Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2- ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.021

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene- 2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Full text of DOI:10.1016/j.bmc.2010.05.021

Bioorganic & Medicinal Chemistry 18 (2010) 4674–4686
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship of 6-arylureido-3-pyrrol-
2-ylmethylideneindolin-2-one derivatives as potent
receptor tyrosine kinase inhibitors
Rahul R. Khanwelkar a, Grace Shiahuy Chen b, Hsiao-Chun Wang a, Chao-Wu Yu a, Chiung-Hua Huang a,
On Lee c, Chih-Hung Chen c, Chrong-Shiong Hwang c, Ching-Huai Ko c, Nien-Tzu Chou c, Mai-Wei Lin c,
Ling-mei Wang c, Yen-Chun Chen c,d, Tzong-Hsiung Hseu d, Chia-Ni Chang c, Hui-Chun Hsu c, Hui-Chi Lin c,
Ying-Chu Shih c, Shuen-Hsiang Chou c, Hsiang-Wen Tseng c, Chih-Peng Liu c, Chia-Mu Tu c, Tsan-Lin Hu c,
Yuan-Jang Tsai c, Ji-Wang Chern a,e,
*
a School of Pharmacy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan
b Department of Applied Chemistry, Providence University, Shalu 43301, Taiwan
c Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu 30011, Taiwan
d Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan
e Department of Life Science, National Taiwan University, Taipei 10027, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor
tyrosine kinases. Investigation of structure–activity relationships at positions 5, 6, and 7 of the oxindole
skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives
that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived
growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency
against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar
range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR
and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl
ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with
the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 March 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 12 May 2010
Keywords:
Inhibitor
Anticancer
Vascular endothelial growth factor receptor
Platelet-derived growth factor receptor
Receptor tyrosine kinase
Structure–activity relationship
1. Introduction
cells, but aberrant RTK activation, in particular that of vascular
endothelial growth factor receptor (VEGFR) and platelet-derived
Angiogenesis refers to the formation of new blood vessels from
the endothelium of preexisting vasculature.1 Pathological angio-
genesis has been implicated in a wide array of vascular hyperprolif-
erative disorders, most notably cancer,1 rheumatoid arthritis,2
diabetic retinopathy,3 endometriosis,4 age-related macular degen-
eration,5 and ocular neovascularization.6 Solid tumors, in particular,
are dependant on angiogenesis for growth beyond a certain critical
size via the induction of new capillaries sprouting from existing
blood vessels, thereby securing a supply of nutrients and oxygen
and enabling waste removal.1,7 In addition, angiogenesis also pro-
motes metastasis.8
growth factor receptor (PDGFR), has been linked to the development
and progression of variety of human cancers.9 The VEGFR family
comprises FLT1 (VEGFR1), KDR (VEGFR2), and FLT4 (VEGFR3),
which play pivotal roles in angiogenesis and contribute to tumor
progression through their ability to mediate tumor angiogenesis
and lymphangiogenesis, and to enhance vascular permeability.10
The PDGFR family comprises PDGFRa, PDGFRb, colony stimulating
factor 1 receptor (CSF1R), c-KIT, and FLT3, which promote tumor cell
growth and metastasis through modification of the tumor microen-
vironment.11 Additionally, mutants of FLT3 and c-KIT are directly
associated with proliferation of acute myeloid leukemia blast cells12
and gastrointestinal stromal tumor cells,13 respectively.
Inhibition of RTK pathways has become an important approach
for the discovery of new anticancer drugs.14 The approval of bev-
acizumab,15 an antibody against VEGF, by the United States Food
and Drug Administration (FDA) for the treatment of first-line met-
astatic colorectal cancer in combination with chemotherapy,16 has
Receptor tyrosine kinases (RTKs) represent a large family of
membrane-bound enzymes that play key roles in tumor growth,
survival, and metastasis. RTK activity is tightly regulated in normal
* Corresponding author. Tel.: +886 223939462; fax: +886 223934221.
E-mail address: jwchern@ntu.edu.tw (J.-W. Chern).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.021

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4675
promoted greater interest in this field. Several selective KDR inhib-
itors, including SU5416,17 PTK787,18 CP-547632,19 SU6668,20 and
ZD6474,21 are in late-stage clinical trials. Although VEGFR and
PDGFR are compelling cancer targets individually, tumors are
capable of secreting multiple angiogenic factors, and they depend
on these factors at different stages of progression.22 As a result of
the complex and redundant cellular signaling network associated
with RTKs, broad-acting and multitargeted RTK inhibitors may be
more advantageous than selective agents, owing to their ability
to block multiple signaling pathways associated with tumor sur-
vival.23 This is evidenced by the FDA’s recent approval of multitar-
geted kinase inhibitors such as imatinib [inhibits Abelson tyrosine
kinase; (Abl), c-KIT protein (CD117), and PDGFR; approved for
chronic myelogenous leukemia,24 gastrointestinal stromal tumors,
and a number of other malignancies], sunitinib [inhibits KDR,
PDGFR2, PDGFRb, c-KIT and FLT3; approved for the treatment of re-
nal cell carcinoma and imatinib-resistant gastrointestinal stromal
tumors],25 sorafenib [inhibits Raf kinase, KDR, PDGFRb, and
c-KIT; approved for the treatment of advanced renal cell carci-
noma],26 and lapatinib [inhibits EGFR, and ErbB-2; approved for
advanced metastatic breast cancer in conjugation with chemother-
apy].27 These agents demonstrate clinical benefits with manage-
able side effects.
ABT-86929a, either have been approved by the FDA or are in late-
stage clinical trials (Fig. 1). Similarly, there are many reports on
oxindole derivatives as potential RTK inhibitors;30 these include
sunitinib,25,30a SU5416,17,30b and SU666820,30c (Fig. 1). However,
structure–activity relationship (SAR) studies on ureido-substituted
oxindoles against VEGFR and PDGFR have been less explored.31 As
part of our efforts toward the discovery and biological evaluation
of new anticancer agents,32 we report here the synthesis and SAR
studies of a series of 6-ureido/thioureido-substituted indolin-2-
ones as potent multitargeted RTK inhibitors. This study has led to
the identification of potential kinase inhibitors for members of
both the VEGFR and PDGFR families.
2. Chemistry
The 5-, 6-, and 7-arylureido-substituted 3-arylmethylidene-2-
oxindole derivatives 13–54 were prepared according to a general
method (Scheme 1). 5-Amino-2-oxindole (6) is commercially
available, whereas a direct reduction of 7-nitro-2-oxindole led to
7-amino-2-oxindole (7) in high yield. Hydrogenation followed by
cyclization of 2,4-dinitrophenyl acetic acid (8) afforded 6-amino-
2-oxindole (9) in moderate yield. Reaction of compounds 6, 7,
and 9 with aryl isocyanates afforded the corresponding arylureid-
oindolin-2-one derivatives 10–12 in excellent yield. Condensation
of derivatives 10–12 with aryl/heteroaryl aldehydes afforded the
corresponding 3-arylmethylidene-2-oxindole derivatives 13–54
in moderate yield.
The activity of inhibitors having a urea moiety,28 especially the
diphenyl urea motif,29 against kinases such as VEGFR, PDGFR,
Tie-2, Raf, and p38 has been described. These candidates, which
include sorafenib,26 PD-173074,28b,c CP-547632,19 KRN633,28c and
R1
H3C
1a (Sunitinib): R1 = CONH(CH2)2N(C2H5)2, R2 = F
1b (SU5416): R1 = R2 = H
1c (SU6668): R1 = (CH2)2COOH, R2 = H
CH3
N
H
O
R2
N
H
H
N
H
N
F
CF3
Cl
H
N
H
N
O
O
N
O
CH3
NH2
S
O
3 (ABT-869)
2 (Sorafenib)
HN
N
NHCH3
H
H
OCH3
N
N
N
N
O
NH2
F
O
N
OCH3
O
N
H
N
N
NH
F
(C2H5)2N
C(CH3)3
O
N
Br
5 (CP-547632)
4 (PD-173074)
H
Figure 1. Structures of RTK inhibitors.
Ar'
O
O
5
H2N
6
Ar
O
Ar
O
O
(b)
(a)
N
H
N
H
N
H
N
H
N
N
N
H
H
H
7
6: 5-NH2
7: 7-NH2
9: 6-NH2
10: 5-sub.
11: 7-sub.
12: 6-sub.
13-18: 5-sub.
19,20: 7-sub.
21-54: 6-sub.
Scheme 1. Reagents and conditions: (a) Ar-NCO, DCM/methanol, 10 °C–rt, 3–4 h; (b) Ar0CHO, pyrrolidine, ethanol, reflux, 1.5–5 h.

4676
R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
arylureido moiety, showed moderate inhibition of KDR and VEG-
3. Results and discussion
3.1. In vitro kinase activity
FR3, but their potency was clearly decreased compared with the
parent compound 36. Overall, substitution on the phenyl ring in-
creased the inhibition of and selectivity for FLT3.
Investigation of substitutions on the pyrrole ring generated
distinct SARs. Structural comparison between 35 and 37 indicated
that the presence of a methyl group at C-30 and a carboxylate group
at C-40 in 35 substantially enhanced kinase inhibition compared
with 37 (Table 2). Further introduction of a methyl group at C-50
on the pyrrole ring (44–47) did not increase inhibition (Table 2),
but these compounds indeed retained the ability to inhibit all the ki-
nases. The only exception was 47 (containing a chloro group), which
had substantially reduced potency for FLT3. Introduction of a meth-
ylene (48 and 49) or ethylene (50) group between the carboxylate
group and pyrrole ring at the C-40 position was tolerated. Surpris-
ingly, 48 showed a tremendous boost (IC50 = 0.9 nM) in potency
against KDR but a drastically decreased potency against FLT3 and
VEGFR3 compared to 44. Compound 48 thus exhibited good selec-
tivity for KDR, as the IC50 values for VEGFR3 and FLT3 were 38-fold
and 60-fold higher, respectively. Further lengthening of the chain on
the pyrrole ring at the C-40 position (50) led to a minor change in the
inhibition profile compared with 49. Shifting of the carboxylate
group from C-40 to C-50 on the pyrrole ring (51–53) resulted in com-
plete loss of inhibition. The potent multikinase inhibitors (35–37,
39–42, 44, 45, and 50) also were investigated for their inhibitory
potential against c-KIT, and they exhibited high potency with IC50
values in the nanomolar range (Table 2).
All prepared compounds were screened for inhibitory activity
against a panel of RTKs that included c-KIT, FLT3, KDR and VEGFR3.
We first estimated the percent inhibition of each kinase in the
presence of 0.1 lM of each compound (Table 1). Derivatives 13
and 14, each containing a 5-arylureido substituent in conjunction
with a 3-phenylmethylidene group on the indolin-2-one ring,
exhibited poor inhibition of all kinases tested. Replacement of
the phenylmethylidene group with pyrrol-2-ylmethylidene at the
3-position, 15, improved the inhibition to 30–60% (e.g., a 50%
inhibitory concentration (IC50) value of 82 nM against VEGFR3
(Table 1). Addition of methyl and phenyl group substituents on
the pyrrole ring of 15, leading to 16, reduced the inhibitory activity.
