6,7-Dimethoxy-Quinazolin-4-yl-Amino-Nicotinamide Derivatives as Potent Inhibitors of VEGF Receptor II

Article abstract of DOI:10.1002/jhet.2750

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor-induced angiogenesis. Inhibition of the VEGF signaling pathway has emerged as one of the most promising new approaches for cancer therapy. A series of 6,7-dimethoxy-quinazolin-4-yl-amino-nicotinamides were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity, and compound 7a was found to be a potent inhibitor of VEGFR-2 in an homogeneous time-resolved fluorescence enzymatic assay with an IC₅₀ as low as 48 nM (comparable activity to ZD-6474).

Full text of DOI:10.1002/jhet.2750

Month 2016
6,7-Dimethoxy-Quinazolin-4-yl-Amino-Nicotinamide Derivatives as
Potent Inhibitors of VEGF Receptor II
Abhishek Ashok, * Kannan Thanukrishnan, Halehatty S. Bhojya Naik, and Soma Ghosh
a
a
b
a
a
Anthem Biosciences Pvt. Ltd, 49 Bommasandra Industrial Area, Bommasandra, Bangalore 560099, Karnataka, India
b
Department 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 March 23, 2016
DOI 10.1002/jhet.2750
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large
enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key pro-
moters of tumor-induced angiogenesis. Inhibition of the VEGF signaling pathway has emerged as one of the
most promising new approaches for cancer therapy. A series of 6,7-dimethoxy-quinazolin-4-yl-amino-
nicotinamides were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). Many com-
pounds display VEGFR-2 inhibitory activity, and compound 7a was found to be a potent inhibitor of
VEGFR-2 in an homogeneous time-resolved fluorescence enzymatic assay with an IC50 as low as 48 nM
(
comparable activity to ZD-6474).
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
In addition, a number of nicotinamide derivatives have
been detailed as potent inhibitors of VEGF [9–13]. We an-
ticipated that one of the vital pharmacophore of ZD6474
and their analogues include the quinazoline ring and it
was thought that it would be worthwhile to synthesize
some new quinazoline derivatives bearing a nicotinamide
derivative at the C-4 position of 6,7-dimetho-
xyquinazoline. Nevertheless, to the best of our knowledge,
although several quinazoline carboxamide series have been
developed as tyrosine kinase inhibitors, nicotinamide
derivatives of 6,7-dimethoxyquinazoline have not been ex-
plored as VEGFR inhibitors. In this communication, we
disclose a series of novel 6,7-dimethoxy-quinazolin-4-yl-
amino-nicotinamide derivatives as potent inhibitors of
VEGF-2.
Tumor-induced angiogenesis is the term that describes
the induction of blood vessels from existing vasculature
of the site of a solid tumor, critical for tumor growth and
dissemination [1,2]. Under hypoxic conditions, tumors in-
duce angiogenesis by secreting vascular endothelial
growth factor (VEGF). VEGF has been considered to play
a major role in angiogenesis, the formation of new vascu-
lature from an existing vascular network [3]. Endothelial
cells lining the walls of neighboring blood vessels bind
VEGF to cell surface VEGF receptor II (also known as
KDR; murine version known as Flk-1), triggering a signal-
ing cascade which leads to endothelial cell proliferation,
migration and increasing vascular permeability in the tu-
mor milieu [4,5]. VEGFR-2 is a membrane-bound tyrosine
kinase receptor requiring ligand binding and dimerization
for transphosphorylation of the receptors and subsequent
substrates [6]. A number of small-molecule inhibitors af-
fect VEFG/VEGFR signaling by directly competing with
the ATP-binding site of the respective intracellular kinase
domain. Of various VEGFR-2 inhibitors, the most successful
marketed agent reportedly working via this mechanism is
Avastin (Genentech, San Francisco, CA) [7,8]. Other drug can-
didates that exhibit this mechanism of action include Novartis’
RESULTS AND DISCUSSION
Chemistry. The new quinazoline derivatives described
herein were synthesized as shown in Scheme 1. Previously
described methods were used for synthesis of 6,7-
dimethoxyquinazoline derivatives
2
[14],
3
[15]
and 4 [16]. Ethyl-4-aminopyridine-3-carboxylate was
commercially available. Reaction of known compound 4
with ethyl-4-aminopyridine-3-carboxylate in 2-propanol
at 80°C yielded compound 5. Compound 5 was
(
Basel, Switzerland) PTK 787 A (Vatalanib) and Astra-
Zeneca’s (Wilmington, DE) ZD6474 B (Vandetanib) (Fig. 1).
