Synthesis and SAR optimization of diketo acid pharmacophore for HCV NS5B polymerase inhibition

Article abstract of DOI:10.1016/j.ejmech.2011.08.028

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Here we report the synthesis and biological evaluation of a new series of α,γ-diketo acids (DKAs) as NS5B polymerase inhibitors. We initiated structure-activity

Full text of DOI:10.1016/j.ejmech.2011.08.028

European Journal of Medicinal Chemistry 46 (2011) 5138e5145
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and SAR optimization of diketo acid pharmacophore for HCV NS5B
polymerase inhibition
,
1
,
,
Aaditya Bhatta , K.R. Gurukumarb 1, Amartya Basub, Maulik R. Patela, Neerja Kaushik-Basub
**
,
Tanaji T. Talelea
,
*
a Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Queens, NY 11439, USA
b Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Here we
report the synthesis and biological evaluation of a new series of -diketo acids (DKAs) as NS5B poly-
merase inhibitors. We initiated structureeactivity relationship (SAR) optimization around the furan
moiety of compound 1a [IC50 ¼ 21.8 M] to achieve more active NS5B inhibitors. This yielded compound
3a [IC50 ¼ 8.2 M] bearing the 5-bromobenzofuran-2-yl moiety, the first promising lead compound of the
series. Varying the furan moiety with thiophene, thiazole and indazole moieties resulted in compound
11a [IC50 ¼ 7.5 M] bearing 3-methylthiophen-2-yl moiety. Finally replacement of the thiophene ring
with a bioisosteric phenyl ring further improved the inhibitory activity as seen in compounds 21a
[IC50 ¼ 5.2 M] and 24a [IC50 ¼ 2.4 M]. Binding mode of compound 24a using glide docking within the
active site of NS5B polymerase will form the basis for future SAR optimization.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 26 July 2011
Received in revised form
18 August 2011
Accepted 19 August 2011
Available online 26 August 2011
a,g
m
m
m
Keywords:
a,g-Diketo acid
Pyrophosphate
NS5B polymerase
Docking
m
m
1. Introduction
crystal structure of HCV NS5B polymerase reveals a classical “right
hand” shape with characteristic fingers, palm and thumb sub-
domains. The combination of crystallographic, biochemical and
mutagenesis studies has facilitated the identification of five distinct
non-nucleoside inhibitor (NNI) binding sites on NS5B [14e17].
Allosteric pockets, AP-1 and AP-2 are located in the thumb sub-
domain, whereas AP-3 and AP-4 are partially overlapped and
located in the palm subdomain, in close proximity to the active site,
and AP-5 is located in the fingers subdomain [17e20]. The NS5B
polymerase active site is located in the palm subdomain, which
contains two Mg2þ ions that are coordinated by conserved aspartic
acid residues Asp220 and Asp318 [18]. The Mg2þ ions serve the dual
role of positioning/stabilizing the pyrophosphate leaving group on
the incoming nucleoside triphosphate (NTP) and activating the 30-
Hepatitis C virus (HCV) infection has emerged as a significant
global public health problem. HCV is responsible for the develop-
ment of malignant chronic liver disease, including liver cirrhosis
and hepatocellular carcinoma, which frequently ends in liver failure
[1e4]. An estimated 200 million cases of HCV infections exist
worldwide, of which over 4.1 million are in the United States [5].
Despite its large medical and economic impact, neither a vaccine
nor a therapy with effective broad spectrum mode of action against
all genotypes of HCV is available [6e8]. Current HCV therapy
comprising of pegylated interferon
a (PEGeIFN-a) in combination
with ribavirin has found limited patient compliance due to severe
adverse effects [9,10]. Therefore, there is an urgent need to develop
novel and efficacious antiviral therapeutics targeting HCV.
The HCV non-structural protein 5B (NS5B), a 66 kDa RNA-
dependent RNA polymerase (RdRp), is an attractive therapeutic
target, since it plays an important role in replicating the HCV RNA
genome and the host lacks its functional equivalent [11e13]. The
OH of the growing RNA for nucleophilic attack on the
group of NTP [14,15].
a-phosphate
Derivatives of 4,5-dihydroxypyrimidine carboxylic acid [19]
and -diketo acids (DKAs) have been reported previously as
a,g
pyrophosphate (PPi) mimetic inhibitors of HIV-1 IN [20] and HCV
NS5B polymerase [19,21]. These compounds function as product-
like analogs and chelate the divalent metal ions at the active site
of NS5B [22,23]. In continuation of our efforts toward identifica-
tion and development of NS5B PPi mimetic inhibitors [24], we
have explored the effect of various substituted aryl/heteroaryl
specificity domain of DKAs on NS5B inhibition. Herein we
* Corresponding author. Tel.: þ1 718 990 5405; fax: þ1 718 990 1877.
** Corresponding author. Tel.: þ1 973 972 8653; fax: þ1 973 972 5594.
E-mail addresses: kaushik@umdnj.edu (N. Kaushik-Basu), talelet@stjohns.edu
(T.T. Talele).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.028

A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
5139
describe the synthesis, SAR, and molecular modeling of optimized
DKA analogs.
2. Results and discussion
2.1. Chemistry
The a,g-diketo esters (1e13, 15e20 and 22e24) were synthe-
sized by Claisen condensation of appropriate acetyl derivative with
diethyl oxalate in the presence of freshly prepared sodium eth-
oxide. The resulting a,g-diketo esters were readily hydrolyzed by
treating with 1N sodium hydroxide to corresponding DKAs
(1ae13a, 15ae20a and 22ae24a) (Scheme 1) [25]. Target
compound 14a was prepared in good yields from the 1H-indazole-
3-carboxylic acid as shown in Scheme 2. The 3-acetyl-1H-indazole
intermediate was synthesized from 1H-indazole-3-carboxylic acid
by initial conversion into Winreb amide using the one-pot reaction:
mixed anhydride method followed by nucleophilic attack of N,O-
dimethylhydroxylamine hydrochloride. The Winreb amide thus
formed was further treated with methyl magnesium bromide to
yield 3-acetyl-1H-indazole [26]. The 3-acetyl-1H-indazole was
Scheme 2. (a) (i) Isobutyl chloroformate, N-methylmorpholine, ꢀ20 ꢁC, 4 h, (ii) N,O-
dimethylhydroxylamine hydrochloride in triethylamine, rt, 6 h; (b) anhydrous THF,
methyl magnesium bromide (12% in THF), ꢀ78 ꢁC, 2 h, rt, 5 h, 55% over two steps; (c)
diethyl oxalate, sodium ethoxide, anhydrous THF, rt, 78%; (d) 1 N NaOH, THF/methanol
1:1, rt, 72%.
than the methyl analog. We also explored the effect of variation in
the position of the substitution around the thiophene ring. Moving
the methyl group from the 5th to the 4th-position (compound 10a),
improved the activity of the compound by w2-fold; whereas
moving it to the 3rd-position yielded compound 11a with w7-fold
higher inhibitory activity. Replacement of the thiophene ring with
thiazole (compound 12a) led to w 1.8-fold decrease in activity.
Introduction of the pyrrole ring in place of the furan ring yielded
compound 13a with similar activity. Introduction of the indazole
moiety in place of the benzofuran (compound 14a) conferred
slightly improved inhibitory activity. Among several corresponding
converted to
a,g-diketo ester 14 and further converted to
compound 14a as described above. Target compound 21a was
prepared from 20a by reacting it with sodium azide in the presence
of the catalytic amount of ammonium chloride (Scheme 3) [27].
2.2. Structureeactivity relationship
The HCV NS5B RdRp inhibitory activity of DKAs is reported in
Table 1. All the DKAs exhibited appreciable inhibition of HCV NS5B
polymerase, with IC50 values ranging from 2.4
mM to 63.9 mM.
a,g-diketo esters tested (data not shown), only compounds 14 and
Having identified compound 1a, a modest NS5B inhibitor as the
initial hit, we sought to explore SAR around the aryl/heteroaryl
specificity domain while keeping the DKA metal chelating phar-
macophore constant. Replacing furan moiety with benzofuran led
to compound 2a which was marginally less active. Since the
17 conferred inhibitory activity comparable to their acid counter-
parts, thus underscoring the observation that carboxylate anion
moiety may be critical for chelating the active site divalent metal
ions.
