Inhibitors of VEGF receptors-1 and -2 based on the 2-((pyridin-4-yl)ethyl) pyridine template

Article abstract of DOI:10.1016/j.bmcl.2005.12.090

We have developed a series of novel potent ((pyridin-4-yl)ethyl)pyridine derivatives active against kinases VEGFR-1 and -2. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying the

Full text of DOI:10.1016/j.bmcl.2005.12.090

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1913–1919
Inhibitors of VEGF receptors-1 and -2 based
on the 2-((pyridin-4-yl)ethyl)pyridine template
Alexander S. Kiselyov,a,* Marina Semenova,b Victor V. Semenovc and Daniel Milligand
aSmall Molecule Drug Discovery, Chemical Diversity, Inc, 11558 Sorrento Valley Road, San Diego, CA 92121, USA
bInstitute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Str., 119334 Moscow, Russia
cZelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky Prospect, 117913 Moscow, Russia
dDepartment of Chemisty, ImClone Systems, 180 Varick Street, New York, NY 10014, USA
Received 30 November 2005; revised 21 December 2005; accepted 22 December 2005
Available online 3 February 2006
Abstract—We have developed a series of novel potent ((pyridin-4-yl)ethyl)pyridine derivatives active against kinases VEGFR-1 and
-2. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying
the arylamino substituent at the 1,3,4-oxadiazole ring. The most specific molecules displayed >10-fold selectivity for VEGFR-2 over
VEGFR-1. Compound activities in vitro and in cell-based assays (IC50 < 100 nM) were similar to those of reported clinical and
development candidates, including PTK787 (VatalanibTM). High permeability of active compounds across the Caco-2 cell monolayer
(>30 · 10À5 cm/min) is indicative of their potential for intestinal absorption upon oral administration.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Angiogenesis, or formation of new blood vessels, is a
highly complex process. Proliferation and migration of
capillary endothelial cells from pre-existing blood vessels
is followed by tissue infiltration and cell assembly into
tubular structures, joining of newly formed tubular
assemblies to closed-circuit vascular systems, and matu-
ration of newly formed capillary vessels. Angiogenesis is
involved in pathological conditions such as tumor
growth and degenerative eye conditions.1 Family of vas-
cular endothelial growth factors (VEGFs),2 endothelial
cell-specific mitogens, has been implicated in regulation
of angiogenesis in vivo. VEGFs mediate their biological
effect through high affinity VEGF receptors which are
expressed on the endothelial cells. These include recep-
tor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2
(kinase insert domain receptor (KDR) or flk1).3
Whereas VEGFR-1 functions are still under investiga-
tion, there is evidence that KDR is a major mediator
of vascular endothelial cell mitogenesis as well as angio-
genesis and microvascular permeability. It is believed
that a direct inhibition of the aberrant KDR kinase
activity results in reduction of tumor angiogenesis and
suppression of tumor growth. Potent, specific, and
non-toxic inhibitors of angiogenesis are powerful clini-
cal tools in oncology and ophthalmology.3
There are reports describing small-molecule inhibitors
that affect VEGF/VEGFR signaling by directly compet-
ing with the ATP binding site of the respective intracel-
lular kinase domain. This event leads to the inhibition of
VEGFR phosphorylation and, ultimately to apoptotic
death of the aberrant endothelial cells. Drug candidates
that exhibit this mechanism of action include PTK787
(VatalanibTM, A) and ZD6474 (VandetanibTM, B). These
are phases III and II clinical candidates, respectively,
against various cancers.4,5 The 6-membered ring of a
phthalazine template in PTK787 has been replaced with
the isosteric anthranyl amide derivatives C and D. Intra-
molecular hydrogen bonding was suggested to be
responsible for the optimal spatial orientation of phar-
macophores, similar to the parent PTK787.6
It has been suggested that the essential pharmacophores
for the VEGFR-2 activity of phthalazines and their ana-
logues include: (i) [6,6]fused (or related) aromatic sys-
tem; (ii) para- or 3,4-di-substituted aniline function in
the position 1 of phthalazine; and (iii) hydrogen bond
acceptor (Lewis’ base: lone pair(s) of a nitrogen- or oxy-
gen atom(s)) attached to the position 4 via an appropri-
ate linker (aryl or fused aryl group).4 In this letter, we
Keywords: Vascular endothelial growth factor receptor 2; VEGFR-2;
VEGFR-1; Receptor tyrosine kinase; Dual kinase inhibitors; Angio-
genesis; ((Pyridin-4-yl)ethyl)pyridines.
