KDR inhibitor with the intramolecular non-bonded interaction: Conformation-activity relationships of novel indole-3-carboxamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.01.063

We previously reported that compound 1, having a similar conformation to PTK787 (2) by forming a pseudo ring structure with an intramolecular non-bonded S-O interaction, exhibited a potent inhibitory activity against VEGFR2 tyrosine kinase (KDR).¹

Full text of DOI:10.1016/j.bmcl.2011.01.063

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1782–1785
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
KDR inhibitor with the intramolecular non-bonded interaction:
Conformation–activity relationships of novel indole-3-carboxamide derivatives
Takahiro Honda a,b, , Hironori Nagahara b, Hiroyuki Mogi a,b, Masakazu Ban a,b, Hiroyuki Aono a,b
⇑
a Research and Development Center, Santen Pharmaceutical Co. Ltd, 8916-16 Takayama-cho Ikoma-shi, Nara 630-0101, Japan
b Graduate School of Materials Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho Ikoma-shi, Nara 630-0192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported that compound 1, having a similar conformation to PTK787 (2) by forming a
pseudo ring structure with an intramolecular non-bonded S–O interaction, exhibited a potent inhibitory
activity against VEGFR2 tyrosine kinase (KDR).1 Applying the ideas of pseudo ring formations, we have
designed three types of novel indole carboxamide derivatives 5–7 with an intramolecular hydrogen
bonding or non-bonded S–O interaction. We describe the design and synthesis of 5–7, and also discuss
the relationships of their KDR inhibitory activity and conformations that were stabilized by their intra-
molecular non-bonded interactions.
Received 30 August 2010
Revised 4 January 2011
Accepted 18 January 2011
Available online 22 January 2011
Keywords:
KDR inhibitor
Intramolecular non-bonded interaction
Non-bonded S–O interaction
Pseudo ring design
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Indole
Intramolecular hydrogen bonding
X-ray crystallography
Angiogenesis is closely related to the proliferation or metastasis
of cancer cells.2 Vascular endothelial growth factor (VEGF) plays a
very important role in the pathological angiogenesis. Thus, the
compounds which hamper this pathway show promise as drugs
for treating cancer. Stimuli to the VEGF/VEGF-receptor signaling
pathway can result in angiogenesis, which is also closely linked
to rheumatoid arthritis (RA) and age-related macular degeneration
(AMD). The clinical studies of Avastin™, a humanized anti-VEGF
monoclonal antibody, revealed that the VEGF/VEGF-receptor sig-
naling inhibitor could be used in treating cancer.3 MacugenR is a
28-base PEGylated aptamer which has been approved as a remedy
for AMD.4 Numerous small molecules for inhibiting VEGF receptor
tyrosine kinase (RTK) have been developed in clinical trials and
some of them have been already marketed for various cancer ther-
apies. These VEGF-RTK inhibitors can be classified by their chemi-
cal structures into types, such as: indolinones, quinazolines,
phthalazines and others.5 Vatalanib (2) is one of the phthalazines
under PIII clinical development for the treatment of cancer.6 An
anthranilamide derivative AAL993 (3)7 is a selective VEGF-RTK
inhibitor of this chemotype. This compound was reported to have
been identified by a database search with a substructure query for
4 which listed phthalazines with an intramolecular hydrogen
bond.8 We previously reported a synthesis of 4-pyridylmethylthio
derivative 1, as a member of this chemotype,9 and found it to have
potent inhibitory activities in a cell-based angiogenesis assay
(IC50 = 250 nM) and in a KDR assay (IC50 = 26 nM).1 The X-ray crys-
tallography of 1 (monohydrate), possessing 2-mercaptonicotina-
mide framework, suggested an intramolecular non-bonded S–O
interaction10 with the shorter S–O distance (2.82 Å) than the sum
(3.32 Å) of van der Waals radii of sulfur and oxygen atoms,11 and
then revealed to have similar conformation to 2 and 3.