The furan-2-ylmethylidine at the 3-position, 17, did not inhibit ki-
nases. Interestingly, an allylic substituent at the 4-positon of
phenylureido moiety (compound 18) was tolerated (compare with
15). The two analogs 19 and 20 with an arylureido moiety at the 7-
position of the 2-oxindole did not inhibit the kinases.
Our attention was drawn by the arylureido substitution at the 6-
position of the oxindole. We first investigated derivatives with an
arylmethylidene group at the 3-position, 21–27. In general, this
series of compounds showed better kinase inhibition than the
5-arylureido-substituted analogs 13 and 14. For most of them, how-
ever, inhibition was <50% (at 0.1 lV). One exception was the vanillin
In view of compound 35 possessing potent inhibitory activity
against both the VEGFR (KDR and VEGFR3) and PDGFR (c-KIT and
FLT3) families, it was subjected to screening for the percent inhibi-
derivative 24, which inhibited c-KIT by 87% and FLT3 by 71%. Deriv-
ative 27, with a 2,3,4-trimethoxyphenyl methylidene substituent,
showed selective inhibition of 70% against c-KIT. We thus
investigated derivatives with a heteroarylmethylidene group at
the 3-position of the oxindole, 28–35. A 3-[5-(3-nitrophenyl)-
furan-2-yl]-methylidene (28–30) or 3-pyridyl (31 and 32) moiety
did not enhance inhibition compared with the arylmethylidene
tion of each kinase at a concentration of 10 lM against a panel of
19 kinases including Polo-like kinases. Compound 35 was found
to maintain some specificity, as it did not inhibit other 14 protein
kinases tested such as AKT, RAF, EGFR, PLK, and MEK (Table 3). In
accord to the inhibitory activity on PDGFR (c-KIT and FLT3), com-
derivatives. Interestingly, 0.1 lM of 33, with a thiophen-3-ylmethy-
pound 35 showed 89% and 80% inhibition on PDGFR
respectively. In addition, compound 35 exhibited high potency on
aurora kinases. At a concentration of 10 M, compound 35 almost
a and PDGFRb,
lidene moiety, inhibited c-KIT and FLT3 by ꢀ80%, which is compara-
ble with sunitinib (1a). Derivatives 34 and 35 each contained a
pyrrolylmethylidene group. Compound 34, with a 3,5-dimethyl-4-
phenyl-substituted pyrrole moiety, did not show good inhibition
l
totally inhibited the activity of Aurora A and Aurora B (Table 3).
IC50 values of 35 for Aurora A and Aurora B were 2.1 nM and
1.2 nM, respectively. As a result, compound 35 is a potent inhibitor
which inhibits Aurora kinases as well as tyrosine kinases that drive
tumor vascularization, including VEGFR (KDR and VEGFR3) and
PDGFR (c-KIT and FLT3) families.
of the kinases, as was the case for 16. Surprisingly, 0.1 lM of com-
pound 35, with a 4-hydroxycarbonyl-3-methylpyrrole moiety,
showed near-complete inhibition of FLT3 and KDR. Also, 35 showed
52% of inhibition against VEGFR3, the best inhibitory activity against
VEGFR3 among all the compounds. The IC50 values of 35 for FLT3,
KDR, and VEGFR3 were 0.5 nM, 0.5 nM, and 0.8 nM, respectively;
these values are much better than those for sunitinib (1a) (Table 2).
The results presented above demonstrated the importance of
the arylureido and pyrrol-2-ylmethylidene groups at positions 6
and 3, respectively, on the indolin-2-one skeleton. Therefore, we
focused on the optimization of the pyrrole ring together with the
6-arylureido moiety. To better understand the SARs, we synthe-
sized compounds 36–53 and tested them for kinase inhibition
(Table 2). The parent compound 36, with no substitution on the
pyrrole or phenyl ring, exhibited high potency with IC50 values of
5.1 nM, 2.3 nM, <1 nM, and 22.4 nM for c-KIT, FLT3, KDR, and VEG-
FR3, respectively. To explore the role of the 6-arylureido moiety,
methoxy, 3,4-dimethoxy, methyl, chloro, phenoxy, phenyl, and
even naphth-2-yl substituents were introduced on the phenyl ring
(37–43). Derivatives 37–43 exhibited selective potency with IC50
values of ꢀ1 nM for FLT3. Substitution on the phenylureido moiety
was well tolerated. Even a bulky group such as naphthalene (com-
pound 43) did not affect inhibition. These results suggested that
the kinases contain a large hydrophobic binding region that inter-
acts with the arylureido moiety of the compounds. In general, com-
pounds 37–43, containing substituents on the phenyl ring of the
3.2. Docking model of 36 to KDR
To better understand the SAR observed, inhibitor 36 was docked
into the active site of KDR in its inactive conformation by molecu-
lar modeling (Fig. 2). The oxindole core mimics the adenine of ATP
in its interaction with the hinge region of KDR, and the phenylure-
ido moiety extends into the hydrophobic pocket of the kinase do-
main. Two hydrogen bonds are formed by the NH group of the
oxindole to Glu917 and the C@O of the oxindole core to Cys919.
Importantly, the nitrogen atom of the pyrrole is located very close
(2.89 Å) to the NH of Cys919. The phenyl group of the phenylurea
unit of 36 accesses the hydrophobic pocket adjacent to ATP-bind-
ing site. The urea unit is nicely bound to the region with the car-
bonyl group situated within H-bonding distance to Asp1046, and
one urea NH forms an H-bond with Glu885.
3.3. In vitro cellular activity
Initially, the potent kinase inhibitors 36, 37, 44, and 45 were
tested for their cytotoxicity toward four human cell lines: MV4-

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4677
Table 1
Inhibition of kinase activity by arylureido-substituted 3-aryl/heteroarylmethylideneindolin-2-one derivatives 13–35a
Aryl/Heteroaryl
O
R
O
N
N
N
H
H
H
13 - 35
b
c
Compd
R
Aryl/heteroaryl
% Inhibition at 0.1 lM
c-KIT
90.1
FLT3
78.8
KDR
74.6
VEGFR3
1a
13
—
5
—
—
—
81.3
12
Cl
E
7.8
2.4
0
0
0
14
15
5
5
OCH3
H
E
Z
12.1
0
30.1
18.9
59.8
5
41.9
15.1
48d
N
H
H3C
Ph
CH3
16
5
H
Z
35
N
H
NO2
17
18
5
5
OCH3
E
Z
—
16.7
42.5
1.7
48
nd
nd
O
CH2CH@CH2
2013
N
H
H3C
COOH
19
20
7
7
OCH3
OCH3
Z
Z
0.9
8
1.1
0
0
nd
nd
CH3
N
H
6.5
S
Cl
21
22
23
6
6
6
H
H
H
E
Z
Z
2.1
26.8
15.9
28.5
11.5
11.3
0
23
0
Cl
NO2
17.5
32.5
OCH3
nd
OH
OCH3
24
25
26
27
28
29
6
6
6
6
6
6
OCH3
OCH3
OCH3
OCH3
H
Z
E
E
E
E
E
86.7
nd
71.4
2.5
5.0
nd
0
nd
nd
42
15
0
OH
OCH3
Cl
5.8
70.2
0
50.3
35.5
19.2
22.9
Cl
H3CO
OCH3
1.6
0
OCH3
NO2
NO2
NO2
O
Cl
50
6.5
20
O
O
30
31
6
6
OCH3
OCH3
E
Z
32.7
42.4
49.3
36.2
0
0
N
1.0
33
(continued on next page)

4678
R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
Table 1 (continued)
b
c
Compd
R
Aryl/heteroaryl
% Inhibition at 0.1 lM
c-KIT
FLT3
KDR
VEGFR3
4
32
33
6
6
OCH3
OCH3
Z
Z
17.8
82.4
18.1
3.6
N
S
76.8
17.5
nd
35.7
0
H3C
H3C
Ph
CH3
COOH
34
6
6
OCH3
OCH3
Z
Z
23.0
nd
7.4
N
H
35
96.5
99.3
52
N
H
a
The values represent the percentage of inhibition of kinases at 0.1
Position of ureido group at the indolin-2-one.
Configuration of the methylidene group.
lM concentration of the compounds. nd: not determined.
b
c
d
IC50 value of 82 nM for compound 15 against VEGFR3 was determined for seven concentrations and in duplicate.
Table 2
Kinase inhibitory activity of 6-arylureido-3-pyrrolylmethylideneindolin-2-ones 35–54a
R1
R2
R3
R4
N
H
O
O
N
N
N
H
H
35 - 54
H
Compd
R1
R2
R3
R4
IC50 (nM)
KDR
c-KIT
FLT3
VEGFR3
1a
MLN 518
2
—
—
—
CH3
H
H
H
H
H
—
—
—
CO2H
H
H
H
H
H
—
—
—
H
H
H
H
H
H
H
H
—
—
—
OCH3
H
OCH3
3,4-(OCH3)2
CH3
Cl
13.1
nd
nd
3.1
5.1
1.5
nd
14.4
9
6.5
86.5
nd
22.6
2070
179.7
0.5
8.9
nd
3.0
0.8
22.4
12.8
92.0
146.8
8.7
35
36
37
38
39
40
41
42
0.5
2.3
1.4
1.2
0.9
1.9
0.8
0.7
<1
30.6
27.0
271.9
13.7
31.5
20.4
H
H
H
H
OPh
Ph
16.2
7.2
68
88
43
H
H
H
nd
1.5
22.1
13
44
45
46
47
48
49
50
51
52
53
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CO2H
CO2H
CO2H
CO2H
CH2CO2H
CH2CO2H
(CH2)2CO2H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CO2H
CO2H
CO2H
H
6.0
3.3
nd
nd
nd
nd
4.5
nd
nd
nd
0.2
14.9
7.3
97.8
51.2
4.0
7.6
3.4
10.2
7.9
0.9
4.7
10
5.8
32.9
39
42
34
OCH3
CH3
Cl
H
OCH3
OCH3
H
Cl
OCH3
11
13
2.7
>1
>1
>1
l
l
l
M
M
M
>1
l
l
l
M
M
M
>1
>1
>1
l
l
l
M
M
M
H
H
>1
>1
54
OCH3
nd
17.3
nd
>1
l
M
COOH
S
a
Value was determined for seven concentrations and in triplicate. nd: not determined.