*
Corresponding author. E-mail: ask@chemdiv.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.090

1914
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1913–1919
Cl
N
Br
HN
HN
HN
N
HN
CF3
O
O
F
1
4
N
N
O
H
N
O
H
N
N
N
A
C
D
B
N
N
N
expand upon our initial findings7 and disclose potent
inhibitors of VEGFR-2 kinase based on a 2-((pyridin-
4-yl)ethyl)pyridine core. We reasoned that this non-
phthalazine template could provide for the proper
pharmacophore arrangement relevant to compounds
PTK787, C and D.
ucts were isolated by flash chromatography (eluent:
hexanes/Et2O, 1:1) to afford analytically pure materials
in 76–92% yield.8
Compounds (5–45, Table 1) were tested in vitro against
isolated VEGFR-2 kinase. Specifically, we measured
their ability to inhibit phosphorylation of a biotinyla-
ted polypeptide substrate (pGAT, CIS Bio Internation-
The desired molecules 5–45 were accessed by a four-step
reaction sequence shown in Scheme 1. Commercially
available methyl picolinic or quinolinic acids 1 were re-
fluxed in anhydrous MeOH with catalytic amount of
concentrated H2SO4 for 1 h followed by the addition
of a 15-fold molar excess of anhydrous hydrazine and
reflux for an additional 2 h. Removal of solvent and ex-
cess hydrazine in vacuo (Caution! Efficient liquid N2
trap!) was followed by the reaction of the resulting crude
hydrazides 2 with a series of aryl thiocyanates (Ar1-
NCS) in dichloroethane at rt for 2 h. Subsequently,
the reaction mixtures were treated in situ with EDCI
(2.5 equiv) at 0 ꢁC, brought to reflux, and stirred for
8 h to promote heterocyclization of the intermediate thi-
osemicarbazides. The targeted amino oxadiazoles 3 were
conveniently isolated by trituration of the concentrated
reaction mixtures with Et2O followed by re-crystalliza-
tion of the resulting solids from EtOH (78–92% yields
for two steps). The collected crystalline materials 3 were
treated with NaH (2.25 equiv) in dry freshly distilled
DMF at 0 ꢁC. The resulting anions were reacted at rt
with a series of aromatic aldehydes (Ar2-CHO) for 8 h
to yield the respective diaryl olefins 4. Compound 4
was isolated in 66–74% by column chromatography
(eluent: hexanes/EtOAc, 2:1; mixtures of trans- and
cis-isomers (ca. 4:1–5:1)) and hydrogenated without sep-
aration in EtOAc for 4 h at rt using 10% Pd/C until both
TLC and LC–MS analyses indicated the complete con-
version of 4 into the targeted molecules 5–45. The prod-
al) in
a homogeneous time-resolved fluorescence
(HTRF) assay at an ATP concentration of 2 lM. The
results were reported as a 50% inhibition concentration
value (IC50). Literature VEGFR-2 inhibitors A–D (Ta-
ble 1) were included as an internal standards for quality
control.6,9
As seen from Table 1, a number of ((pyridin-4-yl)eth-
yl)pyridine derivatives exhibited a robust inhibitory
activity against VEGFR-2. By varying both aryl substit-
uents (anilinic groups) on a 1,3,4-oxadizole function and
(arylethyl)pyridine pharmacophores, we modulated the
compounds’ potency against the enzyme. Initially, we
kept the anilinic function constant (3-CF3-C6H4-NH,
similar to the compound C)4,6 and studied the inhibitory
effect of an arylethyl group and central pyridine tem-
plate on the enzymatic activity of the resultant com-
pounds 5–18 (Table 1). (Pyridin-4-yl)ethyl derivative 5
furnished the best activity with the IC50 value of
64 nM against VEGFR-2. The respective analogue 17
of the compound D (vide supra) showed a considerably
weaker enzymatic potency (0.38 uM). In addition, it did
not display cellular activity, presumably due to poor cell
permeability.10 Furthermore, the nature of pyridine tem-
plate was found to have a profound effect on the com-
pounds potency. Molecules based on 3- and 4-pyridine
cores consistently displayed weaker activity against
VEGFR-2, when compared to the compounds derived
O
Ar1
O
N
1) MeOH, H2SO4
2) NH2NH2
1) Ar1-NCS
2) EDCI
N
NH2
NH
OH
N
O
Het
Het
H
Het
DCE, reflux, 8 h
(78-92% for 2 steps )
1
2
3
Ar1
NH
N
Ar1
N
O
N
N
1) NaH, THF
2) Ar2CHO
N
O
H
H2, 10%Pd-C
Het
Het
DMF, 8 h, RT
(66-74%)
EtOAc, 4 h, RT
(76-92%)
Ar2
Ar2
5-45
4
Scheme 1.