We next explored the influence of substituted phenyl ring
versus the heterocyclic ring on NS5B inhibition. Para-biphenyl
derivative 15a, the first analog explored, was w4-fold less active
than the previously reported unsubstituted phenyl analog
benzofuran ring is in conjugation with the
g-keto group, we
expected that the electron withdrawing bromo group would
increase the acidity of the diketo moiety and its ionized enolic form
would be more suitable for strong metal chelation, thereby
resulting in a relatively more active inhibitor. Consistent with this
hypothesis, compound 3a bearing 5-bromo substitution on the
benzofuran ring exhibited w3.5-fold improvement in inhibitory
activity over compound 2a. Bioisosteric replacement of the
benzofuran moiety by benzothiophene resulted in compound 4a,
with marginal loss of activity with respect to 2a. By contrast,
replacement of benzothiophene (compound 4a) with thiophene
(compound 5a), increased inhibitory activity by w2.2-fold. We
further explored the influence of varying substituents on the
thiophene ring of compound 5a. Substitution of the methyl group
at 5-position of the thiophene ring resulted in compound 6a with
approximately 3-fold decrease in activity than the parent
compound. Bioisosteric replacement of the methyl group with
chloro (compound 7a) or bromo (compound 8a) groups resulted in
w2-fold enhancement of inhibitory activity, whereas 5-iodo
derivative, compound 9a, was found to be relatively less active
(IC50 ¼ 5.7
mM) by Summa and colleagues [28], thus suggesting
possible steric hindrance by the second phenyl ring within the
active site of NS5B polymerase. The 2-nitrophenyl and 2-
fluorophenyl analogs (compounds 16a and 17a, respectively)
were w2-fold more active than 15a, while the 2-methylphenyl
derivative 18a was comparable in activity to the unsubstituted
phenyl analog [28]. Further, the 3-fluorophenyl (compound 19a)
and 3-cyanophenyl (compound 20a) analogs were 7e8 fold less
active, while the 3-(1H-tetrazol-2-yl)phenyl analog (compound
21a) was marginally more active than the unsubstituted phenyl
analog [28]. The 2,6-difluorophenyl derivative 22a exhibited
comparable activity to 2-fluorophenyl analog, thus suggesting that
the second ortho-fluoro substituent may not be important for NS5B
inhibition. The 2,4-dichlorophenyl analog (compound 23a) was 8-
Scheme 1. (a) Diethyl oxalate, sodium ethoxide, anhydrous THF, rt, 65e86%; (b) 1 N
NaOH, THF/methanol 1:1, rt, 70e86%.
Scheme 3. (a) Ammonium chloride, sodium azide, DMF, 110 ꢁC, 6 h, 75%.

5140
A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
Table 1
Structures and NS5B inhibitory activities of target compounds.
Compd
Ar
R
IC50, mM
1a
2a
3a
4a
5a
6a
7a
8a
furan-2-yl
H
H
H
H
H
H
H
H
H
H
H
H
H
C2H5
H
H
H
C2H5
H
H
H
H
H
H
H
H
21.8 ꢂ 1.30
29.6 ꢂ 1.30
8.2 ꢂ 1.20
37.7 ꢂ 1.30
17.3 ꢂ 1.10
55.5 ꢂ 1.20
25.5 ꢂ 0.40
31.4 ꢂ 1.20
63.9 ꢂ 1.30
32.7 ꢂ 1.30
7.5 ꢂ 1.30
30.4 ꢂ 2.10
21.7 ꢂ 1.30
38.4 ꢂ 1.40
24.7 ꢂ 1.30
20.9 ꢂ 1.90
11.3 ꢂ 1.10
12.4 ꢂ 1.50
14.9 ꢂ 1.20
6.9 ꢂ 1.10
49.9 ꢂ 1.10
39.7 ꢂ 1.30
5.2 ꢂ 1.20
17.0 ꢂ 1.20
49.7 ꢂ 1.10
2.4 ꢂ 0.05
benzofuran-2-yl
5-bromobenzofuran-2-yl
benzothiophen-2-yl
thiophen-2-yl
5-methylthiophen-2-yl
5-chlorothiophen-2-yl
5-bromothiophen-2-yl
5-iodothiophen-2-yl
4-methylthiophen-2-yl
3-methylthiophen-2-yl
thiazol-2-yl
pyrrol-2-yl
indazol-3-yl
indazol-3-yl
para-biphenyl
2-nitrophenyl
2-fluorophenyl
2-fluorophenyl
2-methylphenyl
3-fluorophenyl
9a
10a
11a
12a
13a
14
14a
15a
16a
17
17a
18a
19a
20a
21a
22a
23a
24a
Fig. 1. Glide docking model of 24a within the active site of NS5B polymerase. Amino
acid residues are shown in as stick model whereas inhibitor is shown as ball and stick
model. The metal ions (Mnþ2) A and B are shown in cyan color. Dotted black line
indicates hydrogen bonding interaction whereas dotted red line indicates electrostatic
interactions. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
3-cyanophenyl
increase in electron density at the oxygen atom of
a-hydroxy group.
3-(tetrazol-2-yl)phenyl
2,6-difluorophenyl
2,4-dichlorophenyl
2,4-difluorophenyl
The -keto group is located within hydrogen bonding interaction
g
distance from the active site water molecule 2478 (C]
O/HOH2478, 2.3 Å), which in turn forms a hydrogen bond with the
carboxylate group of Asp225. Since 24a forms water mediated
interaction with the strictly conserved Asp225, it is plausible that
the virus would not easily acquire mutations to escape inhibition by
such inhibitors. Indeed, mutations of Asp225 have been shown to
completely inactivate the enzyme [29].
fold less active than the unsubstituted phenyl analog [28], while
2,4-difluorophenyl analog (compound 24a) proved to be the most
active compound in the present investigation.
These studies thus shed light on the effect of various substituted
aryl/heteroaryl groups in DKAs on NS5B inhibition, and have
identified 2,4-difluorophenyl as a promising specificity domain for
further development. To address the issue of poor cell membrane
permeability of DKAs, current efforts are underway to synthesize
tetrazole bioisosteres of the metal chelating carboxyl group in DKA
pharmacophore. This endeavor may potentially develop more
potent compounds with promising cell permeability and physico-
chemical properties. The results of this investigation will be the
subject of a future communication.
3. Conclusions
A new series of a,g-diketo acids linked to varied aryl/heteroaryl
rings was successfully developed as HCV NS5B polymerase inhibi-
tors. The present SAR revealed the tolerance of various aryl/heter-
oaryl systems such as benzofuran, thiophene and benzene as the
specificity domain. The 2,4-difluorophenyl ring as specificity
domain (compound 24a) was found to be the most potent inhibitor
of NS5B polymerase in the present study. Docking model of 24a
within the active site of NS5B polymerase will serve as a rational
basis for future SAR optimization.