11 (myeloid leukemia cells), HepG2 (liver cancer cells), COLO 205
(colon carcinoma cells), and H 460 (lung cancer cells). As shown
in Table 4, all four compounds were highly cytotoxic for MV4-11,
with IC50 values in nanomolar range—comparable with sunitinib
(1a). However, 36, 37, 44, and 45 only showed moderate cytotox-
icity against lines HepG2, COLO 205, and H460. These kinase inhib-
itors exhibited high selective cytotoxicity against acute leukemia
cells. Thus, all the other potent multikinase inhibitors were sub-
jected to the cytotoxicity assay with various acute leukemia cell
lines including MV4-11, MOLM-13, RS4-11, THP-1, U-937, and
Kasumi-1. Interestingly, 6-arylureido-3-pyrrol-2-ylmethylidene-
2-oxindole derivatives 38–43 showed high potency with IC50
values in the range of 1–9 nM for the acute monocytic leukemia
cell lines MV4-11 and MOLM-13 (Table 5). One key structural

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4679
Table 3
Kinase inhibitory activity of 35 against a panel of 19 Kinases
Ma
Kinase
% Inhibition at 10
l
Ma
IC50 (nM)
b
Kinase
% Inhibition at 10
l
AKT1
AKT2
AKT3
ARAF
BRAF
RAF1
0
0
0
0
1.5
0
Aurora A
Aurora B
Aurora C
97.2
98.5
84.2
88.5
79.6
0
2.09
1.19
PDGFR
a
0.98
PDGFRb
PLK1
EGFR
IGF-1R
TIE2/TEK
1.9
0
25.5
PLK2
PLK3
MEK1
MEK2
0
3.5
0.9
0
a
Percent inhibitions were determined in relative to DMSO controls. Compounds were tested in single dose duplicate at a concentration of 10
l
M.
b
Compound 35 and control compound staurosporine were tested in 10-dose IC50 mode with threefold serial dilution starting at 10
lM ATP.
lM. Reactions were carried out at
20
Table 5
Cytotoxicity of compounds against various leukemia cancer cellsa
Compd
IC50
(
lM)
MV4-11
MOLM-13
RS4-11
THP-1
U-937
Kasumi-1
1a
MLN 518
2
35
36
38
39
40
41
42
43
44
45
46
47
48
49
0.009
0.119
0.015
0.017
0.028
0.002
0.003
0.009
0.001
0.002
0.002
0.043
0.015
0.008
0.009
1
nd
0.081
nd
0.230
nd
0.002
0.002
nd
0.002
0.003
0.003
nd
1–5
>5
>5
0.213
>5
>5
0.1
1–5
1
1
1
1.253
>5
0.112
0.123
0.929
>5
0.5–1
1–5
0.310
>5
>5
>5
>5
1–5
>5
nd
0.1
1.310
nd
nd
nd
>5
0.016
0.022
0.015
nd
0.12
nd
0.022
nd
nd
nd
nd
nd
nd
>5
0.204
0.227
1–5
0.1–0.5
0.1–0.5
0.346
>5
>5
0.1
0.1
1–5
nd
>5
0.035
0.038
1–5
nd
0.097
>5
nd
nd
nd
>5
nd
>5
nd
nd
nd
0.019
nd
Figure 2. Model of compound 36 bound to the inactive conformation of KDR
kinase. Docking simulation was performed using the program GOLD and the crystal
structure of KDR kinase (PDB code 2QU5). Hydrogen bonds in magenta-colored
dashed lines are shown between the oxindole core NH and carbonyl of Glu919,
between the oxindole core carbonyl and the Cys919 NH, between the urea carbonyl
and Asp1046 NH, and between the urea NH and Glu885 carboxylate.
0.030
0.008
>5
>5
0.010
>5
50
54
1
>5
>5
a
Value was determined for seven concentrations and in duplicate. nd: not
determined.
Table 4
C-50 methyl group on the pyrrole ring did not affect cytotoxicity
(35 vs 45). Increasing the length of carboxy group at the 40-position
led to a twofold decrease in activity (48 vs 44, 49 vs 45). Interest-
ingly, further elongation by one carbon restored cytotoxicity (50)
against MV4-11. The same trend was found for the MOLM-13 line.
In general, compounds 35–50 exhibited moderate potency against
RS4-11 and THP-1 (except 50, IC50 = 10 nM). Compound 39 had
moderate cytotoxicity against U-937 and Kasumi-1 (IC50 of 0.1
Cytotoxicity of derivatives 36, 37, 44, and 45 against various cancer cellsa
Compd
IC50
(l
M)
COLO 205
>4
1.68
MV4-11
HepG2
H 460
1a
36
37
44
45
0.009
0.028
< 0.05
0.043
0.015
3.81
4.01
3.46
0.52
3.74
>4
3.28
3.01
2.83
3.60
1.4
6.15
3.23
and 0.02 lM, respectively), and 49, which had an IC50 of 0.02 lM
against Kasumi-1. Replacement of the pyrrole moiety with thio-
a
Values were determined for seven concentrations and in duplicate.
phene (54) caused a complete loss of cytotoxicity.
Compound 36 was assessed for its cell permeability and inhibi-
tion of cellular phosphorylation by western blot analysis. Exposure
of Kasumi-1 cells to 36 (0.01–1 lmol/L) for 45 min blocked auto-
phosphorylation of c-KIT in a dose-dependent manner (data not
shown). Additionally, autophosphorylation of FLT3 in RS4-11 cells
and KDR in human umbilical vein endothelial (HUVEC) cells was
feature is that 38–43 lack substituents on the pyrrole moiety. Sub-
stituents on the arylureido group were well tolerated; compounds
38 and 41–43, with a bulky substituent on the arylureido side, all
retained high potency with IC50 values in the range of 1–3 nM for
MV4-11 and MOLM-13. Interestingly, 36, with no substituent on
the arylureido group, showed 10-fold weaker cytotoxicity in this
series of compounds. The substituent on arylureido moiety might
have a hydrophobic effect. Introduction of 30,50-dimethyl and 40-
carboxylate substituents on the pyrrole ring (44) decreased cyto-
toxicity by 1.5-fold against MV4-11 compared with 36. Again,
methoxy (45), methyl (46), and chloro (47) substituents at the 4-
position of the arylureido moiety increased the cytotoxicity against
MV4-11 in this series of compounds (Table 5). It appeared that the
also inhibited by 36 (0.01–1 lmol/L, 45 min). Furthermore, 36
showed substantial ability to enter cells and to compete with
ATP for binding to kinases (data not shown); each of these attri-
butes could potentially contribute to the efficacy of this compound
for the treatment of tumor cells expressing c-KIT, FLT3, and KDR.
As shown in Table 6, cellular kinase phosphorylation was signifi-
cantly inhibited at low concentrations of 36 (IC50 values <10 nM).
Taken together, the in vitro kinase and cytotoxicity results demon-

4680
R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
Table 6
4. Conclusion
Cellular kinase phosphorylation inhibition of 36a
Compd
IC50 (nM)
We developed a new series of 6-arylureido-3-pyrrol-2-yl-indo-
lin-2-one derivatives as multitargeted RTK inhibitors that potently
inhibit members of both the VEGFR and PDGFR families, each of
which plays a major role in angiogenesis. SAR studies of these com-
pounds revealed that the arylureido moiety at the 6-position plays
a key role in kinase inhibition. Incorporation of a pyrrolylmethylid-
ene moiety at the 3-position generated a series of compounds with
potent kinase inhibitory activity and cytotoxicity with IC50 values
in the nanomolar range. Compound 35 is a potent inhibitor against
tyrosine kinases as well as Aurora kinases. Compound 36 demon-
strated significant in vivo efficacy for inhibition of tumor growth.
Results of a virtual docking study of compound 36 to the enzyme
KDR suggested that this compound binds in the ATP-binding pock-
et, with the oxindole core forming a bidentate H-bonding interac-
tion with the hinge region of the enzyme together with the
arylureido group occupying the hydrophobic pocket.
KDR
<1
c-KIT
<10
FLT3
<10
PDGFRb
36
230
a
Each IC50 determination was performed by Western blot analysis.
strate that 36 is a potent and multi-RTK inhibitor that functions to
inhibit kinase phosphorylation both in vitro and in cells.
3.4. Pharmacokinetic studies and in vivo efficacy of 36
Based on the outstanding cytotoxicity of the 6-arylureido-pyrr-
olyl-methylidene-2-oxindole derivatives against acute myeloid
leukemia cell lines, the pharmacokinetic profiles and antitumor
activity of compound 36 was evaluated. To ascertain if the
in vitro effects of 36 correlated with tumor growth inhibition, the
in vivo efficacy of 36 was examined against MV4-11 tumor xeno-
grafts in BALB/c nude mice. Mice were implanted subcutaneously
with MV4-11 tumor cells, and 36 dosing (20 mg/kg per day for se-
ven days) was initiated at post-implantation day 14 when tumor
size was 271 22 mm.3 As shown in Figure 3, MV4-11 tumor
growth was halted, and the overall decrease in tumor volume at
the end of the experiment was 46 2.3%. The average body weight
loss was about 10%, and no animal died during the treatment per-
iod. A pharmacokinetic analysis of 36 was undertaken in rats with
a single intravenous or oral dose (Table 7). Compound 36, when
administered intravenously, showed low-volume distribution and
slow clearance, with a half-life of ꢀ1.1 h. The poor oral pharmaco-
kinetic profile with low bioavailability (2.2%) might be due to the
low solubility of 36.
5. Experimental section
5.1. Gemera; experimental procedures
Melting points were measured using open capillaries on an
electrothermal apparatus and were uncorrected. NMR spectra
were recorded on Bruker AMX series spectrometers at 298 K. Mass
spectra were recorded on a Finnigan MAT TSQ-7000 mass spec-
trometer. Elemental analyses for C, H, N and S were carried out
on a Heraeus VariaEL-III elemental analyzer. The thin layer chro-
matographic analyses were performed using precoated silica gel
plates (60 F254, Merck), and the spots were examined under UV
light. Column chromatography was carried out on Merck Silica
Gel 60 (70–230 mesh). The purity of compounds were tested to
be P95% by LC/MS by HPLC.
5.2. 7-Amino-2-oxindole (7)
A solution of 7-nitrooxindole (3 mmol) and 10% Pd–C (30 wt %)
in ethanol (50 mL) was degassed three times at room temperature.
The mixture was stirred under H2 atmosphere for 4 h. The catalyst
was then removed by filtration over Celite. The filtrate was concen-
trated in vacuo to give crude product, which was washed with
diethyl ether to afford pure compound 7. Yield 77%; mp 228–
229 °C (lit.33 247–249 °C). 1H NMR (DMSO-d6, 200 MHz): d 3.40
(s, 2H), 4.80 (s, 2H), 6.44–6.50 (m, 2H), 6.69 (t, J = 8.0 Hz, 1H),
9.89 (s, 1H).
5.3. 2,4-Diaminophenylacetic acid
Figure 3. Antitumor growth effects of inhibitor 36 in MV4-11 subcutaneous (sc)
xenograft tumor models in BALB/c nude mice. Treatment with 36 at dose of 20 mg/
kg by oral gavage (po) once-daily (qd) was initiated on day 14 and continued to day
20 after sc implantation of MV4-11 human leukemia cells. No mice died during the
treatment. Tumor volumes are expressed as means SEM (n = 4).
A solution of 2,4-dinitrophenylacetic acid (4 mmol) and 10%
Pd–C (10 wt %) in methanol (50 mL) was hydrogenated as in the
method above to give the brown solid 2,4-diamino phenyl acetic
acid. It was stored in inert atmosphere and used immediately for
the synthesis of 6-amino-2-oxindole without further purification.
Table 7
Pharmacokinetic profiles of multikinase inhibitor 36 in Sprague-Dawley ratsa
5.4. 6-Amino-2-oxindole (9)
Parameters
iv (0.5 mg/kg)
po (10 mg/kg)
To a solution of 2,4-diamino phenyl acetic acid (4 mmol) in
methanol (30 mL), 5 mL aqueous 2 N HCl was added. The mixture
was heated at reflux for 4 h. After the removal of methanol, the ob-
tained mixture was adjusted to pH 10 with 10 N aqueous NaOH.
