(1,2,3-Triazol-4-yl)benzenamines: Synthesis and activity against VEGF receptors 1 and 2

Article abstract of DOI:10.1016/j.bmcl.2009.01.046

Derivatives of (1,2,3-triazol-4-yl)benzenamines are described as potent and ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds exhibited VEGFR-2 and VEGFR-1 inhibitory activity comparable

Full text of DOI:10.1016/j.bmcl.2009.01.046

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1344–1348
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(1,2,3-Triazol-4-yl)benzenamines: Synthesis and activity against VEGF
receptors 1 and 2
b
c
Alexander S. Kiselyov a, , Marina Semenova , Victor V. Semenov
*
a deCODE, 2501 Davey Road, Woodridge, IL 60517, USA
b Institute of Developmental Biology, RAS, 26 Vavilov Str., 119334 Moscow, Russia
c Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 117913 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of (1,2,3-triazol-4-yl)benzenamines are described as potent and ATP-competitive inhibitors
of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds exhibited
VEGFR-2 and VEGFR-1 inhibitory activity comparable to that of VatalanibTM in both HTRF enzymatic and
cellular assays.
Received 18 December 2008
Revised 15 January 2009
Accepted 15 January 2009
Available online 20 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Angiogenesis
ATP-competitive kinase inhibitors
Vascular endothelial growth factor receptor
2
VEGFR-2
(1,2,3-Triazol-4-yl) benzenamines
Anti-angiogenesis agents that target malignant vasculature are
of considerable interest due to their perceived potential to target
tumor resistance towards chemo- and radiotherapy.1–3 Vascular
endothelial growth factors (VEGFs) and their respective family of
receptor tyrosine kinases (VEGFRs) are key proteins modulating
angiogenesis, the formation of new vasculature from an existing
vascular network.4–10 These include VEGFR-1 (Flt-1) and VEGFR-2
(Kinase Insert Domain Receptor (KDR) or flk1).10–13 VEGFR-2 is
the major positive signal transducer for endothelial cell prolifera-
tion and differentiation.11 There has been considerable evidence,
including clinical observations, that the abnormal angiogenesis is
implicated in a number of diseases including rheumatoid arthritis,
inflammation, cancer, degenerative eye conditions and others.12,13
Several molecules have been successfully used for sequestering
VEGF leading to a signal blockade via VEGF receptors and, subse-
quently to an inhibition of malignant angiogenesis. One of the
responsible for the optimal spatial orientation of the pharmaco-
phore pieces (Fig. 1).20
The essential pharmacophore elements for the VEGFR-2 activity
of phthalazine-based molecules and their analogues (A-D) in-
clude20 (i) [6,6]fused (or related) aromatic system; (ii) para- or
3,4-di-substituted aniline function in position 1 of the phthalazine
core; (iii) hydrogen bond acceptor (Lewis’ base: lone pair(s) of a
nitrogen- or oxygen atom(s)) attached to position 4 via an appro-
priate linker (aryl or fused aryl group). In this communication,
we expand upon our initial findings19,20 and disclose potent inhib-
itors of VEGFR-2 kinase based on (1,2,3-triazol-4-yl)benzenamines.
We reasoned that this template could provide for the both proper
pharmacophore arrangement and favorable in vitro/cellular po-
tency of the resulting molecules.
A series of (1,2,3-triazol-4-yl)benzenamines 3–28 (Scheme 1,
Table 1) were synthesized as described in Scheme 1.21 Reaction se-
quence involved the initial formation of enamines 1 from the
respective o-nitrotoluene derivatives (R = H, Cl, CF3) in a 79–92%
yield. Pharmacophore group Ar1 was introduced by the 1,3-dipolar
cycloaddition reaction of 1 with a series of aryl azides in MeCN at
reflux to afford the key intermediate nitroaryl 1,2,3-triazoles 2 in a
56–84% isolated yields. Compounds 2 were further converted to
anilines followed by the reductive amination of these in order to
introduce the Ar2 group. Using this methodology, we have pre-
pared a library of 25 derivatives 3–28 (51–72% yields from 2, 24–
52% overall yields from nitrotoluenes).
agents reportedly working via this mechanism is AvastinTM 14
A
.
number of small-molecule inhibitors that affect VEGF/VEGFR sig-
naling by directly competing with the intracellular ATP binding site
of the respective intracellular kinase domain have been reported.