2.3. Glide predicted binding mode of compound 24a
4. Experimental
To understand the binding mechanism of these synthesized
compounds, we generated a Glide docking model on the basis of
the co-crystal structure of rUTP-NS5B polymerase. As expected,
DKA pharmacophore chelates two Mn2þ ions through coordinate
bonding network (Fig. 1). Metal A is coordinated by the carboxylate
anion of the DKA pharmacophore, while metal B is coordinated by
4.1. Chemistry-general
Melting points (mp) were determined on a Thomas-Hoover
capillary melting point apparatus and are uncorrected. The
reagents for organic synthesis were purchased from Aldrich
Chemical Co. (Milwaukee, WI), TCI America (Portland, OR), Alfa
Aesar (Ward Hill, MA), and Acros Organics (Antwerp, Belgium). All
compounds were checked for homogeneity by TLC using silica gel
as a stationary phase. NMR spectra were recorded on a Bruker 400
Avance DPX spectrometer (1H at 400 MHz and 13C at 100 MHz)
outfitted with a z-axis gradient probe. The chemical shifts for 1H
the carboxylate anion in addition to the ionizable tautomeric a-
hydroxy group. The other coordinate bonds to both metals are
contributed by the conserved active site aspartic acid residues e
Asp220, Asp318 and Asp319 and the backbone carbonyl oxygen
atom of Thr221. The 2,4-difluorophenyl moiety is stabilized
through hydrophobic interactions with the methylene groups of
Arg158 and the side chain of Ile160. The 4-fluoro substituent is
located within hydrogen bonding distance from the hydroxyl group
of Ser282 (eF/HO Ser282, 2.5 Å). An intramolecular hydrogen
and 13C are reported in parts per million (
d ppm) downfield from
the tetramethylsilane (TMS) as an internal standard. The 1H NMR
data are reported as follows: chemical shift, multiplicity (s) singlet,
(bs) broad singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet.
The C, H, and N analyses were performed by Atlantic Microlabs, Inc.,
bond between the
g-keto and a-hydroxyl groups forms a pseudo-
six membered ring, a feature favorable to metal chelation due to

A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
5141
(Norcross, GA) and the observed values were within ꢂ0.4% of
4.3.5. Ethyl 4-(5-bromobenzofuran-2-yl)-2-hydroxy-4-oxobut-2-
enoate (3)
calculated values.
Obtained from 2-acetyl-5-bromobenzofuran (0.74 g, 3.12 mmol)
as a yellow solid (0.79 g, 76%); m.p. 260e262 ꢁC; Rf ¼ 0.72 (n-
4.2. General procedure for the preparation of a,g-diketo esters [25]
hexane:EtOAc 70:30); 1H NMR (400 MHz, DMSO-d6, TMS)
d 14.43
(s, 1H), 7.85 (s, 1H), 7.59 (d, J ¼ 7.1 Hz, 1H), 7.55 (d, J ¼ 7.0 Hz, 1H),
7.48 (d, J ¼ 7.9 Hz, 1H), 7.09 (s, 1H), 4.42 (q, J ¼ 7.1 Hz, 2H), 1.43 (t,
J ¼ 7.1 Hz, 3H).
Following the reported procedure [25], sodium ethoxide (0.43 g,
6.30 mmol) was added into a well stirred mixture of appropriate
acetyl derivatives (3.12 mmol) and diethyl oxalate (0.92 g,
6.24 mmol) in anhydrous THF (15 mL) under nitrogen atmosphere.
The reaction mixture was stirred at room temperature and upon
completion of the reaction, was poured into n-hexane (50 mL). The
precipitates were collected and then vigorously stirred in 1N HCl
(30 mL) for 30 min. The resultant solid formed was filtered, washed
with water, and dried under vacuum. The crude mass obtained was
further dissolved in ethyl acetate and reprecipitated with hexane to
obtain desired esters 1e20 and 22e24.
4.3.6. 4-(5-Bromobenzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoic acid
(3a)
Obtained from 3 (0.44 g, 1.3 mmol) as a yellow solid (0.32 g,
80%); m.p. 270e273 ꢁC; Rf ¼ 0.40 (EtOAc:n-hexane 60:40); 1H NMR
(400 MHz, DMSO-d6, TMS)
d
13.74 (bs, 2H), 8.03 (d, J ¼ 6.8 Hz, 2H),
7.72 (d, J ¼ 7.7 Hz, 1H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.00 (s, 1H); 13C NMR
(100 MHz, DMSO-d6, TMS)
d 99.53, 114.08, 114.85, 116.93, 126.31,
129.63, 131.74, 152.27, 154.66, 163.36, 168.28, 179.94. Anal. Calcd. for
C12H7BrO5: C, 46.33; H, 2.27. Found: C, 46.45; H, 2.40.
4.3. General procedure for the preparation of a,g-diketo acids [25]
4.3.7. Ethyl 4-(benzo[b]thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate
(4)
The mixture of 1N NaOH (7 mL) and corresponding ester
derivatives 1e20 and 22e24 (1.3 mmol) in THF/methanol 1:1
(15 mL) was stirred at room temperature for 45 min. Upon
completion, the reaction mixture was concentrated under vacuum.
The viscous concentrate was poured onto crushed ice and acidified
with 1 N HCl to pH 1. The precipitates obtained were filtered,
washed with water and dried under vacuum. The crude mass
obtained was purified by crystallizing it from ethanol which yielded
Obtained from 2-acetylbenzo[b]thiophene (0.55 g, 3.12 mmol)
as a yellow solid (0.68 g, 79%); m.p. 190e194 ꢁC; Rf ¼ 0.65 (n-
hexane:EtOAc 60:40); 1H NMR (400 MHz, DMSO-d6, TMS)
d 13.96
(bs, 1H), 8.71 (s, 1H), 8.08 (d, J ¼ 7.2 Hz, 1H), 8.01 (d, J ¼ 7.2 Hz, 1H),
7.54 (t, J ¼ 7.1 Hz, 1H), 7.50 (t, J ¼ 7.1 Hz, 1H), 7.21 (s, 1H), 4.34 (q,
J ¼ 7.1 Hz, 2H), 1.33 (t, J ¼ 7.1 Hz, 3H).
the desired
a
,g-diketo acids 1ae20a and 22ae24a.
4.3.8. 4-(Benzo[b]thiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid
(4a) [30]
4.3.1. Ethyl 4-(furan-2-yl)-2-hydroxy-4-oxobut-2-enoate (1)
Obtained from 2-acetylfuran (0.34 g, 3.12 mmol) starting
material as white solid (0.55 g, 81%); m.p. 120e122 ꢁC; Rf ¼ 0.52 (n-
Obtained as a yellow solid (0.24 g, 74%) from 4 (0.36 g, 1.3 mmol)
as the starting material; m.p. 212e215 ꢁC; Rf ¼ 0.42 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz, DMSO-d6, TMS)
d 14.14 (bs, 2H),
hexane:EtOAc 60:40); 1H NMR (400 MHz, CDCl3, TMS)
d
14.45 (s,
8.65 (s, 1H), 8.09 (d, J ¼ 7.7 Hz, 1H), 8.02 (d, J ¼ 7.6 Hz, 1H), 7.56 (t,
1H), 7.68 (dd, J ¼ 1.7 Hz, 0.7 Hz, 1H), 7.35 (dd, J ¼ 3.5 Hz, 0.6 Hz, 1H),
6.94 (s, 1H), 6.62 (dd, J ¼ 3.5 Hz, 1.6 Hz, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H),
1.41 (t, J ¼ 7.2 Hz, 3H).
J ¼ 7.4 Hz, 1H), 7.48 (t, J ¼ 7.4 Hz, 1H), 7.16 (s, 1H); 13C NMR
(100 MHz, DMSO-d6, TMS)
d 99.77, 123.65, 125.91, 127.02, 128.61,
132.03, 139.64, 142.15, 142.54, 163.53, 165.01, 187.37. Anal. Calcd. for
C12H8O4S: C, 58.06; H, 3.25. Found: C, 58.33; H, 3.28.
4.3.2. 4-(Furan-2-yl)-2-hydroxy-4-oxobut-2-enoic acid (1a) [30]
Using 1 (0.27 g, 1.3 mmol) as the starting material, 1a was
obtained as a pale yellow solid (0.17 g, 72%); m.p. 145e148 ꢁC;
Rf ¼ 0.42 (EtOAc:n-hexane 60:40); 1H NMR (400 MHz, DMSO-d6,
4.3.9. 2-Hydroxy-4-(thiophen-2-yl)-4-oxobut-2-enoic acid (5a) [31]
Obtained as pink flakes (0.54 g, 76%) using 2-acetylthiophene
(0.39 g, 3.12 mmol) as starting material, followed by subsequent
TMS)
d
14.55 (bs, 2H), 8.09 (s, 1H), 7.67 (s, 1H), 6.79 (dd, J ¼ 3.7 Hz,
hydrolysis of the diketoester; m.p. 170e173 ꢁC; Rf ¼ 0.40 (n-hex-
1.5 Hz, 2H); 13C NMR (100 MHz; DMSO-d6; TMS)
d
99.16 (2), 113.83,
ane:EtOAc 60:40); H NMR (400 MHz, DMSO-d6, TMS)
d
14.40 (bs,
1
119.86,149.48,150.90,163.61 (2). Anal. Calcd. for C8H6O5.1/3H2O: C,
51.07; H, 3.57. Found: C, 51.03; H, 3.41.