Product was extracted in ethyl acetate, and the organic layer was
washed with water. After drying with anhydrous Na2SO4, ethyl
acetate was removed in vacuo to obtain the desired product. Yield
65%; mp 193 °C (lit.34 194–195 °C). 1H NMR (200 MHz, DMSO-d6):
CL (L/h/kg)
T1/2 (h)
Vd (L/kg)
0.4
1.1
0.5
1327.7
NA
13.7
4.1
79.5
590.8
211.1
0.8
AUC0–24 (h ng/mL)
h
Cmax (ng/mL)
Tmax (h)
F (%)
NA
NA
2.2
a
NA: Not available.

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4681
d 3.23 (s, 2H), 5.01 (s, 2H), 6.08–6.11 (m, 2H), 6.79 (d, J = 8.0 Hz,
5.5.7. 1-(4-Methoxyphenyl)-3-(2-oxo-2,3-dihydro-1H-indol-6-
yl)urea (12b)
1H), 10.09 (s, 1H). 13C NMR (50 MHz, DMSO-d6): 36.0, 96.9,
107.5, 112.9, 125.4, 145.2, 149.4, 178.2 ppm.
Yield 87%; mp 290–292 °C. 1H NMR (200 MHz, DMSO-d6): d
3.32 (s, 2H), 3.70 (s, 3H), 6.77 (dd, J = 1.8, 7.7 Hz, 1H), 6.85 (d,
J = 9.2 Hz, 2H), 7.05 (d, J = 8.0 Hz, 1H), 7.23 (br, 1H), 7.33 (d,
J = 9.0 Hz, 2H), 8.39 (s, 1H), 8.57 (s, 1H), 10.30 (s, 1H). 13C NMR
(50 MHz, DMSO-d6): 35.9, 55.7, 100.5, 111.1, 114.5, 119.0, 120.6,
125.0, 133.2, 140.0, 144.6, 153.2, 155.0, 177.4 ppm. Anal.
(C16H15N3O3ꢁ0.3H2O) C, H, N.
5.5. General procedure for the synthesis of 5-, 6-, and 7-aryl-
ureidoindolin-2-one derivatives 10–12
To a solution of 6, 7, or 9 (1 mmol) in 10 mL dichloromethane/
methanol (90:10), the appropriate aromatic/allyl isocyanate
(1.1 mmol) was added slowly at 10 °C. The mixture was stirred at
the room temperature for 4 h. Solid product 10–12 was separated
out from the mixture. It was filtered, washed with dichlorometh-
ane, and air dried.
5.5.8. 1-(3,4-Dimethoxyphenyl)-3-(2-oxo-2,3-dihydro-1H-indol-
6-yl)urea (12c)
Yield 83%; mp 270–271 °C. 1H NMR (400 MHz, DMSO-d6): d
3.37 (s, 2H), 3.69 (s, 3H), 3.72 (s, 3H), 6.76 (dd, J = 2.4, 8.0 Hz,
1H), 6.81–6.86 (m, 2H), 7.05 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 2.4 Hz,
1H), 7.27 (d, J = 2.4 Hz, 1H), 8.41 (s, 1H), 8.55 (s, 1H), 10.28 (s,
1H). 13C NMR (100 MHz, DMSO-d6): 35.9, 55.9, 56.4, 100.5, 104.4,
110.7, 111.1, 113.1, 119.0, 124.9, 133.8, 139.9, 144.5, 144.6,
149.3, 153.1, 177.4 ppm. Anal. (C17H17N3O1ꢁ0.1H2O) C, H, N.
5.5.1. 1-(2-Oxo-2,3-dihydro-1H-indol-5-yl)-3-phenylurea (10a)
Yield 94%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.40
(s, 2H), 6.71 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 6.0 Hz, 1H), 7.16 (dd,
J = 2.0, 8.0 Hz, 1H), 7.25 (t, J = 8.0, 8.2 Hz, 2H), 7.36–7.44 (m,
3H), 8.47 (s, 1H), 8.58 (s, 1H), 10.24 (s, 1H). 13C NMR (50 MHz,
DMSO-d6): d 37.0, 56.0, 109.8, 114.8, 116.9, 118.6, 120.8, 127.1,
133.8, 134.8, 139.1, 153.8, 155.2, 177.1 ppm. Anal. (C15H13N3O2)
C, H, N.
5.5.9. 1-(2-Oxo-2,3-dihydro-1H-indol-6-yl)-3-p-tolylurea (12d)
Yield 89%; mp 271–272 °C. 1H NMR (200 MHz, DMSO-d6): d
2.23 (s, 3H), 3.54 (s, 2H), 6.76 (dd, J = 2.0, 8.0 Hz, 1H), 7.04–7.08
(m, 3H), 7.24 (d, J = 1.6 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 8.47 (s,
1H), 8.60 (s, 1H), 10.32 (s, 1H). Anal. (C6H15N3O2ꢁ0.2H2O) C, H, N.
5.5.2. 1-(4-Chlorophenyl)-3-(2-oxo-2,3-dihydro-1H-indol-5-yl)-
urea (10b)
Yield 99%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.44 (s,
2H), 6.70 (d, J = 8.0 Hz, 1H), 7.16 (dd, J = 2.0, 8.0 Hz, 1H), 7.26 (s,
1H), 7.32 (d, J = 10.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 8.56 (s, 1H),
8.78 (s, 1H), 10.23 (s, 1H). 13C NMR (50 MHz, DMSO-d6): d 37.0,
109.9, 117.1, 118.9, 120.4, 126.0, 127.1, 129.5, 134.3, 139.4,
139.8, 153.5, 177.1 ppm. Anal. (C15H12ClN3O2) C, H, N.
5.5.10. 1-(4-Chlorophenyl)-3-(2-oxo-2,3-dihydro-1H-indol-6-
yl)urea (12e)
Yield 92%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.38 (s,
2H), 6.79 (dd, J = 1.9, 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.21 (d,
J = 1.8 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H),
8.70 (s, 1H), 8.74 (s, 1H), 10.33 (s, 1H). Anal. (C15H12ClN3O2ꢁ0.3H2O)
C, H, N.
5.5.3. 1-(4-Methoxyphenyl)-3-(2-oxo-2,3-dihydro-1H-indol-5-
yl)-urea (10c)
Yield 95%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.44 (s,
2H), 3.70 (s, 3H), 6.70 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H),
7.14 (dd, J = 2.0, 8.0 Hz, 1H), 7.32 (d, J = 9.1 Hz, 3H), 8.36 (s, 2H),
10.22 (s, 1H). 13C NMR (50 MHz, DMSO-d6): d 37.0, 56.0, 109.8,
114.8, 116.9, 118.6, 120.8, 127.1, 133.8, 134.8, 153.8, 155.2,
177.1 ppm. Anal. (C16H15N3O3) C, H, N.
5.5.11. 1-(2-Oxo-2,3-dihydro-1H-indol-6-yl)-3-(4-phenoxy-
phenyl)urea (12f)
Yield 88%; mp 294–295 °C. 1H NMR (200 MHz, DMSO-d6): d
3.38 (s, 2H), 6.78 (dd, J = 1.9, 8.0 Hz, 1H), 6.92–6.99 (m, 4H),
7.04–7.11 (m, 2H), 7.25 (d, J = 1.8 Hz, 1H), 7.31–7.39 (m, 2H),
7.45 (d, J = 8.9 Hz, 2H), 8.61 (s, 1H), 8.64 (s, 1H), 10.33 (s, 1H). Anal.
(C21H17N3O3) C, H, N.
5.5.4. 1-Allyl-3-(2-oxo-2,3-dihydro-1H-indol-5-yl)urea (10d)
Yield 93%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.41 (s,
2H), 3.69 (d, J = 6.0 Hz, 2H), 5.01–5.18 (m, 2H), 5.75–5.94 (m, 1H),
6.14 (t, J = 4.0 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.0 Hz,
1H), 7.31 (s, 1H), 8.29 (s, 1H), 10.19 (s, 1H). 13C NMR (50 MHz,
DMSO-d6): 37.0, 42.4, 109.8, 115.4, 116.6, 118.2, 127.0, 135.4,
137.3, 138.7, 156.2, 177.1 ppm. Anal. (C12H13N3O2) C, H, N.
5.5.12. 1-Biphenyl-4yl-3-(2-oxo-2,3-dihydro-1H-indol-6-yl)urea
(12g)
Yield 87%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.38 (s,
2H), 6.81 (dd, J = 1.7, 8.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.26 (d,
J = 1.3 Hz, 1H), 7.31 (d, J = 7.0 Hz, 1H), 7.43 (t, J = 7.3 Hz, 2H),
7.50–7.64 (m, 6H), 8.70 (s, 1H), 8.71 (s, 1H), 10.35 (s, 1H). Anal.
(C21H17N3O2) C, H, N.
5.5.5. 1-(4-Methoxyphenyl)-3-(2-oxo-2,3-dihydro-1H-indol-7-
yl)urea (11)
Yield 83%; mp 298–299 °C. 1H NMR (200 MHz, DMSO-d6): d
3.51 (s, 2H), 3.70 (s, 3H), 6.83–6.97 (m, 4H), 7.23 (d, J = 7.6 Hz,
1H), 7.39 (d, J = 8.6 Hz, 2H), 8.10 (s, 1H), 8.58 (s, 1H), 10.05 (s,
1H). 13C NMR (50 MHz, DMSO-d6): 36.9, 56.0, 114.8, 120.5, 121.1,
122.2, 122.6, 122.7, 127.5, 133.8, 136.5, 154.0, 155.3, 176.9 ppm.
Anal. (C16H15N3O3ꢁ0.5H2O) C, H, N.
5.5.13. 1-Naphthalen-2-yl-3-(2-oxo-2,3-dihydro-1H-indol-6-yl)-
urea (12h)
Yield 85%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.39 (s,
2H), 6.81 (dd, J = 1.9, 8.0 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H), 7.29–7.48
(m, 4H), 7.75–7.84 (m, 3H), 8.09 (s, 1H), 8.75 (s, 1H), 8.82 (s, 1H),
10.36 (s, 1H). Anal. (C19H15N3O2ꢁ0.3H2O) C, H, N.
5.6. General procedure for the synthesis of arylureido-substi-
tuted 3-arylmethylidene-2-oxindole derivatives 13–54
5.5.6. 1-(2-Oxo-2,3-dihydro-1H-indol-6-yl)-3-phenylurea (12a)
Yield 85%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 3.34 (s,
2H), 6.76 (dd, J = 2.0, 7.8 Hz, 1H), 6.93 (t, J = 7.2, 7.6 Hz, 1H), 7.04
(d, J = 8.0 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 7.6 Hz, 2H),
7.40 (d, J = 7.6 Hz, 2H), 8.54 (s, 1H), 8.60 (s, 1H), 10.28 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): 35.9, 100.5, 110.6, 111.2, 115.1, 118.7,
A catalytic amount of pyrrolidine (0.01 mmol) was added into a
solution of 5-, 6-, or 7-arylureidoindolin-2-one derivatives 10–12
(1 mmol) and aryl/heteroaryl aldehyde (1.1 mmol) in ethanol
(5 mL). The mixture was heated at reflux for 1.5–5 h. After cooling
to room temperature, crude product precipitated from the
118.8, 129.3, 139.7, 140.2, 153.0, 158.2, 177.4 ppm. Anal. (C15H13
-
N3O2ꢁ0.6H2O) C, H, N.

4682
R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
solution and was collected by filtration, washed with ethanol, and
air dried.
