Discovery and evaluation of 3-(5-thien-3-ylpyridin-3-yl)-1H-indoles as a novel class of KDR kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00627-9

We have discovered 3-(5-thien-3-ylpyridin-3-yl)-1H-indoles as potent inhibitors of KDR kinase activity. This communication details the evolution of this novel class from a potent screening lead of vastly different structure with an emphasis on structural modifications that retained activity and provided improvements in key physical properties. The synthesis and in-depth evaluation of these inhibitors are described.

Full text of DOI:10.1016/S0960-894X(03)00627-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2973–2976
Discovery and Evaluation of 3-(5-Thien-3-ylpyridin-3-yl)-1H-
indoles as a Novel Class of KDR Kinase Inhibitors
Mark E. Fraley,* Kenneth L. Arrington, Scott R. Hambaugh, William F. Hoffman,
April M. Cunningham, Mary Beth Young, Randall W. Hungate, Andrew J. Tebben,
Ruth Z. Rutledge, Richard L. Kendall, William R. Huckle, Rosemary C. McFall,
Kathleen E. Coll and Kenneth A. Thomas
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 29 January 2003; accepted 14 February 2003
Abstract—We have discovered 3-(5-thien-3-ylpyridin-3-yl)-1H-indoles as potent inhibitors of KDR kinase activity. This communi-
cation details the evolution of this novel class from a potent screening lead of vastly different structure with an emphasis on struc-
tural modifications that retained activity and provided improvements in key physical properties. The synthesis and in-depth
evaluation of these inhibitors are described.
# 2003 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new capillaries from
established blood vessels, is an essential process for
normal growth and development that has also been
implicated in the pathogenesis of several diseases
including diabetic retinopathy,1 rheumatoid arthritis,2
psoriasis,3 and cancer.4 Specifically, the growth and
metastasis of solid tumors has been shown to be depen-
dent on angiogenesis at an early stage,5 while tumors
that lack adequate vascularization become necrotic or
apoptotic and do not grow beyond a limited size.6
Interest in inhibition of angiogenesis as a new approach
for the treatment of cancer has led to the elucidation of
key underlying molecular mechanisms that control the
angiogenic process. Angiogenesis is regulated by the
expression of a variety of growth factors including vas-
cular endothelial growth factor (VEGF), a selective
mitogen for endothelial cells whose mitogenic signaling
is mediated through the receptor tyrosine kinase KDR
(VEGFR-2).7 Several lines of evidence indicate that
expression and signaling of VEGF are critical for tumor
angiogenesis. Among these, antibodies against VEGF8
and its receptor KDR9 as well as small molecule inhibi-
tors of KDR kinase activity10 have been shown to inhi-
bit angiogenesis in tumor xenograft models. Clinical
trials have been initiated for KDR kinase inhibitors
derived from a number of different structural classes,
including indolin-2-ones, phthalazines, and quinazo-
lines.11
In our efforts to discover novel small molecule inhibi-
tors of KDR kinase activity, a screening of a large non-
directed library of 5-acetamido-2,4-diaryloxazoles pro-
duced lead compound 1 (Fig. 1). Compound 1 exhibited
good intrinsic potency (KDR IC50=16 nM)12 and rea-
sonable cellular activity (cell IC50=320 nM).13
However, we concluded from additional studies that the
poor physical properties of 1, including low aqueous
solubility (<0.005 mg/mL) and high lipophilicity (logP
>4.1),14 would hinder its development. Toward
improving these physical properties, we initially investi-
gated more polar replacements for the 2-hydroxy-3-
naphthyl moiety at C-2 of the oxazole ring system. Of
*Corresponding author. Fax: +1-215-652-6345; e-mail: mark_
fraley@merck.com
Figure 1. Screening lead 1.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00627-9

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M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2973–2976
Table 2. KDR kinase activity of compounds 3a–h
Table 1. KDR kinase activity of compounds 2a–f
Compd
R
H
KDR IC50 (nM)
126
Compd
R
KDR IC50 (nM)
186
3a
2a
3b
115
2b
2c
2d
2e
575
5700
8320
4860
3c
3d
111
60
3e
3f
38
16
2f
4040
3g
3h
64
46
the wide range of aryl and heterocyclic substituents
examined at C-2, all were relatively inactive with the
exception of a small subset of ortho-phenols and, more
interestingly, the 3-pyridyl ring system found in com-
pound 2a (Table 1).15,16 The 3-fold greater potency
provided by the thienyl substituent of 2a compared to
phenyl group within 2b and dramatic loss in potency
upon substitution of this phenyl group (cf 2c–f) were
reminiscent of the SAR established for the pyrazolo[1,5-
a]pyrimidine class of KDR kinase inhibitors.17 We pro-
posed a similar binding mode that placed the pyridyl
ring in the adenine binding region of the ATP active site
and allowed the pyridyl nitrogen to engage in a hydro-
gen bond with Cys 919 of the hinge region.18 We then
envisioned the thienyl group fitting into the sterically
confined hydrophobic region I and the oxazolyl moiety
filling hydrophobic region II.
ylpyridine and 1-[(4-methylphenyl)sulfonyl]-1H-indol-3-
ylboronic acid19 under Suzuki conditions proceeded
smoothly to give 1-[(4-methylphenyl)sulfonyl]-3-(5-
thien-3-ylpyridin-3-yl)-1H-indole. Hydrolysis of the
tosyl group with potassium hydroxide afforded 3a.
Deprotonation of 3a with sodium hydride and alkyl-
ation of the resultant anion with the corresponding
mustard-like amines proceeded in moderate to excellent
yield to furnish compounds 3b–h.20
Because compound 2a showed little improvement in
physical properties (aq soln <0.005 mg/mL, logP >4.0)
over 1, we continued to focus on major structural
modifications. Based on our limited understanding of
the binding mode of 2a, we speculated that potency
would be maintained by replacement of the oxazolyl
appendage at C-3 of the pyridyl ring system with a pla-
nar substituent of lower molecular weight. A survey of a
set of aryl and heterocyclic groups at C-3 culminated in
the identification of the 3-indolyl substituent (3a, Table
2) as the optimal group in terms of retaining activity
and providing an appropriate point of attachment for
solubilizing functionality, via the indolyl nitrogen. We
took advantage of this key feature in the design and
synthesis of compounds 3b–h (Scheme 1). Briefly, palla-
dium-catalyzed cross-coupling of 3-bromo-5-thien-3-
Scheme 1. Synthesis of compounds 3a–h.