These include VatalanibTM (A),15 BAY579352 (B),16 and the isoster-
ic anthranyl amide derivatives C17 and AMG-706 (D).18 Intramolec-
ular hydrogen bonding in C and D was suggested17–19 to be
* Corresponding author. Tel.: +1 630 783 4900.
E-mail address: akiselyov@decode.com (A.S. Kiselyov).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.046

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1344–1348
1345
HN
CF3
Cl
Cl
HN
HN
HN
HN
N
N
N
O
O
NH
N
O
NHMe
O
N
NH
N
N
N
N
A
B
C
D
Figure 1. Selected Inhibitors of VEGFR-2.
N
N
N
N
1
1
N
Ar
N
Ar
iii-iv
R
R
R
NMe2
R
i
ii
(56-84%)
(79-92%)
NO2
(51-72%)
NH
NO2
NO2
2
R = H, Cl, CF3
Ar
1
2
3-28
Scheme 1. Synthesis of (1,2,3-triazol-4-yl)benzenamines 3–28. Reagents and conditions: (i) Me2NCH(OMe)2, DMF, reflux, 40 h; (ii) Ar1N3, MeCN, reflux, 48 h; (iii) Fe, AcOH
aq, reflux, 6 h; (iv) Ar2CHO, iPrOH, reflux, 5 min; NaBH4, iPrOH.
Compounds (3–28, Table 1) were first tested in vitro against
VEGFR-222 measuring their ability to inhibit phosphorylation of a
biotinylated-polypeptide substrate (p-GAT, CIS Bio International)
in a homogenous time-resolved fluorescence (HTRF) assay at an
bined SAR data indicated that (i) substitution pattern on the Ar1
group of the triazole (compare 3 and 10, 6 and 11, 9 and 12) and
ii) nature of the aniline substituent Ar2 (compare 3, 4 and 5, 23–
28) were critical to the VEGFR-2 inhibitory activity of these new
series. In addition, 5-substitution of the central aromatic core
was not tolerated.
Compounds 3–28 were also tested in HTRF format against VEG-
FR-1.22 The results in Table 1 suggest that all VEGFR-2 active
(1,2,3-triazol-4-yl)benzenamine derivatives (e.g., 3, 6, 9, 13, 21,
26, 27) consistently led to good activity against VEGFR-1 with
the IC50 values in 66–210 nM range for the most potent com-
pounds. This outcome could be of benefit in the clinical setting
as both receptors are reported to mediate VEGF signaling in the
aberrant angiogenesis.1–5,20 Further screening of 3–28 against a
number of other receptor (IGF1R, InR, FGFR1, Flt3, EGFR, ErbB2,
c-Met, Ron) and cytosolic (PKA, GSK3b, bcr-Abl, bcr-AblT315,
Cdk1/2, Src, Auroras A and B, Plk1) kinases in HTRF format indi-
cated no significant cross reactivity (PI < 30%, triplicate measure-
ATP concentration of 2 lM. The results were reported as a 50%
inhibition concentration value (IC50). VEGFR-2 inhibitors Vatala-
nibTM and C (Fig. 1) were used as internal standards.