Meanwhile, X-ray co-crystallography analysis of 3 and KDR
uncovered that the amide NH of 3 interacted with the Glu885,
which formed a salt bridge with Lys868 residue, the carbonyl
moiety interacted with the Asp1046 of the DFG-loop and the
4-pyridylmethyl moiety engaged Cys919 of the hinge region
(Fig. 2A).3 The intramolecular hydrogen bonding in 3 was effective
to keep the favorable conformation for interacting at the ATP bind-
ing site of KDR in the so-called ‘DFG-out’ conformation,12 to show
the inhibitory activity. On the basis of these results, we considered
that the anthranilamide in 3 or 2-mercaptonicotinamide frame-
work in 1 could be replacable as a scaffold of this type KDR inhib-
itor, with the indole derivatives (5–7 in Fig. 1) that can form larger
size pseudo rings (Fig. 2B). Then we designed three types of scaf-
folds (Fig. 3), two of which (5, 6) could have similar conformations
and be inhibitory active with an internal hydrogen bond or S–O
interaction, and the rest (7) having a different mode of hydrogen
bond that might cause less or no active. We synthesized them
and investigated the conformation–activity relationships between
the three kind derivatives 5–7.
⇑
Corresponding author.
E-mail address: hondat@ms.naist.jp (T. Honda).
0960-894X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.063

T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1782–1785
1783
HN
HN
Cl
Cl
HN
OMe
OMe
OMe
a
b
O
NO2
O
NH2
HN
S
HN
O
CO2H
CO2H
O
N
N
9
10
8
non-bonded S-O
interaction
N
HN
HN
OMe
HN Ar
N
N
O
O
c
d
NH
NH
1
Vatalanib (2)
N
N
HN
N
CF3
HN
O
11
5
O
H
Scheme 1. Reagents and conditions: (a) HNO3, AcOH, DMF, 85 °C, y. 33%; (b) H2,
10% Pd/C, MeOH, rt y. 65%; (c) 4-pyridinecarboxaldehyde, NaBH3CN, MeOH, rt y.
28%; (d) ArNH2, AlMe3, toluene, reflux, y. 11–42%.
hydrogen
bonding
hydrogen
bonding
H
N
N
A
(4-Pyridylmethylthio)indoles 6 were synthesized by the reac-
tion of methyl (4-pyridylmethylthio)indole-3-carboxylate 13 with
various amines (ArNH2) in a manner similar with (4-pyridylmeth-
ylamino)indoles 5. The methyl ester 13 was furnished from methyl
4-iodoindole-3-carboxylate 12 by the coupling reaction with
4-pyridinemethanethiol hydrochloride using the Pd-Xantphos cat-
alyst system reported by Itoh and Mase.15 In the preparation of
iodide 12 (39% yield) from methyl indole-3-carboxylate 8 by the
reaction using Tl(OCOCF3)3 and KI,16 the diiodide 14 was also pro-
duced in 4% yield.17
AAL993 (3)
4 (A:any atom)
Figure 1. Structures of 4-pyridylmethylthio derivative 1, Vatalanib (2), AAL993 (3)
and substructure (4).
Syntheses of novel 4-substituted indole-3-carboxamides 5–7
are illustrated in Schemes 1–3. (4-Pyridylmethylamino)indoles 5
were synthesized by the reaction of methyl (4-pyridylmethylami-
no)indole-3-carboxylate 11 with various ArNH2 using AlMe3.13
The methyl ester 11 was furnished by the reductive amination of
4-pyridinecarboxaldehyde with 10 which was prepared from com-
mercially available methyl indole-3-carboxylate 8 via nitration14
and reduction.
After the amide 17 was synthesized from commercially avail-
able 4-benzyloxyindole 15 by the Friedel–Crafts type acylation18
with trichloroacetic acid anhydride and dehydrative condensation
with various amines (ArNH2), (4-pyridylmethoxy)indoles 7 were
*
*
A
B
Glu885
Glu885
*
*
*
*
+-O
+-O
NH3
O
NH3
O
*
*
CF3
Lys868
Lys868
R
H
H
N
HN
N
O
O
O
N
*
X
O
H
H
Cys919
*
H
N
Cys919
-
N
*
CO2
*
N
*
N
-
CO2
*
O
H
H
O
N
N
O
O
O
O
Asp1046
Asp1046
SH
SH
*
Hinge
*
Hinge
Figure 2. Schematic drawing of X-ray co-crystallography of 3 and KDR (A). Plausible binding mode of indole derivative 5–7 (B).
Ar
Ar
HN
O
O
HN
N
Ar
HN
HN
HN
O
N
O
H
H
S
N
N
N
pseudo 7-membered ring
pseudo 6-membered ring
pseudo 7-membered ring
5
6
7
Figure 3. Possible interaction in indole derivatives 5–7.