©
2016 Wiley Periodicals, Inc.

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and S. Ghosh
Vol 000
at δ 4.29–4.36, which corresponds to the ethyl ester group.
Again, the doublets at δ 7.95 and δ 8.04 and singlet at δ 8.37
revealed the presence of pyridine group in the molecule.
The IR spectrum of compound 5 showed the presence of
–
1
C=O group due to the appearance of strong band at
633.91cm . Further, the LC–MS showed its molecular
À1
ion peaks at 355.4 (M +H), which are in accordance with
its molecular formula C H N O .
1
8 18 4 4
13
The structure of compound 6 was interpreted by IR,
C
Figure 1. Chemical structures of PTK 787(A) and ZD 6474(B).
NMR, and LC–MS analysis. The appearance of signal at
61.63 in C NMR corresponds to –C=O group of the free
13
1
hydrolyzed by using lithium hydroxide to get the
corresponding acid 6.
The synthesis of compound 7a was attempted by
carboxylic acid on the pyridine moiety, which again was
confirmed by the presence of a strong band at
À1
1669.61cm in the IR. Further, the LC–MS showed its
reaction of compound
6
with 3-bromo-aniline in
molecular ion peaks at 327.1 (M +H), which are in accor-
dance with its molecular formula C H N O .
the presence of N-ethyl-N′-(3-dimethylaminopropyl)
carbodiimide HCl (EDC.HCl), 1-hydroxybenzotriazole
and 4-dimethylaminopyridine at room temperature. Com-
pound 7a was isolated in 75% yield after recrystallization
by using hot 2-propanol. Employing similar conditions,
compounds 7a–7g were synthesized in good yields. The re-
actions were complete within 12h and the products were
precipitated by adding water to the reaction mixture. Puri-
fication of all the compounds was achieved by recrystalli-
zation by using hot 2-propanol.
16 14 4 4
The structure of compounds 7a–7g was confirmed by
1
13
1
IR, H and C NMR, and LC–MS analysis. The
H
NMR of 7a showed a doublet at δ 8.14, doublet of doublet
at δ 8.7, and a singlet at δ 8.86, which are the characteristic
peaks of the pyridine heterocycle present in the molecule.
Also, the appearance of a doublet at δ 7.4, multiplet at δ
7.72–7.75, and a doublet at 8.16 confirmed the presence
of 3-bromo aryl substituent at the amide group. IR spec-
trum showed the presence of –C=O group of amide at
À1
The structures of all the newly synthesized compounds
1668.11cm , which again was confirmed by the appear-
1
13
13
were confirmed by IR, H and C NMR and LC mass
LCMS) spectral studies. The structure of compound 5
ance of signal at 158.08 in the C NMR. The structure
(
of 7a was again confirmed by LC–MS, which showed the
molecular ion peaks at m/z 481.4 (M +H) corresponding
to the molecular formula C H BrN O .
1
was interpreted by IR, H NMR, and LC–MS analysis.
The H NMR showed a triplet at δ 1.31–1.36 and a quartet
1
22
18
5 3
Scheme 1. Synthetic route to synthesize compounds 7a–7g.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Synthesis of 6,7-Dimethoxy-Quinazolin-4-yl-Amino-Nicotinamide Derivatives
Activity of compounds 7a–7g against VEGFR-2.
Compounds 7a–7g were tested against isolated VEGRF-2
kinase in an enzymatic assay to evaluate their ability
to inhibit tyrosine phosphorylation. Inhibition was
determined by measuring the ability to inhibit
phosphorylation level of biotinylated-polypeptide
substrate (p-GAT, CIS Bio International, Cisbio China,
Shanghai, China) in a homogeneous time-resolved
fluorescence (HTRF) assay at an ATP concentration of
CONCLUSION
We have disclosed a series of 6,7-dimethoxy-quinazolin-
4-yl-amino-nicotinamide derivatives as potent inhibitors of
VEGFR-2. The lead compound from this series 7a, was
found to be the most potent by displaying VEGFR-2
inhibitory activity with an IC50 as low as 48nM in an HTRF
enzymatic assay (comparable activity to ZD-6474). The
compounds also provide an opportunity of laying the
foundation for promising molecules of anticancer potency.
2
μM. The results were reported as a 50% inhibition
concentration value (IC ), literature VEGFR-2
a
5
0
inhibitor also being included as an internal standard for
quality control (ZD 6474; IC = 0.045 μM).