4H), 7.40–7.47 (m, 4H), 8.62 (s, 1H, NH), 9.06 (s, 1H), 10.54 (s,
1H), 13.46 (s, 1H). Anal. (C25H24N4O5ꢁ1.1H2O) C, H, N.
5.6.1. (E)-1-[3-Benzylidene-2-oxo-2,3-dihydro-1H-indol-5-yl]-3-
(4-chlorophenyl)urea (13)
5.6.8. (Z)-1-(4-Methoxyphenyl)-3-(2-oxo-3-thiophen-3-ylmeth-
ylene-2,3-dihydro-1H-indol-7-yl)urea (20)
Yield 55%; mp 273–275 °C. 1H NMR (400 MHz, DMSO-d6): d
6.80 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.2 Hz,
1H), 7.42 (d, J = 9.2 Hz, 2H), 7.48–7.53 (m, 3H), 7.61 (s, 1H), 7.70–
7.73 (m, 3H), 8.46 (s, 1H), 8.65 (s, 1H), 10.47 (s, 1H). 13C NMR
(50 MHz, DMSO-d6): d 110.9, 115.1, 120.5, 121.8, 122.1, 126.0,
128.6, 129.5, 129.7, 130.3, 130.7, 134.1, 135.2, 136.7, 139.0,
139.7, 153.5, 169.6. Anal. (C22H16ClN3O2) C, H, N.
Yield 57%; mp 286–287 °C. 1H NMR (200 MHz, DMSO-d6): d
3.70 (s, 3H), 6.88–6.91 (m, 3H), 7.29 (d, J = 8.0 Hz, 1H), 7.37 (d,
J = 8.0 Hz, 2H), 7.48 (s, 1H), 7.53–7.55 (m, 1H), 7.56 (s, 1H), 7.75–
7.77 (m, 1H), 8.04 (s, 1H), 8.12 (s, 1H), 8.56 (s, 1H), 10.30 (s, 1H).
Anal. (C21H17N3O3Sꢁ0.3H2O) C, H, N, S.
5.6.9. (E)-1-[3-(3,4-Dichlorobenzylidene)-2-oxo-2,3-dihydro-1H
-indol-6-yl]-3-phenyl-urea (21)
5.6.2. (E)-1-[3-Benzylidene-2-oxo-2,3-dihydro-1H-indol-5-yl]-3-
(4-methoxyphenyl)-urea (14)
Yield 80%; mp 268–270 °C. 1H NMR (200 MHz, DMSO-d6): d 6.69
(dd, J = 2.0, 8.0 Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 8.3 Hz,
2H), 7.27 (d, J = 3.4 Hz, 1H), 7.36–7.46 (m, 4H, Ar-H), 7.68 (dd,
J = 1.8, 8.0 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H,), 7.92 (d, J = 1.6 Hz, 1H),
8.69 (s, 1H), 8.94 (s, 1H, NH), 10.61 (s, 1H, NH). MS (ESI) m/z 423
[M]+. Anal. (C22H15N3O2Cl2ꢁ0.3H2O) C, H, N.
Yield 70%; mp 286–287 °C. 1H NMR (200 MHz, DMSO-d6): d
3.69 (s, 3H), 6.76–6.85 (m, 3H), 7.28–7.35 (m, 3H), 7.49–7.52 (m,
3H), 7.60 (s, 1H), 7.71–7.75 (m, 3H), 8.43 (s, 1H), 8.47 (s, 1H),
10.45 (s, 1H). Anal. (C23H19N3O3ꢁ0.3H2O) C, H, N.
5.6.3. (Z)-1-[2-Oxo-3-(1H-pyrrol-2-ylmethylene)-2,3-dihydro-
1H-indol-5-yl]-3-phenyl-urea (15)
Yield 70%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 6.32–
6.36 (m, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.90–6.93 (m, 1H), 6.95 (d,
J = 7.3 Hz, 1H), 7.08 (dd, J = 1.9, 8.3 Hz, 1H), 7.26 (t, J = 8.0 Hz,
2H), 7.34–7.37 (m, 1H), 7.46 (d, J = 7.6 Hz, 2H), 7.65 (s, 1H), 7.81
(d, J = 1.9 Hz, 1H), 8.66 (s, 1H), 8.82 (s, 1H), 10.78 (s, 1H), 13.36
(s, 1H). 13C NMR (50 MHz, DMSO-d6): d 110.5, 111.0, 112.3,
118.0, 119.0, 119.1, 121.4, 122.5, 126.3, 126.5, 127.1, 129.7,
130.4, 134.6, 135.2, 140.8, 153.8, 170.2. MS (ESI) m/z 344 [M]+.
Anal. (C20H16N4O2ꢁ0.1H2O) C, H, N.
5.6.10. (Z)-1-[3-(4-Nitrobenzylidene)-2-oxo-2,3-dihydro-1H-
indol-6-yl]-3-phenylurea (22)
Yield 65%; mp 282–284 °C. 1H NMR (200 MHz, DMSO-d6): d
6.65 (dd, J = 2.0, 8.4 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 7.25 (dd,
J = 7.9, 7.9 Hz, 2H), 7.34–7.43 (m, 5H), 7.91 (d, J = 8.4 Hz, 2H),
8.31 (d, J = 8.8 Hz, 2H), 8.68 (s, 1H), 8.97 (s, 1H), 10.64 (s, 1H).
13C NMR (50 MHz, DMSO-d6): 100.8, 111.4, 114.5, 119.2, 123.0,
124.6, 124.8, 129.7, 130.3, 130.7, 131.3, 140.2, 142.8, 143.4,
145.6, 147.0, 153.0, 169.8 ppm. MS (ESI) m/z 400 [M]+. Anal.
(C22H16N4O4ꢁ 0.2H2O) C, H, N.
5.6.4. (Z)-1-[3-(3,5-Dimethyl-4-phenyl-1H-pyrrol-2-ylmethyl-
ene)-2-oxo-2,3-dihydro-1H-indol-5-yl]-3-phenylurea (16)
Yield 60%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 2.29 (s,
3H), 2.34 (s, 3H), 6.85 (d, J = 8.7 Hz, 1H), 6.93 (t, J = 7.4 Hz, 2H), 7.12
(d, J = 7.5 Hz, 1H), 7.26–7.33 (m, 5H), 7.48–7.53 (m, 4H), 7.79 (s,
1H,), 8.51 (s, 1H), 8.71 (s, 1H), 10.74 (s, 1H), 13.71 (s, 1H). 13C
NMR (50 MHz, DMSO-d6): d 11.2, 13.6, 110.3, 111.0, 114.9, 118.7,
119.0, 122.5, 124.1, 125.9, 127.0, 129.0, 129.2, 129.6, 130.4,
134.2, 134.3, 134.6, 135.5, 140.9, 154.0, 170.4 ppm. Anal.
(C28H24N4O2) C, H, N.
5.6.11. (Z)-1-[3-(4-Hydroxy-3-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-3-phenylurea (23)
Yield 77%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.83 (s,
3H), 6.82 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 8.1, 2.0 Hz, 1H), 6.96 (t,
J = 7.3 Hz, 1H), 7.23–7.31 (m, 3H), 7.42–7.54 (m, 4H), 7.66 (d,
J = 8.4 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H), 8.65 (s, 1H), 8.79 (s, 1H),
9.73 (br, 1H), 10.49 (s, 1H). MS (ESI) m/z 401 [M]+. Anal.
(C23H19N3O4ꢁ 0.2H2O) C, H, N.
5.6.5. (E)-1-(4-Methoxyphenyl)-3-{3-[5-(3-nitrophenyl)-furan-
2-ylmethylene]-2-oxo-2,3-dihydro-1H-indol-5-yl}urea (17)
Yield 95%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.69 (s,
3H), 6.73–6.84 (m, 2H), 7.11–7.17 (m, 2H), 7.35 (s, 1H), 7.46 (d,
J = 1.8 Hz, 1H,), 7.53–7.62 (m, 2H), 8.02 (d, J = 8.0 Hz, 1H), 8.37 (d,
J = 8.0 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.69 (s, 2H), 10.49 (s, 1H).
MS (ESI) m/z 496 [M]+. Anal. (C27H20N4O6ꢁ0.5H2O) C, H, N.
5.6.12. (Z)-1-[3-(4-Hydroxy-3-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-3-(4-methoxyphenyl)urea (24)
Yield 60%; mp 276–279 °C. 1H NMR (200 MHz, DMSO-d6): d 3.70
(s, 3H), 3.83 (s, 3H), 6.80–6.91 (m, 4H), 7.18–7.37 (m, 4H), 7.48–
7.52 (m, 1H), 7.65 (d, J = 8.1 Hz, 1H), 8.47 (s, 1H), 8.61 (d,
J = 2.0 Hz, 1H), 8.72 (s, 1H), 9.85 (br, 1H), 10.48 (s, 1H). MS (ESI)
m/z 431 [M]+. Anal. (C24H21N3O5ꢁ0.65H2O) C, H, N.
5.6.6. (Z)-1-[2-Oxo-3-(1H-pyrrol-2-ylmethylene)-2,3-dihydro-
1H-indol-5-yl]-3-allyl-urea (18)
5.6.13. (E)-1-[3-(3-Methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indol-6-yl]-3-(4-methoxyphenyl)urea (25)
Yield 50%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.72 (d,
J = 6.0 Hz, 2H), 5.03–5.20 (m, 2H), 5.77–5.93 (m, 1H), 6.26–6.28 (m,
1H), 6.33–6.36 (m, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.87–6.89 (m, 1H),
7.02 (d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 7.59 (s, 1H), 7.74 (s, 1H), 8.36 (s,
1H), 10.73 (s, 1H), 13.36 (s, 1H). 13C NMR (50 MHz, DMSO-d6): d
42.4, 110.04, 110.4, 112.2, 115.5, 118.2, 118.6, 121.3, 126.1,
126.4, 130.4, 134.6, 135.5, 137.4, 156.3, 170.1. Anal. (C17H16N4O2ꢁ
0.2H2O) C, H, N.
Yield 50%; mp 273–275 °C. 1H NMR (200 MHz, DMSO-d6): d 3.70
(s, 3H), 3.79 (s, 3H), 6.68 (dd, J = 1.9, 8.6 Hz, 1H), 6.86 (d, J = 8.9 Hz,
2H), 7.01 (dd, J = 2.4, 7.6 Hz, 1H), 7.23–7.49 (m, 8H), 8.50 (s, 1H),
8.82 (s, 1H), 10.55 (s, 1H). MS (ESI) m/z 415 [M]+. Anal.
(C24H21N3O4ꢁ 0.2H2O) C, H, N.
5.6.14. (E)-1-[3-(3,4-Dichlorobenzylidene)-2-oxo-2,3-dihydro-
1H-indol-6-yl]-3-(4-methoxyphenyl)urea (26)
5.6.7. (Z)-(5-{7-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-2,4-dimethyl-1H-pyrrol-3-yl)
acetic acid (19)
Yield 85%; mp 261–263 °C. 1H NMR (400 MHz, DMSO-d6): d
3.70 (s, 3H), 6.68 (dd, J = 2.0, 8.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H),
7.33–7.34 (m, 3H), 7.38 (d, J = 2.0 Hz, 1H), 7.67–7.77 (m, 3H),
7.91 (s, 1H), 8.49 (s, 1H), 8.85 (s, 1H), 10.58 (s, 1H). MS (ESI) m/z
453 [M]+. Anal. (C23H17N3O3Cl2ꢁ0.2H2O) C, H, N.
