and
0.17 0.1
similar compounds reported in the literature.5,15
The fact that the distal benzoxyl group is not necessary for this
series of compounds enabled us to reduce the molecular weight of
he scaffold. Initially, compound 8l was used to explore the SAR for
the carboxylic acid derivatives at the 2-position. Coupling the car-
boxylic acid of 8l with simple amines generated corresponding
amides with loss of activity. However, coupling 8l with commer-
Removing a carbon from tyrosine gives 4-hydroxyphenylglycine
analog 9c with threefold less potency. Replacing the 4-hydroxyl-
phenyl in tyrosine with an imidazole resulted in L-histidine analog
cially available
compounds with improved potency and new SAR (Table 3). For
example, the -tyrosine derivative 9a was found to be fourfold
more potent than the corresponding -tyrosine derivative 9b.
a
-amino acids with various side chains generated
9d with ninefold less potency. The most potent compound ob-
tained from this round of optimization was tryptophan analog
9e, which had an IC50 of 50 nM. The carboxylic acid functionality
was found to be important as the corresponding methyl ester 9f
L
D

5366
Y. Deng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5363–5367
Table 2
Table 3
SAR at the distal hydrophobic group
H
N
Ar
O
H
N
Ar
O
N
N
NHR
N
N
OH
O
O
Compd
Ar
R
HCV NS5B D-21 IC50 (lM)
Compd
R
HCV NS5B
IC50 M)
D
-55
HCV NS5B
IC50 M)
D-21
(
l
(l
O
OH
OH
HO
9a
0.07 0.01
O
O
2
1.5 0.1
1.0 0.2
O
O
O
O
HO
HO
8a
8b
3.9 0.2
6.3 1.2
n/a
n/a
9b
9c
9d
9e
0.30 0.03
Ph
O
0.2 0.01
0.64 0.02
0.05 0.01
O
O
O
O
O
O
8c
8d
8e
8f
0.9 0.1
n/a
OH
H
N
O
O
O
HO
HO
O
CF3
0.93 0.03
3.3 0.03
3.3 0.3
n/a
N
H
N
Cl
n/a
O
H
N
9f
0.27 0.05
0.18 0.01
0.23 0.04
3.1 0.2
O
O
Ph
8g
8h
4.0 0.5
1.2 0.1
2.6 0.1
1.0 0.3
O
9g
9h
S
OMe
CF3
O
O
O
O
H
N
O
F
F
HO
8i
8j
1.8 0.1
0.9 0.04
1.5 0.2
OCF3
O
O
OH
HO
HO
2.2
0.1
9i
0.68 0.05
0.06 0.01
0.20 0.03
F
H
N
8k
2.3 0.4
0.38 0.2
9j
O
8l
2.8 0.1
6.4 0.5
0.29 0.05
1.1 0.3
O
S
H
N
O
HO
9k
8m
S

Y. Deng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5363–5367
5367
W. W.; Kati, W. M.; Liu, D.; Liu, Y.; Maring, C. j.; Masse, S.; McDaniel, K. F.;
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showed fivefold loss of potency. The combination of tryptophan
with different functional groups at C-5 position was also briefly ex-
plored. The position of the oxygen in the furan group showed little
impact on the potency as 2-furanyl (compound 9e) and 3-furanyl
groups (compound 9j) have similar potency. However, the corre-
sponding thiophene shows fourfold loss of potency and similar re-
sults was observed for 3-fluorophenyl derivative 9h. Consistent
with the molecular model, the bioisosteric replacement of the C-
2 carboxylate was tolerated as long as a hydrogen bond is main-
tained to the protein (Fig. 2). In fact, the solvent-accessibility of this
group is consistent with the modest effect observed upon elabora-
tion at this position.
6. (a) Beaulieu, P. L. Curr. Top. Med. Chem. 2007, 8, 614; (b) Koch, U.; Narjes, F.
Infect. Dis. Drug Targets 2006, 6, 31.
7. Shipps, G. W.; Deng, Y.; Wang, T.; Popovici-Muller, J.; Curran, P. J.; Rosner, K. E.;
Cooper, A. B.; Girijavallabhan, V.; Butkiewicz, N.; Cable, M. Bioorg. Med. Chem.
