Novel Aryl-Modified Benzoylamino-N-(5,6-dimethoxy-1H-benzoimidazol-2-yl)-heteroamides as Potent Inhibitors of Vascular Endothelial Growth Factor Receptors 1 and 2

Article abstract of DOI:10.1002/jhet.2791

Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the vascular endothelial growth factor (VEGF) pathway. The VEGF and its receptors have been implicated as key factors in tumor angiogenesis and are major targets in cancer therapy. A series of aryl-modified benzoylamino-N-(5,6-dimethoxy-1H-benzoimidazol-2-yl)-heteroamides were synthesized from 2-amino-5,6-dimethoxy benzimidazole and aryl-substituted benzoylamino hetero acids. The new compounds were tested for inhibition of VEGF receptors I and II (VEGFR-1 and VEGFR-2). Compound 6e displayed VEGFR-2 inhibitory activity with a 50% inhibition concentration value as low as 0.020 μM in a homogeneous time-resolved fluorescence enzymatic assay. VEGFR-2 active compounds display good activity against VEGFR-1 as well.

Full text of DOI:10.1002/jhet.2791

Month 2016
Novel Aryl-Modified Benzoylamino-N-(5,6-dimethoxy-1H-
benzoimidazol-2-yl)-heteroamides as Potent Inhibitors of Vascular
Endothelial Growth Factor Receptors 1 and 2
a
a
b
a
*
Abhishek Ashok, Kannan Thanukrishnan, Halehatty S. Bhojya Naik, and Rajendraswami Maridu
aAnthem Biosciences Pvt. Ltd., 49 Bommasandra Industrial Area, Bommasandra, Bangalore 560099, Karnataka, India
bDepartment of PG Studies and Research in Industrial Chemistry, School of Chemical Sciences, Kuvempu University,
Shankaraghatta, Karnataka 577451, India
*
E-mail: abhickm@gmail.com
Additional Supporting Information may be found in the online version of this article.
Received July 13, 2016
DOI 10.1002/jhet.2791
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies
aimed at blocking the vascular endothelial growth factor (VEGF) pathway. The VEGF and its receptors have
been implicated as key factors in tumor angiogenesis and are major targets in cancer therapy. A series of
aryl-modified benzoylamino-N-(5,6-dimethoxy-1H-benzoimidazol-2-yl)-heteroamides were synthesized
from 2-amino-5,6-dimethoxy benzimidazole and aryl-substituted benzoylamino hetero acids. The new
compounds were tested for inhibition of VEGF receptors I and II (VEGFR-1 and VEGFR-2). Compound
6e displayed VEGFR-2 inhibitory activity with a 50% inhibition concentration value as low as 0.020 μM
in a homogeneous time-resolved fluorescence enzymatic assay. VEGFR-2 active compounds display good
activity against VEGFR-1 as well.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
synthesis of novel benzimidazole derivatives remains a
main focus of medicinal research. By looking at the
importance of the presence of methoxy group on the
benzimidazole nucleus contributing to the antiproliferative
activity, it was thought that it would be worthwhile to
synthesize some new benzimidazole derivatives bearing a
dimethoxy group on the benzimidazole moiety with various
heteroamide substituents at the 2-amino position of the
benzimidazole. Nonetheless, to the best of our knowledge,
although several benzimidazole series have been developed
as tyrosine kinase inhibitors, 2-yl-heteroamide derivatives
of benzimidazole have not been explored as VEGF receptor
inhibitors. In this communication, we disclose a series of
novel aryl-modified benzoylamino-N-(5,6-dimethoxy-1H-
benzoimidazol-2-yl)-heteroamides as potent inhibitors
of VEGF receptors 1 and 2.
Angiogenesis, the formation of new blood vessels from
preexisting vessels, is a complex process that normally
occurs in adults only under specific conditions such as
wound healing, inflammation, and menstrual cycle [1,2].
Angiogenesis is an essential step in tumor growth and
metastasis [3]. Significant advances have occurred with
the development of novel antiangiogenic treatments that
target growth factor receptor signaling pathways. One
such pathway involves the vascular endothelial growth
factor (VEGF) signal transduction, which is critical for
the process of neovascularization, otherwise known as
angiogenesis [4,5]. Benzimidazole derivatives have been
explored as anticancer inhibitors of topoisomerase I [6],
Poly [ADP-ribose] polymerase 1 (PARP-1) [7,8], kinase
Chk2 [9,10], Pgp and DNA synthesis [11], and tyrosine
kinases [12–14]. Derivatives of benzimidazoles are active
as human DNA topoisomerase I inhibitors [15–18] and
2-amino benzimidazole derivatives are identified as
potent inhibitors of VEGF receptor 2 (VEGFR-2) kinase
insert domain receptor (KDR) [19]. Studies on
benzimidazole derivatives have shown that the presence
of electron-donating group, such as methoxy, increases
the antiproliferative activity [20,21].
RESULTS AND DISCUSSIONS
Chemistry.
The
new
N-(5,6-dimethoxy-1H-
benzoimidazol-2-yl)-heteroamide derivatives described
herein were synthesized as shown in Scheme 1. Previously
described methods were used for the synthesis of 2 [22], 3
[23], 4 [24], and 5 [25]. The heteroamide compounds 3′a–
3′d and 3″a–3″d were synthesized following an analogous
protocol [26] in good yields as shown in Scheme 2.
Benzimidazole is an important pharmacophore and a
privileged structure in modern drug discovery. The
© 2016 Wiley Periodicals, Inc.

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and R. Maridu
Vol 000
Scheme 1. Synthetic route to synthesize compounds 6a–6h.
Scheme 2. Synthetic route to synthesize compounds 3′a–3′d and 3″a–3″d.
Compounds 3′a–3′d were synthesized in three steps starting
from commercially available 4-amino-nicotinic acid, and
compounds 3″a–3″d were synthesized in two steps
starting from commercially available methyl-3-
aminothiophene-2-carboxylate. The aryl benzoyl chlorides
were commercially available.