Yield 90%; mp 277–278 °C. 1H NMR (200 MHz, DMSO-d6): d
2.23 (s, 3H), 2.32 (s, 3H), 3.30 (s, 2H), 3.70 (s, 3H), 6.87–6.93 (m,

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4683
5.6.15. (E)-1-(4-Methoxyphenyl)-3-[2-oxo-3-(2,3,4-trimethoxy-
benzylidene)-2,3-di-hydro-1H-indol-6-yl]urea (27)
5.6.22. (Z)-1-[3-(3,5-Dimethyl-4-phenyl-1H-pyrrol-2-ylmeth-
ylene)-2-oxo-2,3-dihydro-1H-indol-6-yl]-3-(4-methoxyphenyl)-
urea (34)
Yield 78%; mp 262–264 °C. 1H NMR (400 MHz, DMSO-d6): d 3.68
(s, 3H), 3.76 (s, 3H), 3.79 (s, 3H), 3.85 (s, 3H), 6.65 (dd, J = 1.8, 8.6 Hz,
1H), 6.83 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 1H), 7.30–7.33 (m,
3H), 7.38–7.45 (m, 3H), 8.43 (s, 1H), 8.75 (s, 1H), 10.47 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): 55.7, 56.6, 61.0, 61.9, 100.3, 108.3,
110.7, 114.6, 115.0, 120.6, 121.5, 123.4, 124.9, 126.8, 128.6, 132.9,
142.1, 142.3, 144.2, 152.8, 152.9, 155.1, 155.5, 169.9 ppm. MS
(ESI) m/z 475 [M]+. Anal. (C26H25N3O6ꢁ0.2H2O) C, H, N.
Yield 85%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 2.27 (s,
3H), 2.32 (s, 3H), 3.71 (s, 3H), 6.85–6.87 (m, 3H), 7.29–7.32 (m, 4H),
7.35 (d, J = 8.8 Hz, 2H), 7.43 (dd, J = 8.0, 8.0 Hz, 2H), 7.50 (s, 1H),
7.61 (d, J = 8.4 Hz, 1H), 8.41 (s, 1H), 8.64 (s, 1H), 10.77 (s, 1H),
13.49 (s, 1H). MS (ESI) m/z 478 [M]+. Anal. (C29H26N4O3ꢁ0.3H2O)
C, H, N.
5.6.23. (Z)-5-{6-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-4-methyl-1H-pyrrole-3-
carboxylic acid (35)
5.6.16. (E)-1-{3-[5-(3-Nitrophenyl)-furan-2-ylmethylene]-2-
oxo-2,3-dihydro-1H-indol-6-yl}phenylurea (28)
Yield 81%; mp 278–280 °C. 1H NMR (200 MHz, DMSO-d6): d
6.94–6.98 (m, 2H), 7.19 (s, 1H), 7.25–7.36 (m, 3H), 7.45–7.49 (m,
3H), 7.58 (d, J = 3.8 Hz, 1H), 7.83 (dd, J = 8.0, 8.0 Hz, 1H), 8.24 (dd,
J = 2.0, 8.0 Hz, 1H), 8. 33 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.5 Hz,
1H), 8.73 (s, 1H), 8.88 (s, 1H), 9.22 (s, 1H), 10.60 (s, 1H). MS (ESI)
m/z 466 [M]+. Anal. (C26H18N4O5ꢁ0.3H2O) C, H, N.
Yield 79%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 2.56 (s,
3H), 3.70 (s, 3H), 6.83–6.88 (m, 3H), 7.33–7.37 (m, 3H), 7.52 (s, 1H),
7.68 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H), 8.58 (s, 1H), 8.85 (s,
1H), 10.94 (s, 1H), 13.60 (s, 1H). MS (ESI) m/z 432 [M]+. Anal.
(C23H20N4O5ꢁ0.4H2O) C, H, N.
5.6.24. (Z)-1-[2-Oxo-3-(1H-pyrrol-2-ylmethylene)-2,3-dihydro-
1H-indol-6-yl]-3-phenyl-urea (36)
5.6.17. (E)-1-(4-Chlorophenyl)-3-{3-[5-(3-nitrophenyl)-furan-2-
ylmethylene]-2-oxo-2,3-dihydro-1H-indol-6-yl}urea (29)
Yield 90%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 6.96
(dd, J = 1.8, 8.0 Hz, 1H), 7.19 (s, 1H), 7.33 (d, J = 8.9 Hz, 2H), 7.35
(d, J = 3.5 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H),
7.56 (d, J = 3.7 Hz, 1H), 7.82 (dd, J = 8.0, 8.0 Hz, 1H), 8.23 (dd,
J = 1.5, 9.6 Hz, 1H), 8.32 (d, J = 7.8 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H),
8.70 (s, 1H), 8.84 (s, 1H), 9.08 (s, 1H), 10.60 (s, 1H). MS (ESI) m/z
500 [M]+. Anal. (C26H17N4O5Clꢁ0.3H2O) C, H, N.
Yield 86%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 6.31–
6.32 (m, 1H), 6.74–6.76 (m, 1H), 6.86 (dd, J = 1.8, 8.0 Hz, 1H),
6.96 (dd, J = 7.2, 7.2 Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.29–7.31
(m, 1H), 7.35 (d, J = 1.6 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.50 (d,
J = 9.8 Hz, 1H), 7.56 (s, 1H), 8.65 (s, 1H), 8.77 (s, 1H), 10.86 (s,
1H), 13.21 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 100.5, 110.1,
111.6, 111.7, 115.1, 117.8, 118.7, 119.4, 119.8, 122.4, 124.8,
125.4, 129.3, 130.2, 139.6, 140.1, 140.2, 152.9, 170.2 ppm. MS
(ESI) m/z 344 [M]+. Anal. (C20H16N4O2ꢁ0.1H2O) C, H, N.
5.6.18. (E)-1-(4-Methoxyphenyl)-3-{3-[5-(3-nitrophenyl)-furan-
2-ylmethylene]-2-oxo-2,3-dihydro-1H-indol-6-yl}urea (30)
Yield 83%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.71 (s,
3H), 6.88 (d, J = 9.1 Hz, 2H), 6.95 (s, 1H), 7.19 (s, 1H), 7.49 (d,
J = 2.0 Hz, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.83 (dd, J = 7.7, 8.1 Hz,
1H), 8.23–8.41 (m, 3H), 8.52 (s, 1H), 8.73 (s, 1H), 8.99 (s, 1H),
10.59 (s, 1H). MS (ESI) m/z 496 [M]+. Anal. (C27H20N4O6ꢁ0.7H2O)
C, H, N.
5.6.25. (Z)-1-(4-Methoxyphenyl)-3-[2-oxo-3-(1H-pyrrol-2-
ylmethylene)-2,3-dihydro-1H-indol-6-yl]urea (37)
Yield 75%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.70
(s, 3H), 6.30–6.32 (m, 1H), 6.74–6.88 (m, 4H), 7.29–7.37 (m,
4H), 7.48 (d, J = 9.2 Hz, 1H), 7.55 (s, 1H), 8.46 (s, 1H),
8.70 (s, 1H), 10.85 (s, 1H), 13.20 (s, 1H). 13C NMR (50 MHz,
DMSO-d6): 56.0, 100.7, 110.1, 111.8, 112.0, 114.9, 118.2, 119.5,
119.9, 120.1, 120.9, 125.0, 125.7, 130.5, 133.5, 140.2, 140.6,
153.4, 155.4, 170.6 ppm. MS (ESI) m/z 374 [M]+. Anal.
(C21H18N4O3ꢁ0.2H2O) C, H, N.
5.6.19. (Z)-1-(4-Methoxyphenyl)-3-(2-oxo-3-pyridin-4-ylmeth-
ylene-2,3-dihydro-1H-indol-6-yl)urea (31)
Yield 71%; mp 262–264 °C. 1H NMR (200 MHz, DMSO-d6): d
3.70 (s, 3H), 6.66 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 7.31–
7.36 (m, 4H), 7.39 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 6.2 Hz, 2H), 8.50
(s, 1H), 8.68 (d, J = 6.0 Hz, 2H), 8.89 (s, 1H), 10.64 (s, 1H). 13C
NMR (50 MHz, DMSO-d6): 56.0, 109.9, 111.5, 114.4, 114.9, 121.4,
124.1, 124.5, 129.7, 130.9, 132.9, 143.3, 143.6, 145.2, 150.8,
153.3, 155.6, 169.8 ppm. MS (ESI) m/z 386 [M]+. Anal. (C22H18N4O3ꢁ
0.4H2O) C, H, N.
5.6.26. (Z)-1-(3,4-Dimethoxyphenyl)-3-[2-oxo-3-(1H-pyrrol-2-
ylmethylene)-2,3-dihydro-1H-indol-6-yl]urea (38)
Yield 97%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 3.70 (s,
3H), 3.73 (s, 3H), 6.30–6.32 (m, 1H), 6.74–6.75 (m, 1H), 6.83–6.85
(m, 3H), 7.22 (d, J = 2.0 Hz, 1H), 7.29 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H),
7.48 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 8.46 (s, 1H), 8.66 (s, 1H), 10.80
(s, 1H), 13.19 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 55.9, 56.4,
100.5, 104.5, 110.7, 111.6, 111.7, 113.1, 117.8, 119.3, 119.6,
119.7, 124.7, 125.3, 130.2, 133.8, 139.8, 140.2, 144.6, 149.3,
153.0, 170.2 ppm. MS (ESI) m/z 404 [M]+. Anal. (C22H20N4O4ꢁ
0.2H2O) C, H, N.
5.6.20. (Z)-1-(4-Methoxyphenyl)-3-(2-oxo-3-pyridin-2-ylmeth-
ylene-2,3-dihydro-1H-indol-6-yl)urea (32)
Yield 73%; mp 292–294 °C. 1H NMR (200 MHz, DMSO-d6): d .71
(s, 3H), 6.78 (dd, J = 2.0, 8.0 Hz, 1H), 6.87 (d, J = 9.0 Hz, 2H), 7.33–
7.45 (m, 5H), 7.79 (d, J = 7.7 Hz, 1H), 7.91 (td, J = 1.8, 8 Hz, 1H),
8.53 (s, 1H), 8.84–8.88 (m, 2H), 8.92 (s, 1H), 10.56 (s, 1H). MS
(ESI) m/z 386 [M]+. Anal. (C22H18N4O3ꢁ0.4H2O) C, H, N.
5.6.27. (Z)-1-[2-Oxo-3-(1H-pyrrol-2-ylmethylene)-2,3-dihydro-
1H-indol-6-yl]-3-(p-tolyl)-urea (39)
Yield 87%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 2.21
(s, 3H), 6.27–6.30 (m, 1H), 6.72–6.73 (m, 1H), 6.83 (dd, J = 2.2,
8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 7.30 (d,
J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.52 (s, 1H), 8.49 (s, 1H),
8.68 (s, 1H), 10.79 (s, 1H), 13.17 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): 20.9, 100.5, 111.6, 111.7, 117.8, 118.9,
119.3, 119.6, 119.7, 124.7, 125.3, 129.7, 130.2, 131.2, 137.6,
139.7, 140.2, 152.9, 170.2 ppm. MS (ESI) m/z 358 [M]+. Anal.
