De novo Design and synthesis of N-benzylanilines as new candidates for VEGFR tyrosine kinase inhibitors

Article abstract of DOI:10.1039/b719959g

N-Benzylanilines were designed and synthesized as vascular endothelial growth factor (VEGF)-2 inhibitors using de novo drug design systems based on the X-ray structure of VEGFR-2 kinase domain. Among compounds synthesized, compound 3 showed the most potent inhibitory activity toward VEGFR-2 (KDR) tyrosine kinase and its IC₅₀ value was 0.57 μM.

Full text of DOI:10.1039/b719959g

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de novo Design and synthesis of N-benzylanilines as new candidates for
VEGFR tyrosine kinase inhibitors†
Masaharu Uno, Hyun Seung Ban, Wataru Nabeyama and Hiroyuki Nakamura*
Received 2nd January 2008, Accepted 24th January 2008
First published as an Advance Article on the web 11th February 2008
DOI: 10.1039/b719959g
N-Benzylanilines were designed and synthesized as vascular
endothelial growth factor (VEGF)-2 inhibitors using de novo
drug design systems based on the X-ray structure of VEGFR-
2 kinase domain. Among compounds synthesized, compound
3 showed the most potent inhibitory activity toward VEGFR-
2 (KDR) tyrosine kinase and its IC50 value was 0.57 lM.
(Ludi score = 373) and carboxylic acid (Ludi score = 300) were
chosen for partitions B and C, respectively. (Fig. 1B). Finally,
linking of these four fragments and modification of the obtained
structure due to synthetic compatibility led to candidate molecule
1 (Fig. 1B). According to the docking simulation of 1 toward KDR
kinase domain, five hydrogen bonds would be expected between
the urea and Arg842, the amine and Asn1032, the hydroxyl group
and Leu840, and carboxylic acid and Cys919, and Ligscore2 was
calculated as 5.59. Compound 1 showed the highest Ligscore2
value among the compounds conducted with selected fragments
(data not shown). Therefore, we designed and synthesized a series
of the N-benzylbenzene-1,4-diamine derivatives (1 and 2), and
N-(4-aminophenyl)benzamides (3) based on these observations
(Fig. 2).
Vascular endothelial growth factor (VEGF) is a key growth factor
in tumor angiogenesis. In general, angiogenesis is tightly regulated
and only normally occurs in inflammation, wound healing, and the
female reproductive cycle in the adult.1 Uncontrolled angiogene-
sis involves pathological states such as atherosclerosis, diabetic
retinopathy, rheumatoid arthritis, and solid tumor growth. Vas-
cular endothelial growth factor (VEGF) is a key growth factor
in tumor angiogenesis, therefore, its receptor tyrosine kinases
known as VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) have
been considered as attractive targets for the development of anti-
cancer drugs.2
The development of receptor-based drug design has profoundly
benefited the drug discovery process. Especially, de novo design
based upon a co-crystal structure of a protein–ligand complex
has become a powerful strategy to provide a starting point for
design of a lead compound.3,4 For instance, CDK4 inhibitors,3a
the HIV-1 integrase inhibitors,3b FKBP-12 ligands,3c Factor Xa
inhibitors,3d COX-2 inhibitors,3e and DNA gyrase inhibitors3f have
been developed using de novo systems. In this paper, we designed a
new type of VEGF tyrosine kinase inhibitor using Ludi, a de novo
drug design program,5 based on a crystal structure of tyrosine
kinase domain in complex with a ligand.
Fig. 1 shows the de novo design of the N-benzylbenzene-1,4-
diamine derivatives 1–3. On the basis of an X-ray model of the
KDR binding pocket (PDB code: 1Y6A),6 interaction sites of the
ligand were identified. Furthermore, taking into account other
co-crystal structures of the KDR kinase domain and ligands,
such as AAL993,2a AAZ,6 and 4-amino-furo[2,3-d]pyrimidines,7
we predicted four important interaction sites, Leu840, Arg842,
Cys919, and Asn1032, and partitioned off the binding pocket into
three partitions, A, B, and C (Fig. 1A). Among the fragment
library, phenylurea was chosen as a suitable fragment for partition
A with a high Ludi score (= 212) and two hydrogen bonds were
predicted between two hydrogens of the urea and the carbonyl
group of Arg842. In a similar manner, 3-hydroxybenzylamine
Department of Chemistry, Faculty of Science, Gakushuin University,
1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan. E-mail: hiroyuki.
nakamura@gakushuin.ac.jp; Fax: (+81) (0)3 5992 1029; Tel: (+81) (0)3
3986 0221
† Electronic supplementary information (ESI) available: Spectral data for
new compounds 1–3, 11, 13–17. See DOI: 10.1039/b719959g
Fig. 1 Design process of VEGFR-2 tyrosine kinase inhibitor using the
LUDI de novo drug design systems in the DS modeling 1.2 package based
on the X-ray analysis data of the kinase domain (PDB: 1Y6A).
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water to give the benzoic acid 16, which underwent debenzylation