Lett. 2005, 15, 115.
8. 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.
9. (a) Ikegashira, K.; Oka, T.; Hirashima, S.; Noji, S.; Yamahaka, 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; (b) Hirashima, S.; Oka, T.;
Ikegashira, K.; Noji, S.; Yamanaka, H.; Hara, Y.; Goto, H.; Mizojiri, R.; Nima, Y.;
Noguchi, T.; Ando, I.; Ikeda, S.; Hashimoto, H. Bioorg. Med. Chem. Lett. 2007, 17,
3181.
The most potent inhibitors generated during this optimization
study were subsequently tested in an HCV cell-based replicon as-
say of RNA replication.16 Compound 9e, despite its enzyme po-
tency, had only weak potency in the cell-based assay (ꢀ50
lM),
which led us to believe that this compound might have poor per-
meability likely due to the highly ionizable carboxylic acid group.
Subsequent efforts have focused on replacing the carboxylic acid
with bioisosteres to improve permeability. Moreover, substitution
at unexplored solvent-exposed positions such as N-4 should afford
modulation of physicochemical compound properties with little
effect on intrinsic binding. Further efforts in this series and the re-
sults of cell permeability and activity in the cell-based assays, will
be reported.
10. (a) Senga, K.; Novinson, T.; Wilson, H. R. J. Med. Chem. 1981, 24, 610; (b) Selleri,
S.; Bruni, F.; Costagli, C.; Costanzo, A.; Guerrini, G.; Ciciani, G.; Gratteri, P.;
Bonaccini, C.; Aiello, Petra M.; Besnard, F.; Renard, S.; Costa, B.; Martini, Cla. J.
Med. Chem. 2003, 46, 310.
11. The X-ray structure of an indole-based inhibitor that binds to the finger-loop
region of NS5B (PDB access code: 2brk) was utilized as the frame of Ref. 8. 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 2 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 2,
In conclusion, suitably substituted 4H-pyrazolo[1,5-a]pyrimi-
din-7-one analogs have been shown to be active against the
NS5B RNA-dependent RNA polymerase. This series was obtained
through conformational constraint of the previously reported ami-
nothiazole series. Through the optimization of the 3, 5, and 2 posi-
tions, compounds with double digit nM potency in the in vitro
enzymatic assay were obtained.
while allowing
a flexible adaptation of the protein environment upon
compound binding. The IFD protocol consists of four steps: (1) softened-
potential docking into the rigid receptor to generate an ensemble of poses; (2)
sampling of protein conformations for each ligand pose generated in the first
step; (3) redocking of the ligand into low energy induced-fit structures from
the previous step; and (4) rescoring by accounting for the docking energy.
Extra precision docking was used during the third and fourth steps (Glide XP
(Schrödinger, LLC, New York, NY, 2009). All the options used during IFD were
standard, as provided by the python interface in Maestro 8.5.
Acknowledgments
The authors would like to acknowledge Drs. B.A. Malcolm, B.M.
Baroudy, and Charles Lesburg for insightful discussions.
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13. Briefly, 50
l
L
reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT,
g/mL biotinylated oligoG12, 5 g/mL polyC, 0.5
Ci/mL [3H]-GTP, 10 mM MgCl2, 60 mM NaCl, 100
g/mL BSA, and 6 nM
55) were incubated at room temperature for 50 min in 96-well plates
20 units/mL RNasIN, 0.5
l
l
l
lM
GTP, 1
NS5B (
l
D
with or without test compounds. Assay was terminated by the addition of
50 L 10 mg/mL streptavidin-coated SPA beads supplemented with 100 mM
EDTA, and the incorporation of labeled GTP determined by TopCount
Scintillation Counter. IC50 values were calculated from single experiments
using 11 serial twofold dilutions (0.05–50 M), and data were considered
l
a
l
reliable only when the IC50 value of a positive internal control was within
standard deviation range.
14. Assay was identical to that described in (9) except 5
lM GTP, 20
l
Ci/mL [3H]-
GTP, and 50 nM NS5B (D-21) enzyme form were used in a 3-h reaction at room
temperature.
15. Ishida, T.; Suzuki, T.; Hirashima, S.; Mizutani, K.; Yoshida, A.; Ando, I.; Ikeda, S.;
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