(C21H18N4O2ꢁ0.25H2O) C, H, N.
5.6.21. (Z)-1-(4-Methoxyphenyl)-3-(2-oxo-3-thiophen-3-
ylmethylene-2,3-dihydro-1H-indol-6-yl)urea (33)
Yield 58%; mp 266–267 °C. 1H NMR (200 MHz, DMSO-d6): d
3.70 (s, 3H), 6.75 (dd. J = 2.0, 8.3 Hz, 1H), 6.86 (d, J = 8.9 Hz, 2H),
7.27–7.33 (m, 4H), 7.49 (d, J = 5.0 Hz, 1H), 7.70–7.74 (m, 1H),
8.06 (d, J = 1.5 Hz, 1H), 8.49 (s, 1H), 8.85 (s, 1H), 10.52 (s, 1H).
MS (ESI) m/z 391 [M]+. Anal. (C21H17N3O3S) C, H, N, S.

4684
R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
5.6.28. (Z)-1-(4-Chlorophenyl)-3-[2-oxo-3-(1H-pyrrol-2-ylmeth-
ylene)-2,3-dihydro-1H-indol-6-yl]urea (40)
5.6.34. (Z)-2,4-Dimethyl-5-[2-oxo-6-(3-p-tolylureido)-1,2-
dihydroindol-3-ylidene-methyl]-1H-pyrrole-3-carboxylic acid
(46)
Yield 85%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 6.29–
6.34 (m, 1H), 6.74–6.76 (m, 1H), 6.87 (dd, J = 2.0, 8.0 Hz, 1H),
7.29–7.34 (m, 4H), 7.48 (d, J = 8.9 Hz, 2H), 7.5 (d, J = 8.2 Hz, 1H),
7.56 (s, 1H), 8.80 (s, 1H), 8.81 (s, 1H), 10.86 (s, 1H), 13.20 (s, 1H).
MS (ESI) m/z 378 [M]+. Anal. (C20H15N4O2Clꢁ0.1H2O) C, H, N.
Yield 69%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 2.22 (s,
3H), 2.41 (s, 3H), 2.49 (s, 3H), 6.85 (d, J = 8.0 Hz, 1H), 7.04 (d,
J = 8.0 Hz, 2H), 7.26–7.28 (m, 3H), 7.45 (s, 1H), 7.60 (d, J = 7.0 Hz,
1H), 8.56 (s, 1H), 8.76 (s, 1H), 10.89 (s, 1H), 13.59 (s, 1H). 13C
NMR (50 MHz, DMSO-d6): 12.3, 15.3, 21.2, 110.0, 112.2, 114.8,
117.4, 119.3, 119.9, 122.2, 126.9, 130.1, 131.8, 132.0, 137.8,
139.9, 140.1, 140.6, 153.4, 161.6, 167.2, 170.3 ppm. MS (ESI) m/z
430 [M]+. Anal. (C24H22N4O4ꢁ1.2H2O) C, H, N.
5.6.29. (Z)-1-[2-Oxo-3-(1H-pyrrol-2-ylmethylene)-2,3-dihydro-
1H-indol-6-yl]-3-(4-phenoxyphenyl)urea (41)
Yield 92%; mp 293–295 °C. 1H NMR (200 MHz, DMSO-d6): d
6.30–6.34, (m, 1H), 6.74–6.76 (m, 1H), 6.86 (d, J = 8.3 Hz, 1H),
6.92–6.99 (m, 4H), 7.07 (t, J = 7.4 Hz, 1H), 7.29–7.51 (m, 7H), 7.55
(s, 1H), 8.66 (s, 1H), 8.75 (s, 1H), 10.84 (s, 1H), 13.20 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): 100.5, 111.6, 111.7, 117.8, 118.2,
119.4, 119.6, 119.8, 120.3, 120.5, 123.3, 124.8, 125.3, 130.2,
130.4, 136.2, 139.6, 140.2, 151.2, 153.0, 158.2, 170.2 ppm. MS
(ESI) m/z 436 [M]+. Anal. (C26H20N4O3ꢁ0.1H2O) C, H, N.
5.6.35. (Z)-5-{6-[3-(4-Chlorophenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-2,4-dimethyl-1H-pyrrole-3-
carboxylic acid (47)
Yield 60%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 2.23 (s,
3H), 2.50 (s, 3H), 6.91 (dd, J = 2.0, 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz,
2H), 7.33 (s, 1H), 7.46 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.61 (d,
J = 8.0 Hz, 1H), 9.47 (s, 1H), 9.51 (s, 1H), 10.83 (s, 1H),
13.59 (s, 1H). MS (ESI) m/z 450 [M]+. Anal. (C23H19N4O4Clꢁ0.7H2O)
C, H, N.
5.6.30. (Z)-1-Biphenyl-4-yl-3-[2-oxo-3-(1H-pyrrol-2-ylmeth-
ylene)-2,3-dihydro-1H-indol-6-yl]urea (42)
Yield 97%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 6.30–
6.34 (m, 1H), 6.75–6.77 (m, 1H), 6.88 (dd, J = 1.8, 8.3 Hz, 1H),
7.27–7.36 (m, 3H), 7.41 (d, J = 7.4 Hz, 2H), 7.47–7.65 (m, 9H),
8.77 (s, 1H), 8.82 (s, 1H), 10.87 (s, 1H), 13.21 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): 100.6, 111.6, 111.7, 117.8, 119.1, 119.5,
119.6, 119.8, 124.8, 125.4, 126.6, 127.3, 127.5, 129.4, 130.2,
134.1, 139.6, 139.7, 140.2, 140.4, 152.9, 170.2 ppm. MS (ESI) m/z
420 [M]+. Anal. (C26H20N4O2ꢁ0.2H2O) C, H, N.
5.6.36. (Z)-{2,4-Dimethyl-5-[2-oxo-6-(3-phenylureido)-1,2-
dihydroindol-3-ylidene-methyl]-1H-pyrrol-3-yl} acetic acid
(48)
Yield 85%; mp 226–228 °C. 1H NMR (400 MHz, DMSO-d6): d
2.23 (s, 3H), 2.28 (s, 3H), 3.22 (s, 2H), 6.82–6.86 (m, 1H), 6.90–
6.94 (m, 1H), 7.22–7.27 (m, 2H), 7.33 (d, J = 2.0 Hz, 1H), 7.37 (s,
1H), 7.44–7.48 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H), 9.35 (s, 1H), 9.37
(s, 1H), 10.64 (s, 1H), 13.19 (s, 1H). 13C NMR (50 MHz, DMSO-d6):
10.7, 13.1, 25.0, 110.0, 113.3, 119.0, 119.1, 120.5, 122.4, 125.5,
126.4, 129.5, 130.1, 134.3, 139.2, 139.3, 140.7, 141.2, 144.9,
153.7, 170.7, 176.0 ppm. MS (ESI) m/z 430 [M]+. Anal.
(C24H22N4O4ꢁ0.5H2O) C, H, N.
5.6.31. (Z)-1-Naphthalen-2-yl-3-[2-oxo-3-(1H-pyrrol-2-ylmeth-
ylene)-2,3-dihydro-1H-indol-6-yl]urea (43)
Yield 88%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 6.31–
6.33 (m, 1H), 6.76–6.77 (m, 1H), 6.91 (dd, J = 1.7, 8.0 Hz, 1H),
7.29 (m, 1H), 7.35 (dd, J = 8.0, 8.0 Hz, 1H), 7.40 (dd, J = 2, 8 Hz,
1H), 7.44 (dd, J = 8.0, 8.0 Hz, 1H), 7.48 (dd, J = 2.0, 8.0 Hz, 1H),
7.52 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.83
(d, J = 8.0 Hz, 2H), 8.10 (d, J = 2.4 Hz, 1H), 8.85 (s, 1H), 8.86 (s,
1H), 10.86 (s, 1H), 13.20 (s, 1H). 13C NMR (100 MHz, DMSO-d6):
100.6, 111.6, 111.7, 114.0, 117.8, 119.5, 119.6, 119.8, 120.2,
124.5, 124.9, 125.4, 126.9, 127.5, 128.0, 129.0, 129.6, 130.2,
134.3, 137.8, 139.5, 140.2, 153.0, 170.2 ppm. MS (ESI) m/z 394
[M]+. Anal. (C24H18N4O2) C, H, N.
5.6.37. (Z)-(5-{6-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-2,4-dimethyl-1H-pyrrol-3-yl)
acetic acid (49)
Yield 50%; mp 222–224 °C. 1H NMR (200 MHz, DMSO-d6): d
2.22 (s, 3H), 2.27 (s, 3H), 3.30 (s, 2H), 3.69 (s, 3H), 6.82 (s, 1H),
6.88 (d, J = 8.0 Hz, 2H), 7.32–7.39 (m, 4H), 7.55 (d, J = 8.0 Hz, 1H),
9.08 (s, 1H), 9.28 (s, 1H), 10.73 (s, 1H), 13.25 (s, 1H). 13C NMR
(50 MHz, DMSO-d6): 10.5, 12.9, 31.9, 56.0, 100.5, 111.6, 114.0,
114.8, 118.4, 119.3, 120.3, 120.6, 122.3, 126.5, 129.8, 134.0,
134.1, 139.5, 139.6, 153.7, 155.1, 170.7, 174.7 ppm. MS (ESI) m/z
460 [M]+. Anal. (C25H24N4O5ꢁ1.8H2O) C, H, N.
5.6.32. (Z)-2,4-Dimethyl-5-[2-oxo-6-(3-phenylureido)-1,2-
dihydroindol-3-ylidene-methyl]-1H-pyrrole-3-carboxylic acid
(44)
Yield 84%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 2.52 (s,
6H), 6.92–6.96 (m, 2H), 7.23–7.28 (m, 2H), 7.36 (s, 1H), 7.47–7.50
(m, 3H), 7.65 (d, J = 8.3 Hz, 1H), 9.07 (s, 1H), 9.24 (s, 1H), 10.87 (s,
1H), 13.64 (s, 1H). 13C NMR (50 MHz, DMSO-d6): 12.3, 15.3, 100.8,
111.9, 117.2, 119.0, 119.9, 120.2, 122.1, 122.6, 126.8, 129.6, 131.8,
140.0, 140.1, 140.2, 140.8, 153.4, 156.0, 163.7, 170.9 ppm. MS (ESI)
m/z 416 [M]+. Anal. (C23H20N4O4ꢁ0.7H2O) C, H, N.
5.6.38. (Z)-3-(5-{6-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-2,4-dimethyl-1H-pyrrol-3-yl)
propionic acid (50)
Yield 42%; mp 270–272 °C. 1H NMR (400 MHz, DMSO-d6): d
2.22 (s, 3H), 2.27 (s, 3H), 2.32 (t, J = 8.0 Hz, 2H), 2.63 (t, J = 8.0 Hz,
2H), 3.71 (s, 3H), 6.83–6.86 (m, 3H), 7.28 (d, J = 2.0 Hz, 1H), 7.35
(d, J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.5 (d, J = 8.0 Hz, 1H), 8.50 (s, 1H),
8.70 (s, 1H), 10.67 (s, 1H), 13.19 (s, 1H). MS (ESI) m/z 474 [M]+.