1784
T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1782–1785
HN
6c was shorter than the sum of van der Waals radii of sulfur and
OMe
oxygen atoms (3.32 Å), thus suggesting intramolecular S–O non-
bonded interaction.20 The two terminal aromatic rings were apart
distant in both of 5d and 6c; these were similar to the conforma-
tion of 1, 2, 3. Thus, the compounds (5d and 6c) were found to form
seven-membered or six-membered pseudo ring as we expected.
The X-ray crystallography of 7d (Fig. 6) revealed that the distance
between the hydrogen on nitrogen (N3) and the oxygen (O1) was
1.88 Å, thus intramolecular hydrogen bonding was also made in
7d. The two terminal aromatic rings, however were close (4.33 Å)
HN
OMe
e
f
O
8
S
O
I
N
12
13
H
N
HN
Ar1
HN
OMe
and nearly parallel (22°), which suggests p–p stacking interaction.
g
O
KDR inhibitory activities of indole derivatives 5–7 were evalu-
ated by the reported ELISA assay method,21 and are summarized
in Table 1. While 4-pyridylmethylamino derivative 5a–d inhibited
S
O
I
I
N
KDR weakly (23–25% inhibition at 10
derivatives 6a–d showed more or potent inhibitory activities
(34–92% inhibition at 10 M), in which 6b was most potent with
an IC50 value of 309 nM. 4-Pyridylmethoxy derivatives 7a–d did
not show the activity (4–19% inhibition at 10 M). The activities
lM), 4-pyridylmethylthio
14
6
l
Scheme 2. Reagents and conditions: (e) Tl(OCOCF3)3, CF3CO2H, rt, then KI, H2O, rt y.
41%; (f) 4-pyridinemethanethiol hydrochloride, cat. Xantphos, cat. Pd2(dba)3, DIEA,
1,4-dioxane, 90 °C, 85%; (g) ArNH2, AlMe3, toluene, reflux, y. 33–71%.
l
of the compounds are in order of 7 < 5 < 6 with the identical Ar
group and appear limited to a certain range by every scaffold. This
implies that the other compounds than observed ones (5d, 6c, 7d)
by X-ray crystallography take similar conformations according to
their scaffolds, in their binding to KDR. These results suggested
that the inhibitory activities are driven with their stable conforma-
tions introduced with the non-bonded interactions which were ob-
served in the X-ray crystallographic analyses mentioned above.
From these results, a rationale to the KDR binding of 5–7 will be
given as follows (Figure 2B). In compound 5 or 6 taking the pseudo
ring by intramolecular hydrogen bonding or S–O interaction that
involves the carbonyl oxygen as a member of the pseudo ring,
the amide NH to the Glu885 of KDR, the carbonyl oxygen to
Asp1046 and the pyridine N to Cys919 would effectively work
for the inhibitory activity. Their conformations will be similar to
1, 2 and 3. Though 7 is able to form intramolecular hydrogen bond
and pseudo ring, its carbonyl oxygen being outside the ring would
not be preferable to Glu885 side chain.
In summary, we investigate their conformation–activity rela-
tionships of novel indole-3-carboxamide derivatives 5–7 against
KDR by X-ray crystallography under the pseudo ring idea.22 Com-
pounds 5 and 6 taking the pseudo seven- or six-membered ring
by the intramolecular interaction, whose conformations are similar
to active 1, 2, and 3, showed KDR inhibitory activities, in particular,
the IC50 of 6b was 309 nM. Compound 7 having also a pseudo se-
ven-membered ring did not show the inhibitory activity as its
pseudo ring was deficient of carbonyl oxygen that would bind to
KDR. This study led to novel KDR inhibitor 6b, which was thought
to be a promising lead for potent KDR inhibitor. Further studies of
6b and its derivatives will be presented in due course.
HN
HN
HN
OH
HN Ar
O
h
i
O
OBn
OBn
OBn
15
16
17
HN
HN Ar
O
HN
HN Ar
O
j
k
O
OH
N
18
7
Scheme 3. Reagents and conditions: (h) trichloroacetic acid anhydride, DMF, rt
then 4 M NaOH aq, THF, rt y. 17%; (i) ArNH2, HATU, DIEA, DMF, rt y. 17–76%; (j) 10%
Pd/C, MeOH, rt y. 62–91%; (k) 4-chloromethylpyridine hydrochloride, K2CO3, DMF,
rt y. 19–31%.
prepared from 4-hydroxyindole-3-carboxamide 18, which was ob-
tained from the hydrogenation of 4-benzyloxyindole-3-carboxam-
ide 17.