with TFA to give 3-formyl-5-hydroxybenzoic acid 17 (Scheme 2).
Fig. 2 Design of target compounds.
Synthesis of the aniline fragments that occupied site A is shown
in Scheme 1. Amines 7 and 8 were prepared from 4-nitroaniline
4 with Boc2O or BzCl, and the resulting protected anilines 5
and 6 underwent reduction under Pd/C catalyzed conditions to
afford amines 7 and 8 in 58% and 70% yields, respectively. Aniline
9 was treated with phenylisocyanate to give the corresponding
urea derivative 10, and then deprotection of the Boc group with
trifluoroacetic acid afforded 1-(4-aminophenyl)-3-phenylurea 11.
Scheme 2 Reagents and conditions: (a) MeOH, conc. H2SO4, reflux, 90%.
(b) BnBr, K2CO3, acetone, reflux, 93%. (c) 2 eq. KOH, MeOH, THF, reflux,
60%. (d) BH3·SMe2, THF, 71%. (e) MnO2, AcOEt, reflux, 87%. (f) LiOH,
MeOH–H2O, 92%. (g) TFA, reflux, 72%.
Finally, reductive amination of formyl-5-hydroxybenzoic acid
17 and 3,5-dihydroxybenzaldehyde 18 with amines 8 and 11 pro-
ceeded in the presence of NaCNBH3 to give the N-benzylbenzene-
1,4-diamine derivatives 1–3 in 13–37% yields (Scheme 3).
Scheme 1 Reagents and conditions: (a) Boc2O, CH2Cl2, reflux, 99%.
(b) BzCl, pyridine, cat. DMAP, 60 ◦C, 99%. (c) H2, Pd/C, MeOH. (d)
(Cl3CO)2CO, toluene, 80 ◦C; then 7, 99%. (e) 4 eq. TFA, CH2Cl2, reflux,
91%.
Scheme 3 Synthesis of N-benzylbenzene-1,4-diamine derivatives 1–3: (a)
NaCNBH3, MeOH, 60 ◦C.
Aldehyde 17 was prepared from 5-hydroxyisophthalic acid
12 according to
a
previous report.8 Esterification of 5-
Inhibitory activity of the N-benzylbenzene-1,4-diamine deriva-
tives 1–3 toward VEGFR2 (KDR) tyrosine kinase was examined
by ELISA.9,10 Among the derivatives synthesized, compound 3
showed the most potent inhibitory activity toward KDR tyrosine
kinase and its IC50 value was 0.57 lM (Table 1).
We next examined the inhibitory effect of compound 3 on
activation of VEGFR-2 kinase in cells. As shown in Fig. 3,
immunoblot analysis using HUVEC (human umbilical vein en-
dothelial cells) cells revealed that compound 3 suppressed the
VEGF-induced phosphorylation of KDR in a dose-dependent
hydroxyisophthalic acid with methanol gave the dimethyl ester,
which underwent protection of the phenoxy group with benzylbro-
mide, followed by monohydrolysis using 2 equivalents of KOH to
afford monomethylester 13. Selective reduction of the carboxylic
acid group on compound 13 was carried out using borane dimethyl
sulfide complex to give the benzylalcohol 14. Oxidation of the
hydroxy group on compound 14 with activated MnO2 in ethyl
acetate gave the aldehyde 15. Hydrolysis of the aldehyde 15 was
carried out by treatment with lithium hydroxide in methanol–
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Table 1 Inhibitory activity of the N-benzylbenzene-1,4-diamines 1–3
against KDR
Acknowledgements
This work was supported by a Grant-in-Aid for Science Research
(B) (No. 18350090) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
Compound
IC50/lMa
1
21
Notes and references
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3
15
0.57
a The inhibitory activity of compounds 1–3 toward KDR tyrosine kinase
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Fig. 3 Inhibition of the VEGF-induced phosphorylation of KDR. HU-
VEC cells were preincubated with the indicated concentrations of com-
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manner, although AAL993 exhibited more potent inhibition of
KDR phosphorylation.
10 EIA/RIA stripwellTM plates (Corning) were coated by incubation
◦
overnight at 4 C with 100 ll well−1 of 50 lg ml−1 poly(Glu:Tyr, 4 :
In conclusion, we succeeded in the synthesis of a series of
N-benzylbenzene-1,4-diamine derivatives as new candidates for
KDR inhibitors using de novo drug design systems. Among the
compounds synthesized, we found that compound 3 possessed
significant activity of inhibition of KDR kinase and its IC50 value
was 0.57 lM. Furthermore, compound 3 suppressed the VEGF-
induced phosphorylation of KDR in HUVEC cells. The current
findings suggest that compound 3 would be a candidate for a new
lead compound of KDR inhibitors.
1) peptide (Sigma) in PBS. The kinase reaction was performed in the
plates by addition of 50 ll of kinase buffer (50 mM HEPES, 125 mM
NaCl, 10 mM MgCl2, pH 7.4) containing 100 lM of ATP, 10 ng of
KDR (Invitrogen, catalytic domain of VEGFR2), and the compound
to be tested. After 20 min, the plates were washed three times with wash
buffer (0.1% Tween 20 in PBS) and incubated for 20 min with 50 ll
well−1 of 0.2 lg ml−1 HRP conjugated anti-phosphotyrosine antibody
(Santa Cruz). After two washes, the plates were developed by addition
of 50 ll well−1 tetramethylbenzidine (Sigma) and stopped by addition
of 50 ll well−1 of 2 N H2SO4. The absorbance at 450 nm was measured
by a 96-well plate reader (Tecan).
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 979–981 | 981
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