As can be seen from Table 1, several (1,2,3-triazol-4-yl)benzen-
amines exhibited good inhibitory activity against VEGFR-2 (bolded
entries).22 By varying substituents at both Ar1 and Ar2, it was pos-
sible to modulate compounds’ potency against the enzyme. Nota-
bly, 5-substitution at the core benzene ring in 3–28 (e.g., R = 5-Cl,
5-CF3-) was not tolerated. In studying SAR of the triazole pharma-
cophore (Ar1), we found that compounds featuring meta-substi-
tuted Ar1, as exemplified by m-Cl (3), m-CF3- (6), m-OCF3 (9), m-
iPr (13) displayed sound potency against VEGFR-2 with the IC50
values of 51–110 nM. Para-substitution in this portion of the mol-
ecule was tolerated, however the respective analogues were >4-
fold less active against the enzyme (Table 1, compare 3 and 10, 6
and 11, 9 and 12). In order to further explore steric requirements
for the Ar1 substituent, we prepared a series of (1,2,3-triazol-4-
yl)benzenamines featuring both heterocyclic (Table 1, 16, 17) and
disubstituted Ar1 functionalities (18–22). 2-(Quinolinyl) (16) and
5-(indazolyl) (17) Ar1 groups displayed deleterious effect on the
overall activity of resulting molecules against the kinase. Deriva-
tives featuring 3,4-disubstituted aryl groups featured somewhat
reduced potency when compared to the respective mono-substi-
tuted meta-derivatives (compare 6, 18 and 21). These facts suggest
presence of a relatively tight hydrophobic pocket in the binding
site of VEGFR-2 (vide supra).
ments) at a concentration of 10 lM.
In order to further expand the utility of triazole scaffold, we
have prepared a series of 1H-1,2,3-triazole-4-carboxamides 31–
34 as shown on Scheme 2. Specifically, amides of cyanoacetic acid
(29) underwent cyclization with benzyl azide under basic condi-
tions to furnish the key N-benzyltriazole intermediates 30. N-Ben-
zyl group in 30 was removed by hydrogenation over Pd/C followed
by the reductive amination of resulting NH-triazoles to yield the
targeted molecules 31–34 in a 24–47% overall yields. Disappoint-
ingly, these molecules did not display any significant activity
against VEGFR-2 or VEGFR-1 kinases. For example, compounds
31 and 33 featuring optimized pharmacophores (Table 1, see com-
In the next series of experiments, we optimized the aniline sub-
stituent Ar2 (Table 1, entries 3–8, 23–28). Similar to the earlier
findings,19,20 4-pyridyl group was present in the most active mol-
ecules. 4-Quinolinyl derivatives (26, 27) displayed diminished
activity against VEGFR-2, even when endowed with the optimized
Ar1 functionality. 4-Imidazole derivative 28 was inactive. The com-
pounds 3 and 6) showed IC50 values of ca. 5–7 lM against VEGFR-2
and were essentially inactive in the cell-based assay. Compounds
32 and 34 also did not show any activity against the kinases
(IC50 > 10 lM). We explained this outcome by the considerably dif-
ferent pharmacophore arrangement in the aryltriazole series
exemplified by 3 and in 3,4-substituted triazoles (31–34).

1346
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1344–1348
Table 1
Activity of (1,2,3-triazol-4-yl)benzenamines 3–28 against VEGFR-2 and VEGFR-1 kinase in vitro and in cell-based assays.
N
N
1
N
Ar
NH
2
Ar
3-28
Ar1
Ar2
VEGFR-2, enzymatic, IC50
(l
M)
VEGFR-1, enzymatic, IC50
(
lM)
VEGFR-2, cell-based ELISA, IC50a,b,c
(lM)
a
a
Compound
A, PTK787
VatalanibTM
C
0.054 0.006
(0.042 0.003)b
0.032 0.005
(0.023 0.006)b