The synthesis of compound 6g was attempted by
reaction of compound 5 with 3″c in the presence of
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyriidinium-3-oxide hexafluorophosphate (HATU) and
diisopropylamine at room temperature. The reaction
afforded the cyclized intermediate A 2-(4-methoxyphenyl)-
4H-thieno [3,2-d][1,3]oxazin-4-one (Fig. 1) as the major
product. Synthesis of 6g using other coupling reagents like
N,N′-dicylcohexylcarbodiimide, O-(benzotriazol-1-yl)-N,N,
N′,N′-tetramethyluronium tetrafluoroborate, and propyl-
phosphonic anhydride (T3P) also afforded the cyclized
intermediate A.
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Table 1
Activity of compounds (6a–6h) against VEGFR-2.
Compound
R
Enzymatic, IC50 (μM)a
6a
6b
6c
6d
6e
6f
4-Cl Ph
0.24 Æ 0.03
0.23 Æ 0.07
>10
3,4-diClPh
4-OMePh
4-MePh
Figure 1. Chemical structure of cyclized intermediate A.
>10
4-Cl Ph
0.02 Æ 0.03
0.078 Æ 0.007
0.048 Æ 0.06
>10
3,4-diClPh
4-OMePh
4-MePh
The results obtained emphasized the need of a stronger
base in combination with the coupling reagent for the
synthesis. Use of cesium carbonate (Cs2CO3) as the base
along with HATU afforded the title compounds 6a–6h in
good to moderate yields. The new N-(5,6-dimethoxy-1H-
benzoimidazol-2-yl)-heteroamide derivatives (6a–6h)
were efficiently synthesized by reacting compounds 3′
6g
6h
VEGFR-2, vascular endothelial growth factor receptor 2; IC50, 50% in-
hibition concentration value.
aIC50 values were determined from logarithmic concentration-inhibition
curves (at least eight points) and are given as means of at least two sepa-
rate experiments.
a–3′d and 3″a–3″d and compound
5
with
potency of compounds was modulated against the enzyme.
From the results, it was observed that 3-[(4-chlorobenzoyl)
amino]thiophene-2-carboxamide substituent gave the
highest enzymatic activity (6e; IC50 =20nM). 3-(3,4-
Dichlorobenzoyl)amino and 3-(4-methoxybenzoyl)amino
substituents of thiophene-2-carboxamide benzimi-dazole
derivatives were also highly active against the isolated
VEGFR-2 (6f; IC50 =78nM, 6g; IC50 =48nM). Acceptable
enzymatic inhibition was observed with 3-(4-
chlorobenzoyl)amino and 3-(3,4-dichlorobenzoyl)amino
substituents of pyridine-3-carboxamide benzimidazole
derivatives (6a; IC50 =0.24μM, 6b; IC50 =0.23μM). Other
analogs showed a dramatic decrease in the enzymatic
activity, specifically the para-methyl substituents (6d and
6h) at the aryl benzoylamino substituents of the
heteroamides showed no enzymatic activity.
In general, the aryl-modified benzoylamino substituents
of thiophene-2-carboxamides at the 2-amino position
of 5,6-dimethoxybenzoimidazole were much better
inhibitors than the aryl-modified benzoylamino
substituents of pyridine-3-carboxamides. Also, the halo
substitution at the aryl benzoylamino substituents of the
heteroamides was beneficial for VEGFR-2 inhibition.
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]
pyridinium-3-oxide hexafluorophosphate (HATU) and
Cs2CO3 in N,N-dimethylformamide (DMF) solvent at
80°C. Reactions were complete in 14h and the purification
of compounds was achieved by column chromatography.
The structures of all the newly synthesized compounds
were confirmed by IR, 1H and 13C NMR, and liquid
chromatography (LC) mass spectral studies. The structure
1
of compound 6g was interpreted by IR, H NMR, and
liquid chromatography-mass spectrometry (LCMS)
analysis. The IR spectrum showed the presence of –C=O
group of amide at 1730cmÀ1 confirming the formation of
1
amide group. The H NMR showed a singlet at δ7.03 for
two protons which corresponds to the phenyl group of
benzimidazole nucleus and two doublets at δ7.67 and
δ8.09 confirming the presence of thiophene heterocycle.
Further, 13C NMR showed two strong signals at δ 162.46
and δ 162.80 confirming the presence of two –C = O
groups of amide groups in the molecule. The structure of
6g was again confirmed by LCMS, which showed the
molecular ion peak at m/z 452.9 (M +H), which
corresponds to the molecular formula C22H20N4O5S.
Activity of compounds 6a–6h against VEGFR-2.
All
compounds (6a–6h) were tested against isolated VEGFR-2
by measuring the ability to inhibit phosphorylation of a
biotinylated-polypeptide substrate [poly-Glu–Ala–Tyr
(pGAT), CIS Bio International (Cisbio China, Shanghai,
China)] in a homogeneous time-resolved fluorescence
(HTRF) assay at an adenosine triphosphate (ATP)
concentration of 2μM. The results were reported as a 50%
inhibition concentration value (IC50), a literature VEGFR-
2 inhibitor also being included as an internal standard for
quality control (ZD 6474; IC50 =45 Æ 15 nM).
Table 2
Activity of compounds (6a–6h) against VEGFR-1 and VEGFR-2 tyrosine
kinases.
% inhibition of
% inhibition of
Compound
VEGFR-1 at 10 μMa
VEGFR-2 at 10 μMa
6a
6b
6c
6d
6e
6f
89
57
40
43
91
86
91
40
90
92
35
38
97
100
95
35
The results generated from this article (Table 1) showed
that benzimidazole derivatives exhibited good to excellent
inhibitory activity against VEGFR-2. By varying both
heteroamide and aryl benzoylamino substituents at the 2-
amino position of 5,6-dimethoxybenzimidazole, the
6g
6h
VEGFR-1, vascular endothelial growth factor receptor 1; VEGFR-2,
vascular endothelial growth factor receptor 2.
aAverage of n = 3.
Journal of Heterocyclic Chemistry
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A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and R. Maridu
Vol 000
Activity of compounds 6a–6h against VEGFR-1.