2H), 8.22 (d, J ¼ 3.8 Hz,1H), 8.15 (d, J ¼ 4.9 Hz,1H), 7.30 (t, J ¼ 4.4 Hz,
1H), 7.02 (s, 1H); 13C NMR (100 MHz, DMSO-d6, TMS)
d
115.3, 126.3,
129.5, 136.7, 141.8, 169.2, 178.2, 191.3 Anal. Calcd. For C8H6O4S: C,
48.48; H, 3.05. Found: C, 48.57; H, 3.09.
4.3.3. Ethyl 4-(benzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoate (2)
Obtained from 2-acetylbenzofuran (0.49 g, 3.12 mmol) as yellow
solid (0.65 g, 80%); m.p. 80e83 ꢁC; Rf ¼ 0.43 (n-hexane:EtOAc
4.3.10. Ethyl-2-hydroxy-4-(5-methylthiophen-2-yl)-4-oxobut-2-
enoate (6)
70:30); 1H NMR (400 MHz, DMSO-d6, TMS)
d 12.35 (bs, 1H), 7.89 (s,
1H), 7.79 (d, J ¼ 7.8 Hz, 1H), 7.72 (d, J ¼ 8.2 Hz, 1H), 7.52 (t, J ¼ 7.7 Hz,
1H), 7.35 (t, J ¼ 7.5 Hz, 1H), 6.80 (s, 1H), 4.27 (q, J ¼ 7.0 Hz, 2H), 1.30
(t, J ¼ 7.1 Hz, 3H).
Obtained from 2-acetyl-5-methylthiophene (0.44 g, 3.12 mmol)
as yellow solid (0.61 g, 82%); m.p. 160e162 ꢁC; Rf ¼ 0.70 (n-hex-
ane:EtOAc 80:20); 1H NMR (400 MHz; CDCl3; TMS)
d 14.66 (s, 1H),
7.68 (d, J ¼ 3.8 Hz, 1H), 6.88e6.84 (m, 2H; due to overlap of allylic
(s) and thiophene ring (d) protons), 4.38 (q, J ¼ 7.1 H, 2H), 2.58 (s,
3H), 1.40 (t, J ¼ 7.1 Hz, 3H).
4.3.4. 4-(Benzofuran-2-yl)-2-hydroxy-2-oxobuten-2-enoic acid (2a)
[30]
Obtained from 2 (0.34 g, 1.3 mmol) as a bright yellow solid
(0.25 g, 83%); m.p. 145e147 ꢁC; Rf ¼ 0.31 (EtOAc:n-hexane 60:40);
4.3.11. 2-Hydroxy-4-(5-methylthiophen-2-yl)-4-oxobut-2-enoic acid
(6a)
1H NMR (400 MHz, DMSO-d6, TMS)
d 14.12 (bs, 2H), 8.12 (s,1H), 7.84
(d, J ¼ 7.9 Hz, 1H), 7.77 (d, J ¼ 7.4 Hz, 1H), 7.58 (t, J ¼ 7.8 Hz, 1H), 7.39
Obtained from 6 (0.31 g, 1.3 mmol) as yellow solid (0.20 g, 74%);
(t, J ¼ 7.5 Hz, 1H), 6.03 (s, 1H); 13C NMR (100 MHz, DMSO-d6, TMS)
m.p. 172e174 ꢁC; Rf ¼ 0.53 (EtOAc:n-hexane 60:40); 1H NMR
d
99.62, 123.57, 124.61, 127.31, 127.69, 128.03, 135.61, 146.63, 155.82,
(400 MHz, DMSO-d6, TMS)
d
14.12 (bs, 2H), 8.06 (d, J ¼ 3.8 Hz, 1H),
160.56, 161.59, 163.65. Anal. Calcd. for C12H8O5. 2/5H2O: C, 60.21; H,
3.71. Found: C, 60.05; H, 3.36.
7.03 (d, J ¼ 3.8 Hz, 1H), 6.96 (s, 1H), 2.54 (s, 3H); 13C NMR (100 MHz,
DMSO-d6, TMS) d 16.26, 99.42,128.85,135.46,140.12,152.57,163.58,

5142
A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
164.48, 186.31. Anal. Calcd. for C9H8O4S: C, 50.94; H, 3.80. Found: C,
50.67; H, 3.73.
4.3.19. 2-Hydroxy-4-(4-methylthiophen-2-yl)-4-oxobut-2-enoic acid
(10a)
Obtained as yellow solid (0.19 g, 72%) using 10 (0.31 g, 1.3 mmol)
4.3.12. Ethyl 2-hydroxy-4-(5-chlorothiophene-2-yl)-4-oxobut-2-
enoate (7)
as the starting material; m.p. 170e173 ꢁC; Rf ¼ 0.44 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz, DMSO-d6, TMS)
d 14.14 (bs, 2H),
Obtained as buff powder (0.62 g, 76%) using 2-acetyl-5-
chlorothiophene (0.49 g, 3.12 mmol) as the starting material; m.p.
185e188 ꢁC; Rf ¼ 0.52 (EtOAc:n-hexane 60:40); 1H NMR (400 MHz;
8.05 (s, 1H), 7.75 (s, 1H), 6.96 (s, 1H), 2.25 (s, 3H); 13C NMR
(100 MHz, DMSO-d6, TMS) 15.57, 99.57, 132.93, 136.46, 140.06,
d
141.73, 163.52, 164.94, 186.46. Anal. Calcd. for C9H8O4S: C, 50.94; H,
3.80. Found: C, 51.11; H, 3.80.
CDCl3; TMS)
d
14.33 (s,1H), 7.64 (d, J ¼ 4.1 Hz,1H), 7.02 (d, J ¼ 4.1 Hz,
1H), 6.83 (s, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 1.40 (t, J ¼ 7.1 Hz, 3H).
4.3.20. 2-Hydroxy-4-(3-methylthiophen-2-yl)-4-oxobut-2-enoic acid
(11a)
4.3.13. 2-Hydroxy-4-(5-chlorothiophene-2-yl)-4-oxobut-2-enoic acid
(7a) [32]
Obtained as brown solid (0.23 g, 86%) using 2-acetyl-3-
methylthiophene (0.44 g, 3.12 mmol) as the starting material fol-
lowed by alkaline hydrolysis of the ester using the procedure as
above; m.p. 147e151 ꢁC; Rf ¼ 0.49 (EtOAc:n-hexane 90:10); 1H NMR
Obtained as yellow solid (0.25 g, 82%) using 7 (0.34 g, 1.3 mmol)
as the starting material; m.p. 210e213 ꢁC; Rf ¼ 0.35 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz, DMSO-d6, TMS)
d 14.52 (bs, 2H),
8.16 (d, J ¼ 4.1 Hz, 1H), 7.34 (d, J ¼ 4.2 Hz, 1H), 7.00 (s, 1H); 13C NMR
(400 MHz, DMSO-d6, TMS)
d
14.15 (bs, 2H), 7.98 (d, J ¼ 4.9 Hz, 1H),
(100 MHz, DMSO-d6, TMS)
d
99.07, 130.04, 134.81, 139.65, 141.70,
7,17 (d, J ¼ 4.9 Hz, 1H), 6.69 (s, 1H), 2.55 (s, 3H); 13C NMR (100 MHz,
163.46, 164.66, 185.69. Anal. Calcd. for C8H5ClO4S. 1/3H2O: C, 40.34;
H, 2.40. Found: C, 40.64; H, 2.46.