EXPERIMENTAL
5
0
As can be seen from Table 1, a number of compounds
exhibited low nanomolar potency against isolated
VEGFR-2 enzyme. Of note were compounds that
contained a halo-substituted aryl moiety at the amide po-
sition of the nictotinamide derivative. As such, the 3-
bromo, 3-chloro-4-fluoro and 4-fluoro aryl substituted
compounds gave rise to very active analogues with excel-
lent inhibition against the isolated enzyme with an
enzymatic IC50 of 48, 75, and 79nM respectively (entries
Chemistry.
Chemicals were obtained from Sigma-
Aldrich Co. (Bengaluru, India) TLC experiments were
performed on alumina-backed silica gel 40F 254 plates
(Merck, Darmstadt, Germany). The plates were illuminated
under UV (254nm) and KMnO . Melting points were
determined by using Buchi B-540 (Buchi India (p) Ltd.,
4
1
13
Bengaluru, India) and are uncorrected. All H and
NMR spectra were recorded on a Bruker AM-300 and AM-
C
1
1
4
6
-2, 4). Indeed, 7a (entry 1), with an enzymatic IC50 of
8 nM performed comparably to the compound ZD-
474 in these assays (IC = 45nM). Importantly, it was
400 (300 MHz, 400 MHz for H NMR and 75, 100 MHz
13
for C NMR), Bruker BioSpin Corp., Germany. Molecular
weights of unknown compounds were checked by LC–MS
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: s, singlet; d, doublet; t, triplet; dd, doublet of
doublet; m, multiplet; bs, broad singlet. IR Spectra were
recorded by using a Bruker Alpha FTIR spectrometer by
using a diamond 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
±0.4% of the calculated amounts.
5
0
also observed that the thiophene-2-yl-methyl substituent
at the amidic position (entry 5) also gave the active com-
pound with enzymatic IC50 of 17nM. Interestingly, a
cyclohexyl substituent at the amidic position (entry 4)
was tolerated giving rise to a moderately active com-
pound (IC = 0.39 μM). Additionally, the 1H- indazole-
5
0
6
-yl compound also displayed reasonable activity (entry
6
). The cyclopropyl substituent led to diminished potency
against the enzyme (entry 7). In general, the aryl-
substituted carboxamides (7a, 7b, 7d, and 7e and 7f) were
much better inhibitors than the cycloalkyl-substituted
carboxamides (7c and 7g).
4-(6,7-Dimethyl-quinazolin-4-ylamino)-nicotinicacid
ethylester (5).
To a suspension of 4-Chloro-6,7-
dimethoxyquinazoline 4 (2.0 g, 9 mmol) in 2-propanol
(
(
20 mL) was added ethyl-4-aminopyridine-3-carboxylate
1.47 g, 9 mmol) at 25°C. The reaction mixture was
Table 1
allowed to stir at 80°C for 4 h. After completion of reac-
tion, reaction mixture was cooled to 0°C. The residue
Activity of compounds (7a–7g) against VEGFR-2 tyrosine kinase.
Enzymatic,
IC50 (μM)
was filtered and dried to get product 5 as beige solid (3 g,
a
À1
Entry
Compound
R
9
5%). mp: 298.5–298.7°C. IR (ATR, cm ) ʋ: 3385.22
(
N–H), 3026.28 (Ar–CH), 1633.91 (C=O), 1569.74 (Ar–
1
2
3
4
5
6
7
7a
7b
7c
7d
7e
7f
3-Br Ph
3-Cl, 4-F, Ph
Cyclohexyl
0.048
0.075
0.39
0.079
0.017
0.12
1
C=C), 1217.20 (C–O). H NMR (DMSO-d 300MHz) δ
(ppm): 1.31–1.36 (t, J= 7.2 Hz, 3H, –OCH CH ), 3.99 (s,
3
J = 6.9 Hz, 2H, –OCH ), 7.37 (s, 1H, Ar–H), 7.95 (d,
J = 8.7 Hz, 2H, Ar–H), 8.04 (d, J= 8.4 Hz, 2 H, Ar–H),
6
2
3
4-F Ph
H –OCH ), 4.02 (s, 3H, –OCH ), 4.29–4.36 (q,
3 3
Thiophene-2-yl-methyl
1H-indazole-6-yl
Cyclopropyl
2
7g
>10
8
.37 (s, 1H, pyridine H), 8.89 (s, 1H, –N=CH), 11.51
a
Determined against isolated enzyme for at least two independent exper-
13
(bs, 1H, –NH). C NMR (DMSO-d 100MHz) δ: 14.68
iments (at least 8 points per experiment). Positive control, ZD-6474
6
IC50 = 0.045 μM.