0.087 0.011
4.23 0.51
>10
0.051 0.05
>10
>10
0.066 0.005
0.39 0.09
0.59 0.13
1.22 0.26
0.11 0.01
0.27 0.05
0.61 0.09
0.77 0.18
0.68 0.14
0.44 0.11
0.93 0.18
0.63 0.15
0.14 0.03
0.98 0.21
>10
0.14 0.02
(0.11 0.03)b
0.17 0.05
(0.130 0.081)b
0.13 0.025
>10
>10
0.066 0.07
>10
0.021 0.03
(0.016 0.001)b
0.09 0.01
(0.0012 0.0002)b
0.16 0.05d
>10
>10
0.096 0.14
>10
3
4
5
6
7
8
9
3-Cl-(C6H4)
3-Cl-(C6H4)
3-Cl-(C6H4)
3-F3C-(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3CO(C6H4)
4-Cl-(C6H4)
4-F3C-(C6H4)
4-F3CO(C6H4)
3-iPr-(C6H4)
3-tBu-(C6H4)
4-tBu-(C6H4)
2-(Quinolinyl)
5-(Indazolyl)
3-F3C,4-Cl(C6H4)
3-Cl,4-F3C(C6H4)
3,4-di-Cl(C6H4)
4-Me-3-F3C(C6H3)
3-Me-4-F3C(C6H3)
3-Cl-(C6H4)
4-Pyridine
3-Pyridine
2-Pyridine
4-Pyridine
3-Pyridine
2-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
3,4-di-F-(C6H3)
5-Piperonyl
5-Piperonyl
4-Quinolyl
4-Quinolyl
4-Imidazolo
>10
>10
0.072 0.01
0.13 0.025
0.66 0.12
1.13 0.19
0.13 0.03
0.25 0.08
0.53 0.07
0.64 0.11
0.65 0.13
0.51 0.10
1.13 0.21
0.65 0.12
0.11 0.02
0.86 0.20
>10
0.085 0.02
0.27 0.01d
1.92 0.36
3.11 0.45
0.15 0.05
0.33 0.11
1.63 0.34
1.19 0.22
2.33 0.28
0.78 0.16
2.92 0.44
1.47 0.27
0.18 0.03
2.02 0.35
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3-Cl-(C6H4)
3-CF3-(C6H4)
3-Cl-(C6H4)
3-F3C(C6H4)
3-Cl-(C6H4)
>10
>10
0.19 0.08
0.16 0.04
>10
>10
>10
0.17 0.02
0.21 0.05
>10
>10
>10
0.27 0.06
0.31 0.09
>10
a
IC50 values were determined from the logarithmic concentration-inhibition point (10 points).22 The important values are given as the mean of at least two duplicate
experiments.
b
c
Lit. IC50 values, as measured at 8 l
M ATP.15,17
Lit. data correspond to the inhibition of VEGF-induced phosphorylation of VEGFR-2 in CHO cells.15,17
Molecules with best enzymatic and cellular potencies against VEGFR-2 are bolded.22,23
d
We reasoned that proper alignment of the lower portion of the
pocket of the ATP-binding site of the kinase.19,20 There is a possibil-
molecule, namely nitrogen atoms (Lewis’ base, hydrogen bond
acceptors) of 4-pyridyl or 4-quinolyl groups (Table 1, e.g., 3 and
30) with the Arg1302 moiety in the ATP-binding pocket of VEG-
FR-2 is required for the activity. Docking of 3 into the cocrystal
structure 2P2l of a biphenyl analogue D with VEGFR2 revealed that
the 1,2,3-triazole function of 3 is accommodated in a well-defined
binding pocket lined up with Ala866 and Lys868. This allows for a
better fit of a meta-substituted aryl moiety in the hydrophobic
ity for it to displace a water molecule from the active site of kinase
(Fig. 2a). In addition, we concluded that N-atoms of the triazole
ring could make a contact with the backbone NH of Arg1046. These
features are absent in the 1,2,3-triazole derivative 31. Lack of activ-
ity for 31–34 likely results from the improper alignment of its
amide substituent in the hydrophobic pocket of the kinase. Mini-
mization studies suggest that the positioning of Ar1 pharmaco-
phore for 3 and 31 differs considerably (Fig. 2b). In addition,
1
Ar
1
Ar
HN
1
Ar
HN
N
O
(31) Ar = m-Cl-C6H4-; Ar = 4-Py
1
2
NH
O
ii-iv
i
N
N
(32) Ar = p-Cl-C6H4-; Ar = 4-Py
1
2
O
N
N
H
(33) Ar = m-CF3-C6H4-; Ar = 4-Py
(33-69%)
(48-75%)
1
2
N
N
H
NH2
(34) Ar = p-CF3-C6H4-; Ar = 4-Py
1
2
NC
2
Ar
Ph
29
30
31-34
Scheme 2. Reagents and conditions: (i) PhCH2N3, KOH, EtOH, 80 °C, 1 h; (ii) H2, Pd/C, 80 °C, 4 h; (iii) Ar2CHO, 4Å mol. sieves, benzene, 90 °C, 4 h; (iv) NaBH4, iPrOH, 100 °C, 2 h.