DMSO-d6, TMS) d 17.24, 100.93, 133.96, 134.07, 134.20, 146.80,
163.48, 165.98, 186.76. Anal. Calcd. for C9H8O4S: C, 50.94; H, 3.80.
Found: C, 51.11; H, 3.80.
4.3.14. Ethyl 4-(5-bromothiophen-2-yl)-2-hydroxy-4-oxobut-2-enoate
(8)
4.3.21. Ethyl 2-hydroxy-4-(4-thiazole-2-yl)-4-oxobut-2-enoate (12)
Obtained as brown flakes (0.50 g, 71%) using 2-acetylthiazole
(0.39 g, 3.12 mmol) as starting material; m.p. 190e192 ꢁC;
Rf ¼ 0.61 (n-hexane:EtOAc 70:30); 1H NMR (400 MHz; CDCl3; TMS)
Obtained from 2-acetyl-5-bromothiophene (0.64 g, 3.12 mmol)
as yellow solid (0.76 g, 81%); m.p. 180e183 ꢁC; Rf ¼ 0.65 (n-hex-
ane:EtOAc 70:30); 1H NMR (400 MHz, DMSO-d6, TMS)
d 11.11 (bs,
1H), 8.09 (s, 1H), 7.45 (d, J ¼ 3.4 Hz, 1H), 6.98 (s, 1H), 4.29 (q,
d
14.08 (s, 1H), 8.08 (d, J ¼ 3.0 Hz,1H), 7.78 (d, J ¼ 3.0 Hz,1H), 7.40 (s,
J ¼ 6.8 Hz, 2H), 1.30 (t, J ¼ 7.0 Hz, 3H).
1H), 4.40 (q, J ¼ 7.1 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz, 3H).
4.3.15. 4-(5-Bromothiophen-2-yl)-2-hydroxy-4-oxobut-2-enoic acid
(8a)
4.3.22. 2-Hydroxy-4-(4-thiazole-2-yl)-4-oxobut-2-enoic acid (12a)
Obtained as brown solid (0.19 g, 72%) using 12 (0.29 g, 1.3 mmol)
as the starting material; m.p. 210e213 ꢁC; Rf ¼ 0.45 (EtOAc:n-
Obtained as yellow solid (0.29 g, 82%) using 8 (0.39 g, 1.3 mmol)
as the starting material; m.p. 210e212 ꢁC; Rf ¼ 0.41 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz; CDCl3; TMS)
d 13.41 (bs, 2H),
hexane 80:20); 1H NMR (400 MHz, DMSO-d6, TMS)
d
14.07 (bs, 2H),
8.28 (d, J ¼ 3.1 Hz, 1H), 8.19 (d, J ¼ 2.9 Hz, 1H), 7,15 (s, 1H); 13C NMR
8.09 (d, J ¼ 4.0 Hz, 1H), 7.45 (d, J ¼ 4.0 Hz, 1H), 6.99 (s, 1H); 13C NMR
(100 MHz, DMSO-d6, TMS) d 99.63, 129.90, 134.72, 137.34, 163.54,
(100 MHz, DMSO-d6, TMS)
d
99.14, 124.08, 133.42, 135.33, 144.31,
165.14,186.61. Anal. Calcd. for C7H5NO4S. 1/5CH3COOC2H5: C, 43.21;
H, 3.07; N, 6.86. Found: C, 42.89; H, 2.67; N, 7.14.
163.45, 164.90, 185.43. Anal. Calcd. for C8H8BrO4S. 1/4C3H6O: C,
35.37; H, 2.27. Found: C, 35.33; H, 1.93.
4.3.23. Ethyl 2-hydroxy-4-oxo-4-(1H-pyrrol-2-yl)-but-2-enoate (13)
Obtained as green solid (0.46 g, 70%) using 2-acetyl-1H-pyrrole
(0.34 g, 3.12 mmol) as the starting material; m.p. 130e134 ꢁC;
Rf ¼ 0.65 (n-hexane:EtOAc 80:20); 1H NMR (400 MHz; CDCl3; TMS)
4.3.16. Ethyl-2-hydroxy-4-(5-iodothiophen-2-yl)-4-oxobut-2-enoate
(9)
Obtained from 2-acetyl-5-iodothiophene (0.78 g, 3.12 mmol) as
a brown solid (0.76 g, 69%); m.p. 225e229 ꢁC; Rf ¼ 0.70 (n-hex-
d 12.75 (bs, 1H), 10.25 (s, 1H), 6.98 (s, 2H), 6.03 (s, 1H), 5.29 (s, 1H),
1
ane:EtOAc 70:30); H NMR (400 MHz, CDCl3, TMS)
d
14.43 (s, 1H),
4.23 (q, J ¼ 7.2 Hz, 2H), 1.21 (t, J ¼ 7.1 Hz, 3H).
7.46 (d, J ¼ 3.9 Hz, 1H), 7.35 (d, J ¼ 3.9 Hz, 1H), 6.82 (s, 1H), 4.39 (q,
J ¼ 7.1 Hz, 2H), 1.40 (t, J ¼ 7.1 Hz, 3H).
4.3.24. 2-Hydroxy-4-oxo-4-(1H-pyrrol-2-yl)-but-2-enoic acid (13a)
[30]
4.3.17. 2-Hydroxy-4-(5-iodothiophen-2-yl)-4-oxobut-2-enoic acid
(9a)
Obtained as white solid (0.19 g, 83%) using 13 (0.27 g, 1.3 mmol)
as the starting material; m.p. 157e160 ꢁC; Rf ¼ 0.52 (EtOAc:n-
Obtained as brown solid (0.32 g, 76%) using 9 (0.46 g, 1.3 mmol)
hexane 70:30); 1H NMR (400 MHz; CDCl3; TMS)
d 14.00 (bs, 2H),
12.26 (s, 1H), 7.28 (s, 2H), 6.87 (s, 1H), 6.31 (s, 1H). Anal. Calcd. for
C8H7NO4.2/5H2O: C, 51.31; H, 4.17; N, 7.44. Found: C, 51.61; H, 4.51;
N, 7.73.
as the starting material; m.p. 235e238 ꢁC; Rf ¼ 0.40 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz, DMSO-d6, TMS)
d 14.36 (bs, 2H),
7.91 (d, J ¼ 3.5 Hz, 1H), 7.55 (d, J ¼ 3.2 Hz, 1H), 6.97 (s, 1H); 13C NMR
(100 MHz, DMSO-d6, TMS)
d 91.78, 99.33, 135.92, 139.67, 148.14,
163.51 (2), 165.33. Anal. Calcd. for C8H8IO4S. 1/6C4H8O: C, 30.97; H,
1.90. Found: C, 30.64; H, 1.75.
4.3.25. 3-Acetyl-1H-indazole [26]
Following a prenominal variation in the reported procedure [26],
isobutyl chloroformate (0.79 g, 5.88 mmol) and N-methylmorpho-
line (0.59 g, 5.88 mmol) were added to a solution of 1H-indazole-3-
carboxylic acid (0.6 g, 3.70 mmol) in anhydrous THF (15 mL) under
nitrogen atmosphere at ꢀ20 ꢁC, and the mixture was stirred for 4 h.
To this mixture was added N,O-dimethylhydroxylamine hydro-
chloride (0.54 g, 5.55 mmol) suspended in 5 mL triethylamine. The
reaction was then stirred at room temperature for 6 h, concentrated
under vacuum and suspended in 20 mL n-hexane. The white
precipitates (Winreb amide) formed were filtered off, dried and
4.3.18. Ethyl-2-hydroxy-4-(4-methylthiophen-2-yl)-4-oxobut-2-
enoate (10)
Obtained as yellow solid (0.54 g, 73%) using 2-acetyl-4-
methylthiophene (0.44 g, 3.12 mmol) as the starting material;
m.p. 155e158 ꢁC; Rf ¼ 0.67 (n-hexane:EtOAc 60:40); 1H NMR
(400 MHz, DMSO-d6, TMS)
d 14.32 (bs, 1H), 8.09 (s, 1H), 7.77 (s,
1H), 6.99 (s, 1H), 4.31 (q, J ¼ 7.1 Hz, 2H), 2.27 (s, 3H), 1.31 (t,
J ¼ 7.1 Hz, 3H).