Compounds 6a–6h were also tested in an HTRF format
against VEGFR-1, a receptor tyrosine kinase related to
VEGFR-2. The results in Table 2 indicate that the
compounds inhibit VEGFR-1 at a compound screening
concentration of 10 μM. The data generated show that
VEGFR-2 active compounds display good activity
against VEGFR-1 as well. A percentage inhibition of
<40% at 10 μM is considered to be inactive.
of aqueous Na2CO3 solution and was concentrated under
reduced pressure to remove the water. The residue was
triturated with hot ethanol (15mL), and the ethanolic
solution was filtered and concentrated under reduced
pressure to afford the title compound 5 (2.3g, 64%) as
yellow solid. IR (ATR, cmÀ1) ʋ: 3425, 2367, 1642, 1566,
1359, 1198. 1H NMR (400MHz, DMSO-d6) δ (ppm):
2.23 (bs, 1H, NH), 7.94 (bs, 2H, NH2), 6.95 (s, 2H, ArH),
3.76 (s, 6H, OCH3). 13C NMR (100MHz, DMSO-d6) δ:
150.6, 146.3, 123.7, 97.04. ESI-MS m/z 193.8 (M+H).
General procedure for the synthesis of 4-aryl-modified
bezoylamino-nicotinic acid methyl ester 2′a–2′d.
To a
CONCLUSION
solution of 4-amino-nicotinic acid methyl ester (1g,
6.5 mmol) in N,N-dimethyl acetamide (10 mL) was added
triethyl amine (4.58mL, 32.5 mmol) at room temperature,
and the reaction mass was allowed to stir at room
temperature for 10min. The corresponding benzoyl
chlorides (11.7mmol) were then added to the reaction
mass and stirred for 1h at room temperature. Reaction
mass diluted with water (20 mL) and the solids
precipitated were filtered and dried under suction to
afford the title compounds 2′a–2′d as solids.
We have disclosed a series of novel aryl-modified
benzoylamino-N-(5,6-dimethoxy-1H-benzoimidazol-2-yl)-
heteroamides as potent inhibitors of VEGFR-2. These
compounds are also inhibitors of the VEGFR-1 receptor.
The compounds also provide an opportunity of laying the
foundation for promising molecules of anticancer potency.
Methyl
4-[(4-chlorobenzoyl)amino]pyridine-3-carboxylate
EXPERIMENTAL
(2′a). Starting from 2.06 g of 4-chlorobenzoyl chloride
(a), the title compound 2′a (1.6 g, 84%) was obtained as
off-white solid, mp 170–172°C. IR (ATR, cmÀ1) ʋ: 3266,
2924, 1694, 1589, 1223, 1095. 1H NMR (400MHz,
DMSO-d6) δ (ppm): 11.71 (bs, 1H, NH), 9.07 (s, 1H,
CH-pyridine), 8.71 (d, J 5.6, 1H, CH-pyridine), 8.5 (d, J
6.0, 1H, CH-pyridine), 7.74–7.41 (dt, J1 8.4, J2 2.0, 2H,
ArH), 7.98–7.96 (dt, J1 9.2, J2 2.0, 2H, ArH), 3.93 (s, 3H,
OCH3). 13C NMR (100MHz, DMSO-d6) δ: 165.3, 162.8,
151.1, 141.8, 149.2, 138.6, 129.2, 128.8, 120.1, 110.8,
108.3, 52.4. ESI-MS m/z 291.9 (M +H).
Chemistry.
Chemicals were obtained from Sigma-
Aldrich Co. (Sigma Aldrich, Bengaluru, India) TLC
experiments were performed on alumina-backed silica gel
40F254 plates (Merck, Darmstadt, Germany). The plates
were illuminated under UV (254 nm) and KMnO4. Melting
points were determined using Buchi B-540 apparatus
(Buchi India (p) Ltd, Bengaluru, India). All H and 13C
1
NMR spectra were recorded on a Bruker Avance III HD
400-MHz NMR, Bruker BioSpin Corp., Silberstreifen 4,
Rheinstetten, Germany. Molecular weights of unknown
compounds were checked by LCMS 6200 series Agilent
Technology (Agilent, Bengaluru, India). Chemical shifts
are reported in ppm (δ) with reference to internal standard
TMS. The signals are designated as follows: singlet (s),
doublet (d), triplet (t), doublet of doublet (dd), doublet of
triplet (dt), multiplet (m), and broad singlet (brs). IR
spectra were recorded using a Bruker Alpha FTIR spec-
trometer (Bruker, Rheinstetten, Germany) using a diamond
attenuated total reflectance (ATR) single reflectance
module (24 scans). Elemental analysis was carried out with
a Perkin-Elmer model 240-C apparatus (Thermofisher,
Bengaluru, India). The results of elemental analysis (C,
H, and N) were within Æ0.4% of the calculated amounts.
The aryl benzoyl chlorides were commercially available.
Methyl
4-[(3,4-dichlorobenzoyl)amino]pyridine-3-
carboxylate (2′b).
Starting from 2.47 g of 3,4-
dichlorobenzoyl chloride (b), the title compound 2′b
(1.6 g, 78%) was obtained as off-white solid, mp 185–
186°C IR (ATR, cmÀ1) ʋ: 3265, 2925, 1689, 1588, 1230,
1090. 1H NMR (400MHz, DMSO-d6) δ (ppm): 11.63
(bs, 1H, NH), 9.06 (s, 1H, CH-pyridine), 8.73 (d, J 6.0,
1H, CH-pyridine), 8.41 (d, J 6.0, 1H, CH-pyridine), 8.15
(d, J 1.6, 1H, ArH), 7.94 (d, J 8.4, 1H, ArH), 7.91 (dd,
J1 8.4, J2 2.0, 1H, ArH), 3.93 (s, 3H, OCH3). 13C NMR
(100 MHz, DMSO-d6) δ: 165.4, 162.8, 151.3, 141.9,
149.4, 136.4, 133.7, 130.9, 126.4, 120.3, 111.7, 109.2,
52.4. ESI-MS m/z 326.1 (M + H).
Methyl 4-[(4-methoxybenzoyl)amino]pyridine-3-carboxylate
(2′c).