(–CH , –OCH CH ), 56.97 (CH , –OCH ), 57.55 (CH ,
3 2 3 3 3 3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and S. Ghosh
Vol 000
–
OCH ), 61.22 (CH , –OCH CH ), 100.47 (quinazoline
coupling of compound 6 with 3-bromo-aniline in the pres-
ence of EDC.HCl. It was obtained as an off-white solid
(550 mg, 75%). mp: 211.7–220.8°C. IR (ATR, cm ) υ:
3
2
2
3
C), 104.56 (quinazoline C), 108.19 (pyridine C), 124.45
pyridine C), 127.12 (quinazoline C), 130.16 (quinazoline
C), 136.76 (pyridine C), 142.10 (quinazoline C), 149.19
pyridine C), 150.78 (quinazoline C), 156.93 (quinazoline,
À1
(
3437.19 (N–H), 3418.07 (amide N–H), 3010.82 (Ar–
(
CH), 1668.11 (C=O), 1582.89 (Ar-C=C), 1234.20 (C–O).
1
N=CHN), 158.45 (C=O), and 165.69 (quinazoline, N=C–
NH). LC–MS (ESI, m/z): 355.4 (M + H).
H NMR (DMSO-d6 300MHz) δ (ppm): 3.94 (s, 3H
–OCH ), 4.09 (s, 3H, –OCH ), 7.28 (s, 1H, Ar–H), 7.4 (d,
3
3
4
-(6,7-Dimethyl-quinazolin-4-ylamino)-nicotinic acid (6).
J = 5.1 Hz, 1H, Ar–H), 7.55 (bs, 2H), 7.68 (s, 1H, Ar–H),
7.72–7.75 (m, 2H, Ar–H), 8.16 (d, J= 5.7 Hz, 1H, Ar–H),
Lithium hydroxide (0.71 g, 16.9 mmol) at 0°C was added
to a solution of compound 5 (2.0 g, 5.6mmol) in tetrahy-
drofuran (8 mL) and water (2 mL) and the reaction mixture
was slowly warmed to room temperature (25°C) over a pe-
riod of 12h. After the completion of the reaction, water
8.41 (d, J = 6Hz, 1H, pyridine H), 8.7 (dd, J =6.3 Hz,
1
J = 5.1 Hz, 1H, pyridine H), 8.86 (s, 1H, pyridine-H),
2
1
3
9.27 (s, 1H, ÀN=CH), 11.15 (bs, 1H, –NH). C NMR
(DMSO-d6 100MHz) δ: 56.22 (CH , –OCH ), 57.17
3
3
(
50 mL) was added to the reaction mixture, and the vola-
(CH3, –OCH3), 101.05 (quinazoline C), 105.50
(quinazoline C), 107.42 (pyridine C), 113.71 (quinazoline
C), 119.93 (pyridine C), 120.07 (Ar–C), 123.52 (Ar–C),
123.64 (Ar–C), 127.78 (Ar–C), 131.2 (Ar–C), 140.26
(Ar–C), 147.76 (quinazoline C), 149.12 (pyridine C),
151.98 (quinazoline C), 152.56 (pyridine C), 152.68 (pyr-
idine C), 154.99 (quinazoline C), 156.43 (quinazoline,
N=CHN), 158.08 (C, –CONH), and 163.69 (quinazoline,
N=C–NH). LC–MS (ESI, m/z): 481.4 (M+ H). Anal.
