Inhibition of c-KIT, VEGFR-2 (KDR), and ABCG2 by analogues of OSI-930

Article abstract of DOI:10.1016/j.bmcl.2011.08.070

The quinoline domain of OSI-930, a dual inhibitor of receptor tyrosine kinases (RTKs) c-Kit and KDR, was modified in an effort to further understand the SAR of OSI-930, and the binding site characteristics of c-Kit and KDR. A series of 16 compounds with h

Full text of DOI:10.1016/j.bmcl.2011.08.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6495–6499
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of c-Kit, VEGFR-2 (KDR), and ABCG2 by analogues of OSI-930
Jay P. Patel a, Ye-Hong Kuang a,b, Zhe-Sheng Chen a, Vijaya L. Korlipara a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Queens, NY 11439, USA
b Department of Dermatology, Xiang Ya Hospital, Central South University, Changsha 41008, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The quinoline domain of OSI-930, a dual inhibitor of receptor tyrosine kinases (RTKs) c-Kit and KDR, was
modified in an effort to further understand the SAR of OSI-930, and the binding site characteristics of c-
Kit and KDR. A series of 16 compounds with heteroatom substituted pyridyl and phenyl ring systems was
synthesized and evaluated against a panel of kinases including c-Kit and KDR. Aminopyridyl derivative 6
Received 16 July 2011
Revised 12 August 2011
Accepted 15 August 2011
Available online 22 August 2011
was found to be the most active member of the series with 91% and 57% inhibition of c-Kit at 10
lM and
1
l
M, respectively and 88% and 50% inhibition of KDR at 10 M and 1 M, respectively. The target com-
l
l
Keywords:
Tyrosine kinase inhibitors
ABCG2 pump
pounds were also tested for their ability to inhibit efflux of mitoxantrone through inhibition of ATP
dependent ABCG2 pump. Nitropyridyl derivative 5 and o-nitrophenyl derivative 7 exhibited complete
inhibition of the ABCG2 pump with IC50 values of 13.67
l
M and 16.67
lM, respectively.
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibition of receptor tyrosine kinases (RTKs) has emerged as a
promising approach for the treatment of many types of human
cancers. Epidermal growth factor receptor (EGFR) inhibition for
the treatment of non-small cell lung cancer, c-Kit inhibition for
the treatment of gastrointestinal stromal tumors (GIST), and Abl
inhibition for the treatment of acute as well as chronic myeloid
leukemia has found success in the recent years with introduction
of several drugs in this arena.1 Imatinib, a Bcr-Abl kinase and c-
Kit kinase inhibitor; erlotinib, an EGFR inhibitor; lapatinib, an EGFR
and vascular endothelial growth factor receptor (VEGFR) inhibitor;
and OSI-930, a dual c-Kit and VEGFR-2 inhibitors are few of the
many tyrosine kinase inhibitors, either currently available in mar-
ket or in clinical trials for the treatment of cancer (Fig. 1).1
Studies have shown that c-Kit, a stem cell factor receptor, is
over-expressed or mutated, in small cell lung cancer, mast cell leu-
kemia, seminoma, acute myeloid leukemia and most commonly in
GIST patients.2 VEGFR-2 (KDR) has been shown to play an impor-
tant role in the regulation of tumor angiogenesis.3,4 Thus inhibition
of both these RTKs can result in improved antitumor efficacy
through inhibition of cell proliferation and anti-angiogenic effect.
A number of tyrosine kinase inhibitors including imatinib,
AG1478, erlotinib, and lapatinib have been shown to interact with
ATP-binding cassette (ABC) transporters such as the ABCG2 and
block its efflux function, thus increasing the intracellular accumu-
lation of anticancer drugs and reversing ABCG2-mediated multi-
drug resistance (MDR).5–9 Thus tyrosine kinase inhibitors have
the added potential to contribute to cancer chemotherapy through
the reversal of MDR caused by the overexpression of ATP-binding
cassette transporters.
OSI-930 (Fig. 1), a dual c-Kit and KDR inhibitor with IC50 values
of 29 and 5 nM, respectively was chosen as a lead compound.10,11
Target compounds exploring the structure–activity relationship
of the southern part of OSI-930 were designed. Modifications
involved replacement of the quinoline group with phenyl and
pyridyl ring systems substituted with functional groups that
N
O
O
N
HN
N
N
O
O
N
CH
HN
C
HN
O
N
N
Erlotinib
Imatinib
N
N
H
N
S
O
HN
OCF3
O
NH
NH
O
Cl
O
S
F
N
O
OSI-930
Lapatinib
⇑
Corresponding author. Tel.: +1 718 990 5369; fax: +1 718 990 1877.