The results of X-ray crystallographic analyses of selected com-
pounds (5d and 6c) are shown as ORTEP drawings in Figures 4
and 5.19 Compound 5d had the distance between the hydrogen
on nitrogen (N3) and the oxygen (O1) of 1.97 Å, which would be
intramolecular hydrogen bonding. The S–O distance (3.01 Å) of
o
A
1.97
H
N
O
NH
N
N
H
5d
Figure 4. ORTEP drawing of compound 5d. Hydrogen atoms on carbon atoms are omitted for the sake of clarity.

T. Honda et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1782–1785
1785
o
A
N
3.01
S
O
NH
N
H
6c
Figure 5. ORTEP drawing of compound 6c. Hydrogen atoms on carbon atoms are omitted for the sake of clarity.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.063.
π π
-
stacking
References and notes
N
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H
N
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O
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A
1.88
N
H
7d
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Figure 6. ORTEP drawing of compound 7d. Hydrogen atoms on carbon atoms are
omitted for the sake of clarity.
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Table 1
KDR inhibitory activitya at 10
lM of compounds 5–7
Compounds
Inhibition
(%)
Compounds
Inhibition
(%)
5a (Ar = 4-Cl-Ph)
5b (Ar = 3-CF3-Ph)
5c (Ar = 5-indan)
5d (Ar = 4-t-Bu-Ph)
5e (Ar = 3-Cl-Ph)
5f (Ar = 3-Me-Ph)
5g (Ar = 3,5-Me-Ph)
5h (Ar = 4-OCF3-Ph)
5i (Ar = 3-isoquinolin)
6a (Ar = 4-Cl-Ph)
6b (Ar = 3-CF3-Ph)
23
25
27
6c (Ar = 5-indan)
6d (Ar = 4-t-Bu-Ph)
6e (Ar = 3-Cl-Ph)
59
51
35
52
35
35
74
9
25
18
18
19
6f (Ar = 3-Me-Ph)
6g (Ar = 3,5-Me-Ph)
6h (Ar = 4-OCF3-Ph)
6i (Ar = 3-isoquinolin)
7a (Ar = 4-Cl-Ph)
14
15. Itoh, T.; Mase, T. Org. Lett. 2004, 6, 4587.
55
34
7b (Ar = 3-CF3-Ph)
7c (Ar = 5-indan)
7d (Ar = 4-t-Bu-Ph)
4
19
6
16. Yamada, F.; Tamura, M.; Somei, M. Heterocycles 1998, 49, 451.
17. Structure of compound 14 was confirmed by X-ray crystallographic analysis.
See Supplementary data.
92 (309 nM)b
18. Cipiciani, A.; Clementi, S.; Giulietti, G.; Marino, G.; Savelli, G.; Linda, P. J. Chem.
Soc. Perkin 2 1982, 523.
19. X-ray crystallographic analyses data of them (CCDC Number #790833 for 5d,
#790834 for 6c and #790835 for 7d) were deposited with Cambridge
Crystallographic Data Center.
a
KDR inhibitory activity was measured with a kinase aasay development kit
purchased from CARNA BIOSCIENCE Co. Ltd.
b
Parenthesis is a value of IC50
.
20. Recently IKKb inhibitors possessing 1-6 non-bonded S–O interactions were
reported by the researchers of Takeda Pharm. Co. Ltd Sugiyama, H.; Yoshida,
M.; Mori, K.; Kawamoto, T.; Sogabe, S.; Takagi, T.; Oki, H.; Tanaka, T.; Kimura,
H.; Ikeura, Y. Chem. Pharm. Bull. 2007, 55, 613.
Acknowledgments
21. KDR inhibitory activity assay was performed by the ELISA method by
measuring the phosphorylation level of biotinylated peptide including Tyr in
the sequence.
22. (a) Furet, P.; Caravatti, G.; Gaugano, V.; Lang, M.; Meyer, T.; Shoepfer, J. Bioorg.
Med. Chem. Lett. 2008, 18, 897; (b) Nakamura, H.; Sasaki, Y.; Uno, M.; Yoshikawa,
T.; Asano, T.; Ban, H. S.; Fukazawa, H.; Shibuya, M.; Uehara, Y. Bioorg. Med. Chem.
Lett. 2006, 16, 5127.
We greatly thank Shouhei Katao of Nara Institute of Science and
Technology for X-ray crystallographic analyses of compounds 5d,
6c and 7d. Also, we thank Dr. Ken-ichi Fujimura and Dr. Koushi
Fujisawa of the Research and Development Center at Santen Phar-
maceutical Co. Ltd for their helpful suggestions.