Calcd. for C H BrN O (480.32): C, 55.01; H, 3.78; N,
tiles were evaporated under reduced pressure. Aqueous
layer was acidified with 1.5N HCl to pH ~2 and was ex-
tracted with ethyl acetate (2 ×50mL). Organic layer was
washed with water (1× 50mL), saturated brine solution
(
1 X 50mL) and dried over anhydrous sodium sulfate.
Solvents were evaporated under reduced pressure to get
product 6 as an off-white solid (1.32g, 72%). mp: 270.3–
2
À1
70.5°C. IR (ATR, cm ) υ: 3168.49 (N–H), 2968.85
(
(
Ar–CH), 1669.61 (C=O), 1598.39 (Ar–C=C), 1220.18
C–O). H NMR (DMSO-d 400MHz) δ (ppm): 4.0 (s,
22
18
5 3
1
14.58. Found: C, 55.14; H 3.92; N, 14.72.
6
3
7
H –OCH ), 4.01 (s, 3H, –OCH ), 7.29 (s, 1H, Ar–H),
.90 (d, J = 8.8Hz, 2H, Ar–H), 8.04 (d, J= 8.8Hz, 2H,
4-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(3-chloro-4-
fluoro-phenyl)-nicotinamide (7b). This compound was pre-
pared by coupling of compound 6 with 3-chloro-4-fluoro-
aniline in the presence of EDC.HCl. It was obtained as
an off-white solid (488mg, 70%). mp: 240.6–241.7°C. IR
3
3
Ar–H), 8.15 (s, 1H, pyridine H), 8.85 (s, 1H, –N=CH),
1
3
1
5
1.02 (bs, 1H, –NH). C NMR (DMSO-d 75 MHz) δ:
6.99 (CH , –OCH ), 57.66 (CH , –OCH ), 100.61
6
3
3
3
3
À1
(
quinazoline C), 104.77 (quinazoline C), 109.1 (pyridine
C), 126.01 (pyridine C), 128.1 (quinazoline C), 131.1
quinazoline C), 137.15 (pyridine C), 142.9 (quinazoline
C), 149.93 (pyridine C), 150.7 (pyridine C), 151.03
quinazoline C), 152.3 (pyridine C), 157.32 (quinazoline,
(ATR, cm ) υ: 3432.75 (N–H), 3280.16 (amide N–H),
3091.39 (Ar–CH), 1666.66 (C=O), 1555.10 (Ar-C=C),
1
(
1236.13 (C–O). H NMR (DMSO-d 300MHz) δ (ppm):
6
3.94 (s, 3H –OCH ), 4.09 (s, 3H, –OCH ), 7.27 (s, 1H,
3
3
(
Ar–H), 7.46 (s, 1H, Ar–H), 7.68 (s, 1H, Ar–H), 7.71–
7.76 (m, 1H, Ar–H), 8.14 (dd, J = 6.5 Hz, J =4.5 Hz,
N=CHN), 161.63 (C, -COOH), and 165.93 (quinazoline,
N=C–NH). LC–MS (ESI, m/z): 327.1 (M + H).
1
2
1H, Ar–H), 8.43 (d, J =6.3 Hz, 1H, pyridine), 8.74 (dd,
J = 6.3 Hz, J = 4.8 Hz, 1H, pyridine-H), 8.88 (s, 1H, pyri-
General procedure for the synthesis of 4-(6,7-dimethoxy-
1
2
13
quinazolin-4-ylamino)-N-nicotinamide derivatives (7a–7g).
dine H), 9.27 (s, 1H, –N=CH), 11.27 (bs, 1H, –NH).
C
N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide
HCl
NMR (DMSO-d6 100MHz) δ: 57.13 (CH , –OCH ),
3
3
(
(
(
EDC.HCl) (442 mg, 2.3 mmol), 1-hydroxybenzotriazole
353mg, 2.3mmol), and 4-dimethylaminopyridine
37 mg, 0.3mmol) at 0°C under nitrogen atmosphere were
57.34 (CH , –OCH ), 101.06 (quinazoline C), 105.6
3 3
(quinazoline C), 107.44 (pyridine C), 113.71 (quinazoline
C), 115.5 (pyridine C), 117.55 (Ar–C), 119.59 (Ar–C),
121.80 (Ar–C), 122.93 (Ar–C), 135.82 (Ar–C), 147.73
(quinazoline C), 149.3 (pyridine C), 152 (quinazoline C),
152.11 (pyridine C), 152.32 (pyridine C), 152.57–152.67
(Ar–C, d, J = 10), 155.62 (quinazoline C), 156.43
(quinazoline, N=CHN), 158.08 (C, –CONH), and 163.58
(quinazoline, N=C–NH). LC–MS (ESI, m/z): 454.4 (M
+ H). Anal. Calcd. for C H ClFN O (453.86): C,
added to a solution of compound 6 (500 mg, 1.5 mmol) in
N,N-dimethylformamide (2.5 mL). After 5 min, corre-
sponding amine (1.5 mmol) was added to the reaction mix-
ture at 0°C and the reaction mixture was slowly allowed to
reach room temperature (25°C) and stirred at room temper-
ature over a period of 12 h. The resulting reaction mass was
poured into crushed ice to precipitate the product. The
precipitated solid was filtered and dried under reduced
pressure and was recrystallized with hot 2-propanol to
yield the title compounds (7a–7g).
22
17
5 3
58.22; H, 3.78; N, 15.43. Found: C, 58.04; H 3.59; N,
15.22.
4-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(cyclohexyl)-nic-
otinamide (7c). This compound was prepared by coupling
of compound 6 with cyclohexylamine in the presence of
4
-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(3-bromo-phe-
nyl)-nicotinamide (7a). This compound was prepared by
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Synthesis of 6,7-Dimethoxy-Quinazolin-4-yl-Amino-Nicotinamide Derivatives
À1
EDC.HCl. It was obtained as a pale yellow solid (356mg,
IR (ATR, cm ) ʋ: 3479.40 (N–H), 3378.07 (amide N–H),
À1
5
7%). mp: 172.9–173.8°C. IR (ATR, cm ) ʋ: 3454.58
3015.48 (Ar–CH), 1655.83 (C=O), 1579.20 (C=C–Ar),
1235.34 (C–O). H NMR (DMSO-d 300MHz) δ (ppm):
3.92 (s, 3H –OCH ), 4.08 (s, 3H, –OCH ), 4.71 (d,
1
(
1
N–H), 3274.94 (amide N–H), 3015.33 (Ar–CH),
612.46 (C=O), 1554.2 (Ar-C=C), 1231.17 (C–O). H
6
1
3
3
NMR (DMSO-d 300MHz) δ (ppm): 1.27–1.44 (m, 4H,
cyclohexane), 1.71–1.87 (m, 4H, cyclohexane), 3.76–3.83
J = 5.7 Hz, 2H, –CH2 thiophene), 7.01–7.02 (m, 1H,
thiophene-H), 7.12–7.17 (m, 1H, thiophene H), 7.27 (s,
1H, Ar–H), 7.67 (s, 1H, Ar–H), 7.72 (bs, 2H, –NH), 8.27
6
(
m, 1H, cyclohexane), 3.92 (s, 3H, –OCH ), 4.08 (s, 3H,
3
–
OCH ), 7.25 (s, 1H, Ar–H), 7.59 (bs, 2H, –NH), 7.67
(d, J = 6.3 Hz, 1H, pyridine H), 8.67 (dd, J = 6.3Hz,
3
1
(
(
s, 1H, Ar–H), 8.27 (d, J =6.3 Hz, 1H, pyridine H), 8.67
dd, J = 6.0Hz, J = 4.8Hz, 1H, pyridine H), 8.83 (s, 1H,
J = 4.8 Hz, 1H, pyridine H), 8.87 (s, 1H, pyridine H),
9.25 (s, 1H, –N=CH), 9.93 (bs, 1H, –NH). C NMR
2
13
1
2
1
3
pyridine H), 9.27 (s, 1H, ÀN=CH). C NMR (DMSO-d6
00MHz) δ: 22.3 (cyclohexane C), 27.4 (cyclohexane
C), 33.6 (cyclohexane C), 46.1 (cyclohexane C), 56.89
CH , –OCH ), 57.13 (CH , –OCH ), 101.07 (quinazoline
(DMSO-d 100 MHz) δ: 38.11 (–CH , thiophene), 56.22
6
2
1
(CH , –OCH ), 57.10 (CH , –OCH ), 101.13 (quinazoline
3 3 3 3
C), 105.49 (quinazoline C), 107.37 (pyridine C), 113.57
(quinazoline C), 115.90 (pyridine C), 125.73 (thiophene
C), 126.40 (thiophene C), 127.19 (thiophene C), 141.60
(thiophene C), 148.43(quinazoline C), 149.15 (pyridine
C), 151.92 (quinazoline C), 152.52 (pyridine C), 152.62
(pyridine C), 155.01 (quinazoline C), 156.32 (quinazoline,
N=CHN), 158.04 (C, –CONH), 164.79 (quinazoline,
N=C–NH). LC–MS (ESI, m/z): 422.4 (M+ H). Anal.