M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2973–2976
Table 5. Rat pharmacokinetic data for compounds 3d–f a
2975
%F
Compd
Cl (mL/min/kg)
t1=2 (h)
Vdss (L/kg)
3d
3e
3f
65
15
13
1.9
1.6
1.8
7.2
1.8
1.7
35
28
33
aCompound dosed 5 mg/kg iv as a solution in DMSO and 20 mg/kg po
as a suspension in aqueous 0.5% methyl cellulose (pH 5).
Table 6. KDR kinase selectivity (fold)a of 3f
PDGFRb
Flt-1
20
Flt-4
3
FGFR-1
246
FGFR-2
136
c-Src
147
7
aExpressed as the biochemical IC50 (nM) ratios to KDR.
Figure 2. Proposed binding mode of 3f.
may be due, in part, to high protein binding, as mea-
sured for 3f (99.8% bound to human plasma protein).
The addition of the alkyl amines to 3a was well-toler-
ated for compounds 3b and 3c, and modestly potency-
enhancing for 3d–h, with 3f exhibiting the greatest
potency (KDR IC50=16 nM). The three-carbon meth-
ylene spacer in 3e and 3g conferred 2-fold greater
potency relative to the two-carbon linker in analogues
3c and 3b, respectively. That inhibitory activity was
relatively insensitive to the nature of the basic side chain
is consistent with the proposed binding mode depicted
in Figure 2 wherein the side chain extends away from
the binding cleft and is solvent exposed. Further SAR
studies supported this model. As was the case in the
oxazolyl series (compounds 2a–f), replacement of the
thienyl group with a substituted phenyl ring led to
nearly complete loss of activity in this series, pre-
sumably the result of steric clashes within hydrophobic
region I. The indolyl ring system proved more tolerant
of substitution in terms of retaining activity. For exam-
ple, placing a chloro, cyano, or methoxyl group at the
indolyl C-5 position of 3f resulted in only a 3-fold
decrease in potency.
Physical properties were determined for selected com-
pounds (Table 4). Gratifyingly, compounds 3d–f
showed greater aqueous solubility and lower lipophili-
city relative to compounds 1 and 2a, leading us to eval-
uate their pharmacokinetic properties.
Compounds 3d–f exhibited moderate pharmacokinetic
behavior in rats with t1=2 ranging from 1.6 to 1.9 h and
oral bioavailability ca. 30% (Table 5).
The selectivity of 3f for inhibition of KDR kinase versus
several closely related receptor tyrosine kinases and a
non-receptor tyrosine kinase (c-Src) is presented in
Table 6 and representative of the class. In general, these
compounds showed low levels of selectivity for KDR
against the highly KDR-homologous kinases PDGFRb,
Flt-1, and Flt-4 and higher levels against FGF-1, FGF-
2, and c-Src.
In conclusion, we have described the evolution of a
novel 3-(5-thien-3-ylpyridin-3-yl)-1H-indolyl class of
KDR kinase inhibitors from a potent oxazole-based
lead. Compounds within this new series retained high
activity and exhibited improved physical properties and
reasonable pharmacokinetics in rats.
Cell data indicated that the basic side chain was
required for achieving submicromolar levels of inhibi-
tory activity in this series (Table 3). A modest shift (5-
to 20-fold) was observed in cellular IC50 compared to
the corresponding biochemical IC50 value. This effect
Table 3. Cell activity for selected compounds
Acknowledgements
Compd
KDR IC50 (nM)
Cell IC50 (nM)
We thank Dr. Art Coddington, Dr. Chuck Ross, and
Dr. Harri Ramjit for mass spectral analyses, Matt
Zrada and Ken Anderson for solubility and logP deter-
minations, and Dr. George Hartman and Elaine Walker
for editing and assistance in the preparation of this
manuscript.
3a
3d
3e
3f
126
60
38
16
37% inhib. @ 2500 nM
1410
495
328
256
3h
46
Table 4. Physical properties of compounds 3d–f
References and Notes
Compd
LogP
Solubility @ pH 5.2
(mg/mL)
Solubility @ pH 7.4
(mg/mL)
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