Table 1. Activity of ((pyridin-4-yl)ethyl)pyridines and quinolines 5–45 against VEGFR-1 and -2
Ar1
Ar1
N
N
O
Ar1
Ar1
N
N
N
O
N
N
Ar1
N
O
N
N
N
H
N
H
N
H
N
H
N
H
O
O
N
Het
N
N
N
Ar2
Ar2
Ar2
Ar2
Ar2
5-45
Templates: 2-Py
3-Py
4-Py
2-Quin
Compound
Pyridine
ring
Ar1
Ar2
VEGFR-2, enzymatic,
a
IC50 (lM)
VEGFR-1, enzymatic,
a
IC50 (lM)
VEGFR-2, cell-based
a
ELISA, IC50 (lM)
A, PTK787
B, ZD6474
C
0.054 0.006 (0.042 0.003)
0.022 0.003 (0.017 0.003)
0.032 0.005 (0.023 0.006)
0.015 0.004 (0.009 0.001)
0.064 0.008
0.56 0.07
0.72 0.07
>10
0.14 0.02 (0.11 0.03)
0.10 0.01 (0.09 0.01)
0.17 0.05 (0.130 0.081)
0.16 0.05 (0.13 0.03)
0.22 0.04
0.76 0.08
1.22 0.16
>10
0.021 0.03 (0.016 0.001)
1.66 0.11 (2.70 0.17)
0.09 0.01 (0.0012 0.0002)
0.05 0.01 (0.0012 0.0001)
0.11 0.02
1.11 0.10
2.54 0.32
>10
D
5
2-Py
3-Py
4-Py
2-Quin
2-Py
2-Py
2-Py
2-Py
3-Py
2-Py
2-Py
2-Py
2-Py
2-Quin
2-Py
2-Py
3-Py
3-Py
4-Py
2-Quin
2-Py
2-Py
2-Py
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
2-Py
3-Py
4-Py
2-Py
2-Py
2-Py
2-Py
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-F3C(C6H4)
3-Quinolino-
3-Quinolino-
4-t-Bu(C6H4)
4-i-Pr(C6H4)
4-t-Bu(C6H4)
4-i-Pr(C6H4)
4-t-Bu(C6H4)
4-t-Bu(C6H4)
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
3-Pyridine
2-Pyridine
Piperonyl
6
7
8
9
>10
>10
>10
>10
>10
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
>10
>10
>10
>10
>10
>10
3,4-Di-F(C6H3)
Piperonyl
5-Indazole
2-Quinoline
5-Quinoline
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
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
4-Pyridine
>10
3.87 0.30
>10
>10
>10
>10
>10
>10
>10
>10
>10
N/Ab,10
5.64 0.51
0.38 0.04
>10
0.043 0.008c
0.051 0.009
0.19 0.02
0.25 0.03
0.31 0.03
>10
0.44 0.06
>10
ND
0.77 0.11
0.62 0.10
0.22 0.02
0.27 0.03
0.48 0.04
>10
0.063 0.01
0.075 0.01
0.32 0.03
0.39 0.04
0.52 0.06
ND
4-ClF2CO(C6H4)
4-F3CO(C6H4)
4-F3C(C6H4)
0.031 0.006
0.044 0.007
0.058 0.008
0.22 0.02
0.35 0.04
0.29 0.04
0.44 0.05
0.36 0.007
0.44 0.04
0.069 0.009
0.47 0.05
0.55 0.06
0.77 0.09
0.17 0.02
>10
0.73 0.01
0.56 0.05
0.32 0.05
0.81 0.06
0.93 0.06
0.84 0.07
0.53 0.06
0.47 0.05
0.67 0.08
0.26 0.04
0.71 0.08
0.74 0.07
1.07 0.11
0.50 0.07
>10
0.044 0.007
0.051 0.007
0.076 0.009
0.53 0.06
0.66 0.07
0.72 0.08
0.59 0.06
0.51 0.06
0.74 0.10
0.11 0.01
0.86 0.12
0.76 0.11
2.14 0.23
0.29 0.02
ND
4-ClF2CO(C6H4)
4-F3CO(C6H4)
4-F3C(C6H4)
4-ClF2CO(C6H4)
4-F3CO(C6H4)
4-F3C(C6H4)
3-F3CO(C6H4)
3-F3CO(C6H4)
3-F3CO(C6H4)
3-Me(C6H4)
3-MeO(C6H4)
2-MeO(C6H4)
2-Me(C6H4)
>10
>10
ND
(continued on next page)

1916
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1913–1919
from the 2-pyridine scaffold (e.g., compare 5–7, Table
1). This pattern was observed throughout our
structure–activity relationship studies of the title
series (e.g., compare 19 and 20 with 21–23 and 25–27
with 28–33, Table 1). The related 2-quinoline template
did not yield enzymatically active compounds, despite
the optimized substitution pattern (see entries 8, 18,
and 24).
Good inhibitory activity of 5 was explained by the
proper alignment of the 4-substituent, namely pyri-
dine-type nitrogen atom (Lewis’ base: hydrogen bond
acceptor) with the Arg1302 moiety in the ATP binding
pocket of VEGFR-2. We further reasoned that in 9 and
10 the nitrogen of the pyridine ring is likely to be misa-
ligned with the Arg1302. Similarly, indazole, 2- and 5-
quinoline functions in 14–16 could be too large to fit
into a respective binding pocket.11 2-Pyridyl template
may be responsible for picking an additional hydrogen
bonding with the amide bond of Val899 improving
overall potency of the respective analogues against the
kinase. On the contrary, lone nitrogen pair in the deriv-
atives of 3- and 4-pyridine templates may cause unfa-
vorable electrostatic interactions in the ATP binding
site of the kinase, as evidenced from the reported bind-
ing mode of PTK787 and its analogues to VEGFR2.11
Similarly, 2-quinoline core may be too bulky to be
properly accommodated in the binding pocket due to
the close proximity of Leu840.MMFF94 Force Field
minimization studies suggested good overlap between
active series described in this paper and the develop-
ment candidates PTK787 and C (Fig. 1).11 Following
these data, we decided to continue optimization of mol-
ecules derived from 4-pyridyl aldehyde (19–45, Table 1).
In the next step, we focused on studying SAR of the
anilinic portion of the molecule attached to the 1,3,4-
oxadiazole moiety. The molecules substituted with
para-t-Bu- and para-i-Pr- groups displayed good
Figure 1. Structural overlap between inhibitor 27 (gold), PTK787 A
(green), and C (green).

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1913–1919
1917
Table 2. Passive diffusion potential across Caco-2 cell monolayer for
selected compounds
potency with the IC50 values of 43 and 51 nM in the
enzymatic assay (e.g., 19 and 20). The 4-ClF2CO-group
(25) resulted in improved activity (IC50 = 31 nM). Both
4-F3CO- and 4-F3C-derivatives (26 and 27) also inhibit-
ed VEGFR-2 (IC50s of 44 and 58 nM). 3-F3CO- (34)
and 3-MeO- (38) derivatives afforded enzymatic poten-
cies of 69 and 170 nM, respectively. Again, a similar
substitution pattern for 3- and 4-pyridine templates
yielded compounds with dramatically reduced activities
(35 and 36; 0.47 and 0.55 uM, respectively). Small meta-
substituents resulted in considerably less active mole-
cules (37, IC50 = 0.77 lM). ortho-Substitution abolished
enzymatic inhibition (39 and 40; IC50 > 10 lM for
both). 3,4-Di-substituted aryl groups also yielded potent
compounds. Examples include 3,4-di-Cl- (41;
IC50 = 79 nM), 3,4-di-MeO (42, IC50 = 67 nM), 4-Me-
3-CF3- (43, IC50 = 87 nM), and piperonyl groups (44;
IC50 = 85 nM). Larger anilinic substituents also led to
the diminished potency against the enzyme (45). Specif-
ically, para-phenyl- and phenoxy- (data not shown)
derivatives displayed weak to no enzymatic activity.
We speculated that these functions cannot be properly
accommodated in the tight hydrophobic pocket of
VEGFR-2.11 Three selected compounds, namely 19,
25, and 27 were found to be ATP-competitive inhibitors
of VEGFR-2 in the radioassay.12
Compound
Intrinsic permeability,
Pm value · 10À5 cm/min
Absorption potential
C
38.6 (lit. 45.2)6
High
Med
D
17.3 (lit. 21.7)6
32.4
2.7
5
High
Low
17
19
20
25
26
27
28
30
34
41
42
43
44
52.7
42.8
High
High
High
36.9
24.7
Med/high
High
Med
43.8
18.7
15.4
42.6
Med
High
High
28.7
25.2
Med/high
High
Med/high
49.3
23.6
Table 3. Compounds 19, 25, and 27 are ATP-competitive inhibitors of
VEGFR-2
Compound
Ki at IC50 (lM)
Ki at IC90 (lM)
19
25
27
0.13
0.10
0.12
0.18
0.08
0.15
Compounds 5–45 were also tested via HTRF format
against VEGFR-1. The results in Table 1 indicate that
many VEGFR-2 active ((pyridin-4-yl)ethyl)pyridines
display good activity against VEGFR-1 as well. For
the most potent compounds, the IC50 values were in
0.14–0.65 lM range. This outcome could be of benefit
in the clinical setting as both receptors are reported to
mediate VEGF signaling in angiogenesis.1,13 Notably,
several compounds containing bulky para-substituted
anilinic functions at the 1,3,4-oxadiazole pharmaco-
phore (19, 20, 25, and 26) yielded over 10-fold selectivity
for the VEGFR-2 versus VEGFR-1 kinase. This obser-
vation suggests that it is possible to develop VEGFR-2-
specific inhibitors lacking VEGFR-1 activity. Structural
reasons for this VEGFR-2 specificity are under further
investigation. Screening of 5–45 against a number of
other receptor (IGF1R, InR, FGFR1, Flt3, ErbB1,
ErbB2, and c-Met) and cytosolic (PKA, GSK3b, PKB/
Akt, bcr-Abl, and Cdk2/5) kinases revealed no signifi-
cant-cross-reactivity (PI > 30%, triplicate measure-
ments) at a screening concentration of 10 lM.
In summary, we have developed a series of novel potent
((pyridin-4-yl)ethyl)pyridine derivatives active against
kinases VEGFR-1 and -2. Both specific and dual
ATP-competitive inhibitors of VEGFR-2 were identi-
fied. Kinase selectivity could be controlled by varying
the arylamino substituent at the 1,3,4-oxadiazole ring.
Several most specific molecules displayed >10-fold selec-
tivity for VEGFR-2 over VEGFR-1. Compound activi-
ties in both in vitro and cell-based assays
(IC50 < 100 nM) were similar to those of the reported
clinical and development candidates, including
PTK787 (VatalanibTM). The analogues presented in this
letter are potentially useful in the treatment of condi-
tions such as cancer. Further details on their biological
properties, such as functional activity, together with
murine oral exposure data will be presented in due
course (Table 3).
Active in vitro inhibitors of VEGFR-2 were further
characterized in a cell-based phosphorylation ELISA
(Table 1).14 In general, good in vitro-to-cell-based activ-
ity correlation has been found for these compounds. In
our hands, the best compounds displayed 44–110 nM
activity in inhibiting cell-based phosphorylation of
VEGFR-2. This fact indicates that a number of ((pyri-
din-4-yl)ethyl)pyridine derivatives, including 19, 20,
25–27, and 41–44, could be further developed for in vivo
studies as both VEGFR-2-specific and VEGFR-1/2-dual
inhibitors. High permeability of active compounds
across Caco-2 cell monolayer (>30 · 10À5 cm/min) is
indicative of their potential for intestinal absorption
upon oral administration (Table 2).10
References and notes
1. (a) Risau, W. Nature 1997, 386, 671; (b) Hanahan, D.;
Folkman, J. Cell 1996, 86, 353; (c) Folkman, J.; Klags-
burn, M. Science 1987, 235, 442; (d) Folkman, J. J. Natl.
Cancer Inst. 1991, 82, 4; (e) Kerbel, E. S. Carcinogenesis
2000, 21, 505.
2. (a) Ferrara, N.; Gerber, H.-P.; LeCouter, J. Nat. Med.
2003, 9, 669; (b) Carmeliet, P.; Jain, R. Nat. Med. 2003, 9,
653; (c) Veikkola, T.; Karkkainen, M.; Claesson-Welsh,
L.; Alitalo, K. Cancer Res. 2000, 60, 203.
3. (a) Klagsbrun, M.; D’Amore, P. Annu. Rev. Physiol. 1991,
53, 217; (b) Neufeld, G.; Cohen, T.; Gengrinovitch, S.;
Poltorak, Z. FASEB J. 1999, 13, 9; (c) Zachary, I.
Biochem. Soc. Trans. 2003, 31, 1171; (d) Klagsbrun, M.;

1918
A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1913–1919
Moses, M. A. Chem. Biol. 1999, 6, R217; The anti- C21H16F3N5O: C, 61.31; H, 3.92; N, 17.02. Found: C,
angiogenic antibody AvastinTM (Bevacizumab) has recently
been approved to treat colorectal cancer see (e) Culy, C.
Drugs Today 2005, 41, 23; The anti-angiogenic aptamer
MacugenTM (Pegaptanib sodium) has recently been
approved to treat neovascular age-related macular degen-
eration; see (f) Fine, S. L.; Martin, D. F.; Kirkpatrick, P.
Nat. Rev. Drug Disc. 2005, 4, 187.
61.12; H, 4.06, N, 16.88.
Compound 30: 5-(3-(2-(pyridin-4-yl)ethyl)pyridin-4-yl)-N-
(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine; mp
226–228 ꢁC; 1H NMR (400 MHz, DMSO-d6) d, ppm:
2.72 (m, 2H, CH2), 2.96 (m, 2H, CH2), 5.28 (br s, exch
D2O, 1H, NH), 6.66 (d, J = 7.2 Hz, 2H), 7.24 (d,
J = 5.2 Hz, 2H), 7.22 (d, J = 7.2 Hz, 2H), 7.53 (d,
J = 4.8 Hz, 1H), 8.48 (d, J = 5.2 Hz, 2H), 8.61 (s, 1H),
8.70 (d,J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d, ppm: 29.9, 36.4, 106.2, 116.3, 118.5, 120.7, 122.6, 124.3,
125.4, 137.2, 142.7, 145.9, 147.0, 148.3, 149.1, 149.5, 163.8;
ESI MS (M+1): 412, (MÀ1): 410; HRMS, exact mass
calcd for C21H16F3N5O: 411.1307. Found: 411.1298.
Elemental analysis: calcd for C21H16F3N5O: C, 61.31; H,
3.92; N, 17.02. Found: C, 61.07; H, 4.11, N, 16.81.
Compound 33: 5-(4-(2-(pyridin-4-yl)ethyl)pyridin-3-yl)-N-
(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine; mp
218–220 ꢁC; 1H NMR (400 MHz, DMSO-d6) d, ppm: 2.84
(m, 2H, CH2), 2.95 (m, 2H, CH2), 5.33 (br s, exch D2O, 1H,
NH), 6.59 (d, J = 7.2 Hz, 2H), 7.23 (d, J = 5.2 Hz, 2H), 7.28
(d, J = 7.2 Hz, 2H), 7.39 (d, J = 4.8 Hz, 1H), 8.53 (d,
4. 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.
5. (a) Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem.
2002, 45, 1300; (b) Wedge, S. R.; Kendrew, J.; Hennequin,
L. F.; Valentine, P. J.; Barry, S. T.; Brave, S. R.; Smith, N.
R.; James, N. H.; Dukes, M.; Curwen, J. O.; Chester, R.;
Jackson, J. A.; Boffey, S. J.; Kilburn, L. L.; Barnett, S.;
Richmond, G. H. P.; Wadsworth, P. F.; Walker, M.;
Bigley, A. L.; Taylor, S. T.; Cooper, L.; Beck, S.;
Juergensmeier, J. M.; Ogilvie, D. J. Cancer Res. 2005,
65, 4389.
J = 5.2 Hz, 2H), 8.66 (d, J = 4.8 Hz, 1H), 8.87 (s, 1H); 13
C
NMR (100 MHz, DMSO-d6) d, ppm: 29.5, 36.6, 106.1,
115.9, 120.6,122.9, 124.5, 124.9, 125.7, 132.4, 145.9, 147.0,
148.5, 149.1, 149.6, 150.8, 164.1; ESI MS (M+1): 412,
(MÀ1): 410; HRMS, exact mass calcd for C21H16F3N5O:
411.1307. Found: 411.1301. Elemental analysis: calcd for
C21H16F3N5O: C, 61.31; H, 3.92; N, 17.02. Found: C, 61.15;
H, 3.75, N, 16.86.
6. (a) Manley, P. W.; Furet, P.; Bold, G.; Bruggen, J.;
¨
Mestan, J.; Meyer, T.; Schnell, C.; Wood, J. J. Med.
Chem. 2002, 45, 5697; (b) Manley, P. W.; Bold, G.;
Fendrich, G.; Furet, P.; Mestan, J.; Meyer, T.; Meyhack,
B.; Strauss, A.; Wood, J. Cell. Mol. Biol. Lett. 2003, 8,
532; (c) Altmann, K.-H.; Bold, G.; Furet, P.; Manley, P.
W.; Wood, J. M.; Ferrari, S.; Hofmann, F.; Mestan, J.;
Huth, A.; Kruger, M.; Seidelmann, D.; Menrad, A.;
¨
Haberey, M.; Thierauch, K.-H., U.S. Patent 6,878,720 B2,
2005.
9. VEGFR-2 kinase inhibition was determined by measuring
the phosphorylation level of poly-Glu-Ala-Tyr-biotin
(pGAT-biotin) peptide in the HTRF assay. Into a 96-well
Costar plate was added 2 ll/well of 25· compound in a
100% DMSO (final concentration in the 50 ll kinase
reaction was typically 1 nM to 10 lM). Next, 38 ll of
reaction buffer (25 mM Hepes, pH 7.5, 5 mM MgCl2, 5 mM
MnCl2, 2 mM DTT, and 1 mg/ml BSA) containing
0.5 mmol pGAT-biotin and 3–4 ng KDR enzyme was
added to each well. After 5–10 min preincubation, the
kinase reaction was initiated by the addition of 10 ll of
10 lM ATP in the reaction buffer, after which the plate was
incubated at room temperature for 45 min. The reaction
was stopped by addition of 50 ll KF buffer (50 mM Hepes,
pH 7.5 and 0.5 M KF, and 1 mg/ml BSA) containing
100 mM EDTA and 0.36 lg/ml PY20K (Eu-cryptate
labeled anti-phosphotyrosine antibody, CIS Bio Interna-
tional) was added and after an additional 2 h incubation at
rt, the plate was analyzed in a RUBYstar HTRF Reader.
10. Poor passive diffusion potential (intrinsic permeability,
Pm = 2.7 · 10À5 cm/min) for 17 across Caco-2 cell mono-
layer was predictive of poor cell permeability. In general,
good correlation between cell-based activity and Pm values
was observed for all active compounds (Table 2).
11. (a) McTigue, M. A.; Wickersham, J. A.; Pinko, C.;
Showalter, R. E.; Parast, C. V.; Tempczyk-Russell, A.;
Gehring, M. R.; Mroczkowski, B.; Kan, C. C.; Villafran-
ca, J. E.; Appelt, K. Structure 1999, 7, 319; (b) Berman, H.
M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Protein Data
Bank, Nucleic Acids Res. 2000, 28, 235.
12. Competition assays were conducted with varying concen-
trations (0–100 uM) of ATP. Specifically, five different
concentrations of 32P ATP were incubated with VEGFR-2
in the absence, IC50 or IC90 concentration of the inhibitors
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
7. Piatnitski, E. L.; Duncton, M.; Katoch-Rouse, R.; Sher-
man, D.; Kiselyov, A. S.; Milligan, D.; Balagtas, C.;
Wong, W.; Kawakami, J.; Doody, J. Bioorg. Med. Chem.
Lett. 2005, 15, 4696.
8. Analytical data for selected compounds. Compound 24: N-
(4-tert-butylphenyl)-5-(2-(2-(pyridin-4-yl)ethyl)quinolin-3-
yl)-1,3,4- oxadiazol-2-amine; mp 242–244 ꢁC; 1H NMR
(400 MHz, DMSO-d6) d, ppm: 1.29 (s, 9H, t-Bu), 2.86 (m,
2H, CH2), 3.19 (m, 2H, CH2), 5.27 (br s, exch D2O, 1H,
NH), 6.52 (d, J = 7.2 Hz, 2H), 7.06 (d, J = 7.2 Hz, 2H),
7.25 (d, J = 5.6 Hz, 2H), 7.42 (m, 1H), 7.66 (m, 1H), 7.72
(d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H),
8.69 (d, J = 5.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
d, ppm: 30.8, 33.8, 38.2, 39.7, 107.3, 115.3, 122.9, 124.8,
125.4, 125.7, 126.5, 127.0, 127.9, 128.2, 129.3, 132.1, 134.6,
139.5, 139.8, 144.8, 148.3, 149.5, 159.7, 164.4; ESI MS
(M+1): 451, (MÀ1): 449; HRMS, exact mass calcd for
C28H27N5O: 449.2216. Found: 449.2208. Elemental anal-
ysis: calcd for C28H27N5O: C, 74.81; H, 6.05; N, 15.58.
Found: C, 74.59; H, 5.87, N, 15.33.
Compound 27: 5-(2-(2-(pyridin-4-yl)ethyl)pyridin-3-yl)-N-
(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine; mp
211–213 ꢁC; 1H NMR (400 MHz, cDMSO-d6) d, ppm:
2.94 (m, 2H, CH2), 3.36 (m, 2H, CH2), 5.35 (br s, exch
D2O, 1H, NH), 6.62 (d, J = 7.2 Hz, 2H), 7.11 (dd,
J1 = 8.0 Hz, J2 = 4.8 Hz, 1H), 7.19 (d, J = 7.2 Hz, 2H),
7.33 (d, J = 5.2 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H), 8.55 (d,
J = 5.2 Hz, 2H), 8.73 (d, J = 4.8 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d, ppm: 33.5, 38.0, 108.5, 115.9,
120.6, 122.2, 123.7, 124.6, 125.9, 130.1, 133.8, 145.2, 146.0,
148.3,148.9, 156.7, 165.3; ESI MS (M+1): 412, (MÀ1):
410; HRMS, exact mass calcd for C21H16F3N5O:
411.1307. Found: 411.1302. Elemental analysis: calcd for

A. S. Kiselyov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1913–1919
1919
parameters using GraphPad software. Determined Ki
values for the three selected compounds are listed in Table 3.
13. Eskens, F. Br. J. Cancer 2004, 90, 1.
and rocked for 1 h at 4 ꢁC. (ii) ELISA for detection of
tyrosine-phosphorylated chimeric receptor: 96-well ELISA
plates were coated using 100 ll/well of 10 lg/ml of
aFGFR1 antibody and incubated overnight at 4 ꢁC.
aFGFR1 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, aFGFR1-coated ELISA plates were washed three
times with PBS + 0.1% Tween-20, blocked by addition of
200 ll/well of a 3% BSA in PBS for 1 h, and washed again.
Lysate (80 ll) WAS 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 bound phosphorylated chimeric receptor, 100 ll/
well of anti-phosphotyrosine antibodies (RC20:HRP),
Transduction Laboratories was added (final concentration
0.5 lg/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 ll/well of equal
amounts of the Kirkegaard & Perry Laboratories (KPL)
substrates A and B. The reaction was stopped by addition
of 50 ll/well of a 0.1 N H2SO4 and absorbance was
measured at 450 nm.
14. Cell-based assay for VEGFR-2 inhibition: (i) Transfection
of 293 cells with DNA expressing FGFR1/VEGFR-2
chimera: a chimeric construct containing the extracellular
portion of FGFR1 and the intracellular portion of
VEGFR-2 was transiently transfected into 293 adenovi-
rus-transfected kidney cells. DNA for transfection was
diluted to a 5 lg/ml final concentration in a serum-free
medium and incubated at room temperature for 30 min
with 40 ll/ml of Lipofectamine 2000, also in serum-free
media. Two hundred and fifty microliters of the Lipofect-
amine/DNA mixture was added to 293 cells suspended at
5· 105 cells/ml. Suspension (200 ll/well) was added to a
96-well plate and incubated overnight. Within 24 h, media
were 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, and 1 mM Na3VO4)