E-mail address: korlipav@stjohns.edu (V.L. Korlipara).
Figure 1. Examples of tyrosine kinase inhibitors on market or clinical trials.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.070

6496
J. P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6495–6499
O
OCF3
O
H2N
S
a
S
OCH3
NH2
+
N
H
OCF3
NH2
2
3
1
CHO
NO2
b
N
4
OCF3
O
OCF3
O
S
S
N
H
N
H
NH
c
NH
NH2
NO2
N
N
6
5
Scheme 1. Reagents and conditions: (a) (CH3)3Al, anhydrous toluene, rt (16 h) followed by reflux for 24 h; (b) NaBH4, trifluoroacetic acid, EtOAc; (c) hydrazine monoformate
Zinc Dust, MeOH.
impart different steric, electronic, solubility, and H-bonding char-
acteristics. The functional groups chosen included those that were
rich in heteroatoms with the potential to form H-bond interactions
with the backbone cysteine residues in the hinge regions of c-Kit
and KDR. In this report we describe the synthesis of compounds
5–20, their activity against a panel of kinases including c-Kit and
KDR, and the ABCG2 pump.
3-Amino-N-(4(trifluoromethoxy)phenyl)thiophene-2-carboxam-
ide 3 was obtained by activation of 4-trifluoromethoxyaniline with
trimethylaluminum, followed by reaction of the activated amine-
trimethylaluminum adduct with methyl 3-aminothiophene-2-
carboxylate under reflux conditions (Scheme 1).12 The nitropyridyl
derivative 5, nitrophenyl derivatives 7–9 and hydroxyphenyl
derivative 10 were obtained by the reductive alkylation of 3-ami-
nothiophene carboxamide derivative 3 with 3-nitroisonicotinalde-
hyde 4 which was prepared according to the literature procedure13
or the commercially available substituted benzaldehydes (Schemes
1 and 2).14–16
Nitro derivatives 5 and 7–9 were reduced to the corresponding
amines 6 and 11–13 by using either Zn dust method17 or PtO2
catalyzed hydrogenation method.18 The amine derivatives 11–13
were subsequently treated with acetic anhydride or di-2-pyridyl
thionocarbonate in order to obtain acetamides 14–1619 and
isothiocyanates 17–19,20 respectively (Scheme 2). Treatment of
m-amino derivative 12 with bromoacetyl bromide to form the bro-
moacetamide derivative 2119 resulted in a product whose NMR
spectrum showed all the expected protons including a sharp sin-
glet for benzylic CH2 protons at 5.16 ppm and a singlet for alkyl
CH2 protons of bromoacetyl group at 4.02 ppm. A singlet at
4.56 ppm integrating for two protons that did not correspond to
any of the expected protons was also observed. LCMS data showed
molecular ion peak of 568 instead of the expected molecular ion
peak of 528. Based on this information, it was determined that
the secondary amine at position 3 was also bromoacetylated which
then underwent cyclization to yield compound 20. The singlet at
position 4.56 ppm presumably corresponds to the two protons of
cyclic –CH2 group.
Target compounds were also tested against other members of type
III and type V kinase families to which c-Kit and KDR, respectively,
belong to. All target compounds were tested against a panel of
eight kinases which included c-Kit, PDGFR
a, PDGFRb, Flt3, KDR,
VEGFR-1, VEGFR-3 and Tie-2 at 1 M and 10
l
lM concentrations.
Summary of c-Kit and KDR inhibition results of the target
compounds exhibiting greater than 40% inhibition is presented in
Table 1 and their selectivity versus other kinases is presented in
discussion. Acetamidopyridyl derivative 22 was used as
a
reference.
As shown in Table 1, nitropyridyl derivative 5 and the amino-
pyridyl derivative 6 were among the best in inhibiting c-Kit and/
KDR. Nitro analogue 5 showed 80% inhibition of c-Kit at 10
concentration. Amino analogue 6 exhibited 57% and 91% inhibition
of c-Kit at 1 and 10 M, respectively, whereas in case of KDR it
showed 50% and 88% inhibition at 1 and 10 M, respectively. Com-
pound 6 weakly inhibited Flt3 (41%), VEGFR-3 (41%) and PDGFRb
(47%) at 10 M concentration. Thus compound 6 is somewhat sim-
ilar in potency to the reference compound 22 at 10 M. Compound
22 displayed 81% inhibition of c-Kit at 1 M and complete inhibi-
tion at 10 M. It inhibits KDR completely at both concentrations.
In addition, compound 22 also inhibited all other kinases in the test
group completely at 10 M. Compound 6 showed weaker inhibi-
lM
l
l
l
l
l
l
l
tion of other kinases, and thus appears to be more selective for
c-Kit and KDR than the reference compound.
Compounds in the phenyl series were generally less effective in
inhibiting c-Kit and KDR, as well as the other kinases in the test
group. m-Acetamide analogue 15 showed 58% inhibition of c-Kit
at 10 lM, suggesting that acetamide group of compound 15 may
be interacting with the c-Kit enzyme active site in a similar manner
as the acetamide group of reference compound 22. Lack of pyridyl
N in 15 is contributing to its reduced activity compared to com-
pound 22.
Another compound in phenyl series which showed kinase inhi-
bition was p-hydroxy analogue 10 which possesses H-bond accep-
tor group similar to the pyridyl derivatives 5, 6 and 22 except that
it is one atom away from the aromatic ring. At 10
10 showed 68% and 54% inhibition of c-Kit and KDR, respectively.
It showed 44% inhibition of PDGFRb at 10 M. p-Amino analogue
lM, compound
Target compounds 5–20 were tested against c-Kit and KDR to
evaluate their ability to inhibit these enzymes by Z’-LYTE assay.21
l

J. P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6495–6499
OCF3
6497
O
S
N
H
OCF3
O
S
R= o -NO2, 7
R = m -NO2, 8
a
NH
N
H
R
NH2
9
R = p -NO2,
R = p -OH, 10
3
b
OCF3
O
S
OCF3
O
N
H
S
f
x
N
H
NH
NH
o -NH2, 11
m -NH2, 12
p -NH2, 13
NH2
NHCOCH2Br
21
c
e
d
OCF3
O
S
OCF3
O
OCF3
O
S
S
N
N
H
N
N
H
NH
NH
O
NHCOCH3
NCS
NHCOCH2Br
17
18
19
o -NCS,
m -NCS,
p -NCS,
20
14
15
16
o -NHCOCH3,
o -NHCOCH3,
p -NHCOCH3,
Scheme 2. Reagents and conditions: (a) NaBH4/trifluoroacetic acid, EtOAc, substituted benzaldehydes; (b) PtO2/H2/MeOH, 40 psi/30 min; (c) acetic anhydride, chloroform;
(d) di-2-pyridylthienocarbonate, chloroform; (e) BrCH2COBr, NaHCO3, distilled THF, 0 °C.
13 on the other hand showed less than 40% inhibition in all en-
zymes at both tested concentrations. Interestingly, cyclic bromo-
acetamide analogue 20 showed 41% inhibition of c-Kit at 10 lM.
target compounds with OSI-930 or the reference compound 22 as
shown in Figures 2 and 3 demonstrates the relative placement of
H-bonding groups in these molecules.
All other nitro (7–9), amino (11–13), acetamide (14 and 16) and
isothiocyanate (17–19) analogues in phenyl series showed less
than 40% inhibition of the kinases. Taken together, the kinase inhi-
bition data reinforces the importance of H-bond acceptor group in
the southern domain and in a precise location. Overlay of various
Table 1
Percent inhibition of c-Kit and KDR by OSI-930 analogues
Compound
X
-R
Concentration
M)
% Inhibition
(
l
c-Kit
KDR
(VEGFR-2)
*
*
5
N
N
C
20-NO2
1
10
1
10
1
10
1
10
1
10
1
*
80
57
6
20-NH2
50
91
88
*
*
10
15
20
22
40-OH
68
54
*
*
C
30-NHCOCH3
*
*
*
58
*
C
Cyclized deriv.
30-NHCOCH2Br
30-NHCOCH3
41
81
102
N
101
101
10
*
Indicates less than 40% inhibition. All experiments were performed in duplicates.
Figure 2. Overlay of OSI-930 and compounds 5, 6, and 22.