Calcd. for C H N O S (421.48): C, 59.84; H, 4.54; N,
(
3
3
3
3
C), 106.1 (quinazoline C), 107.63 (pyridine C), 114.1
quinazoline C), 119.73 (pyridine C), 147.79 (quinazoline
C), 149.32 (pyridine C), 151.93 (quinazoline C), 152.61
pyridine C), 152.73 (pyridine C), 155.01 (quinazoline
(
(
C), 156.61 (quinazoline, N=CHN), 158.13 (C, –CONH),
and 165.12 (quinazoline, N=C–NH). LC–MS (ESI, m/z):
4
6
1
08.5 (M +H). Anal. Calcd. for C H N O (407.48): C,
22 25 5 3
4.85; H, 6.18; N, 17.19. Found: C, 64.87; H 6.07; N,
7.08.
21 19 5 3
16.62. Found: C, 59.77; H 4.38; N, 16.54.
4-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(1H-indazole-6-
4
-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(4-fluoro-phe-
yl)-nicotinamide (7f).
This compound was prepared by
nyl)-nicotinamide (7d). This compound was prepared by
coupling of compound 6 with 4-fluoroaniline in the pres-
coupling of compound 6 with 1H-indazole-6-ylamine in
the presence of EDC.HCl. It was obtained as a brown solid
(237 mg, 35%). mp: 211.7–215.5°C. IR (ATR, cm ) υ:
À1
ence of EDC.HCl. It was obtained as an off-white solid
À1
(
418 mg, 65%). mp: 266.2–270.3°C. IR (ATR, cm ) υ:
3428.78 (amide N–H), 3185.12 (N–H), 3159 (Ar–CH),
1
3
426.82 (N–H), 3316.91 (amide N–H), 3044.05 (Ar–
1664.98 (C=O), 1553.22 (C=C–Ar), 1231.25 (C–O). H
CH), 1665.87(C=O), 1511.32 (C=C–Ar), 1221.70 (C–O).
H NMR (DMSO-d6 300MHz) δ (ppm): 3.92 (s, 3H
NMR (DMSO-d6 300MHz) δ (ppm): 3.96 (s, 3H
–OCH ), 4.09 (s, 3H, –OCH ), 7.28 (s, 1H, Ar–H), 7.42–
1
3
3
–
OCH ), 4.02 (s, 3H, –OCH ), 7.27 (s, 1H, Ar–H), 7.66
7.46 (m, 1H, Ar–H), 7.6 (bs, 2H, –NH), 7.68 (s, 1H, Ar–
H), 7.7 (d, J= 8.4 Hz, 1H, Ar–H), 8.03 (s, 1H, Ar–H), 8.3
(s, 1H, indazole H), 8.49 (d, J = 6.0 Hz, 1H, pyridine H),
8.74 (dd, J =6.3 Hz, J = 4.8 Hz, 1H, pyridine H), 8.94
3
3
(bs, 2H, –NH), 7.68 (s, 1H, Ar–H), 7.87 (d, J= 8.7 Hz,
2
H, Ar–H), 8.02 (d, J =8.4Hz, 2H, Ar–H), 8.42 (d,
J =6.0 Hz, 1H, pyridine H), 8.72 (dd, J = 6.3 Hz,
1
1
2
J = 4.8Hz, 1H, pyridine H), 8.86 (s, 1H, pyridine H),
(s, 1H, pyridine-H), 9.27 (s, 1H, –N=CH), 11.27 (bs, 1H,
–NH), 13.14 (bs, 1H, –NH). C NMR (DMSO-d6
2
13
13
9.25 (s, 1H, –N=CH), 11.27 (bs, 1H, –NH). C NMR
(
(
(
DMSO-d6 100 MHz) δ: 56.82 (CH , –OCH ), 57.17
100 MHz) δ: 56.19 (CH , –OCH ), 57.19 (CH , –OCH ),
3
3
3
3
3
3
CH , –OCH –OCH ), 101 (quinazoline C), 105.83
101.04 (quinazoline C), 101.58 (indazole C), 105.45
(quinazoline C), 107.40 (pyridine C), 113.70 (quinazoline
C), 115.46 (pyridine C), 120.40 (indazole C), 121.04
(indazole C), 127.67 (indazole C), 133.76 (indazole C),
136.81 (indazole C), 144.61 (indazole C), 147.79
(quinazoline C), 149.09 (pyridine C), 151.92 (quinazoline
C), 152.56 (pyridine C), 152.66 (pyridine C), 154.97
(quinazoline C), 156.42 (quinazoline, N=CHN), 158.05
(C, –CONH), and 163.63 (quinazoline, N=C–NH). LC–
MS (ESI, m/z): 442.5 (M + H). Anal. Calcd. for
C H N O (441.45): C, 62.58; H, 4.34; N, 22.21. Found:
3
3
3
quinazoline C), 107.48 (pyridine C), 112.9 (quinazoline
C), 115.31 (Ar–C), 119.73 (pyridine C), 120.1 (Ar–C),
39.2 (Ar–C), 147.91 (quinazoline C), 149.41 (pyridine
1
C), 150.01 (quinazoline C), 150.32–150.41 (Ar–C, d,
J =9), 152.66 (pyridine C), 152.91 (pyridine C), 154.33
(
(
(
(
quinazoline C), 156.14 (quinazoline, N=CHN), 159.12
C, –CONH), and 165 (quinazoline, N=C–NH). LC–MS
ESI, m/z): 420.4 (M+ H). Anal. Calcd. for C H FN O
2
2
18
5 3
419.42): C, 63.00; H, 4.33; N, 16.70. Found: C, 62.89;
H 4.20; N, 16.86.
23 19 7 3
4
-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(thiophen-2-yl-
methyl)-nicotinamide (7e). This compound was prepared
by coupling of compound with thiophen-2-yl-
C, 62.69; H 4.42; N, 22.30.
4-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-(cyclopropyl)-
nicotinamide (7 g). This compound was prepared by cou-
pling of compound 6 with cyclopropylamine in the pres-
ence of EDC.HCl. It was obtained as an off-white solid
6
methylamine in the presence of EDC.HCl. It was obtained
as a pale brown solid (530 mg, 82%). mp: 176.6–180.6°C.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Ashok, K. Thanukrishnan, H. S. Bhojya Naik, and S. Ghosh
Vol 000
À1
(
3
400 mg, 72%). mp: 212.0–221.8°C. IR (ATR, cm ) υ:
SV-XL (modified APC-labeled streptavidin, CIS Bio
International) in KF buffer is added, and after additional
403.34 (N–H), 3157.52 (amide N–H), 2997.85 (Ar–
CH), 1632.01 (C=O), 1500 (C=C–Ar), 1234.47 (C–O).
H NMR (DMSO-d 300 MHz) δ (ppm): 0.63–0.70 (m,
H, cyclopropane), 0.76–0.82 (m, 2H, cyclopropane),
2
h incubation at room temperature, the plate is read in a
1
6
RUBYstar HTRF Reader.
2
2
4
–
.91–2.97 (m, 1H, cyclopropane), 3.92 (s, 3H –OCH3),
.08 (s, 3H, –OCH ), 7.24 (s, 1H, Ar–H), 7.6 5(bs, 2H,
3
Acknowledgments. We are thankful to Anthem Biosciences
management, Anthem Biosciences, Bangalore, India, for their
invaluable support and allocation of resources for this work. We
would like to thank analytical chemistry team, Department of
Analytical Chemistry, Anthem Biosciences, Bangalore, India,
for having carried out all the analytical work. Also, we would
like to thank molecular biology team of Anthem Biosciences for
executing the VEGFR-2 activity studies.
NH), 7.67 (s, 1H, Ar–H), 8.25 (d, J= 6.0Hz, 1H, pyridine
H), 8.66 (dd, J = 6.3Hz, J = 4.8Hz, 1H, pyridine H), 8.85
1
2
1
3
(
(
(
s, 1H, pyridine-H), 9.25 (s, 1H, –N=CH). C NMR
DMSO-d6 100 MHz) δ: 5.9 (cyclopropane C), 20.6
cyclopropane C), 56.63 (CH , –OCH ), 57.1 (CH ,
3
3
3
–OCH ), 101.1 (quinazoline C), 105.8 (quinazoline C),
3
1
07.21 (pyridine C), 115.1 (quinazoline C), 118.79 (pyri-
dine C), 147.76 (quinazoline C), 149.75 (pyridine C),
51.93 (quinazoline C), 152.81 (pyridine C), 152.76 (pyr-
1
REFERENCES AND NOTES
idine C), 155.04 (quinazoline C), 156.95 (quinazoline,
N=CHN), 158.36 (C, –CONH), and 164.39 (quinazoline,
N=C–NH). LC–MS (ESI, m/z): 366.5 (M + H). Anal.
Calcd. for C H N O (365.39): C, 62.46; H, 5.24; N,
[
[
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nM to 10 μM) is added into a black 96-well Costar
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1
4
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4
5min. The reaction is stopped by addition of 50 μL KF
buffer (50 mM Hepes, pH7.5, 0.5M KF, and 1 mg/mL
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet