Design and chemical synthesis of [1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel class of VEGFR-2 kinase inhibitors

Article abstract of DOI:10.1016/j.tetlet.2009.04.062

The Letter describes a facile approach to 7,8-dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel class of potent inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). The synthetic sequence is centered around preparation of the k

Full text of DOI:10.1016/j.tetlet.2009.04.062

Tetrahedron Letters 50 (2009) 3809–3812
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design and chemical synthesis of [1,2,4]triazol[1,5-c]pyrimidin-5-yl amines,
a novel class of VEGFR-2 kinase inhibitors
b
c
c
Alexander S. Kiselyov a, , Eugene L. Piatnitski Chekler , Natalia B. Chernisheva , Lev K. Salamandra ,
*
Victor V. Semenov c
a deCODE Chemistry, 2501 Davey Road, Woodridge, IL 60517, USA
b Department of Chemistry, ImClone Systems Inc., 180 Varick Street, New York, NY 10014, USA
c N. D. Zelinsky Institute of Organic Chemistry, RAN, 47 Leninsky Prospekt, 117918, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
The Letter describes a facile approach to 7,8-dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel
class of potent inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). The synthetic
sequence is centered around preparation of the key 3(5)-cyanomethyl-1,2,4-triazole intermediates and
their Knoevenagel condensation with aromatic aldehydes. A subsequent three-step conversion of Knoe-
venagel adducts involving a reduction of vinyl nitriles followed by the reaction of the resulting amines
with aryl isothiocyanates and cyclization of the respective thioureas yielded targeted heterocycles as a
1:1 mixture of tautomers. A representative molecule featured sound activity against VEGFR-2 in both
enzymatic and cellular assays.
Received 11 March 2009
Revised 1 April 2009
Accepted 6 April 2009
Available online 21 April 2009
Keywords:
Knoevenagel condensation
VEGFR-2
7,8-Dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-
yl amines
Ó 2009 Elsevier Ltd. All rights reserved.
Cyclization
Angiogenesis
Kinase inhibitor
Angiogenesis, or formation of new blood vessels, is involved in
embryonic development1,2 and wound healing as well as in patho-
logical conditions such as tumor growth and degenerative eye con-
ditions.3–6 The family of vascular endothelial growth factors
(VEGFs) has been implicated in regulation of angiogenesis in vivo.
VEGFsmediatetheirbiological effectthroughhigh-affinityreceptors
(VEGFRs).7–11 Modulation of VEGF/VEGFR signalingactivityoffersan
attractive target for inhibition of an aberrant angiogenesis and sup-
pression of tumor growth.12–14 A number of small-molecule inhibi-
tors are known to affect VEGF/VEGFR signaling. These include
sition 4 via an appropriate linker (aryl or fused aryl group).21–23 In
this research article, we report a synthetic approach towards 7,8-
dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines as inhibitors of
VEGFR-2 kinase. We reasoned that this template could provide a
proper pharmacophore arrangement relevant to compounds A–D
(Fig. 2). MMFF94 Force Field minimization studies suggested good
overlap between our proposed chemical series and the develop-
ment candidate VatalanibTM A (Fig. 2a and b).24
Our synthetic effort was based on the development of expedi-
tious route to the key intermediate nitriles 6a and 6b (Scheme
1). We reasoned that the thiourea derivatives 8a–8d resulting from
the reduction of Knoevenagel products 6a and 6b to furnish the
respective amines 7a and 7b, would yield the targeted molecules
9a–9d upon ring closure reaction. In the synthesis of unsubstituted
triazoles (R = H), we initially prepared 2 in 84% yield by reacting
ethyl ethoxycarbonylacetimidate 1 with formyl hydrazine (Scheme
1). It was subsequently heated in vacuo to afford a cyclization
product 3 in 78% yield. The triazole derivative 3 was treated with
ammonia to result in the intermediate triazolyl acetamide that
was further dehydrated using P2O5/dry sand/absolute pyridine to
furnish the targeted heteroaryl acetonitrile 5a in 33% combined
yield. The respective 5-methyl-3-cyanomethyl-1,2,4-triazole 5b
was prepared in 60% yield by reaction of commercially available
cyanoacetic acid hydrazide and acetic acid iminoester prepared
PTK787 (Vatalanib A),15–17 its analogue BAY579352 (B), and the
TM
isosteric anthranyl amide derivatives C and AMG-706 (D).12 Intra-
molecular hydrogen bonding in C and D was suggested to be respon-
sible for the optimal spatial orientation of the pharmacophore
pieces, similar to that of the parent PTK787 (Fig. 1).18
The essential pharmacophore elements19,20 for the VEGFR-2
activity of phthalazine-based molecules and their analogues (A-
D) include (i) [6,6]fused (or related) aromatic system; (ii) para-,
meta-, or 3,4-di-substituted aniline functionality in position 1 of
the phthalazine core (A, Fig. 1); (iii) hydrogen bond acceptor (Le-
wis’ base:lone pairs of nitrogen- or oxygen atoms) attached to po-
* Corresponding author. Tel.: +1 630 783 4600.
E-mail address: akiselyov@decode.com (A.S. Kiselyov).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.062

3810
A. S. Kiselyov et al. / Tetrahedron Letters 50 (2009) 3809–3812
CF3
HN
Cl
Cl
HN
1
HN
HN
HN
N
N
N
O
O
4 N
O
NHMe
O
NH
N
NH
N
N
N
N
A
C
B
D
Figure 1. Selected inhibitors of vascular endothelial growth factor receptor II (VEGFR-2) based on phthalazine core (A) and its isosteric analogues.
EtOOC
a
EtOOC
HN
EtOOC
HN
N
i.
ii.
iii, iv
v
NC
N
R
- H2O
N
H
3
OEt
NHNHCHO
2
N
1
N
R
NC
N
H
O
5a,b
NHNH2
4
N
H2N
N
N
NC
viii
vi
vii
R
N
N
HN
N
R
N
H
Hydrophobic pocket
Ar
6a,b
7a,b
b
Ala866
Lys868
HN
Val848
Val899
Phe918
Leu840
N
N
H
N
N
N
Ar
N
S
R
HN
Ar
N
HN
ix
N
Leu1035
N
R
Arg1032
N
N
NH
N
R
+
H N
N
HN
NH
-
a, R = H, Ar = 3-CF3-C6H4
9a-d
N
8a-d
-
b, R = H, Ar = 4-Cl-C6H4
-
Figure 2. Structural overlap between (a) 9b (blue) and VatalanibTM (gray); (b)
Pharmacophore hypothesis for the mode of binding of 9b to VEGFR-2: central
phenyl ring is surrounded by Leu840, Val848, Val899, Phe918, and Leu1035; the
triazole ring is in close proximity with the Ala866 and Lys868, and pyridine nitrogen
is near Arg1032.
c, R = Me, Ar = 4-Br-C6H4
-
d, R = Me, Ar = 3-CF3-C6H4
Scheme 1. Reagents and conditions: (i) H2NN–CHO, MeOH, rt, 4 days (84%); (ii)
125–130 °C at 29 mm Hg, 20 min (78%); (iii) aqueous NH3, 17 h; (iv) P2O5, dry
pyridine, reflux, 5 h (33% for iii and iv); (v) MeCN, HCl (gas), MeOH, À4 °C, 4 days;
MeOH, 40 °C–55 °C, 9.5 h (60%); (vi) 4-PyCHO, piperidine, i-PrOH, 4 Å molecular
sieves, reflux, 6 h; (vii) NH3, i-PrOH; H2/Raney-Ni, 50 atm, 50 °C, 20 h (71–75%);
(viii) ArNCS, t-BuOH, reflux, 10 min (20–95%); (ix) HgO (yellow), i-PrOH, reflux, 3 h
(39–79%).
in situ from acetonitrile.25 Presence of the nitrile group was con-
firmed by both mass spectrometry and infrared spectrometry
(strong IR band at 2256 cmÀ1). Knoevenagel condensation of ni-
triles 5a and 5b with 4-pyridylaldehyde afforded the key interme-
diate nitriles 6a and 6b predominantly as trans-isomers in 71–75%
yield. These intermediates were hydrogenated in the presence of
Raney-Ni to give amines 7a and 7b that were used in the next step
without purification. Specifically, 7a and 7b were reacted with cor-
responding aryl isothiocyanates to furnish respective thioureas
8a–8d in 20–95% yield. Notably, thioureas eliminate corresponding
isothiocyanate fragments in the mass spectrometer (direct electron
impact). The final step of the synthesis was thermal cyclization of
thioureas in the presence of yellow HgO to afford heterocycles 9a–
9d in 39–79% yields. We were not able to convert molecules 9a–9d
to the respective [6.5]fused aromatic derivatives under a variety of
experimental conditions (DDQ or MnO2-mediated oxidation, Pd-
catalyzed aromatization, etc.).
regiochemistry of the cyclization is well documented.26 In a repre-
sentative example (Fig. 3), upon cyclization of 8c, one NH-triazole
(13.2–13.7 Hz) and both thiourea NH-protons signals (7.76 (d,
J = 8.6 Hz, 1H, NH), 9.6 (s, 1H, NH)) of the starting material disap-
peared to afford 9c. Tautomers 9c were confirmed by 13C NMR,
1H–1H NOESY, and 1H–13C HSQC. Notably, the resulting 7,8-dihy-
dro[1,2,4]triazolo[1,5-c]pyrimidin-5-amines exist as ca. 1:1 mix-
ture of tautomers in DMSO. For example, proton NMR spectrum
of 9c features a double set of signals for both methyl substituent
in the triazole ring and the Ar group protons.
7,8-Dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines 9a–9d
were screened in vitro for kinase activity. The synthesized
analogues were subjected to biological evaluation in VEGFR-2
enzymatic and cellular assays. Compound 9a demonstrated the
highest activity in both VEGFR-2 kinase enzymatic assay19–23
Chemical structure of the [6.5]-fused triazoles was unambigu-
ously assigned using NMR spectroscopy. The N1 versus N4 triazole

A. S. Kiselyov et al. / Tetrahedron Letters 50 (2009) 3809–3812
3811
Br
Br
Br
HN
N
HN
N
N
N
N
N
N
N
N
NH HN
S
N
N
NH
N
N
8c
9c
8c, NH-protons
H
Br
N
S
N
CH
3
CH3
N
N
N
H
N
H
N
N
N
H
7.76
9.60
13.24
8
13
12
11
10
9
7
9c isomers, NH- protons
3'
Br
2'
N
Triazol CH3
2.32 and 2,38
H
N
N
N
H3C
N
Br
H
N
N
N
N
N
N
H
C
3
2,3-Ar-H
3'-H-Ar
N
2'-H-Ar
9.31
6.91
2
9
7
8
Figure 3. 1HNMR spectra of the starting thiourea 8c and the product 9c (1:1 mixture of tautomers).
(IC50 = 62 nM)
and
cellular
phosphorylation
assay19–23
Supplementary data
(IC50 = 78 nM). In comparison, clinical candidate PTK787 (Vatala-
TM
nib A) showed IC50 values of 54 nM and 21 nM in these assays,
Experimental details, proton NMR spectra, mass spectrometry
data and elemental analysis data. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.04.062.
respectively. These results confirm our previous observations19–23
that the interaction of nitrogen lone pair of the 4-pyridyl moiety
with Arg1032 (Fig. 2) is critical for VEGFR-2 kinase activity.
In summary, we have achieved a convenient synthesis of 7,8-
dihydro[1,2,4]triazol[1,5-c]pyrimidin-5-yl amines, a novel class of
kinase inhibitors. Representative compound 9a exhibited nano-
molar activity for VEGFR-2 kinase. The discovery of a novel chem-
ical series active against VEGFR-2 may yield additional derivatives
with affinity for other kinases. Furthermore, the scaffold’s resis-
tance to aromatization under a variety of conditions suggests po-
tential for its metabolic stability in vivo. Currently, we continue
with evaluation of the active compounds in a series of biological
assays. These data will be reported elsewhere.
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