A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
5143
immediately transferred to a three neck flask containing 10 mL
anhydrous THF cooled to ꢀ78 ꢁC. To this was added methyl
magnesium bromide (12% in THF) (19 mL, 18.5 mmol). The reaction
was allowed to stir at ꢀ78 ꢁC for 2 h and then at room temperature
for 5 h. The completion of the reaction was monitored by TLC. The
reaction was quenched by slow addition of saturated aqueous
solution of ammonium chloride followed by extraction with ethyl
acetate (3 ꢃ 20 mL). The combined organic layer was dried over
magnesium sulfate and concentrated under vacuum. The resulting
viscous mass was purified by column chromatography using
hexane: ethyl acetate (85:15) solvent system to obtain desired acetyl
derivative (0.32 g, 55%); m.p.182e185 ꢁC (Lit. m.p.184e186 ꢁC) [26];
Rf ¼ 0.71 (n-hexane:EtOAc 70:30); 1H NMR (400 MHz; CDCl3; TMS)
hexane 60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d
12.99 (bs, 2H),
8.11 (d, J ¼ 8.1 Hz, 1H), 7.84 (t, J ¼ 7.5 Hz, 1H), 7.75 (t, J ¼ 7.2 Hz, 1H),
7.69 (d, J ¼ 7.5 Hz, 1H), 6.40 (s, 1H); 13C NMR (100 MHz; DMSO-d6;
TMS)
d 103.40, 124.67 (2), 129.36, 132.15, 134.53, 135.06, 147.11,
164.17 (2). Anal. Calcd. for C10H7NO6: C, 50.63; H, 2.98; N, 5.91.
Found: C, 50.57; H, 2.98; N, 5.73.
4.3.32. Ethyl 4-(2-fluorophenyl)-2-hydroxy-4-oxobut-2-enoate (17)
Obtained as white solid (0.56 g, 76%) using 2-
fluoroacetophenone (0.43 g, 3.12 mmol) as the starting material;
m.p. 176e178 ꢁC, Rf ¼ 0.65 (n-hexane:EtOAc 60:40); 1H NMR
(400 MHz; DMSO-d6; TMS)
d
14.34 (bs, 1H), 7.89 (t, J ¼ 7.2 Hz, 1H),
7.71 (q, J ¼ 6.6 Hz, 1H), 7.45e7.34 (m, 2H), 6.85 (s, 1H), 4.29 (q,
J ¼ 7.0 Hz, 2H), 1.29 (t, J ¼ 7.0 Hz, 3H). Anal. Calcd. for C12H11FO4.1/
5H2O: C, 59.60; H, 4.75. Found: C, 59.59; H, 4.47.
d
13.85 (s, 1H), 8.18 (d, J ¼ 7.2 Hz, 1H), 7.67 (d, J ¼ 7.2 Hz, 1H), 7.46 (t,
J ¼ 7.3 Hz, 1H), 7.32 (t, J ¼ 7.3 Hz, 1H), 2.65 (s, 3H).
4.3.26. Ethyl 2-hydroxy-4-(1H-indazol-3-yl)-4-oxobut-2-enoate (14)
Obtained as yellow solid (0.63 g, 78%) by using 3-acetyl-1H-
indazole (0.49 g, 3.12 mmol) as the starting material; m.p.
182e184 ꢁC; Rf ¼ 0.60 (n-hexane:EtOAc 70:30); 1H NMR (400 MHz;
4.3.33. 4-(2-Fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (17a)
[30]
Obtained as white solid (0.22 g, 82%) using 17 (0.31 g, 1.3 mmol)
as the starting material; m.p.192e195 ꢁC, Rf ¼ 0.59 (n-hexane:EtOAc
DMSO; TMS)
d
14.26 (s, 1H), 14.04 (s, 1H), 8.25 (d, J ¼ 8.1 Hz, 1H),
70:30); 1H NMR (400 MHz; DMSO-d6; TMS)
d 14.29 (bs, 2H), 7.91 (t,
7.74 (d, J ¼ 8.2 Hz, 1H), 7.52 (t, J ¼ 7.6 Hz, 1H), 7.39 (t, J ¼ 7.5 Hz, 1H),
7.35 (s, 1H), 4.33 (q, J ¼ 7.1 Hz, 2H), 1.32 (t, J ¼ 7.1 Hz, 3H). Anal.
Calcd. for C13H12N2O4.1/4H2O : C, 58.98; H, 4.76; N, 10.58. Found: C,
59.08; H, 4.70; N, 10.56.
J ¼ 7.3 Hz,1H), 7.71 (q, J ¼ 6.7 Hz,1H), 7.39 (dd, J ¼ 6.7 Hz, 5.6 Hz, 2H),
6.90 (s, 1H); 13C NMR (100 MHz, DMSO-d6, TMS)
d
102.28, 117.47,
123.83, 125.69, 130.70, 136.12, 159.94, 163.48, 170.16, 187.44. Anal.
Calcd. for C10H7FO4: C, 57.13; H, 3.36. Found: C, 56.95; H, 3.45.
4.3.27. 2-Hydroxy-4-(1H-indazole-3-yl)-4-oxobut-2-enoic acid (14a)
Obtained as yellow solid (0.22 g, 72%) using 14 (0.34 g,1.3 mmol)
as the starting material; m.p. 224e226 ꢁC; Rf ¼ 0.42 (EtOAc:n-
4.3.34. 2-Hydroxy-4-oxo-4-o-tolylbut-2-enoic acid (18a) [30]
Obtained as white solid (0.19 g, 71%) using 2-
methylacetophenone (0.42 g, 3.12 mmol) as the starting material
followed by subsequent hydrolysis of the ester; m.p. 167e171 ꢁC;
Rf ¼ 0.68 (n-hexane:EtOAc 60:40); 1H NMR (400 MHz; DMSO-d6;
hexane 60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d 14.24 (s, 3H),
8.24 (d, J ¼ 8.1 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz, 1H), 7.50 (t, J ¼ 7.6 Hz, 1H),
7.38 (t, J ¼ 7.4 Hz, 1H), 7.33 (s, 1H). 13C NMR (100 MHz, DMSO-d6,
TMS)
d
14.12 (bs, 2H), 7.69 (d, J ¼ 7.2 Hz, 1H), 7.48 (t, J ¼ 7.4 Hz, 1H),
TMS)
d
100.50, 111.89, 121.89 (2), 124.38, 127.71, 141.58, 141.90,
7.37e7.32 (m, 2H), 6.72 (s, 1H), 2.47 (s, 3H); 13C NMR (100 MHz,
DMSO-d6, TMS) 21.00, 102.04, 126.67, 129.40, 132.20, 132.53,
163.46, 163.70, 189.63. Anal. Calcd. for C11H8N2O4: C, 56.90; H, 3.47;
N, 12.06. Found: C, 56.78; H, 3.51; N, 11.96.
d
136.15,138.46,161.88 (2), 163.66. Anal. Calcd. for C11H10O4: C, 64.07;
H, 4.89. Found: C, 63.95; H, 4.67.
4.3.28. Ethyl 2-hydroxy-4-(biphenyl-4-yl)-4-oxobut-2-enoate (15)
Obtained as white solid (0.66 g, 72%) using 4-acetylbiphenyl
(0.61 g, 3.12 mmol) as the starting material; m.p. 168e170 ꢁC;
Rf ¼ 0.54 (n-hexane:EtOAc 60:40); 1H NMR (400 MHz; CDCl3; TMS)
4.3.35. Ethyl 4-(3-fluorophenyl)-2-hydroxy-4-oxobut-2-enoate (19)
Obtained as white solid (0.54 g, 73%) using 3-
fluoroacetophenone (0.43 g, 3.12 mmol) as the starting material;
m.p. 165e168 ꢁC; Rf ¼ 0.61 (n-hexane:EtOAc 60:40); 1H NMR
d
15.39 (s, 1H), 8.07 (d, J ¼ 8.5 Hz, 2H), 7.73 (d, J ¼ 8.5 Hz, 2H),
7.67e7.62 (m, 2H), 7.54e7.39 (m, 3H), 7.12 (s, 1H), 4.41 (q, J ¼ 7.1 Hz,
2H), 1.42 (t, J ¼ 7.1 Hz, 3H).
(400 MHz; CDCl3; TMS)
d
15.23 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H),
7.72e7.69 (m,1H), 7.52 (q, J ¼ 7.2 Hz,1H), 7.34 (t, J ¼ 7.2 Hz,1H), 7.09
(s, 1H), 4.42 (q, J ¼ 7.1 Hz, 2H), 1.43 (t, J ¼ 7.2 Hz, 3H).
4.3.29. 2-Hydroxy-4-(biphenyl-4-yl)-4-oxobut-2-enoic acid (15a) [30]
Obtained as white solid (0.29 g, 85%) using 15 (0.38 g, 1.3 mmol)
as the starting material; m.p. 182e186 ꢁC; Rf ¼ 0.40 (n-hex-
4.3.36. 4-(3-Fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (19a)
Obtained as white solid (0.21 g, 77%) using 19 (0.31 g,1.3 mmol) as
the starting material; m.p. 187e189 ꢁC; Rf ¼ 0.47 (EtOAc:n-hexane
ane:EtOAc 60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d 15.25 (s,
1H), 14.25 (s, 1H), 8.18 (d, J ¼ 7.9 Hz, 2H), 7.90 (d, J ¼ 7.8 Hz, 2H), 7.81
60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d 14.26 (bs, 2H), 7.92 (d,
(d, J ¼ 7.8 Hz, 2H), 7.60e7.40 (m, 3H), 7.17 (s, 1H); 13C NMR
J ¼ 7.7 Hz,1H), 7.86 (d, J ¼ 7.6 Hz,1H), 7.62 (q, J ¼ 7.2 Hz,1H), 7.54 (m,
(100 MHz; DMSO-d6; TMS)
d
98.3, 127.5 (2), 127.7 (2), 129.1 (2),
1H), 7.10 (s, 1H); 13C NMR (100 MHz, DMSO-d6, TMS)
d
98.60, 114.88,
129.6 (2), 133.9, 139.1, 145.8, 163.6, 170.8, 190.2 (2). Anal. Calcd. for
C16H12O4. 1/6H2O: C, 67.36; H, 4.59. Found: C, 67.20; H, 4.51.
121.32, 124.52, 131.73, 137.45, 161.56, 163.49, 170.88, 189.26. Anal.
Calcd. for C10H7FO4.1/5H2O:C, 56.19; H, 3.49. Found: C, 56.51; H, 3.32.
4.3.30. Ethyl 2-hydroxy-4-(2-nitrophenyl)-4-oxobut-2-enoate (16)
Obtained as white solid (0.54 g, 65%) using 2-nitroacetophenone
(0.51 g, 3.12 mmol) as the starting material; m.p. 178e182 ꢁC;
Rf ¼ 0.59 (n-hexane:EtOAc 80:20); 1H NMR (400 MHz; DMSO-d6;
4.3.37. Ethyl 4-(3-cyanophenyl)-2-hydroxy-4-oxobut-2-enoate (20)
Obtained as white solid (0.58 g, 76%) using 3-
cyanoacetophenone (0.45 g, 3.12 mmol) as the starting material;
m.p. 178e181 ꢁC, Rf ¼ 0.67 (n-hexane:EtOAc 70:30); 1H NMR
TMS)
d
10.81 (bs, 1H), 8.10 (d, J ¼ 8.0 Hz, 1H), 7.84 (t, J ¼ 7.2 Hz, 1H),
(400 MHz; DMSO-d6; TMS) d 14.49 (bs, 1H), 8.56 (s, 1H), 8.34 (d,
7.75 (t, J ¼ 7.6 Hz, 1H), 7.69 (d, J ¼ 6.6 Hz, 1H), 6.49 (s, 1H), 4.25 (q,
J ¼ 7.8 Hz, 1H), 8.14 (d, J ¼ 7.7 Hz, 1H), 7.77 (t, J ¼ 7.9 Hz, 1H), 7.19 (s,
J ¼ 6.9 Hz, 2H), 1.26 (t, J ¼ 7.0 Hz, 3H).
1H), 4.32 (q, J ¼ 7.1 Hz, 2H), 1.32 (t, J ¼ 7.0 Hz, 3H).
4.3.31. 2-Hydroxy-4-(2-nitrophenyl)-4-oxobut-2-enoic acid (16a) [30]
Obtained as pink solid (0.23 g, 75%) using 16 (0.34 g, 1.3 mmol)
as the starting material; m.p. 195e198 ꢁC; Rf ¼ 0.47 (EtOAc:n-
4.3.38. 4-(3-Cyanophenyl)-2-hydroxy-4-oxobut-2-enoic acid (20a)
Obtained as white solid (0.22 g, 78%) using 20 (0.32 g, 1.3 mmol)
as the starting material; m.p. 201e205 ꢁC, Rf ¼ 0.47 (EtOAc:n-

5144
A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
hexane 60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d
14.24 (bs, 2H),
4.3.44. Ethyl 4-(2,4-difluorophenyl)-2-hydroxy-4-oxobut-2-enoate
(24)
8.55 (s, 1H), 8.34 (d, J ¼ 7.9 Hz, 1H), 8.13 (d, J ¼ 7.3 Hz, 1H), 7.77 (t,
J ¼ 7.7 Hz, 1H), 7.17 (s, 1H). 13C NMR (100 MHz, DMSO-d6, TMS)
Obtained as white powder (0.58 g, 72%) using 2,4-
difluoroacetophenone (0.49 g, 3.12 mmol) as the starting mate-
rial; m.p. 190e192 ꢁC; Rf ¼ 0.73 (n-hexane:EtOAc 60:40); 1H NMR
d
98.81, 112.83, 118.43, 130.79, 132.16, 132.63, 136.21, 137.31, 163.40,
170.95, 188.59. Anal. Calcd. for C11H7NO4.1/5H2O: C, 59.84; H, 3.38;
N, 6.34. Found: C, 60.16; H, 3.34; N, 6.23.
(400 MHz; DMSO-d6; TMS)
7.48 (t, J ¼ 7.1 Hz, 1H), 7.29 (t, J ¼ 7.1 Hz, 1H), 6.87 (s, 1H), 4.29 (q,
J ¼ 7.1 Hz, 2H), 1.29 (t, J ¼ 7.0 Hz, 3H).
d
15.26 (bs, 1H), 7.99 (q, J ¼ 7.9 Hz, 1H),
4.3.39. 4-(3-(1H-Tetrazol-5-yl)phenyl)-2-hydroxy-4-oxobut-2-enoic
acid (21a)
According to reported procedure [27], NaN3 (0.16 g, 2.51 mmol)
was added to a well stirred reaction mixture of 20a (0.3 g,
1.38 mmol) and ammonium chloride (0.1 g, 1.88 mmol) in anhy-
drous DMF. The reaction was then heated to 110 ꢁC and intermit-
tently monitored by TLC. Upon completion, the reaction mixture
was concentrated under vacuum. Water was added and the
aqueous layer was acidified with HCl to pH 1. The resulting
precipitates were filtered off, washed with water and dried under
vacuum. The crude product was purified by precipitating it using
hexane: ethyl acetate system to obtain the tetrazole derivative as
buff solid (0.27 g, 75%); m.p. 210e213 ꢁC, Rf ¼ 0.31 (EtOAc:n-hexane
4.3.45. 4-(2,4-Difluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid
(24a) [30]
Obtained as white solid (0.21 g, 70%) using 24 (0.33 g, 1.3 mmol)
as the starting material; m.p. 210e214 ꢁC, Rf ¼ 0.51 (EtOAc:n-
hexane 60:40); 1H NMR (400 MHz; DMSO-d6; TMS)
d 14.29 (bs, 2H),
8.00 (q, J ¼ 8.0 Hz, 1H), 7.48 (dd, J ¼ 7.7 Hz, 7.4 Hz, 1H), 7.29 (dt,
J ¼ 7.6 Hz, 7.3 Hz, 1H), 6.86 (s, 1H); 13C NMR (100 MHz, DMSO-d6,
TMS)
d 102.18, 105.94, 113.27, 120.96, 132.88, 141.99, 163.46, 167.06,
169.43, 186.78. Anal. Calcd. for C10H6F2O4: C, 52.64; H, 2.65. Found:
C, 52.90; H, 2.81.