6498
J. P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6495–6499
KDR along with other kinases belonging to the same families to
evaluate specificity in kinase inhibition. Nitropyridyl derivative 5
exhibited 80% inhibition of c-Kit at 10 M concentration and was
not effective against other kinases. Aminopyridyl derivative 6
showed 91% and 88% inhibition at 10 M and 51% and 50% inhibi-
tion at 1 M of c-Kit and KDR, respectively. Compound 6 exhibited
l
l
l
weak or no inhibition of other kinases. Analogues in the phenyl
series were generally less active. m-Acetamide derivative 15 with
the structural resemblance to the reference compound 22 showed
58% inhibition of c-Kit at 10 lM. The significantly weaker activity
of 15 versus 22 demonstrated the importance of the H-bond accep-
tor group in the ring. p-Hydroxy derivative 10 possesses the ability
to serve as a H-bond acceptor similar to the reference compound
22 with a slightly altered location of the electronegative atom.
Although it exhibited inhibition of c-Kit and KDR, it was weaker
than the reference compound. The kinase assay results demon-
strate the importance of a H-bond acceptor group in the southern
region of the target compounds and further suggest that moving
this H-bond acceptor functionality by even a one atom distance
can significantly impact the activity. Although all target com-
pounds were less active in inhibiting c-Kit and KDR than reference
compound 22, the most potent compound in the series 6 was
found to be more selective for c-Kit and KDR. Nitropyridyl and
nitrophenyl derivatives 5 and 7 significantly potentiate the sensi-
tivity of anticancer drug mitoxantrone in ABCG2-overexpressing
cells. These molecules do not influence the sensitivity of the paren-
tal cells.
Figure 3. Overlay of compounds 10, 13, and 22.
Table 2
Effect of analogs of OSI-930 on the cytotoxicity of mitoxantrone in the ABCG2-
transfected multidrug resistant cells
Acknowledgments
Treatment
HEK293/pcDNA3.1
HEK/ABCG2-482-R2
IC50
FRa
IC50
FRa
We thank Dr. Susan Bates (NCI, Bethesda, MD) for providing
HEK-293/pcDNA and HEK-ABCG2-R2 multidrug resistant cells
and OSI Pharmaceuticals Inc. for providing us with the reference
compound 22 and funding the Invitrogen’s Z’-LYTE assay. Jay Patel
thanks OSI Pharmaceuticals Inc. for providing him the summer stu-
dentship award. Ye-Hong Kuang thanks the State Scholarship Fund
of China (No. 2008637062) for supporting the study at St. John’s
University.
Mitoxantrone
15.41 4.68
7.46 6.63
15.55 2.64
7.66 3.44
13.64 2.13
13.31 1.37
7.06 1.42
9.38 0.6
21.55 12.4
20.44 9.06
10.62 2.59
9.12 3.19
14.09 0.36
11.84 7.98
14.39 3.63
150.82 18.6
12.94 5.73
1
0.44
240.8 4.00
13.67 10.44
51.67 0.42
16.67 14.17
51.27 11.81
31.52 21.4
29.57 12.18
34.56 29.4
57.7 25.24
55.66 12.89
35.89 2.22
28.74 10.58
46.28 1.6
15.33
0.82
3.52
0.99
3.37
1.92
1.89
2.05
3.67
3.65
2.42
1.85
3.13
2.79
7.14
138.2
1.03
+5 (10
+6 (10
+7 (10
+8 (10
+9 (10
l
l
l
l
l
M)
M)
M)
M)
M)
1.09
0.49
0.91
0.89
0.47
0.64
1.59
1.48
0.7
0.59
0.96
0.72
0.94
10.46
0.83
+10 (10
+11 (10
+12 (10
+13 (10
+14 (10
+15 (10
+16 (10
+17 (10
+18 (10
+19 (10
l
l
l
l
l
l
l
l
l
l
M)
M)
M)
M)
M)
M)
M)
M)
M)
M)
Supplementary data
41.31 1.83
89.9 99.33
1967.29 417.9
16.42 7.69
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.070.
+FTC (2.5 lM)
References and notes
Cell survival was determined by MTT assay. Data are the means SD of at least
three independent experiments performed in triplicate.
Fold-resistance was the IC50 value for mitoxantrone of HEK/ABCG2-482-R2 cells
divided by IC50 value of HEK293/pcDNA3 cells in the absence or presence of analogs
of OSI-930 or FTC.
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