Starting from 2 g of 4-methoxybenzoyl chloride
5,6-Dimethoxy-1H-1,3-benzodiazol-2-amine (5). Cyanogen
bromide (3.7g, 0.035mol) and NaHCO3 (4.9g, 0.058mol)
were added in one portion to a solution of 4,5-
dimethoxybenzene-1,2-diamine 4 (5 g, 0.029mol) in 12mL
of water at room temperature, and stirring was continued for
24h. The reaction mixture was basified to pH=8 using 1M
(c), the title compound 2′c (1.69, 90%) was obtained as
off-white solid, mp 190–191°C IR (ATR, cmÀ1) ʋ: 3252,
2922, 1685, 1584, 1228, 1092. 1H NMR (400MHz,
DMSO-d6) δ (ppm): 11.70 (bs, 1H, NH), 9.06 (s, 1H,
CH-pyridine), 8.69 (d, J 5.6, 1H, CH-pyridine), 8.51 (d, J
6.0, 1H, CH-pyridine), 7.97–8.01 (dt, J1 8.8, J2 2.4, 2H,
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ArH), 7.09–7.13 (dt, J1 8.8, J2 2.4, 2H, ArH), 3.94 (s, 3H,
OCH3), 3.89 (s, 3H, ArOCH3). 13C NMR (100MHz,
DMSO-d6) δ: 165.3, 162.8, 151.4, 141.8, 149.4, 130.2,
124.3, 125.4, 113.6, 111.7, 109.3, 54.5, 52.1. ESI-MS m/
z 287.3 (M + H).
Methyl 4-[(4-methylbenzoyl)amino]pyridine-3-carboxylate
chloride (c), the title compound 2″c (1.37, 74%) was
obtained as off-white solid, mp 132–133°C. IR (ATR,
cmÀ1) ʋ: 3332, 2924, 1693, 1671, 1579, 1477, 1452,
1
1283, 1110. H NMR (400 MHz, CDCl3) δ (ppm): 11.13
(bs, 1H, NH), 8.3 (d, J 5.2, 1H, CH-thiophene), 7.53 (d,
J 5.6, 1H, CH-thiophene), 6.99–7.03 (dt, 2H, J1 9.2, J2
2.8, 2H, ArH), 7.97–8.01 (dt, 2H, J1 8.8, J2 2.8, 2H,
ArH), 3.94 (s, 3H, OCH3), 3.89 (s, 3H, ArOCH3). 13C
NMR (100MHz, CDCl3) δ: 165.3, 162.9, 145.5, 131.9,
129.4, 125.9, 122.3, 114.1, 110, 55.5, 52.1. ESI-MS m/z
292.3 (M + H).
(2′d).
Starting from 1.82g of 4-methylbenzoyl chloride
(d), the title compound 2′d (1.84g, 87%)was obtained as
off-white solid, mp 164–165°C. IR (ATR, cmÀ1) ʋ: 3261,
2923, 1689, 1584, 1230, 1090. 1H NMR (400MHz,
DMSO-d6) δ (ppm): 11.71 (bs, 1H, NH), 9.06 (s, 1H, CH-
pyridine), 8.71 (d, J 6.0, 1H, CH-pyridine), 8.51 (d, J 6.0,
1H, CH-pyridine), 6.93–6.89 (dt, J1 8.8, J2 2.4, 2H, ArH),
7.93–7.89 (dt, J1 8.4, J2 2.8, 2H, ArH), 3.93 (s, 3H, OCH3),
2.4 (s, 3H, ArCH3). 13C NMR (100MHz, DMSO-d6) δ:
165.4, 162.7, 151.4, 141.8, 149.2, 130.4, 127.5, 124.9,
120.1, 113.8, 110.1, 108.2, 52.2. ESI-MS m/z 271.3 (M+H).
Methyl 3-[(4-methylbenzoyl)amino]thiophene-2-carbox-ylate
(2″d). Starting from 1.76g of 4-methylbenzoyl chloride
(d), the title compound 2″d (1 g, 60%) was obtained as off-
white solid, mp 108–109°C. IR (ATR, cmÀ1) ʋ: 3331,
2924, 1670, 1578, 1468, 1450, 1291, 1098. 1H NMR
(400 MHz, CDCl3) δ (ppm): 11.10 (bs, 1H, NH), 8.2 (d, J
5.2, 1H, CH-thiophene), 7.53 (d, J 5.6, 1H, CH-thiophene),
6.92–6.88 (dt, 2H, J1 8.8, J2 2.4, 2H, ArH), 7.94–7.90 (dt,
2H, J1 8.4, J2 2.8, 2H, ArH), 3.94 (s, 3H, OCH3), 2.42 (s,
3H, ArCH3). 13C NMR (100MHz, CDCl3) δ: 165.3, 162.9,
145.2, 130.9, 127.8, 124.3, 122.2, 113.4, 110.6, 52.5, 20.8.
ESI-MS m/z 276.3 (M+ H).
General procedure for the synthesis of 3-aryl-modified
bezoylamino-thiophene-2-methylcarboxylates 2″a–2″d.
To a solution of methyl-3-aminothiophene-2-carboxylate
(1 g, 6.36 mmol) in N,N-dimethyl acetamide (10 mL) was
added triethyl amine (4.44 mL, 31.8mmol) at room
temperature, and the reaction mass was allowed to stir at
room temperature for 10 min. The corresponding benzoyl
chlorides (11.44 mmol) were then added to the reaction
mass and stirred for 1 h at room temperature. Reaction
mass diluted with water (20 mL) and the solids
precipitated were filtered and dried under suction to
General procedure for the synthesis of 4-aryl-modified
bezoylamino-nicotinic acids 3′a–3′d. To a solution of 4-
aryl-modified bezoylamino-nicotinic acid methyl ester 2′
a–2′d (0.5 g, 1 eq) in THF (5 mL) and water (3 mL) was
added lithiumhydroxide monohydrate (1.8 eq) at room
temperature and was allowed to stir at room temperature
for 16h. The reaction mass was concentrated under
reduced pressure, and the residue was diluted with water
(10 mL). The aqueous layer was extracted with methyl
tertiary butyl ether (MTBE) (5 mL), and the organic layer
separated was discarded. The aqueous was acidified to
pH = 2 using acetic acid; solids precipitated were filtered,
washed with water (5mL), and dried under suction to
afford the title compounds 2″a–2″d as solids.
Methyl 3-[(4-chlorobenzoyl)amino]thiophene-2-carboxylate
(2″a).