70:30); 1H NMR (400 MHz; DMSO-d6; TMS)
d
15.45 (bs, 2H), 8.67 (s,
5. NS5B inhibition assay
1H), 8.35 (d, J ¼ 7.6 Hz,1H), 8.26 (d, J ¼ 7.6 Hz, 1H), 7.81 (t, J ¼ 7.7 Hz,
1H), 7.17 (s, 1H). [Note: The absence of the tetrazole ring eNH
proton was attributed to the protopic tautomerism. Moreover, the
existence of the proton was confirmed by alkylation of the tetrazole
ring by treating it with benzyl chloride under basic conditions (data
not shown)]. Anal. Calcd. for C11H8N4O4: C, 50.77; H, 3.10; N, 21.53.
Found: C, 50.63; H, 3.24; N, 21.66.
For evaluating the biological activity of the compounds, the
enzymatic reaction mixtures containing 20 mM TriseHCl (pH 7.0),
100 mM NaCl, 100 mM sodium glutamate, 0.5 mM DTT, 0.01% BSA,
0.01% Tween-20, 5% glycerol, 20 U/mL of RNase Out, 0.5
rA/U12, 25 M UTP, 2e5 Ci [
32P]UTP, 300e500 ng of NS5BC
and 1.0 mM MnCl2 with or without inhibitors in a total volume of
25
L were incubated for 1 h at 30 ꢁC. The concentration of DMSO in
mM of poly
m
m
a-
D
21
m
4.3.40. Ethyl 4-(2,6-difluorophenyl)-2-hydroxy-4-oxobut-2-enoate
(22)
all reactions was maintained constant at 10%. Reactions were
terminated by the addition of ice-cold 5% (v/v) trichloroacetic acid
(TCA) containing 0.5 mM pyrophosphate. The quenched reaction
mixtures were incubated at ꢀ20 ꢁC for 1 h to precipitate out
denatured polymeric RNA products, transferred to GF-B filters,
washed twice with 5% (v/v) TCA containing 0.5 mM pyrophosphate
to remove unincorporated UTP, and rinsed three times with water
and once with ethanol before vacuum drying. The amount of
radioactive UMP incorporated into RNA products was quantified on
a liquid scintillation counter (Packard). Activity of NS5B in the
absence of the inhibitor but containing an equivalent amount of
DMSO (control reaction) was set at 100% and that in the presence of
the inhibitor was quantified relative to this control. The concen-
tration of inhibitors inhibiting 50% of NS5B RdRp activity (IC50) was
calculated from the inhibition curves as a function of inhibitor
concentration and values obtained represent an average of at least
two independent measurements in duplicate.
Obtained as white powder (0.68 g, 85%) using 2,6-
difluoroacetophenone (0.49 g, 3.12 mmol) as the starting material;
m.p. 156e158 ꢁC; Rf ¼ 0.60 (n-hexane:EtOAc 70:30); 1H NMR
(400 MHz; CDCl3; TMS)
d 14.38 (s, 1H), 7.52e7.46 (m, 1H), 7.01 (t,
J¼ 8.6Hz,2H), 6.79(s,1H), 4.40(q,J¼ 7.1 Hz, 2H),1.40 (t, J¼ 7.1Hz,3H).
4.3.41. 2-Hydroxy-4-(2,6-difluorophenyl)-4-oxobut-2-enoic acid (22a)
[30]
Obtained as white powder (0.23 g, 79%) using 22 (0.33 g,
1.3 mmol) as the starting material; m.p. 179e181 ꢁC; Rf ¼ 0.53 (n-
hexane:EtOAc 70:30); 1H NMR (400 MHz; DMSO-d6; TMS)
d 13.09
(bs, 2H), 7.62e7.60 (m, 1H), 7.23 (t, J ¼ 7.9 Hz, 2H), 6.41 (s, 1H); 13C
NMR (100 MHz; DMSO-d6; TMS)
d 104.66, 112.66, 112.90, 116.54,
133.80, 158.35, 158.42, 160.85, 160.92, 164.10. Anal. Calcd. for
C10H6F2O4. 2/3H2O: C, 49.99; H, 2.97. Found: C, 49.85; H, 2.60.
4.3.42. Ethyl 4-(2,4-dichlorophenyl)-2-hydroxy-4-oxobut-2-enoate
(23)
6. Molecular modeling
Obtained as white solid (0.64 g, 71%) using 2,4-
dichloroacetophenone (0.58 g, 3.12 mmol) as the starting mate-
rial; m.p. 134e136 ꢁC; Rf ¼ 0.62 (n-hexane:EtOAc 80:20); 1H NMR
Molecular docking computations were carried out on a Dell
Precision 470n workstation with the RHEL 4.0 operating system
using Glide 5.5 (Schrodinger, LLC, New York, NY). 3D Structures of
the entire target DKAs were constructed using the fragment
dictionary of Maestro 9.0 (Schrodinger, LLC, New York, NY) and
geometry was optimized by Macromodel program v9.7 using the
OPLS-AA force field with the steepest descent followed by trun-
cated Newton conjugate gradient protocol. Each ligand structure
was subsequently processed with the LigPrep v2.3 program
(Schrodinger, LLC, New York, NY) to assign the protonation states
appropriate for physiological pH, using the “ionizer” subprogram. A
maximum of 100 conformers were generated for each ligand using
a combination of Monte Carlo Multiple Minimum and low mode
conformational searching in conjugation with OPLS_2005 force
field (Schrodinger, LLC, New York, NY). Minimized structures were
filtered with a maximum relative energy difference of 5 kcal/mol to
(400 MHz; CDCl3; TMS)
d
14.57 (s, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.50
(s, 1H), 7.36 (d, J ¼ 8.5 Hz, 1H), 6.95 (s, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H),
1.39 (t, J ¼ 7.1 Hz, 3H).
4.3.43. 4-(2,4-Dichlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid
(23a) [30]
Obtained as white solid (0.26 g, 78%) using 23 (0.37 g, 1.3 mmol)
as the starting material; m.p. 165e168 ꢁC; Rf ¼ 0.62 (DCM:MeOH
98:2); 1H NMR (400 MHz; DMSO-d6; TMS)
d 13.97 (bs, 2H), 7.78 (s,
1H), 7.67 (d, J ¼ 7.7 Hz, 1H), 7.57 (dd, J ¼ 7.9 Hz, 7.7 Hz, 1H), 6.55 (s,
1H); 13C NMR (100 MHz, DMSO-d6, TMS)
d
103.19, 128.28 (2),
130.43, 131.77 (2), 132.11, 135.90, 136.98, 163.83. Anal. Calcd. for
C10H6Cl2O4: C, 46.16; H, 2.33. Found: C, 46.19; H, 2.39.

A. Bhatt et al. / European Journal of Medicinal Chemistry 46 (2011) 5138e5145
5145
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Author contributions
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This work was supported by the National Institute of Health
Research Grant CA153147 to N.K-B., and St. John’s University Seed
Grant No. 579-1110 to T. T. T.
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