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1344–1348
1347
a
b
c
Hydrophobic Pocket
Ala866
Cl
Lys868
Val848
N
N
N
Val899
Phe918
NH
Leu840
Leu1035
Arg1032
NH2
N
+
H2N
N
H
Figure 2. Structural overlap between (a) 3 (green) and biphenyl analogue of D (blue) from the 2P2 l co-crystal structure with VEGFR; (b) structural overlap of compounds 3
and 31; (c) pharmacophore hypothesis for the mode of binding of (1,2,3-triazol-4-yl)benzenamine 3 within the ATP binding pocket of VEGFR-2. Central aromatic ring is
surrounded by Leu840, Val848, Val899, Phe918 and Leu1035. The amide is in close proximity with the Ala 866 and Lys868, and pyridine nitrogen is near Arg1032.
6. Olsson, A.-K.; Dimberg, A.; Kreuger, J.; Claesson-Welsh, L. Nat. Rev. Mol. Cell Biol.
2006, 5, 359.
7. Zachary, I. Biochem. Soc. Trans. 2003, 31, 1171.
Table 2
Compounds 3, 6 and 13 are ATP-competitive inhibitors of VEGFR-2.
Compound
Ki at IC50
(lM)
Ki at IC90 (lM)
8. Perona, R. Clin. Transl. Oncol. 2006, 8, 77.
9. Jin, T.; Nakatani, H.; Taguchi, T. World J. Gastroenterol. 2006, 12, 703.
10. Itakura, J.; Ishiwata, T.; Shen, B. Int. J. Cancer 2000, 85, 27.
11. Saaristo, A.; Karpanen, T.; Alitalo, K. Oncogene 2000, 19, 6122.
12. Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W. Nature Rev. Drug Discov.
2004, 3, 391.
3
6
13
0.12
0.09
0.16
0.11
0.08
0.14
13. Fine, S. L.; Martin, D. F.; Kirkpatrick, P. Nature Rev. Drug Disc. 2005, 4, 187.
14. Culy, C. Drugs Today 2005, 41, 23.
acidic proton of the pyrrole NH in the triazole ring of 31–34 may
cause unfavorable interactions in the binding pocket lined up with
Leu840, Phe918 and Leu1035 residues. Observed structure–activity
relationship of the novel (1,2,3-triazol-4-yl)benzenamines 3–28
are in line with the proposed pharmacophore hypothesis (Fig. 2c).
Active in vitro inhibitors of VEGFR-2 were further characterized
in a cell-based phosphorylation ELISA assay (Table 1).23 In general,
good in vitro-to-cell based activity correlation has been found for
these compounds. In our hands, the best compounds displayed
85–310 nM activity in inhibiting cell-based phosphorylation of
VEGFR-2. Further, competition assays were conducted for the se-
15. Bold, G.; Altmann, K-H.; Jorg, F.; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld,
B.; Bruggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Pascal, F.; Hofmann, F.;
Martiny-Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.; Acemoglu, F.; Boss,
E.; Emmenegger, R.; Lasser, L.; Masso, E.; Roth, R.; Schlachter, C.; Vetterli, W.;
Wyss, D.; Wood, J. M. J. Med. Chem. 2000, 43, 2310.
16. Chang, Y.S.; Cortes, C.; Polony, B. Proc. Am. Assoc. Cancer Res. (AACR) 2005, 46,
Abstr 2030.
17. Manley, P. W.; Furet, P.; Bold, G.; Brüggen, J.; Mestan, J.; Meyer, T.; Schnell, C.;
Wood, J. J. Med. Chem. 2002, 45, 5697.
18. Kaufman, S.; Starnes, C.; Coxon, A. Proc. Am. Assoc. Cancer Res. (AACR) 2006, 47,
Abstr 3792.
19. Kiselyov, A. S.; Piatnitski, L. E.; Samet, A. V.; Kisly, V. P.; Semenov, V. V. Bioorg.
Med. Chem. Lett. 2007, 17, 1369. and references cited therein.