Starting from 2 g of 4-chlorobenzoyl chloride
(a), the title compound 2″a (135 g, 72%) was obtained as
off-white solid, mp 115–116°C. IR (ATR, cmÀ1) ʋ: 3307,
2925, 1662, 1581, 1471, 1445, 1291, 1096. 1H NMR
(400 MHz, CDCl3) δ (ppm): 11.2 (bs, 1H, NH), 8.28 (d,
J 5.4, 1H, CH-thiophene), 7.55 (d, J 5.6, 1H, CH-
thiophene), 7.52–7.48 (dt, 2H, J1 8.4, J2 2.4, 2H, ArH),
7.94–7.98 (dt, 2H, J1 8.4, J2 2.4, 2H, ArH), 3.94 (s, 3H,
OCH3). 13C NMR (100 MHz, CDCl3) δ: 165.3, 163.1,
145, 138.7, 132, 129.2, 128.8, 122.3, 110.6, 52.2. ESI-
MS m/z 296.7 (M+ H).
afford the title compounds 3′a–3′d as solids.
4-[(4-Chlorobenzoyl)amino]pyridine-3-carboxylic acid (3′
a). Off-white solid (0.27g, 58%), mp 260–262°C. IR
(ATR, cmÀ1) ʋ: 3051, 2845, 1687, 1559, 1426, 1287,
1092. 1H NMR (400MHz, DMSO-d6) δ (ppm): 13.17
(bs, 1H, COOH), 9.08 (s, 1H, CH-pyridine), 8.71 (d, J
5.6, 1H, CH-pyridine), 8.16 (d, J 6.0, 1H, CH-pyridine),
7.71 (d, J 8.8, 2H, ArH), 7.96 (d, J 8.8, 2H, ArH). 13C
NMR (100MHz, DMSO-d6) δ: 164.3, 162.2, 149.5,
147.7, 141.5, 137.6, 132.3, 129.2, 129.1, 121.5, 117.7,
Methyl
3-[(3,4-dichlorobenzoyl)amino]thiophene-2-
carboxylate (2″b).
Starting from 2.38 g of 3,4-
dichlorobenzoyl chloride (b), the title compound 2″b
(1.36 g, 65%) was obtained as off-white solid, mp
124–125°C. IR (ATR, cmÀ1) ʋ: 3330, 2924, 1671, 1573,
1
1442, 1290, 1102. H NMR (400 MHz, CDCl3) δ (ppm):
118.2, 107. ESI-MS m/z 275.6 (M-H).
4-[(3,4-Dichlorobenzoyl)amino]pyridine-3-carboxylic acid
(3′b). Off-white solid (0.285 g, 60%), mp 265–266°C.
11.2 (bs, 1H, NH), 8.25 (d, J 5.4, 1H, CH-thiophene), 8.13
(d, J 2.1, 1H, ArH), 7.82 (dd, J1 8.4, J2 2.2, 1H, ArH), 7.60
(d, J 8.4, 1H, ArH), 7.56 (d, J 5.2, 1H, CH-thiophene),
3.95 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) δ:
165.2, 161.9, 144.6, 136.9, 133.6, 133.5, 132, 130.9, 130,
126.1, 122.2, 110.9, 52.2. ESI-MS m/z 331.3 (M + H).
IR (ATR, cmÀ1) ʋ: 3066, 2826, 1683, 1559, 1425, 1282,
1092. 1H NMR (400MHz, DMSO-d6) δ (ppm): 13.18
(bs, 1H, COOH), 9.08 (s, 1H, CH-pyridine), 8.64 (d, J
6.0, 1H, CH-pyridine), 8.16 (d, J 5.6, 1H, CH-pyridine),
8.06 (d, J 2.0, 1H, ArH), 7.89 (dd, J1 8.4, J2 2.0, 1H, ArH),
7.78 (d, J 8.4, 1H, ArH). 13C NMR (100MHz, DMSO-d6)
Methyl
3-[(4-methoxybenzoyl)amino]thiophene-2-carbox-
ylate (2″c).
Starting from 1.94 g of 4-methoxybenzoyl
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and R. Maridu
Vol 000
δ: 164.4, 162.2, 149.6, 147.2, 141.4, 133.4, 130.7, 124.8,
3-[(4-Methoxybenzoyl)amino]thiophene-2-carboxylic
acid
(3″c). Pale-yellow solid (0.27g, 58%), mp 207–208°C.
IR (ATR, cmÀ1) ʋ: 3345, 2936, 2627, 1679, 1645, 1568,
1285, 1118. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.59 (bs, 1H, COOH), 11.15 (bs, 1H, NH), 8.10 (d, J 5.4,
1H, CH-thiophene), 7.91 (d, J 5.2, 1H, CH-thiophene),
7.88 (d, J 8.8, 2H, ArH), 7.13 (d, J 8.8, 2H, ArH), 3.85 (s,
3H, ArOCH3). 13C NMR (100MHz, DMSO-d6) δ: 164.9,
162.1, 143.4, 131.8, 130.7, 128.4, 124.8, 121.1, 113.7,
113.2, 111, 54.95. ESI-MS m/z 276.3 (M-H).
124.7, 114.2, 118.2, 107.2. ESI-MS m/z 310.1 (M-H).
4-[(4-Methoxybenzoyl)amino]pyridine-3-carboxylic acid (3′
c). Off-white solid (0.29 g, 62%), mp 278–279°C. IR
(ATR, cmÀ1) ʋ: 3062, 2835, 1685, 1559, 1424, 1286,
1
1090. H NMR (400MHz, DMSO-d6) δ (ppm): 13.17 (bs,
1H, COOH), 9.08 (s, 1H, CH-pyridine), 8.68 (d, J 6.0, 1H,
CH-pyridine), 8.14 (d, J 5.6, 1H, CH-pyridine), 7.88 (d, J
8.8, 2H, ArH), 7.18 (d, J 8.8, 2H, ArH), 3.85 (s, 3H,
ArOCH3). 13C NMR (100MHz, DMSO-d6) δ: 164.3,
162.8, 149.4, 147.2, 141.8, 130.1, 128.8, 124.2, 120.4,
3-[(4-Methylbenzoyl)amino]thiophene-2-carboxylic
acid
(3″d). Pale-yellow solid (0.3 g, 64%), mp 175–176°C.