Anal. (C26H16N4O5ꢁ0.4H2O) C, H, N.
5.6.33. (Z)-5-{6-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-2,4-dimethyl-1H-pyrrole-3-
carboxylic acid (45)
Yield 85%; mp >300 °C. 1H NMR (200 MHz, DMSO-d6): d 3.67 (s,
6H), 3.71 (s, 3H), 6.84–6.88 (m, 3H), 7.34–7.37 (m, 3H), 7.50 (s,
1H), 7.75 (d, J = 8.0 Hz, 1H), 8.59 (s, 1H), 8.84 (s, 1H), 10.90
(s, 1H), 11.50–12.40 (br, 1H), 13.67 (s, 1H). 13C NMR (50 MHz,
DMSO-d6): 12.3, 15.3, 56.0, 100.7, 111.8, 114.8, 117.5, 119.3, 119.7,
120.3, 120.9, 122.0, 126.9, 131.8, 133.6, 140.1, 140.2, 140.3, 153.5,
155.3, 170.9, 177.8 ppm. MS (ESI) m/z 446 [M]+. Anal. (C24H22N4O5)
C, H, N.
5.6.39. (Z)-4-Methyl-5-[2-oxo-6-(3-phenylureido)-1,2-dihydro-
indol-3-ylidenemethyl]-1H-pyrrole-2-carboxylic acid (51)
Yield 55%; mp 218–220 °C. 1H NMR (400 MHz, DMSO-d6): d 2.30
(s, 3H), 6.38–6.43 (m, 1H), 6.93–7.09 (m, 2H), 7.26 (s, 2H), 7.41 (s,
2H), 7.50–7.61 (m, 3H), 9.82 (s, 1H, NH), 9.93 (s, 1H), 10.75 (s, 1H),
13.18 (s, 1H). MS (ESI) m/z 402 [M]+. Anal. (C22H18N4O4ꢁ0.9H2O) C,
H, N.

R. R. Khanwelkar et al. / Bioorg. Med. Chem. 18 (2010) 4674–4686
4685
5.6.40. (Z)-5-{6[3-(4-Chlorophenyl)-ureido]-2-oxo-1,2-dihydro-
indol-3-ylidene-methyl}-4-methyl-1H-pyrrole-2-carboxylic
acid (52)
fetal bovine serum. Unless otherwise indicated, cell culture re-
agents were obtained from GIBCO™/Invitrogen Life Technologies
(USA). All the cells were grown and maintained in a humidified
atmosphere at 37 °C and 5% CO2.
Yield 78%; mp 202–204 °C. 1H NMR (400 MHz, DMSO-d6): d 2.30
(s, 3H), 6.44 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H),
7.33 (s, 1H), 7.42 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.62 (s, 1H), 10.02
(s, 1H), 10.08 (s, 1H), 10.74 (s, 1H), 13.17 (s, 1H). 13C NMR (50 MHz,
DMSO-d6): 12.4, 100.9, 110.0, 112.1, 114.7, 117.8, 119.8, 120.0,
120.4, 120.5, 122.1, 125.7, 128.0, 129.4, 135.2, 140.4, 140.5,
153.8, 166.2, 170.5 ppm. MS (ESI) m/z 436 [M]+. Anal.
(C22H17N4O4Clꢁ0.9H2O) C, H, N.
5.9. Cell proliferation assays
Each cell line was plated in 96-well microtiter plates (10,000
cells per well), and serial dilutions of indicated compounds were
added. At the end of the incubation period (72 h at 37 °C), cell via-
bility was determined by a tetrazolium dye, MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Promega, USA).
The formazan crystals were dissolved in DMSO, and the absorbance
at 600 nm was recorded using an ELISA plate reader. IC50 values
were calculated using nonlinear regression and defined as the con-
centration needed for a 50% reduction in absorbance of treated ver-
sus untreated control cells.
5.6.41. (Z)-5-{6-[3-(4-Methoxyphenyl)-ureido]-2-oxo-1,2-
dihydroindol-3-ylidene-methyl}-1H-pyrrole-2-carboxylic acid
(53)
Yield 62%; mp 244–246 °C. 1H NMR (200 MHz, DMSO-d6): d
3.71 (s, 3H), 6.60–6.61 (m, 1H), 6.67–6.68 (m, 1H), 6.86 (d,
J = 10.0 Hz, 2H), 6.99 (dd, J = 2.0, 8.0 Hz, 1H), 7.39–7.51 (m, 5H),
9.51 (s, 1H), 9.72 (s, 1H), 10.81 (s, 1H), 13.19 (s, 1H). MS (ESI) m/
z 418 [M]+. Anal. (C22H18N4O5ꢁ0.9H2O) C, H, N.
5.10. Western blot analysis
5.6.42. (Z)-5-{6-[3-(4-Methoxyphenyl)-urido]-2-oxo-1,2-
dihydro-indol-3-ylidene-methyl}thiophen-2-carboxylic acid
(54)
Cells were plated at 2–5 ꢂ 105 cells per well in six-well plates
(Orange Scientific, Belgium). Cells were then cultured for 24 h,
and then incubated in serum-free medium overnight before
preparation of cell lysates. Cells were treated with compounds
in serum-free medium for 45 min, followed by addition of li-
gands, stem cell factor (50 ng/ml), VEGF (VEGF-162, 50 ng/mL),
FLT-3 ligand (40 ng/mL), which were obtained from R&D Systems
(USA) for 5–15 min. Lysates were prepared by the addition of cell
lysis buffer (Cell Signaling Technology, USA); the lysates were
clarified by centrifugation (14,000g, 10 min, 4 °C). Lysate protein
content was quantified using the Pierce protein assay kit (USA)
according to the manufacturer’s instructions with bovine serum
albumin as the reference standard. Proteins were separated by
electrophoresis on SDS-polyacrylamide gels, electroblotted onto
NC membranes (Hybond-C Extra, Amersham Biosciences, USA),
and then probed using primary anti-phosphotyrosine antibody
(PY99, Santa Cruz Biotechnology, USA). Western blots were
developed using enhanced chemiluminescence with horseradish
peroxidase–labeled secondary antibodies and the SuperSignalÒ
HRP substrate (Pierce, USA). The membranes were stripped
(Western Blot Stripping Buffer, Pierce, USA), washed, and re-
probed with anti-c-KIT, anti-KDR (Santa Cruz Biotechnology),
and anti-FLT3 (R&D Systems) and developed as described above.
The IC50 value of each compound was calculated using nonlinear
regression with GraphPad Prism v5.0.
Yield 98%; mp >300 °C. 1H NMR (400 MHz, DMSO-d6): d 3.71 (s,
3H), 6.84 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 12.0 Hz, 2H), 7.47 (s, 1H), 7.53 (s, 1H), 7.72 (d, J = 4.0 Hz, 1H),
7.78 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 8.56 (s, 1H), 8.97
(s, 1H), 10.63 (s, 1H). 13C NMR (100 MHz, DMSO-d6): 55.7, 100.0,
110.9, 113.6, 114.3, 115.1, 120.3, 124.0, 124.3, 124.9, 129.3,
134.6, 137.0, 140.2, 144.1, 144.4, 153.9, 154.5, 166.5, 170.7 ppm.
MS (ESI) m/z 435 [M]+. Anal. (C22H17N3O5S) C, H, N, S.
5.7. In vitro kinase assays
The IC50 values for the inhibition of kinases VEGFR3, FLT3, KDR,
and c-kit by compounds were determined in a time-resolved fluo-
rescence format (Cell Signaling Technology, USA), measuring the
inhibition of phosphate transfer to a substrate by the respective
enzyme. VEGFR3, FLT3, KDR, and c-KIT were assayed in 60 mM
HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), pH
7.5, 5 mM MgCl2, 5 mM MnCl2, 3
itol, 1.5 M biotinylated peptide substrate (MYDKEYYS, EGPW-
LEEEEEAYGWMDF, ANASPEC, biotinylated gastrin (1–17)) and
20 M ATP. Reactions were incubated at room temperature for
lM Na3VO4, 1.25 mM dithiothre-
l
l
30 min, and the phosphorylated peptide captured on streptavi-
din-coated microtiter plates (PerkinElmer Life Sciences, USA) con-
taining reaction stop buffer (50 mM EDTA, pH 8). Phosphorylated
peptide was measured with the ULTRA Evolution Multi-Detection
Microplate Reader from Tecan (Tecan Group Ltd, Switzerland)
using phosphotyrosine mouse monoclonal antibody (P-Try-100)
and europium-labeled anti-mouse IgG. The IC50 value of compound
was calculated using nonlinear regression with SigmaPlot 10.0.
5.11. In vivo xenograft tumor model
The procedure for establishing tumor xenografts and the dosing
of 36 were carried out in accordance with Industrial Technology
Research institutional animal care and use committee. Female
nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl, 6–8 weeks old)
were purchased from the National Laboratory Animal Center (Tai-
wan). Female BALB/c nude mice were implanted subcutaneously
in the right flank with 1 ꢂ 107 MV4-11 cells (human leukemia
FLT3-ITD) per mouse. Treatments were initiated when tumor sizes
were 200–300 mm3. Mice were randomly assigned into cohorts
(four mice per group for efficacy studies). Compound 36 at
20 mg/kg, once-daily (qd), and vehicle (10% NMP + 20% Cremophor
EL + 70% saline) were given via oral gavage (po.) for seven days.
Tumor volumes were assessed every day, and body weights were
assessed twice weekly. Caliper measurements of tumors were
converted into mean tumor volume using the formula:
0.5 ꢂ [length ꢂ (width)2].
5.8. Cell lines
Human MV4-11 (FLT3-ITD), MOLM-13 (FLT3-ITD), RS4-11
(FLT3-WT), THP-1 (FLT3-WT) leukemic cells and HepG2, COLO205,
and H460 solid tumor cells were obtained from American Tissue
Culture Collection. The cells were grown in RPMI 1640 containing
10% fetal bovine serum, 1 mmol/L sodium pyruvate, and
10 mmol/L HEPES (pH 7.4). All the cells were grown and maintained
in a humidified atmosphere at 37 °C and 5% CO2. Kasumi-1 (BCRC-
60505) and human umbilical vein endothelial cells (H-UV001) were
obtained from the Bioresource Collection and Research Center (Tai-
wan) and cultured in RPMI 1640 and Medium 199 containing 10%

4686
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Theuer, A.; Towbin, H.; Wenger, F.; Woods-Cook, K.; Menrad, A.; Siemeister, G.;
5.12. In vivo pharmacokinetics
Schirner, M.; Thierauch, K.-H.; Schneider, M. R.; Drevs, J.; Martiny-Baron, G.;
Totzke, F.; Marmé, D. Cancer Res. 2000, 60, 2178.
The pharmacokinetic profile was investigated in overnight
fasted male Sprague-Dawley rats following administration of a sin-
gle intravenous or oral dose of 36. Compound 36 was administered
as a solution in 20% N-methyl-2-pyrrolidone, 10% cremophor EL, in
0.9% normal saline. At 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h post-dose,
blood was collected and processed to plasma by centrifugation
and stored at ꢃ80 °C until analysis. Samples were analyzed by li-
quid chromatography coupled with tandem mass spectrometry
(LC/MS/MS) technique. Pharmacokinetics were analyzed by Win-
Nonlin 5.2 (Pharsight, CA) via noncompartmental analysis.
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