20. Kiselyov, A. S.; Balakin, K.; Tkachenko, S. E. Expert Opin. Invest. Drugs 2007, 16,
83.
lected molecules with varying concentration (0–100 lM) of ATP.
21. Analytical data for selected molecules:2-(1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-
yl)-N-(pyridin-4-ylmethyl)benzenamine (3): mp 174–175 °C; 1H NMR (400 MHz,
DMSO-d6) d, ppm: 4.28 (d, J = 8.0 Hz, 2H), 6.50 (d, J = 7.2 Hz, 1H), 6.61 (br s,
exch D2O, 1H, NH), 6.93 (m, 1H), 7.06 (d, J = 7.6 Hz, 1H), 7.22 (m, 1H), 7.28 (d,
J = 7.6 Hz, 1H), 7.35 (s, 1H), 7.40 (m, 1H), 7.46 (d, J = 7.2 Hz, 2H), 7.94 (s, 1H),
8.55 (d, J = 7.2 Hz, 2H), 8.73 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d,
ppm: 45.8, 109.6, 113.7, 118.0, 124.3, 125.6, 127.9, 128.2, 128.5, 129.4, 129.8,
132.1, 134.2, 134.7, 146.4, 147.4, 148.7, 150.1; ESI MS (M+1): 363, (M-1): 361.
Elemental analysis: Calcd for C20H16ClN5: C, 66.39; H, 4.46; N, 19.36. Found: C,
66.17; H, 4.58, N, 19.13.
Specifically, five different concentrations of 32P ATP were incu-
bated with VEGFR-2 along with varying concentrations (absence,
IC50, IC90) of the inhibitors 3, 6 and 13 for 45 min at RT. A double
reciprocal graph of the degree of phosphorylation (1/cpm) against
ATP-concentration (1/[ATP]) was plotted. The data were analyzed
by a non-linear least-squares program to determine kinetic param-
eters using GraphPad software. Determined Ki values for the three
selected compounds are listed in Table 2. These data suggest an
ATP-competitive mechanism for the interaction of these inhibitors
with VEGFR-2.
N-(Pyridin-4-ylmethyl)-2-(1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-
yl)benzenamine (6): mp 196–197 °C; 1H NMR (400 MHz, DMSO-d6) d, ppm:
4.31 (d, J = 8.0 Hz, 2H), 6.48 (d, J = 7.2 Hz, 1H), 6.58 (br s, exch D2O, 1H, NH),
6.94 (m, 1H), 7.21 (m, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.33 (s, 1H), 7.36–7.41 (m,
4H), 7.97 (s, 1H), 8.56 (d, J = 7.6 Hz, 2H), 8.78 (d, J = 7.2 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d, ppm: 46.1, 109.4, 117.9, 120.1, 124.0, 124.3, 125.3,
127.8, 128.6, 128.9, 129.1, 129.7, 130.7, 131.5, 133.4, 146.0, 147.9, 148.5,
150.1; ESI MS (M+1): 396, (M-1): 394. Elemental analysis: Calcd for
C21H16F3N5: C, 63.79; H, 4.08; N, 17.71. Found: C, 63.52; H, 3.87, N, 17.49.2-
(1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)-N-(quinolin-4-ylmethyl)benzenamine
(26): mp 231–233 °C; 1H NMR (400 MHz, DMSO-d6) d, ppm: 4.31 (d, J = 7.6 Hz,
2H), 6.49 (d, J = 7.2 Hz, 1H), 6.65 (br s, exch D2O, 1H, NH), 6.91 (m, 1H), 7.03 (d,
J = 7.6 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.18 (m, 1H), 7.28–7.30 (m, 2H), 7.42 (m,
1H), 7.54 (m, 1H), 7.68 (m, 1H), 7.98 (d, J = 7.6 Hz, 1H), 8.07 (s, 1H), 8.21 (d,
J = 7.2 Hz, 1H), 8.71 (d, J = 7.2 Hz, 1H), 8.91 (d, J = 7.2 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d, ppm: 44.5, 109.4, 113.8, 118.1, 121.8, 124.3, 125.2,
126.2, 126.9, 127.5, 128.3, 128.9, 128.3,129.1, 129.3, 129.8, 130.2, 130.6, 132.3,
134.2, 146.4, 146.6, 149.1, 150.8; ESI MS (M+1): 413, (M-1): 411; Elemental
analysis: Calcd for C24H18ClN5: C, 69.98; H, 4.40; N, 17.00. Found: C, 69.76; H,
4.28; N, 16.81.
In summary, we have described a series of (1,2,3-triazol-4-
yl)benzenamines as potent inhibitors of both VEGFR-2 and VEG-
FR-1 receptors. Enzymatic and cellular activities of representative
molecules are comparable to the clinical candidates VatalanibTM
and AMG-706. This outcome could be of benefit in the clinical set-
ting as both receptors are reported to mediate VEGF signaling in
the angiogenesis. Notably, 1H-1,2,3-triazole-4-carboxamide ana-
logues did not show any appreciable activity against the kinases
suggesting strict electronic and spatial demands for the proper
alignment of ligand in a binding pocket of the enzymes.
References and notes
N-((1H-imidazol-4-yl)methyl)-2-(1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)ben-
zenamine (28): mp 163–164 °C; 1H NMR (400 MHz, DMSO-d6) d, ppm: 4.30 (d,
J = 7.6 Hz, 2H), 6.51 (d, J = 7.2 Hz, 1H), 6.59 (br s, exch D2O, 1H, NH), 6.77 (s,
1H), 6.90 (m, 1H), 7.08 (d, J = 7.6 Hz, 1H), 7.21 (m, 1H), 7.29 (d, J = 7.6 Hz, 1H),
7.32 (s, 1H), 7.43 (m, 1H), 7.48 (s, 1H), 8.12 (s, 1H), 8.73 (d, J = 7.2 Hz, 1H), 12.9
(br s, exch D2O, 1H, NH imidazole); 13C NMR (100 MHz, DMSO-d6) d, ppm: 43.9,
1. Bailar, J. C., III; Gornick, H. L. N. Engl. J. Med. 1997, 336, 1569.
2. Boehm, T.; Folkman, J.; Browder, T. Nature 1997, 390, 404.
3. Risau, W. Nature 1997, 386, 671.
4. Klagsbrun, M.; Moses, M. A. Chem. Biol. 1999, 6, R217.
5. Hanahan, D.; Folkman, J. Cell 1996, 86, 353.

1348
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1344–1348
109.2, 113.6, 118.2, 120.2, 125.1, 127.6, 127.9, 128.5, 129.7, 130.2, 131.6, 132.2,
chimeric construct containing the extracellular portion of FGFR1 and the
intracellular portion of VEGFR-2 was transiently transfected into 293
adenovirus-transfected kidney cells. DNA for transfection was diluted to a
135.6, 134.4, 146.7, 148.1, 148.7; ESI MS (M+1): 352, (M-1): 350. Elemental
analysis: Calcd for C18H15ClN6: C, 61.63; H, 4.31; N, 23.96. Found: C, 61.38; H,
4.12, N, 23.69.
5
lg/ml final concentration in a serum-free medium and incubated at room
N-(3-Chlorophenyl)-5-(pyridin-4-ylmethylamino)-1H-1,2,3-triazole-4-carbox-
amide (31): mp 181–183 °C (decomp.); 1H NMR (400 MHz, DMSO-d6) d, ppm:
4.31 (d, J = 8.0 Hz, 2H), 6.78 (br s, exch D2O, 1H, NH), 7.13 (d, J = 7.6 Hz, 1H),
7.37 (m, 1H), 7.45 (d, J = 7.2 Hz, 2H), 7.78 (d, J = 7.6 Hz, 1H), 7.98 (s, 1H), 8.65 (d,
J = 7.2 Hz, 2H), 10.20 (br s, exch D2O, 1H, NH), 11.8 (s, exch D2O, 1H, NH
triazole); 13C NMR (100 MHz, DMSO-d6) d, ppm: 45.8, 119.6, 121.7, 124.4,
124.6, 124.9, 130.2, 133.9, 137.1, 142.9, 147.5, 150.2, 163.2; ESI MS (M + 1):
330, (M-1): 328; Elemental analysis: Calcd for C15H13ClN6O: C, 54.80; H, 3.99;
N, 25.56. Found: C, 54.59; H, 3.82, N, 25.38.
temperature for 30 min with 40 l/mL of Lipofectamine 2000, also in serum-
l
free media. 250 ll of the Lipofectamine/DNA mixture was added to 293 cells
suspended at 5 Â 105 cells/ml. Suspension (200
ll/well) was added to a 96-
well plate and incubated overnight. Within 24 h, media was removed and
100 ll of media with 10% fetal bovine serum was added to the now adherent
cells followed by an additional 24 h incubation. Test compounds were added to
the individual wells (final DMSO concentration was 0.1%). Cells were lysed by
re-suspension in 100 ll Lysis buffer (150 mM NaCl, 50 mM Hepes pH 7.5, 0.5%
Triton X-100, 10 mM NaPPi, 50 mM NaF, 1 mM Na3VO4) and rocked for 1 h at
4 °C.
22. VEGFR tyrosine kinase inhibition is determined by measuring the
phosphorylation level of poly-Glu-Ala-Tyr-biotin (pGAT-biotin) peptide in a
Homogenous Time-Resolved Fluorescence (HTRF) assay. Into a black 96-well
(ii) ELISA for detection of tyrosine-phosphorylated chimeric receptor: 96-well
ELISA plates were coated using 100
and incubated overnight at 4 °C. FGFR1 was prepared in a buffer made with
16 ml of a 0.2 M Na2CO3 and 34 ml of a 0.2 M NaHCO3 with pH adjusted to 9.6.
Concurrent with lysis of the transfected cells, FGFR1 coated ELISA plates were
washed three times with PBS + 0.1% Tween 20, blocked by addition of 200 l/
ll/well of 10 lg/ml of aFGFR1 antibody,
Costar plate is added
concentration in the 50
2
l
l
l/well of 25X compound in 100% DMSO (final
a
l kinase reaction is typically 1 nM to 10 M). Next,
l
38
ll of reaction buffer (25 mM Hepes pH 7.5, 5 mM MgCl2, 5 mM MnCl2,
a
2 mM DTT, 1 mg/ml BSA) containing 0.5 mmol pGAT-biotin and 3–4 ng KDR
enzyme is added to each well. After 5–10 min preincubation, the kinase
l
well of a 3% BSA in PBS for 1 h and washed again. Eighty microliters lysate were
then transferred to the coated and blocked wells and incubated for 1 h at 4 °C.
The plates were washed three times with PBS + 0.1% Tween 20. To detect
reaction is initiated by the addition of 10
after which the plate is incubated at room temperature for 45 min. The
reaction is stopped by addition of 50 l of KF buffer, (50 mM Hepes, pH 7.5,
0.5 M KF, 1 mg/ml BSA) containing 100 mM EDTA and 0.36 g/ml PY20 K (Eu-
ll of 10 lM ATP in reaction buffer,
l
bound phosphorylated chimeric receptor, 100
antibodies (RC20:HRP, Transduction Laboratories) were added (final
concentration 0.5 g/ml in PBS) and incubated for 1 h. The plates were
washed six times with PBS + 0.1% Tween 20. Enzymatic activity of HRP was
detected by adding 50 l/well of equal amounts of the Kirkegaard & Perry
Laboratories (KPL) Substrate A and Substrate B. The reaction was stopped by
addition of 50 l/well of a 0.1 N H2SO4, absorbance was measured at 450 nm.
ll/well of anti-phosphotyrosine
l
cryptate labeled anti-phosphotyrosine antibody, CIS bio international) is added
and after an additional 2 h incubation at room temperature, the plate is read in
a RUBYstar HTRF Reader.
l
l
23. Cell-based assay for VEGFR-2 inhibition:
(i) Transfection of 293 cells with DNA expressing FGFR1/VEGFR-2 chimera: A
l