IR (ATR, cmÀ1) ʋ: 3340, 2930, 2635, 1685, 1647, 1570,
1289, 1120. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.59 (bs, 1H, COOH), 11.10 (bs, 1H, NH), 8.10 (d, J
5.4, 1H, CH-thiophene), 7.91 (d, J 5.4, 1H, CH-
thiophene), 7.81 (d, J 8.8, 2H, ArH), 7.08 (d, J 8.8, 2H,
ArH), 2.38 (s, 3H, ArCH3). 13C NMR (100 MHz,
DMSO-d6) δ: 165.2, 162.2, 141.4, 130.2, 129.4, 124.3,
121.2, 112.8, 111.8, 20.8. ESI-MS m/z 260.3 (M-H).
114.3, 118.3, 107.1, 55.4, 54.6. ESI-MS m/z 271.3 (M-H).
4-[(4-Methylbenzoyl)amino]pyridine-3-carboxylic acid (3′
d).
Off-white solid (0.26 g, 55%), mp 252–253°C. IR
(ATR, cmÀ1) ʋ: 3066, 2842, 1687, 1559, 1426, 1282,
1092. 1H NMR (400MHz, DMSO-d6) δ (ppm): 13.17
(bs, 1H, COOH), 9.08 (s, 1H, CH-pyridine), 8.68 (d, J
6.0, 1H, CH-pyridine), 8.14 (d, J 5.6, 1H, CH-pyridine),
7.82 (d, J 8.8, 2H, ArH), 7.10 (d, J 8.8, 2H, ArH), 3.85
(s, 3H, ArOCH3), 2.38 (s, 3H, ArOCH3). 13C NMR
(100 MHz, DMSO-d6) δ: 164.2, 162.3, 149.7, 147.2,
141.8, 130.5, 125.3, 124.8, 118.4, 113.2, 110.7, 107.2,
21.2. ESI-MS m/z 255.3 (M-H).
General procedure for the synthesis of aryl-modified
benzoylamino-N-(5,6-dimethoxy-1H-benzoimidazol-2-yl)-hete-
roamides 6a–6h. To a solution of 2-amino benzimidazole
derivative 5 (0.5g, 2.58mmol) and heteroamide compounds
3′a–3′d and 3″a–3″d (1eq) in DMF (3mL) was added
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]
pyridinium-3-oxide hexafluoropho-sphate (HATU) (1.28g,
3.36mmol) followed by cesium carbonate (2.53g,
7.76mmol) at 0–5°C under nitrogen atmosphere. The
reaction mass was heated at 80°C for 14h. Reaction mass
was diluted with water (10mL) and was extracted with ethyl
acetate (2×15mL). The organic layer was washed with
water (2×10mL), dried over Na2SO4, and concentrated
under reduced pressure to give the title compounds 6a–6h
General procedure for the synthesis of 4-aryl-modified
bezoylamino-thiophene-2 acids 3″a–3″d. To a solution of
3-aryl-modified bezoylamino-thiophene-2-methylcarboxy-
lates 2″a–2″d (0.5g, 1eq) in THF (5mL) and water (3mL)
was added lithiumhydroxide monohydrate (1.8eq) at room
temperature and was allowed to stir at room temperature for
16h. The reaction mass was concentrated under reduced
pressure, and the residue was diluted with water (10mL).
The aqueous layer was extracted with MTBE (5mL), and
the organic layer separated was discarded. The aqueous was
acidified to pH=2 using acetic acid; solids precipitated were
filtered, washed with water (5mL), and dried under suction
to afford the title compounds 3″a–3″d as solids.
as oil, which was purified by column chromatography.
4-[(4-Chlorobenzoyl)amino]-N-(5,6-dimethoxy-1H-benzim-
idazol-2-yl)pyridine-3-carboxamide (6a).
Starting from
3-[(4-Chlorobenzoyl)amino]thiophene-2-carboxylic
acid
(3″a). Off-white solid (0.275 g, 58%), mp 184–185°C.
IR (ATR, cmÀ1) ʋ: 3332, 2925, 2637, 1681, 1648, 1567,
1291, 1120. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.54 (bs, 1H, COOH), 11.26 (bs, 1H, NH), 8.06 (d, J
5.2, 1H, CH-thiophene), 7.68 (d, J 8.8, 2H, ArH), 7.93
(d, J 8.8, 2H, ArH), 7.91 (d, J 5.4, 1H, CH-thiophene).
13C NMR (100 MHz, DMSO-d6) δ: 166.3, 161.9, 141.5,
137.1, 132.7, 131.2, 129.8, 129.2, 129.1, 128.8, 121.5,
0.71 g of heteroamide derivative 3′a, the title compound
6a (0.44 g, 38%) was obtained as off-white solid after
purification by flash chromatography (silica, 0.5:4
EtOAc/hexane elution), mp 325–326°C. IR (ATR, cmÀ1
)
1
ʋ: 3431, 3418, 3010, 1668, 1685, 1582, 1234. H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, OCH3), 7.03
(s, 2H, CH-benzimidazole), 7.73 (d, J 8.8, 2H, ArCH),
7.98 (d, J 8.8, 2H, ArCH), 8.41 (d, J 5.8, 1H, CH-
pyridine), 8.69 (d, J 5.7, 1H, CH-pyridine), 9.06 (s, 1H,
CH-pyridine), 12.34 (bs, 3H, NH). 13C NMR (100MHz,
DMSO-d6) δ: 55.9, 96.2, 108.4, 118.7, 119.2, 119.9,
123.8, 133.0, 137.1, 140.2, 141.1, 146.4, 149.1, 151.7,
152.1, 162.4. ESI-MS m/z 452.4 (M + H). Anal. Calcd for
117.7. ESI-MS m/z 280.6 (M-H).
3-[(3,4-Dichlorobenzoyl)amino]thiophene-2-carboxylic acid
(3″b). Off-white solid (0.26 g, 55%), mp 192–193°C. IR
(ATR, cmÀ1) ʋ: 3330, 2937, 2635, 1687, 1647, 1571,
1288, 1120. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.52 (bs, 1H, COOH), 11.28 (bs, 1H, NH), 8.12 (d, J 1.9,
1H, ArH), 8.02 (d, J 5.4, 1H, CH-thiophene), 7.95–7.84
(m, 3H, CH-thiophene and ArH). 13C NMR (100MHz,
DMSO-d6) δ: 166.2, 162.1, 141.6, 138.2, 133.4, 132.6,
130.9, 129.7, 128.2, 121.1, 110.4. ESI-MS m/z 315.1(M-H).
C22H18ClN5O4 (451.87): C 58.48, H 4.02, N 15.50.
Found: C 58.56, H 4.06, N 15.53.
4-[(3,4-Dichlorobenzoyl)amino]-N-(5,6-dimethoxy-1H-benzi-
midazol-2-yl)pyridine-3-carboxamide (6b).
Starting from
0.8 g of heteroamide derivative 3′b, the title compound 6b
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Synthesis of Benzimidazole-2-yl-heteroamide Derivatives
(0.5g, 40%) was obtained as off-white solid after
ʋ: 3438, 3218, 2992, 1732, 1648, 1603, 1572, 1027, 897.
1H NMR (400MHz, DMSO-d6) δ (ppm): 3.79 (s, 6H,
OCH3), 7.03 (s, 2H, CH-benzimidazole), 7.7 (d, J 8.8, 2H,
ArCH), 7.68 (d, J 5.6, 1H, CH-thiophene), 8.01 (d, J 8.8,
2H, ArCH), 8.09 (d, J 5.6, 1H, CH-thiophene), 12.38–
12.42 (bs, 3H, NH). 13C NMR (100MHz, DMSO-d6) δ:
56.2, 96.3, 118.7, 121.8, 122.4, 123.8, 128.4, 129.2, 129.3,
129.4, 133, 137.1, 146.5, 151.7, 162.3. ESI-MS m/z 457.5
(M + H). Anal. Calcd for C21H17ClN4O4S (456.91): C
55.20, H 3.75, N 12.26. Found: C 55.21, H 3.69, N 12.35.
3-[(3,4-Dichlorobenzoyl)amino]-N->(5,6-dimethoxy-1H-benzi-
purification by flash chromatography (silica, 0.75:4
EtOAc/hexane elution), mp 340–341°C. IR (ATR, cmÀ1
)
1
ʋ: 3432, 3280, 3091, 1666, 1680, 1555, 1236. H NMR
(400MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, OCH3), 7.03
(s, 2H, CH-benzimidazole), 7.89 (dd, J1 8.4, J2 2.0, 1H,
ArCH), 7.96 (d, J 8.4, 1H, ArCH), 8.01 (s, 1H, ArCH),
8.41 (d, J 5.8, 1H, CH-pyridine), 8.69 (d, J 5.7, 1H,
CH-pyridine), 9.06 (s, 1H, CH-pyridine), 12.34 (bs, 3H,
NH). 13C NMR (100MHz, DMSO-d6) δ: 55.9, 96.2,
108.4, 118.7, 119.3, 119.9, 121.8, 123.8, 134.2, 138.5,
141.1, 141.3, 146.4, 149.1, 151.7, 152.1, 162.4. ESI-MS
m/z 486.9 (M+H). Anal. Calcd for C22H17Cl2N5O4
(486.32): C 54.34, H 3.52, N 14.40. Found: C 54.32, H
3.68, N 14.44.
midazol-2-yl)thiophene-2-carboxamide (6f).
Starting from
0.81g of heteroamide derivative 3″b, the title compound
6f (0.59g, 47%) was obtained as off-white solid after
purification by flash chromatography (silica, 1:4
4-[(4-Methoxybenzoyl)amino]-N-(5,6-dimethoxy-1H-benzi-
EtOAc/hexane elution), mp 305–306°C. IR (ATR, cmÀ1
)
1
midazol-2-yl)pyridine-3-carboxamide (6c).
Starting from
ʋ: 3442, 3221, 2990, 1734, 1649, 1604, 1576, 910. H
NMR (400 MHz, DMSO-d6) δ (ppm): 3.79 (s, 6H, OCH3),
7.03 (s, 2H, CH-benzimidazole), 7.68 (d, J 5.6, 1H, CH-
thiophene), 7.7 (dd, J1 8.4, J2 2.0, 1H, ArCH), 7.92 (d, J
8.4, 1H, ArCH), 8.09 (d, J 5.6, 1H, CH-thiophene), 8.21
(s, 1H, ArCH), 12.38–12.42 (bs, 3H, NH). 13C NMR
(100MHz, DMSO-d6) δ: 56.2, 96.3, 119.3, 121.8, 122.4,
123.9, 128.9, 129.2, 129.4, 134.2, 138.4, 146.5, 151.7,
162.3, 162.7. ESI-MS m/z 491.9 (M+H). Anal. Calcd for
C21H16Cl2N4O4S (491.36): C 51.33, H 3.28, N 11.40.
0.7g of heteroamide derivative 3′c, the title compound 6c
(0.48 g, 42%) was obtained as off-white solid after
purification by flash chromatography (silica, 0.5:4
EtOAc/hexane elution), mp 352–353°C. IR (ATR, cmÀ1
)
1
ʋ: 3440, 3290, 3080, 1670, 1650, 1530, 1210. H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, OCH3), 7.03
(s, 2H, CH-benzimidazole), 7.10 (d, J 8.8, 2H, ArCH),
7.95 (d, J 8.8, 2H, ArCH), 8.41 (d, J 5.8, 1H, CH-
pyridine), 8.69 (d, J 5.7, 1H, CH-pyridine), 9.06 (s, 1H,
CH-pyridine), 12.34 (bs, 3H, NH). 13C NMR (100MHz,
DMSO-d6) δ: 55.1, 55.9, 96.2, 108.4, 113.2, 114.2,
119.9, 123.8, 125.2, 126.1, 140.8, 141.1, 146.4, 149.1,
151.7, 152.1, 162.4. ESI-MS m/z 447.9 (M +H). Anal.
Calcd for C23H21N5O5 (447.45): C 61.74, H 4.73, N
Found: C 51.42, H 3.11, N 11.58.
3-[(4-Methoxybenzoyl)amino]-N-(5,6-dimethoxy-1H-benzi-
midazol-2-yl)thiophene-2-carboxamide (6g).
Starting from
0.71g of heteroamide derivative 3″c, the title compound 6g
(0.37g, 32%) was obtained as off-white solid after
purification by flash chromatography (silica, 0.75:4
EtOAc/hexane elution), mp 314–315°C. IR (ATR, cmÀ1) ʋ:
15.65. Found: C 61.85, H 4.77, N 15.78.
4-[(4-Methylbenzoyl)amino]-N-(5,6-dimethoxy-1H-benzim-
1
idazol-2-yl)pyridine-3-carboxamide (6d).
Starting from
3429, 3217, 2961, 1730, 1647, 1604, 1567, 1024, 895. H
0.66g of heteroamide derivative 3′d, the title compound
6d (0.38 g, 45%) was obtained as off-white solid after
purification by flash chromatography (silica, 1:4
NMR (400MHz, DMSO-d6) δ (ppm): 3.79 (s, 6H, OCH3),
3.87 (s, 3H, ArOCH3), 7.03 (s, 2H, CH-benzimidazole), 7.14
(d, J 8.8, 2H, ArCH), 7.67 (d, J 5.6, 1H, CH-thiophene), 7.98
(d, J 8.8, 2H, ArCH), 8.09 (d, J 5.6, 1H, CH-thiophene),
12.38–12.42 (bs, 3H, NH). 13C NMR (100MHz, DMSO-d6)
δ: 55.6, 56.2, 96.3, 113.6, 114.4, 121.8, 122.4, 126.2, 129.2,
129.3, 141.3, 146.5, 151.8, 162.4, 162.8. ESI-MS m/z 452.9
(M+H). Anal. Calcd for C22H20N4O5S (452.49): C 58.40, H
4.46, N 12.38. Found: C 58.54, H 4.49, N 12.47.
EtOAc/hexane elution), mp 312–313°C. IR (ATR, cmÀ1
)
1
ʋ: 3440, 3412, 3070, 1670, 1690, 1570, 1230. H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, OCH3), 7.03
(s, 2H, CH-benzimidazole), 7.07 (d, J 8.8, 2H, ArCH),
7.94 (d, J 8.8, 2H, ArCH), 8.41 (d, J 5.8, 1H, CH-
pyridine), 8.69 (d, J 5.7, 1H, CH-pyridine), 9.06 (s, 1H,
CH-pyridine), 12.34 (bs, 3H, NH). 13C NMR (100MHz,
DMSO-d6) δ: 21.1, 56.2, 96.2, 108.4, 110.2, 111.4,
119.9, 121.8, 122.5, 123.8, 138.4, 141.1, 146.4, 149.1,
151.7, 152.1, 162.4. ESI-MS m/z 431.9 (M +H). Anal.
Calcd for C23H21N5O4 (431.45): C 64.03, H 4.91, N
3-[(4-Methylbenzoyl)amino]-N-(5,6-dimethoxy-1H-benzim-
idazol-2-yl)thiophene-2-carboxamide (6h).
Starting from
0.67 g of heteroamide derivative 3″d, the title compound
6h (0.4 g, 36%) was obtained as off-white solid after
purification by flash chromatography (silica, 0.5:4
EtOAc/hexane elution), mp 281–282°C. IR (ATR, cmÀ1
)
16.23. Found: C 63.98, H 4.87, N 16.18.
3-[(4-Chlorobenzoyl)amino]-N-(5,6-dimethoxy-1H-benzim-
1
ʋ: 3423, 3215, 2950, 1724, 1647, 1552, 1020, 890. H
NMR (400MHz, DMSO-d6) δ (ppm): 2.4 (s, 3H,
ArCH3), 3.79 (s, 6H, OCH3), 7.03 (s, 2H, CH-
benzimidazole), 7.12 (d, J 8.8, 2H, ArCH), 7.67 (d, J 5.6,
1H, CH-thiophene), 7.96 (d, J 8.8, 2H, ArCH), 8.09 (d, J
5.6, 1H, CH-thiophene), 12.38–12.42 (bs, 3H, NH). 13C
idazol-2-yl)thiophene-2-carboxamide (6e).
Starting from
0.72g of heteroamide derivative 3″a, the title compound
6e (0.37 g, 32%) was obtained as off-white solid after
purification by flash chromatography (silica, 0.75:4
EtOAc/hexane elution), mp 295–296°C. IR (ATR, cmÀ1
)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and R. Maridu
Vol 000
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HTRF assay.
The VEGF receptor tyrosine kinase
inhibition is determined by measuring the phosphorylation
level of pGAT-biotin peptide in an HTRF assay. Into a
black 96-well Costar plate is added 2μL/well of 25×
compound in 100% DMSO (final concentration in the 50-μ
L kinase reaction is typically 1nM to 10μM). Next, 38μL
of reaction buffer (25mM of Hepes, pH7.5, 5mM of
MgCl2, 5mM of MnCl2, 2mM of dithiothreitol (DTT), and
1mg/mL of Bovine Serum Albumin (BSA)) containing
0.5mmol of pGAT-biotin and 3–4ng of KDR enzyme is
added to each well. After 5–10min of preincubation, the
kinase reaction is initiated by the addition of 10μL of 10-μ
M ATP in reaction buffer, after which the plate is
incubated at room temperature for 45min. The reaction is
stopped by addition of 50μL of Potassium Fluoride (KF)
buffer (50mM of Hepes, pH7.5, 0.5M of KF, and
1mg/mL of BSA) containing 100mM of EDTA and
0.36μg/mL of PY20K (Eu-cryptate-labeled antiphos-
photyrosine antibody, CIS Bio International). After 30min,
100μL of 10-nM Streptavidin-XL (SV-XL) (modified
Allophycocyanin (APC)-labeled streptavidin, CIS Bio
International) in KF buffer is added, and after additional 2h
incubation at room temperature, the plate is read in a
RUBYstar HTRF reader (BMG, Labtech, Allmendingen,
Ortenberg, Germany).
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valuable support and allocation of resources for this work. We
would like to thank analytical chemistry team, Department of An-
alytical Chemistry, Anthem Biosciences, Bangalore, India, for
having carried out all the analytical work. Also, we would like
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet