Design, synthesis, and evaluations of substituted 3-[(3- or 4- carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases

Article abstract of DOI:10.1021/jm9904295

Receptor tyrosine kinases (RTKs) have been implicated as therapeutic targets for the treatment of human diseases including cancers, inflammatory diseases, cardiovascular diseases including arterial restenosis, and fibrotic diseases of the lung, liver, and kidney. Three classes of 3-substituted indolin-2-ones containing propionic acid functionality attached to the pyrrole ring at the C-3 position of the core have been identified as catalytic inhibitors of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) RTKs. Some of the compounds were found to inhibit the tyrosine kinase activity associated with isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver tyrosine kinase 1 (Flk-1)/kinase insert domain- containing receptor (KDR)], fibroblast growth factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinase with IC₅₀ values at nanomolar level. Thus, compound 1 showed inhibition against VEGF-R2 (Flk-1/KDR) and FGF-R1 tyrosine kinase activity with IC₅₀ values of 20 and 30 nM, respectively, while compound 16f inhibited the PDGF-R tyrosine kinase activity with IC₅₀ value of 10 nM. Structural models and structure-activity relationship analysis of these compounds for the target receptors are discussed. The cellular activities of these compounds were profiled using cellular proliferation assays as measured by bromodeoxyuridine (BrdU) incorporation. Specific and potent inhibition of cell growth was observed for some of these compounds. These data provide evidence that these compounds can be used to inhibit the function of these target receptors.

Full text of DOI:10.1021/jm9904295

5120
J . Med. Chem. 1999, 42, 5120-5130
Design , Syn th esis, a n d Eva lu a tion s of Su bstitu ted 3-[(3- or
4-Ca r boxyeth ylp yr r ol-2-yl)m eth ylid en yl]in d olin -2-on es a s In h ibitor s of VEGF ,
F GF , a n d P DGF Recep tor Tyr osin e Kin a ses
Li Sun,* Ngoc Tran, Congxin Liang, Flora Tang, Audie Rice, Randall Schreck, Kara Waltz, Laura K. Shawver,
Gerald McMahon, and Cho Tang*
SUGEN, Inc., 230 East Grand Avenue, South San Francisco, California 94080-4811
Received August 23, 1999
Receptor tyrosine kinases (RTKs) have been implicated as therapeutic targets for the treatment
of human diseases including cancers, inflammatory diseases, cardiovascular diseases including
arterial restenosis, and fibrotic diseases of the lung, liver, and kidney. Three classes of
3-substituted indolin-2-ones containing propionic acid functionality attached to the pyrrole ring
at the C-3 position of the core have been identified as catalytic inhibitors of the vascular
endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth
factor (PDGF) RTKs. Some of the compounds were found to inhibit the tyrosine kinase activity
associated with isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver
tyrosine kinase 1 (Flk-1)/kinase insert domain-containing receptor (KDR)], fibroblast growth
factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinase
with IC50 values at nanomolar level. Thus, compound 1 showed inhibition against VEGF-R2
(Flk-1/KDR) and FGF-R1 tyrosine kinase activity with IC50 values of 20 and 30 nM, respectively,
while compound 16f inhibited the PDGF-R tyrosine kinase activity with IC50 value of 10 nM.
Structural models and structure-activity relationship analysis of these compounds for the target
receptors are discussed. The cellular activities of these compounds were profiled using cellular
proliferation assays as measured by bromodeoxyuridine (BrdU) incorporation. Specific and
potent inhibition of cell growth was observed for some of these compounds. These data provide
evidence that these compounds can be used to inhibit the function of these target receptors.
In tr od u ction
Compound 1 was cocrystallized with the catalytic
domain of FGF-R1 (flg-1)7 and was found to inhibit
tyrosine phosphorylation of VEGF-R2 (Flk-1/KDR) and
PDGF-R in NIH 3T3 cells with IC50 values of 0.4 and
60.9 µM, respectively.6 The cocrystal structure revealed
that the indolin-2-one core occupied the adenine binding
site of ATP and that 1 induced receptor conformational
changes as a result of interactions between the propionic
acid moiety of 1 and Asn568 in the sugar binding
region.7 This study suggested that the substitution on
the core might confer kinase selectivity of these ATP
mimetic 3-substituted indolin-2-ones. The information
deduced from this study is useful for further modifica-
tion of 3-[(substituted pyrrol-2-yl)methylidenyl]indolin-
2-ones as inhibitors against FGF-R1 and other highly
homologous RTKs, such as VEGF-R2 (Flk-1/KDR).
Many growth factors and cytokines mediate cellular
signaling through the activation of tyrosine kinases. In
human disease, tyrosine kinases have been implicated
as potential therapeutic targets for cancers, cardiovas-
cular disease, inflammatory diseases, fibrotic diseases,
and other diseases resulting from chronic tissue injury.
More recently, synthetic compounds have been identi-
fied that can block the function of specific tyrosine
kinases and preclinical data are emerging to support
the use of these compounds in clinical studies. In the
area of cancer, receptor tyrosine kinases play important
roles in the process of tumor development and spread.1-3
In this regard, RTKs have been involved in tumor
growth, survival, metastasis, and angiogenesis.4,5
We previously reported a series of 3-substituted
indolin-2-ones with potent and selective inhibitory
activity toward different RTKs.6 The selectivity of these
compounds against particular RTKs depended on the
substituents on the indolin-2-one core, especially at the
C-3 position. Of particular interest, 3-[(substituted
pyrrol-2-yl)methylidenyl]indolin-2-ones showed selective
inhibitory activity against the VEGF-R2 (Flk-1/KDR)
tyrosine autophosphorylation at the cellular level.6 From
this series of compounds, SU5402 (compound 1 in Table
1) and SU5416 (compound 2 in Table 3) have been
utilized as specific inhibitors of the FGF-R and VEGF-
R, respectively.
In addition, compound 2 was derived from a previous
chemical series and is currently under clinical evalua-
tion for the treatment of human cancers.8 Compound 2
was found to inhibit tyrosine autophosphorylation of
VEGF-R2 (Flk-1/KDR) and PDGF-R tyrosine kinases
(TKs) in NIH 3T3 mouse fibroblast cells with IC50 values
of 1.04 and 20.26 µM, respectively.6 Preclinical studies
have indicated that 2 did not inhibit tumor cell growth
in vitro but did specifically inhibit endothelial cell
proliferation facilitated by VEGF (IC50 ) 40 nM).
Consistent with its anti-angiogenic properties, com-
pound 2 also inhibited tumor growth in vivo in a wide
range of tumor models.8
* To whom correspondence should be addressed.
Our goal in the present study was to explore the
10.1021/jm9904295 CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/19/1999

Substituted Indolin-2-ones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5121
onic acid functionality were designed. First, modification
of the substitutions at the C-4, C-5, or C-6 positions
resulted in a series of 1 analogues (9a -d in Table 1).
Second, swapping the propionic acid moiety at the C-3′
position with the methyl group at the C-4′ position of 1
gave the corresponding regioisomer, compound 11a
(Table 2). In addition, analogues of 11a with different
substitutions on the indolin-2-one core were also pre-
pared (compounds 11b-h in Table 2). Third, combining
the structural features of 2 (a potent VEGF-R inhibitor)
and compound 11a yielded compound 16a and its
analogues (Table 3).
Ch em istr y
F igu r e 1. Compound 11a is shown to be docked within a
homology model of the PDGF-R. The receptor peptide backbone
is represented by ribbons. Key nonconserved residues sur-
rounding 11a are shown with carbon atoms colored gray.
Corresponding residues of the FGF-R1 and VEGF-R2 (Flk-1/
KDR) are indicated. Compound 11a is designated with carbon
atoms colored yellow. Hydrogen bonds are denoted between
11a and arginine (dotted lines).
In general, compounds were prepared following con-
densation of substituted indolin-2-ones with aldehydes
in the presence of base as described previously.6 Indolin-
2-one and 6-methoxy-indolin-2-one are commercially
available, whereas 4-methyl-, 5-bromo-, 5-aminosulfo-
nyl-, 6-aryl-, and 5-carboxyindolin-2-ones and all of the
aldehydes were prepared using the following methods.
4-Methyl- and 5-bromoindolin-2-one were prepared by
the method described previously.6 5-(Aminosulfonyl)-
indolin-2-ones (compounds 4 in Scheme 1) were pre-
pared by amidation of 5-(chlorosulfonyl)indolin-2-one
(compound 3 in Scheme 1) that was prepared by
sulfonylation of indolin-2-one with chlorosulfonic acid
(Scheme 1). 6-Arylindolin-2-ones (8a -e) were prepared
from 5-bromo-2-fluoronitrobenzene. Suzuki coupling of
the commercially available aryl boronic acid with 5-bro-
mo-2-fluoronitrobenzene gave 5-aryl-2-fluoronitroben-
zene (compounds 5a -e in Scheme 1). Displacement of
o-fluoro substitution of 5a -e with dimethyl malonate
followed by hydrolytic decarboxylation with 6 N aqueous
hydrochloric acid and reductive cyclization gave 6-arylin-
dolin-2-ones (8a -e) (Scheme 1).10 5-Carboxyindolin-2-
one was synthesized according to the reported method.11
Analogues of 1 (9a -d , Table 1) were prepared as
depicted elsewhere.12 Compound 11a analogues (Scheme
2 and Table 2) were prepared by condensing the
substituted indolin-2-ones with 4-carboxyethyl-3-meth-
ylpyrrol-2-carboxaldehyde (10), which was prepared as
described previously (Scheme 2).13 Analogues of com-
pound 16a (Scheme 3 and Table 3) were synthesized
by condensing substituted indolin-2-ones and 3-(5-
formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid (com-
pound 15 in Scheme 3), which was prepared from
4-(2-methoxycarbonylethyl)-3,5-dimethyl-1-H-pyrrole-2-car-
boxylic acid benzyl ester via hydrogenolysis, decarboxy-
lation, formylation, and hydrolysis (Scheme 3). As
reported in our previous report, analogues of 3-[(sub-
stituted pyrrol-2-yl)methylidenyl]indolin-2-ones were
shown to exist exclusively as cis isomers (Z-isomer).6
We suggested that this may be due in part to intramo-
lecular hydrogen bonding between the NH-1′ and CdO
at the C-2 position of the indolin-2-one core. All of the
compounds in this study are assumed to preferably exist
as cis isomers.
influence of various chemical modifications of 3-[(sub-
stituted pyrrol-2-yl)methylindenyl]indolin-2-ones on their
inhibitory activity and selectivity against VEGF, FGF,
and PDGF RTKs. The structure-activity relationship
of these compounds with respect to both kinase activity
and ligand-dependent events in cells is discussed.
Design
All of the compounds included in this study were
based, in part, on chemical features of 1. The cocrys-
tallographic structure of 1 and the catalytic domain of
FGF-R1 revealed important ligand-receptor interac-
tions as well as areas for further modifications.7 First,
it showed the presence of three crystallographic water
molecules in proximity to the C-5 position of the indolin-
2-one core, suggesting that there is more space in this
area for hydrophilic substitutions (such as carboxylic
acid and aminosulfonyl functionalities). Second, a com-
parison of a cocrystallographic study with FGF-R1 and
PD1730749 suggested that introduction of a lipophilic
phenyl (or substituted phenyl) substituent at the C-6
position of the 1 core might enhance inhibitory activity
against FGF-R1.
For VEGF-R and PDGF-R, homology models were
built based on the FGF-R1 crystal structure. Since
VEGF-R like FGF-R1 also has an asparagine residue
in the sugar binding region (Figure 1), the propionic
acid-asparagine interaction observed in the 1 cocrystal
structure would likely be preserved upon binding to the
VEGF-R. In the case of PDGF-R, the corresponding
residue in the sugar-binding region is aspartic acid
(Figure 1). Although favorable interaction between the
propionic acid of 1 and the aspartic acid of PDGF-R
could exist if one of the acids is deprotonated, such
interaction would be less favorable than the propionic
acid-asparagine interaction observed in the 1/FGF-R1
cocrystal. On the other hand, PDGF-R has a basic
arginine residue in proximaty to the C-4′ position on
the pyrrole ring of 1. Thus, to enhance PDGF-R inhibi-
tory activities, the propionic acid side chain of 1 could
be moved to the C-4′ position. In this manner, three
types of 3-substituted indolin-2-ones containing propi-
Resu lts a n d Discu ssion
Compounds were evaluated for their inhibitory activ-
ity toward tyrosine phosphorylation activity associated
with isolated VEGF-R2 (Flk-1/KDR), FGF-R1, PDGF-
R, and epidermal growth factor receptor (EGF-R) ty-

5122 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Sun et al.
Ta ble 1. Inhibition of Tyrosine Kinase Activities and Ligand-Dependent Cell Proliferation Using 1 Analogues
3'
compounds
R
inhibition of tyrosine kinase activity (IC50, µM)a
inhibition of cell proliferation (IC50, µM)b
ID
VEGF-R2
FGF-R1
PDGF-Râ
EGF-R
VEGF
FGF
PDGF
EGF
1
H
4-CH3
5-Br
6-(3-OCH3phenyl)
6-(3-OC2H5phenyl)
0.02
0.20
0.35
28.3
1.0
0.03
0.03
0.08
1.2
0.51
2.11
0.62
0.45
2.38
>100
>100
>100
>100
>100
0.05
0.10
0.04
0.61
0.46
2.80
0.25
0.49
0.14
0.27
28.4
46.4
6.70
8.29
11.3
>50
28.8
>50
49.2
>50
9a
9b
9c
9d
4.5
a
IC50 values for VEGF-R2 (Flk-1/KDR) and FGF-R1 were determined by at least two separate tests and reported as mean values.
b
IC50 values for PDGF- and EGF-dependent cell proliferations were determined by at least two separate tests and reported as mean
values.
Ta ble 2. Inhibition of Tyrosine Kinase Activities and Ligand-Dependent Cell Proliferation Using Compounds 11a -h
4'
compounds
R
inhibition of tyrosine kinase activity (IC50, µM)a
inhibition of cell proliferation (IC50, µM)b
ID
VEGF-R2
FGF-R1
PDGF-Râ
EGF-R
VEGF
FGF
PDGF
EGF
11a
11b
11c
11d
11e
11f
11g
11h
H
2.14
0.24
0.92
1.35
0.30
0.09
1.47
0.45
3.68
0.77
0.46
3.89
1.05
0.36
2.13
1.54
0.14
4.19
5.98
0.14
0.16
0.17
1.42
3.08
>100
>100
>100
>100
>100
>100
>100
>100
0.40
>50
10.1
58.4
>50
>50
1.40
0.15
0.18
0.65
2.78
>50
>50
>50
>50
21.8
30.5
19.2
22.8
5-COOH
5-SO2NH2
6-OCH3
6-phenyl
6-(3-OCH3phenyl)
6-(2-OCH3phenyl)
6-(4-OCH3phenyl)
>50
>50
31.6
1.00
>50
1.20
0.81
0.18
0.04
1.80
0.13
0.04
1.60
a
b
IC50 values for VEGF-R2 (Flk-1) and FGF-R1 were determined by at least two separate tests and reported as mean values. IC50
values for PDGF- and EGF-dependent cell proliferations were determined by at least two separate tests and reported as mean values.
Ta ble 3. Inhibition of Tyrosine Kinase Activities and Ligand-Dependent Cell Proliferation Using Compounds 16a -i
'
compounds
R
inhibition of tyrosine kinase activity (IC50, µM)a
inhibition of cell proliferation (IC50, µM)b
ID
VEGF-R2
FGF-R1
PDGF-Râ
EGF-R
VEGF
FGF
PDGF
EGF
2
0.70
2.43
1.73
0.07
1.26
8.29
0.14
0.30
4.37
0.52
7.08
3.04
2.05
0.28
0.28
5.40
2.29
1.40
7.33
5.76
10.50
0.06
0.06
1.21
1.53
0.55
0.01
0.10
2.22
1.0
>100
>100
>100
>100
84.8
>100
>100
>100
>100
>100
0.04
0.41
0.07
>50
42.8
0.67
10.0
0.99
3.0
50.0
4.54
16.5
3.65
>50
>50
2.35
0.11
0.23
0.94
0.68
>50
>50
>50
>50
>50
>50
21.9
24.1
31.3
>50
16a
16b
16c
16d
16e
16f
16g
16h
16i
H
5-Br
5-COOH
5-SO2NH2
6-OCH3
9.30
3.60
>50
>50
25.1
10.0
1.50
6-phenyl
6-(3-OCH3phenyl)
6-(2-OCH3phenyl)
6-(4-OCH3phenyl)
4.00
5.64
4.20
a
b
IC50 values for VEGF-R2 (Flk-1) and FGF-R1 were determined by at least two separate tests and reported as mean values. IC50
values for PDGF- and EGF-dependent cell proliferations were determined by at least two separate tests and reported as mean values.
rosine kinases. Tyrosine autophosphorylation assays
were used to assess the inhibitory activities of com-
pounds toward PDGF-R and EGF-R tyrosine kinases,
whereas peptide-base transphosphorylation assays
were performed for VEGF-R2 (Flk-1/KDR) and FGF-R1
kinase activity. Compounds were also evaluated in
ligand-stimulated cell proliferation assays using human
umbilical vein endothelial cells (HUVECs) and NIH3T3

Substituted Indolin-2-ones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5123
Sch em e 1. Synthesis of Substituted Indolin-2-onesa,b
an Asn at the position similar to Asn568 in FGF-R1, it
is not surprising that 1 has been shown to exhibit
similar potency for this receptor kinase as well (Figure
1). In the case of PDGF-R, the corresponding residue is
either aspartic acid or aspartate, depending on the
ionization status of 1 (Figure 1). Nonetheless, the
interaction between the propionic acid of 1 and aspartic
acid would be less favorable than that between this
propionic acid and asparagine. The reduced potency of
1 toward PDGF-R tyrosine kinase is consistent with this
model. Methylation at the C-4 position (9a in Table 1)
of 1 retained potency against FGF-R1 but led to 10- and
4-fold decrease in potency toward VEGF-R2 (Flk-1/KDR)
and PDGF-R, respectively. Bromination at the C-5
position of 1 had little effect on inhibitory activity
against either PDGF-R or FGF-R, but considerably
decreased the potency toward VEGF-R2 (Flk-1) (9b in
Table 1). These results have supported a model where
FGF-R1 can better accommodate substitutions at both
the C-4 and C-5 positions of the indolin-2-one core than
either VEGF-R2 (Flk-1) or PDGF-R. In this model, FGF-
R1 contains an alanine residue in the vicinity of the C-5
position of indolin-2-one, whereas VEGF-R2 (Flk-1) and
PDGF-R contain larger cysteine residues at the com-
parable position (Figure 1).
c
a
4: (a) HSO3Cl, room temperature to 68 °C, 2.5 h. (b) NH4OH,
b
ethanol, room temperature, overnight. 8a -e: (a) ArB(OH)2,
(Ph3P)4Pd,NaHCO3,toluene-ethanol,reflux,2 h.(b)CH2(COOCH3)2,
NaH, DMSO, 100 °C, 2 h. (c) 6 N HClaq, overnight. (d) Fe, HOAc,
reflux, 2 h.
Sch em e 2. Synthesis of Compounds 11a -h a
Aryl substitutions at the C-6 position of the 1 core
retained or slightly decreased the potency against
PDGF-R but led to a significant decrease in potency
toward VEGF-R2 (Flk-1) and FGF-R1 (compounds 9c
and 9d in Table 1). The decreased activity of 9c and 9d
against FGF-R1 was unexpected since superposition of
1 with PD173074, which is a highly potent and selective
inhibitor of FGF-R1,9 suggested that such phenyl sub-
stitution might enhance FGF-R1 inhibitory activity by
increasing further side chain interactions.
a
(a) Piperidine, ethanol, reflux, 4 h, then 2 N HClaq
.
Sch em e 3. Synthesis of Compounds 16a -ia
All of these 1 analogues showed potent inhibitory
activity against VEGF- and FGF-induced cell prolifera-
tion but weak potency toward PDGF-induced cell pro-
liferation. However, these results were not correlated
to the data from kinase assays. For instance, compound
9a was found to be 141- and 40-fold more potent than
9c when tested against VEGF-R2 (Flk-1) and FGF-R1,
respectively. However, both compounds showed compa-
rable potencies in the cell proliferation assays. This is
probably due to other factors such as cell membrane
penetration, chemical stability, metabolic stability, or
the ability to affect other cellular targets including
protein kinases. Therefore, advance of these compounds
for in vivo assessment will largely rely on clarifying the
role of the above factors in the cellular inhibitory activity
for these inhibitors. Compound 9b was the best com-
pound from this series that showed high potency against
all three targets in both kinase and cell proliferation
assays when compared to EGF-R.
a
(a) Pd/C, H2, methanol, room temperature, 2 h. (b) NaOAc,
100 °C, 3 days. (c) ClCHdN+(CH3)2Cl-, ClCH2CH2Cl, room tem-
perature, 2 h. (d) NaOHaq, room temperature. (e) NaOHaq, reflux,
2 h. (f) Substituted indolin-2-ones, piperidine, ethanol, reflux,
overnight, then 2 N HClaq
.
mouse fibroblast cells. IC50 values were defined as the
concentration of a compound required to achieve 50%
inhibition of tyrosine kinase activity or the ligand-
stimulated growth compared to vehicle-treated controls
(DMSO). Compounds with IC50 values greater than 50
µM were considered inactive. The results from these
assays are summarized in Tables 1-3. The SAR analy-
ses are discussed separately for each class of compounds
in this report.
1. An a logu es of Com p ou n d 1. Compound 1 is a
prototype compound that exhibits good potency against
VEGF-R2 (Flk-1/KDR), FGF-R1, and PDGF-R tyrosine
kinases with IC50 values of 0.02, 0.03, and 0.51 µM,
respectively (Table 1). Since VEGF-R2 (Flk-1) also has
2. An a logu es of Com p ou n d 11a . The regioisomer
of 1, 11a (Table 2), was found to be 107- and 123-fold
less potent than 1 toward VEGF-R2 (Flk-1) and FGF-
R1, respectively, and 4-fold more potent when tested
against the PDGF-R (IC50 ) 0.14 µM, Table 2). Modeling
studies have suggested that differences in the inhibitory
target profiles of 11a and 1 may be attributed to
different compound-receptor interactions. Specifically,
the propionic acid moiety of 1 can interact with aspar-
agine within the catalytic domain of VEGF-R2 (Flk-1)

5124 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Sun et al.
or FGF-R1 as has been observed previously in the
1/FGF-R1 cocrystal structure.7 Also it may interact,
though less favorably, with the aspartic acid/aspartate
of PDGF-R as discussed previously. On the other hand,
the propionic acid moiety of 11a , attached to the C-4′
position of the core, might interact with a lysine within
the catalytic domain of VEGF-R2 (Flk-1) or FGF-R1 and
an arginine residue of PDGF-R (Figure 1). In particular,
the guanidine group of the arginine residue in PDGF-R
is aligned to interact with the carboxylate of 11a with
hydrogen bond distance of about 2 Å. For VEGF-R2 (Flk-
1) and FGF-R1, the lysine residue exhibiting a shorter
side chain than arginine in PDGF-R would be in a less
favorable position to interact with the carboxylate of 11a
(the hydrogen bond distance would be about 2.8 Å).
These considerations may help to explain, in part, the
finding that 11a was found to be more potent against
PDGF-R than against VEGF-R or FGF-R.
eration was observed for the 11a series as for the 1
series. For example, 11g was over 31- and 790-fold more
potent than 11d when tested against VEGF- and FGF-
dependent cell proliferation, respectively, despite their
similar kinase inhibitory profile on these RTKs. In
addition, 11g is slightly more potent against PDGF-
dependent cell proliferation but 10-fold less potent in
the kinase assay than 11d . The compound 11f was the
only compound of this series that showed potent inhibi-
tory activity against both kinase and ligand-dependent
cell proliferation. In this regard, it inhibited VEGF-,
FGF-, and PDGF-dependent cell proliferation with IC50
values of 0.18, 0.13, and 0.18 µM, respectively (Table
2).
3. An a logu es of Com p ou n d 16a . Combining the
chemical structural features of compounds 11a (propi-
onic acid at the C-4′ position of the pyrrole ring) and 2
(3,5-dimethylpyrrole side chain), the 16a series (Table
3) was prepared. Compound 16a showed equal potency
compared to 11a when tested against VEGF-R2 (Flk-1)
and FGF-R1 but was found to be slightly more potent
than 11a toward PDGF-R (IC50 ) 0.06 µM) (Table 3).
This result suggested that the extra methyl substituent
at the C-5′ position on the pyrrole ring of 16a might
provide an extra interaction to PDGF-R. In general,
analogues of 16a shared some structure-activity rela-
tionships with the 11a series. In this regard, a small
lipophilic bromo substituent at the C-5 position of 16b
did not change the kinase profile when compared to the
parent compound 16a . On the other hand, the hydro-
philic substituents (i.e., carboxyl or aminosulfonyl group
in 16c and 16d , respectively) increased the inhibitory
potency against both VEGF-R2 (Flk-1/KDR) and FGF-
R, but decreased the potency toward PDGF-R. Phenyl
and 3-methoxyphenyl substitution at the C-6 position
on the indolin-2-one core (16f and 16g in Table 3)
increased potency toward VEGF-R2 (Flk-1) and FGF-
R1 and retained or slightly decreased the potency
against PDGF-R. Compound 16f represented the most
potent inhibitor of PDGF-R in this study (IC50 ) 0.01
µM). In addition, 2-methoxyphenyl substitution in com-
pound 16h exhibited decreased inhibitory activity against
all three RTKs when compared to 16a .
Hydrophilic substituents at the C-5 position of 11a
(i.e., carboxyl or aminosulfonyl group in 11b or 11c,
respectively) were shown to lead to increased potency
toward both VEGF-R2 (Flk-1) and FGF-R1 and de-
creased the potency against the PDGF-R tyrosine kinase
(Table 2). It is conceivable that these substitutions may
displace water molecules in the vicinity of the C-5
position that were observed in the 1/FGF-R1 cocrystal
structure7 and hence might lead to increased potencies
toward these RTKs.
In addition, lipophilic substituents at the C-6 position
on the indolin-2-one core maintained or reduced the
inhibitory activity against the PDGF-R as in the case
of 1 series. In this regard, methoxy, phenyl, and
3-methoxyphenyl groups at the C-6 position of the
indolin-2-one core (compounds 11d -f) retained the
potency against the PDGF-R, whereas C-6 substituents
of 11g and 11h led to 10- and 22-fold decreased potency,
respectively, when compared to 11a . However, the effect
of a C-6 aryl substitution in the 11a series with respect
to potency toward the VEGF-R2 (Flk-1) and FGF-R1
kinases (compounds 11e-h ) was found to be opposite
that observed for the 1 series. In this regard, C-6 aryl
substitutions in the 11a series increased the inhibitory
activity as it relates to VEGF-R2 (Flk-1) and FGF-R1
(Table 2) whereas C-6 aryl substituents in the 1 series
reduced the potency (Table 1). We explain these findings
by suggesting that the C-6 aryl substitutions may move
the indolin-2-one core slightly toward the entrance of
the ATP binding site. This movement may weaken the
propionic acid/asparagine interaction in 1 series, but
strengthen the propionic acid/lysine interaction in the
11a series. Compound 11f was found to be the most
potent inhibitor of this series as it relates to inhibition
of all three of these RTKs with IC50 values of 0.09, 0.36,
and 0.17 µM for VEGF-R2 (Flk-1), FGF-R1, and PDGF-
R, respectively.
Lastly, most of the compounds from the 16a series
were found to have inhibitory activity against ligand-
dependent cell proliferation stimulated by VEGF, FGF,
and PDGF. This is in contrast to their low activity when
tested against EGF-dependent cell proliferation. A
comparison of this series to the corresponding analogues
of 11a indicated that the 16a series showed decreased
potency toward VEGF- and FGF-dependent cell prolif-
eration and retained similar inhibitory activity against
PDGF-dependent cell proliferation. Compounds 16c and
16d were inactive in all cell-base assays due to their
poor membrane permeability. Similarly, there is no
correlation between the kinase and cell proliferation
results.
Most of the compounds from the 11a series were also
found to show potent inhibitory activity toward VEGF-,
FGF-, and PDGF-dependent cell proliferation. The
exceptions to this are compounds 11b and 11c, which
were shown to be inactive using these cell-based assays
while exhibiting potency in the kinase assays. This
result may be due to poor cell permeability since these
compounds are highly polar. A similar lack of correlation
between kinase inhibition and inhibition of cell prolif-
Con clu sion
In this report, we described a series of 3-substituted
indolin-2-ones containing a propionic acid functionality
and their inhibitory activities toward VEGF-R2 (Flk-1/
KDR), FGF-R1, and PDGF-R tyrosine kinases. These
in vitro results suggest potential utilities for the treat-

Substituted Indolin-2-ones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5125
ment of cancers, inflammatory diseases, cardiovascular
diseases, and fibrotic diseases of the lung, liver, and
kidney. The chemical structural requirements for in-
hibiting these RTKs have been explored.
arterial restenosis. On the other hand, multiple signal
transduction pathways and their respective RTKs could
be involved in a given disease (i.e., tumor angiogenesis).
In this case, compounds inhibiting all three RTKs
discussed in this report (i.e., 11f in Table 2) may be
advantageous as long as such inhibition does not
compromise safety. Further evaluation of compounds
from these series’ for in vivo efficacy in various disease
models is ongoing. In vivo efficacy of these compounds
in animal models will also depend on pharmacokinetic
properties including aqueous solubility, relative oral
bioavailability, metabolic stability, and tissue distribu-
tion.
Our previous report has indicated that 2 showed good
potency toward inhibition of VEGF-R2 (Flk-1), whereas
1, unlike 2, was also a potent inhibitor of FGF-R1.6,7 In
this case, potency of 1 toward FGF-R1 tyrosine kinase
activity was attributed, in part, to the presence of a
propionic acid side chain of 1 at the C-3′ position on the
pyrrole ring.7 In this report, we designed, synthesized,
and evaluated a series of analogues possessing struc-
tural features contained in these two prototype com-
pounds in order to assess the impact of these structural
modifications on the profile of kinase inhibitory activi-
ties. As described above, the compounds were classified
into three groups, 1-related compounds (Table 1), ana-
logues of 1 regioisomer, 11a (Table 2), and hybrid
compounds (16a series in Table 3) containing structure
features of both 2 and 11a .
Exp er im en ta l Section
1H NMR spectra were recorded by Acorn NMR using a
Nicolet NT300 or a Nicolet NT360. Tetramethylsilane (TMS)
was used as an internal standard, and chemical shifts are
reported in parts per million (δ) downfield from TMS. Coupling
constants are reported in hertz. Mass spectra (electron spray)
were recorded by SYNPEP CORP, using an API I PLUS
spectrometer. Elemental analyses were performed by Gal-
braith Laboratories, Inc. Elemental analysis results are within
+0.4% of the theoretical values.
5-Am in osu lfon yl-2-in d olin -2-on e (4). To a 100 mL flask
charged with 27 mL of chlorosulfonic acid was added slowly
13.3 g (100 mmol) of indolin-2-one. The reaction temperature
was maintained below 30 °C during the addition. After the
addition, the reaction mixture was stirred at room temperature
for 1.5 h, heated to 68 °C for 1 h, cooled, and poured into water.
The precipitate was washed with water and dried in a vacuum
oven to give 11.0 g of 5-chlorosulfonyl-2-indolin-2-one (3) (50%
yield), which was used without further purification. Compound
3 (2.1 g, 9.1 mmol) was added to 10 mL of ammonium
hydroxide in 10 mL of ethanol and stirred at room temperature
overnight. The mixture was concentrated and the solid col-
lected by vacuum filtration to give 0.4 g (20% yield) of
5-aminosulfonyl-2-indolin-2-one as an off-white solid (4): 1H
NMR (360 MHz, DMSO-d6) δ 10.67 (s, 1H, NH-1), 7.63-7.66
(m, 2H, H-4,6), 7.13 (s, 2H, H2NSO2-5), 6.91 (d, J ) 8.04 Hz,
1H, H-7), and 3.56 (s, 2H, CH2-3); MS m/z (relative intensity,
%) 211 ([M - 1]+, 100).
6-P h en yl-2-in d olin -2-on e (8a ). Tetrakis(triphenylphos-
phine)palladium (0.8 g, 0.7 mmol) was added to a mixture of
3.1 g (25.4 mmol) of benzeneboronic acid, 5 g (22.7 mmol) of
5-bromo-2-fluoronitrobenzene and 22 mL of 2 M sodium
carbonate solution in 50 mL of toluene and 50 mL of ethanol.
The mixture was refluxed for 2 h, concentrated, and the
residue extracted twice with ethyl acetate. The ethyl acetate
layer was washed with water and brine, dried, and concen-
trated to give a yellow oil. The oil was chromatographed on
silica gel eluting with 5% ethyl acetate in hexane to give 4.75
g (96% yield) of 4-fluoro-3-nitrobiphenyl (5a ) as a yellow oil.
Dimethyl malonate (10 mL, 87.5 mmol) in 25 mL of dimethyl
sulfoxide was added dropwise to 3.5 g (145.8 mmol) of sodium
hydride suspended in 25 mL of dimethyl sulfoxide and the
mixture was heated at 100 °C for 10 min. The mixture was
cooled to room temperature and 4.7 g (21.6 mmol) of 5a in 25
mL of dimethyl sulfoxide was added. The mixture was heated
at 100 °C for 2 h, cooled, and quenched with 300 mL of
saturated ammonium chloride solution. The mixture was
extracted three times with ethyl acetate and the combined
organic layers washed with water and brine and evaporated
to give crude dimethyl-3-nitrobiphenyl-4-malonate (6a ) as a
yellow oil.
The following chemical structural requirements for
these 3-substituted indolin-2-ones to inhibit VEGF-R2
(Flk-1), FGF-R1, and PDGF-R tyrosine kinases were
deduced from the enzymatic assay results and rational-
ized by protein kinase structural models. First, it was
shown that a solubilizing propionic acid was found to
be required for high potency against these RTKs.
Structural models suggested that the propionic acid side
chain could interact favorably with asparagine residues
in VEGF-R2 (Flk-1) and FGF-R1 for 1 analogues or with
basic residues (lysine or arginine) in all three target
RTKs for the 11a and 16a analogues. Second, regarding
the location of the propionic acid, FGF-R1 and VEGF-
R2 (Flk-1) clearly favor substitution at the C-3′ position,
while PDGF-R prefers the C-4′ position of the pyrrole
ring. Third, the hydrophilic substituent (i.e., aminosul-
fonyl or carboxyl group) at the C-5 position of the
indolin-2-one core is favorable for the inhibitory activity
against both VEGF-R2 (Flk-1) and FGF-R1 (i.e., 11b
and 11c vs 11a and 16c and 16d vs 16a ). This is in
agreement with the cocrystallographic study of 1/FGF-
R1, which indicated that three water molecules localized
near the C-5 position of the indolin-2-one core. There-
fore, hydrophilic substituent at the C-5 position should
enhance the affinity of the inhibitor to FGF-R1 as well
as VEGF-R2 (Flk-1), given the high homology existing
between the two RTKs. Finally, The effect of C-6 aryl
substitutions depends on the location of the propionic
acid side chain. For the 1 series, it reduced the inhibi-
tory activities against FGF-R1 and VEGF-R2 (Flk-1).
However, for the 11a and 16a analogues, it increased
potency toward these receptors, especially when the
substituent is phenyl or 3-methoxyphenyl.
In summary, we have prepared a series of compounds
that can inhibit VEGF-R2 (Flk-1/KDR), FGF-R1, and
PDGF-R tyrosine kinases. Since all three RTKs play
crucial roles in many disease processes such as cancer,
arterial restenoesis, diabetic retinopathies, atheroscle-
rosis, fibrosis, and rheumatoid arthritis, inhibitors of
these RTKs may have therapeutic potential. For in-
stance, inhibitors that selectively inhibit VEGF-R2 (Flk-
1) and Flt-1 could be developed for the treatment of
rheumatoid arthritis whereas compounds with selective
inhibitory activity against PDGF-R may be useful for
Crude 6a was refluxed in 30 mL of 6 N hydrochloric acid
for 24 h. The precipitate was collected by filtration, washed
with water, and dried to give 4.5 g (80% based on 6a ) of
3-nitrobiphenyl-4-acetic acid (7a ) as a cream-colored solid.
Iron chips (2.6 g, 46.6 mmol) was added all at once to 4.5 g
(17.5 mmol) of 7a in 40 mL of acetic acid. The mixture was
refluxed for 2 h, concentrated to dryness, and taken up in ethyl

5126 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Sun et al.
acetate. The solids were removed by filtration and the filtrate
was washed twice with 1 N hydrochloric acid and brine and
dried over anhydrous sodium sulfate. The filtrate was con-
centrated to give 3.4 g (93% yield) of 6-phenyl-2-indolin-2-one
drous sodium sulfate, and concentrated to give crude dimethyl
3′-methoxy-3-nitrobiphenyl-4-malonate (6c) as a pale yellow
solid.
Crude 6c was heated at 110 °C in 45 mL of 6 N hydrochloric
acid for 4 days and cooled. The precipitate was collected by
filtration, washed with water and hexane, and dried to give
5.3 g of crude 3′-methoxy-3-nitrobiphenyl-4-acetic acid (7c) as
a light tan solid.
1
(8a ) as a light brown solid: H NMR (360 MHz, DMSO-d6) δ
10.40 (s, br, 1H, NH-1), 7.57-7.60 (m, 2H, H-2′,6′), 7.42-7.46
(m, 2H, H-3′,5′), 7.34 (dt, J ) 1.96, 7.23 Hz, 1H, H-4′), 7.27 (d,
J ) 7.69 Hz, 1H, H-4), 7.19 (dd, J ) 1.59, 7.69 Hz, 1H, H-5),
7.01 (d, J ) 1.59 Hz, 1H, H-7), 3.49 (s, 2H, CH2-3); MS m/z
(relative intensity, %) 210 ([M + 1]+, 100).
Compound 7c (5.2 g, 18.1 mmol) was dissolved in methanol
and hydrogenated over 0.8 g of 10% palladium on carbon for
3 h at room temperature. The catalyst was removed by
filtration and washed with methanol, and the filtrates were
combined and concentrated to give a brown solid. The solid
was chromatographed on silica gel eluting with ethyl acetate-
hexaneacetic acid to give 3.0 g (73% yield based on 5c) of 6-(3-
methoxypheny)-2-indolin-2-one (9c) as a pink solid: 1H NMR
(360 MHz, DMSO-d6) δ 10.39 (s, br, 1H, NH-1), 7.35 (t, J )
7.99 Hz, 1H, H-5′), 7.26 (d, J ) 7.79 Hz, 1H, H-4), 7.19 (dd, J
) 1.30, 7.79 Hz, 1H, H-5), 7.51 (td, J ) 0.79, 7.99 Hz, 1H,
H-4′), 7.09 (t, J ) 2.34 Hz, 1H, H-2′), 7.01 (d, J ) 1.30 Hz, 1H,
H-7), 6.92 (ddd, J ) 0.79, 2.34, 7.99 Hz, 1H, H-6′), 3.80 (s, 3H,
OCH3-3′), 3.49 (s, 2H, CH2-3); MS m/z (relative intensity, %)
240 ([M + 1]+, 100).
6-(4-Met h oxyp h en yl)-2-in d olin -2-on e (8d ). Tetrakis-
(triphenylphosphine)palladium (1 g, 0.9 mmol) was added to
a mixture of 5 g (32.9 mmol) of 4-methoxyphenylboronic acid,
6.6 g (30 mmol) of 5-bromo-2-fluoronitrobenzene, and 30 mL
of 2 M sodium carbonate solution in 50 mL of toluene and 50
mL of ethanol. The mixture was refluxed for 2 h, concentrated,
and the residue extracted twice with ethyl acetate. The ethyl
acetate layer was washed with water and brine, dried, and
concentrated to give a brown oily solid. The solid was chro-
matographed on silica gel eluting with 5% ethyl acetate in
hexane to give crude 4-fluoro-4′-methoxy-3-nitrobiphenyl (5d )
as a pale yellow solid.
Dimethyl malonate (10 mL, 87.5 mmol) was added dropwise
to 2.0 g (83.3 mmol) of sodium hydride suspended in 60 mL of
dimethyl sulfoxide. The mixture was heated to 100 °C for 10
min and cooled to room temperature. Crude 5d (5.2 g, 21.0
mmol) in 50 mL of dimethyl sulfoxide was added and the
mixture was heated at 100 °C for 2 h. The reaction mixture
was cooled and quenched with 300 mL of saturated ammonium
chloride solution and extracted three times with ethyl acetate.
The extracts were combined, washed with saturated am-
monium chloride, water, and brine, dried over anhydrous
sodium sulfate, and concentrated to give crude dimethyl 4′-
methoxy-3-nitrobiphenyl-4-malonate (6d ) as a yellow oil.
Crude 6d was heated at 100 °C in 60 mL of 6 N hydrochloric
acid for 15 h and cooled. The precipitate was collected by
filtration, washed with water and hexane, and dried to give
7.2 g of crude 4′-methoxy-3-nitrobiphenyl-4-acetic acid (7d ) as
a light tan solid.
Iron chips (3.6 g, 62.1 mmol) was added in one portion to
7.2 g (25.1 mmol) of 7d in 50 mL of glacial acetic acid and
heated at 100 °C overnight. The reaction mixture was con-
centrated to dryness, sonicated in ethyl acetate, and filtered
to remove the insolubles. The filtrate was washed twice with
1 N hydrochloric acid, brine, dried over anhydrous sodium
sulfate, and concentrated to give 2.7 g (38% yield based on
5-bromo-2-fluoronitrobenzene) of 6-(4-methoxyphenyl)-2-indo-
lin-2-one (8d ) as a rose-colored solid: 1H NMR (360 MHz,
DMSO-d6) δ 10.38 (s, br, 1H, NH-1), 7.50-7.54 (m, 2H, H-2′6′),
7.23 (d, J ) 7.72 Hz, 1H, H-4), 7.14 (dd, J ) 1.38, 7.72 Hz,
1H, H-5), 6.98-7.02 (m, 2H, H-3′,5′), 6.97 (d, J ) 1.38 Hz, 1H,
H-7), 3.78 (s, 3H, OCH3-4′), 3.47 (s, 2H, CH2-3); MS m/z
(relative intensity, %) 240 ([M + 1]+, 100].
6-(2-Met h oxyp h en yl)-2-in d olin -2-on e (8b ). Tetrakis-
(triphenylphosphine)palladium (1 g, 0.9 mmol) was added to
a mixture of 5 g (32.9 mmol) of 2-methoxyphenylboronic acid,
6.6 g (30 mmol) of 5-bromo-2-fluoronitrobenzene, and 30 mL
of 2 M sodium carbonate solution in 50 mL of toluene and 50
mL of ethanol. The mixture was refluxed for 2 h, concentrated,
and the residue extracted twice with ethyl acetate. The ethyl
acetate layer was washed with water and brine, dried, and
concentrated to give dark green oil which solidified on standing
to give 6 g of crude 4-fluoro-2′-methoxy-3-nitrobiphenyl (5b).
Dimethyl malonate (14 mL, 122.5 mmol) was added drop-
wise to 2.9 g (120.8 mmol) of sodium hydride suspended in 50
mL of dimethyl sulfoxide. The mixture was heated at 100 °C
for 15 min and cooled to room temperature. Crude 5b in 60
mL of dimethyl sulfoxide was added, and the mixture was
heated at 100 °C for 2 h. The reaction mixture was cooled and
quenched with 300 mL of saturated ammonium chloride
solution and extracted twice with ethyl acetate. The extracts
were combined, washed with saturated ammonium chloride,
water, and brine, dried over anhydrous sodium sulfate, and
concentrated to give crude dimethyl 2′-methoxy-3-nitrobiphe-
nyl-4-malonate (6b) as a yellow oil.
Crude 6b was heated at 100 °C in 50 mL of 6 N hydrochloric
acid for 24 h and cooled. The precipitate was collected by
filtration, washed with water and hexane, and dried to give
9.8 g of 2′-methoxy-3-nitrobiphenyl-4-acetic acid (7b) as a light
tan solid.
Iron chips (5 g, 89.6 mmol) was added in one portion to 9.8
g (34.1 mmol) of 7b in 50 mL of glacial acetic acid was heated
to 100 °C for 3 h. The reaction mixture was concentrated to
dryness, sonicated in ethyl acetate and filtered to remove the
insolubles. The filtrate was washed twice with 1 N hydrochloric
acid, water, brine, dried over anhydrous sodium sulfate and
concentrated. The residue was chromatographed on silica gel
eluting with ethyl acetate-hexane (1:2) to give 5.4 g (75% yield
based on 5-bromo-2-fluoronitrobenzene) of 6-(2-methoxyphe-
nyl)-2-indolin-2-one (8b) as a rose-colored solid: 1H NMR (360
MHz, DMSO-d6) δ 10.32 (s, br, 1H, NH-1), 7.31 (dt, J ) 1.86,
7.39 Hz, 1H, H-4′), 7.23 (dd, J ) 1.86, 7.39 Hz, 1H, H-6′), 7.28
(d, J ) 7.65 Hz, 1H, H-4), 7.08 (dd, J ) 1.16, 7.39 Hz, 1H,
H-3′), 6.70 (dt, J ) 1.16, 7.39 Hz, 1H, H-5′), 6.98 (dd, J ) 1.69,
7.65 Hz, 1H, H-5), 6.91 (d, J ) 1.69 Hz, 1H, H-7), 3.74 (s, 3H,
OCH3-2′), 3.47 (s, 2H, CH2-3); MS m/z (relative intensity, %)
240 ([M + 1]+ , 100).
6-(3-Met h oxyp h en yl)-2-in d olin -2-on e (8c). Tetrakis-
(triphenylphosphine)palladium (0.7 g, 0.6 mmol) was added
to a mixture of 5 g (32.9 mmol) of 3-methoxyphenylboronic
acid, 3.8 g (17.3 mmol) of 5-bromo-2-fluoronitrobenzene and
11 mL of 2 M sodium carbonate solution in 100 mL of toluene.
The mixture was refluxed for 2 h, diluted with water, and
extracted with ethyl acetate. The ethyl acetate was washed
with saturated sodium bicarbonate, brine, dried, and concen-
trated to give an oily solid. The solid was chromatographed
on silica gel eluting with ethyl acetate-hexane to give 4.3 g
of crude 4-fluoro-3′-methoxy-3-nitrobiphenyl (5c).
Dimethyl malonate (9.7 mL, 84.9 mmol) was added dropwise
to 2.0 g (83.3 mmol) of sodium hydride suspended in 50 mL of
dimethyl sulfoxide. The mixture was heated to 100 °C for 35
min and cooled to room temperature. Compound 5c (4.2 g, 17.0
mmol) in 50 mL of dimethyl sulfoxide was added and the
mixture was heated at 100 °C for 1 h. The reaction mixture
was cooled and quenched with 300 mL of saturated ammonium
chloride solution and extracted twice with ethyl acetate. The
extracts were combined, washed with brine, dried over anhy-
6-(3-Eth oxyp h en yl)-2-in d olin -2-on e (8e). Tetrakis(triph-
enylphosphine)palladium (0.8 g. 0.7 mmol) was added to a
mixture of 4.2 g (25.3 mmol) of 3-ethoxyphenylboronic acid,
5.0 g (22.7 mmol) of 5-bromo-2-fluoronitrobenzene and 22 mL
of 2 M sodium carbonate solution in 50 mL of toluene and 50
mL of ethanol. The mixture was refluxed for 2 h and concen-
trated, water was added, and the mixture was extracted twice
with ethyl acetate. The ethyl acetate layer was washed with

Substituted Indolin-2-ones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5127
water and brine, dried, and concentrated. The residue was
chromatographed on silica gel eluting with 5% ethyl acetate
in hexane to give 5.3 g of crude 4-fluoro-3′-ethoxy-3-nitrobi-
phenyl (5e) as a yellow oil.
20 mL of ethanol. The solid was dissolved in 90 mL of 2.5 N
sodium hydroxide and stirred at 70-80 °C for 3 h. The mixture
was cooled to room temperature and acidified to pH 2 with
0.5 N hydrochloric acid. The precipitate was collected by
vacuum filtration and washed thoroughly with water to give
crude 5-carboxyindolin-2-one as a dark brown solid. After
standing overnight, the filtrate yielded 2 g of 5-carboxy-indolin-
2-one as a yellow solid. The crude dark brown product was
dissolved in hot methanol, the insoluble material was removed
by filtration, and the filtrate was concentrated to give another
5.6 g of 5-carboxyindolin-2-one as a brown solid. The combined
Dimethyl malonate (11.4 mL, 100 mmol) was added drop-
wise to 4.0 g (167 mmol) of sodium hydride suspended in 20
mL of dimethyl sulfoxide. The mixture was heated to 100 °C
for 10 min and cooled to room temperature. Crude 5e (5.3 g,
20.2 mmol) in 25 mL of dimethyl sulfoxide was added, and
the mixture was heated at 100 °C for 2 h. The reaction mixture
was cooled and quenched with 300 mL of saturated ammonium
chloride solution and extracted three times with ethyl acetate.
The extracts were combined, washed with water and brine,
dried over anhydrous sodium sulfate, and concentrated to give
crude dimethyl 3′-ethoxy-3-nitrobiphenyl-4-malonate (6e) as
yellow oil.
Crude 6e was heated at 100 °C in 60 mL of 6 N hydrochloric
acid for a total of 4 days and cooled. The precipitate was
collected by filtration, washed with water and hexane, and
dried to give 4.7 g (77% yield based on 5-bromo-2-fluoroni-
trobenzene) of crude 3′-ethoxy-3-nitrobiphenyl-4-acetic acid as
a light tan solid (7e).
Iron chips (2.4 g, 41 mmol) was added in one portion to 4.6
g (15.3 mmol) of 7e in 40 mL of glacial acetic acid and refluxed
for 2 h. The reaction mixture was concentrated to dryness,
treated repeatedly with ethyl acetate, and filtered to remove
the insoluble. The filtrate was washed twice with 1 N
hydrochloric acid, brine, dried over anhydrous sodium sulfate,
and concentrated to give 3.5 g (91% yield) of 8e as a light
brown solid: 1H NMR (360 MHz, DMSO-d6) δ 10.40 (s, br, 1H,
NH-1), 7.33 (t, J ) 7.92 Hz, 1H, H-5′), 7.25 (d, J ) 7.70 Hz,
1H, H-4), 7.19 (dd, J ) 1.34, 7.70 Hz, 1H, H-5), 7.13 (d, br, J
) 7.92 Hz, 1H, H-4′), 7.07 (t, J ) 2.62 Hz, 1H, H-2′), 7.00 (d,
J ) 1.34 Hz, 1H, H-7), 6.90 (dd, J ) 2.62, 7.92 Hz, 1H, H-6′),
4.08 (q, J ) 7.00 Hz, 2H, OCH2CH3-3′), 3.49 (s, 2H, CH2-3)
1.34 (t, J ) 7.00 Hz, OCH2CH3-3′); MS m/z (relative intensity,
%) 254 ([M + 1]+, 100).
3-[4-Meth yl-5-(2-oxo-1,2-dih ydr oin dol-3-yliden em eth yl)-
1H-p yr r ol-3-yl]p r op ion ic Acid (11a ). 3-(5-Formyl-4-methyl-
1H-pyrrol-3-yl)propionic acid (10) (9.0 g, 50 mmol), which was
prepared as previously reported,13 and 6.0 g (45 mmol) of
indolin-2-one in 50 mL of ethanol were heated to 70 °C for 4
h. Acetic acid (12 mL) was slowly added, resulting in a copious
precipitate. The mixture was refluxed for 5 min and cooled to
room temperature, and the precipitate was collected by
vacuum filtration and washed with 30 mL of ethanol. The
precipitate was slurry-washed at reflux in 30 mL of ethanol,
cooled to room temperature, collected by vacuum filtration,
washed with 20 mL of ethanol, and dried under vacuum to
give 11.9 g (89% yield) of 3-[4-methyl-5-(2-oxo-1,2-dihydroindol-
3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid as an orange
solid: 1H NMR (360 MHz, DMSO-d6) δ 13.29 (s, br, 1H, NH-
1′), 12.05 (s, br, 1H, CH2CH2COOH-4′), 10.78 (s, br, 1H, NH-
1), 7.73 (d, J ) 7.43 Hz, 1H, H-4), 7.61 (s, 1H, H-vinyl), 7.13
(s, 1H, H-5′), 7.10 (t, J ) 7.43 Hz, 1H, H-6), 6.97 (t, J ) 7.43
Hz, 1H, H-5), 6.85 (d, J ) 7.43 Hz, 1H, H-7), 2.65 (t, J ) 7.30
Hz, 2H, CH2CH2COOH-4′), 2.46 (t, J ) 7.30 Hz, 2H, CH2CH2-
COOH-4′), 2.25 (s, 3H, CH3-3′); MS m/z (relative intensity, %)
297 (M+, 100). Anal. (C17H16N2O3‚0.25H2O) C, H, N.
1
yield was 85%. H NMR (360 MHz, DMSO-d6) δ 12.56 (s, br,
1H, COOH-5), 10.70 (s, 1H, NH-1), 7.81 (dd, J ) 0.68. 8.23
Hz, 1H, H-6), 7.74 (s, br, 1H, H-4), 6.87 (d, J ) 8.23 Hz, 1H,
H-7), and 3.53 (s, 2H, CH2-3).
The reaction mixture of 88.6 mg (0.5 mmol) of 5-carboxy-
indolin-2-one, 90.6 mg (0.5 mmol) of 10, and 75 µL (0.75 mmol)
of piperidine in 2.0 mL of ethanol was heated at 95 °C for 5 h
and cooled to room temperature. The precipitate was filtered,
washed with cold ethanol, 2 N hydrochloric acid, and water,
and dried to give 42 mg (25%) of 3-[4-(2-carboxyethyl)-3-
methyl-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-
5-carboxylic acid (11b) as a dark yellow solid: 1H NMR (360
MHz, DMSO-d6) δ 13.27 (s, br, 1H, NH-1′), 12.28 (s, br, 2H,
COOH-5 and CH2CH2COOH-4′), 11.11 (s, 1H, NH-1), 8.34 (d,
J ) 1.36 Hz, 1H, H-4), 7.78 (s, 1H, H-vinyl), 7.75 (dd, J ) 1.36,
8.20 Hz, 1H, H-6), 7.19 (d, J ) 3.07 Hz, 1H, H-5′), 6.93 (d, J
) 8.20 Hz, 1H, H-7), 2.65 (t, J ) 7.56 Hz, 2H, CH2CH2COOH-
4′), 2.46 (t, J ) 7.56 Hz, 2H, CH2CH2COOH-4′), 2.29 (s, 3H,
CH3-3′); MS m/z (relative intensity, %) 341 ([M + 1]+, 7). Anal.
(C18H16N2O5‚0.5H2O) C, H, N.
3-[4-Met h yl-5-(2-oxo-5-su lfa m oyl-1,2-d ih yd r oin d ol-3-
ylid en em eth yl)-1H-p yr r ol-3-yl]p r op ion ic Acid (11c). The
reaction mixture of 106 mg (0.5 mmol) of 5-aminosulfonyl-
indolin-2-one (4), 90.6 mg (0.5 mmol) of 10, and 75 µL (0.75
mmol) of piperidine in 2.0 mL of ethanol was heated at 95 °C
for 5 h and cooled to room temperature. The precipitate was
filtered, washed with cold ethanol, 2 N hydrochloric acid, and
water, and dried to give 132 mg (70%) of 3-[4-methyl-5-(2-oxo-
5-sulfamoyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-
1
propionic acid (11c): H NMR (360 MHz, DMSO-d6) δ 13.28
(s, br, 1H, NH-1′), 12.03 (s, br, 1H, CH2CH2COOH-4′), 11.15
(s, br, 1H, NH-1), 8.20 (d, J ) 1.60 Hz, 1H, H-4), 7.73 (s, 1H,
H-vinyl), 7.59 (dd, J ) 1.60, 8.17 Hz, 1H, H-6), 7.23 (d, J )
2.85 Hz, 1H, H-5′), 7.10 (s, 2H, NH2SO2-5), 6.99 (d, J ) 8.17
Hz, 1H, H-7), 2.66 (t, J ) 7.41 Hz, 2H, CH2CH2COOH-4′), 2.47
(t, J ) 7.41 Hz, 2H, CH2CH2COOH-4′), 2.29 (s, 3H, CH3-3′).
Anal. (C17H17N3O5S‚H2O) C, H, N.
3-[5-(6-Me t h oxy-2-oxo-1,2-d ih yd r oin d ol-3-ylid e n e -
m et h yl)-4-m et h yl-1H -p yr r ol-3-yl]p r op ion ic Acid (11d ).
This compound was prepared using the same procedure
described in the preparation of 11c with a yield of 43%: 1H
NMR (360 MHz, DMSO-d6) δ 13.10 (s, br, 1H, NH-1′), 12.04
(s, br, 1H, CH2CH2COOH-4′), 10.76 (s, br, 1H, NH-1), 7.63 (d,
J ) 8.29 Hz, 1H, H-4), 7.46 (s, 1H, H-vinyl), 7.07 (d, J ) 3.03
Hz, 1H, H-5′), 6.55 (dd, J ) 2.32, 8.29 Hz, 1H, H-5), 6.43 (d, J
) 2.32 Hz, 1H, H-7), 3.74 (s, 3H, OCH3-6), 2.63 (t, J ) 7.31
Hz, 2H, CH2CH2COOH-4′), 2.45 (t, J ) 7.31 Hz, 2H, CH2CH2-
COOH-4′), 2.23 (s, 3H, CH3-3′); MS m/z (relative intensity, %)
327 ([M + 1]+, 100). Anal. (C18H18N2O4‚0.5H2O) C, H, N.
3-[4-(2-Ca r boxyeth yl)-3-m eth yl-1H-p yr r ol-2-ylm eth yl-
en e]-2-oxo-2,3-dih ydr o-1H-in dole-5-car boxylic Acid (11b).
Indolin-2-one (6.7 g, 50 mmol) was added to a stirred suspen-
sion of 23 g (172.5 mmol) of aluminum chloride in 30 mL of
dichloroethane in an ice bath. Chloroacetyl chloride (11.3 g,
100 mmol) was slowly added and hydrogen chloride gas was
evolved. After 10 min of stirring, the reaction was warmed to
40-50 °C for 1.5 h. The mixture was cooled to room temper-
ature and poured into ice water. The precipitate was collected
by vacuum filtration, washed with water, and dried under
vacuum to give 10.3 g (98%) of 5-chloroacetylindolin-2-one as
an off-white solid. A suspension of 9.3 g (44.4 mmol) of
5-chloroacetylindolin-2-one was stirred in 90 mL of pyridine
at 80-90 °C for 3 h and then cooled to room temperature. The
precipitate was collected by vacuum filtration and washed with
3-[4-Methyl-5-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidene-
m eth yl)-1H-p yr r ol-3-yl]p r op ion ic Acid (11e). This com-
pound was prepared using the same procedure described in
the preparation of 11c with a yield of 78%: 1H NMR (360 MHz,
DMSO-d6) δ 13.29 (s, br, 1H, NH-1′), 12.01 (s, vbr, 1H, CH2-
CH2COOH-4′), 10.89 (s, br, 1H, NH-1), 7.83 (d, J ) 7.92, 1H,
H-4), 7.65 (s, 1H, H-vinyl), 7.63 (d, J ) 7.51 Hz, 2H, H-2′′,6′′),
7.45 (t, J ) 7.51 Hz, 2H, H-3′′,5′′), 7.33 (t, J ) 7.51 Hz, 1H,
H-4′′), 7.28 (dd, J ) 1.93, 7.92 Hz, 1H, H-5), 7.16 (d, J ) 1.93
Hz, 1H, H-7), 7.09 (d, J ) 1.71 Hz, 1H, H-5′), 2.66 (t, J ) 7.47
Hz, 2H, CH2CH2COOH-4′), 2.46 (t, J ) 7.47 Hz, 2H, CH2CH2-
COOH-4′), and 2.27 (s, 3H, CH3-3′); MS m/z (relative intensity,
%) 373 (M+, 100). Anal. (C23H20N2O3‚H2O) C, H, N.
3-{5-[6-(3-Met h oxy-p h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-

5128 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Sun et al.
ylid en em eth yl]-4-m eth yl-1H-p yr r ol-3-yl}p r op ion ic Acid
(11f). This compound was prepared using the same procedure
as for preparation of 11c with a yield of 97%: H NMR (360
indolin-2-one, and 0.05 g (0.6 mmol) of piperidine were refluxed
in 5 mL of ethanol overnight. The mixture was concentrated
to dryness and chromatographed on a silica gel column eluting
with ethyl acetate-hexane-acetic acid to give 80 mg (23%
yield) of 3-[2,4-dimethyl-5-(2-oxo-1,2-dihydroindol-3-ylidenem-
ethyl)-1H-pyrrol-3-yl]propionic acid (16a ) as a mustard yellow
solid: 1H NMR (360 MHz, DMSO-d6) δ 13.38 (s, br, 1H, NH-
1′), 12.05 (s, br, 1H, CH2CH2COOH-4′), 10.70 (s, br, 1H, NH-
1), 7.69 (d, J ) 7.39 Hz, 1H, H-4), 7.53 (s, 1H, H-vinyl), 7.06
(t, J ) 7.39 Hz, 1H, H-6), 6.95 (t, J ) 7.39 Hz, 1H, H-5), 6.85
(d, J ) 7.39 Hz, 1H, H-7), 2.63 (t, J ) 7.45 Hz, 2H, CH2CH2-
COOH-4′), 2.34 (t, J ) 7.45 Hz, 2H, CH2CH2COOH-4′), 2.28
(s, 3H, CH3), 2.24 (s, 3H, CH3); MS m/z (relative intensity, %)
311 ([M + 1]+, 100). Anal. (C18H18N2O3) C, H, N.
3-[5-(5-Br om o-2-oxo-1,2-dih ydr oin dol-3-yliden em eth yl)-
2,4-d im eth yl-1H-p yr r ol-3-yl]p r op ion ic Acid (16b). The
reaction mixture of 106.0 mg (0.5 mmol) of 5-bromo-indolin-
2-one, 97.5 mg (0.5 mmol) of 15, and 75 µL (0.75 mmol) of
piperidine in 2.0 mL of ethanol was heated at 95 °C for 5 h
and cooled to room temperature. The precipitate was filtered,
washed with cold ethanol, 2 N hydrogen chloride, and water,
and dried to give 171 mg (88%) of 3-[5-(5-bromo-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrol-3-yl]-
propionic acid as a dark yellow solid: 1H NMR (360 MHz,
DMSO-d6) δ 12.04 (s, br, 1H, CH2CH2COOH-4′), 10.80 (s, br,
1H, NH-1), 8.00 (d, J ) 2.06 Hz, 1H, H-4), 7.67 (s, 1H, H-vinyl),
7.19 (dd, J ) 2.06, 8.50 Hz, 1H, H-6), 6.79 (d, J ) 8.50 Hz,
1H, H-7), 2.64 (t, J ) 7.63 Hz, 2H, CH2CH2COOH-4′), 2.35 (t,
J ) 7.63 Hz, 2H, CH2CH2COOH-4′), 2.29 (s, 3H, CH3), 2.27
(s, 3H, CH3); MS m/z (relative intensity, %) 389 (M+, 100).
Anal. (C18H17BrN2O3) C, H, N.
3-[4-(2-C a r b o xye t h yl)-3,5-d im e t h yl-1H -p y r r o l-2-y l
m eth ylen e]-2-oxo-2,3-dih ydr o-1H-in dole-5-car boxylic Acid
(16c). This compound was prepared using the same procedure
as for preparation of 16b with a yield of 65%: 1H NMR (300
MHz, DMSO-d6) δ 13.39 (s, 1H, NH-1′), 12.31 (s, br, 2H,
COOH-5 and CH2CH2COOH-4′), 11.07 (s, 1H, NH-1), 8.04 (s,
1H, H-4), 7.72 (d, J ) 8.07 Hz, 1H, H-6), 7.70 (s, 1H, H-vinyl),
6.93 (d, J ) 8.07 Hz, 1H, H-7), 2.64 (t, J ) 7.37 Hz, 2H, CH2-
CH2COOH-4′), 2.34 (t, J ) 7.37 Hz, 2H, CH2CH2COOH-4′),
2.30 (s, 3H, CH3), 2.28 (s, 3H, CH3); MS m/z (relative intensity,
%) 354 (M+, 100). Anal. (C19H18N2O5‚0.5H2O) C, H, N.
3-[2,4-Dim eth yl-5-(2-oxo-5-su lfa m oyl-1,2-d ih yd r oin d ol-
3-ylid en em et h yl)-1H -p yr r ol-3-yl]p r op ion ic Acid (16d ).
This compound was prepared using the same procedure as for
preparation of 16b with a yield of 60%: 1H NMR (300 MHz,
DMSO-d6) δ 13.40 (s, 1H, NH-1′), 12.09 (s, br, 1H, CH2CH2-
COOH-4′), 11.10 (s, 1H, NH-1), 8.16 (s, 1H, H-4), 7.65 (s, 1H,
H-vinyl), 7.55 (d, J ) 8.21 Hz, 1H, H-6), 7.10 (s, br, 2H, H2-
NSO2-5), 6.98 (d, J ) 8.21 Hz, 1H, H-7), 2.64 (t, J ) 7.22 Hz,
2H, CH2CH2COOH-4′), 2.38 (t, J ) 7.22 Hz, 2H, CH2CH2-
COOH-4′), 2.31 (s, 3H, CH3), 2.28 (s, 3H, CH3); MS m/z
(relative intensity, %) 389 (M+, 100). Anal. (C18H19N3O5S‚
0.8H2O) C, H, N.
1
MHz, DMSO-d6) δ 13.29 (s, br, 1H, NH-1′), 12.07 (s, br, 1H,
CH2CH2COOH-4′), 10.88 (s, br, 1H, NH-1), 7.82 (d, J ) 7.77,
1H, H-4), 7.65 (s, 1H, H-vinyl), 7.36 (t, J ) 8.06 Hz, 1H, H-5′′),
7.29 (dd, J ) 1.40, 7.77 Hz, 1H, H-5), 7.20 (d, J ) 8.06 Hz,
1H, H-6′′), 7.16 (d, J ) 2.23 Hz, 1H, H-2′′), 7.14-7.15 (m, br,
1H, H-5′), 7.09 (d, J ) 1.40 Hz, 1H, H-7), 6.91 (dd, J ) 2.23,
8.06 Hz, 1H, H-4′′), 3.82 (s, 3H, OCH3-3′′), 2.65 (t, J ) 7.55
Hz, 2H, CH2CH2COOH-4′), 2.57 (t, J ) 7.55 Hz, 2H, CH2CH2-
COOH-4′), 2.27 (s, 3H, CH3-3′); MS m/z (relative intensity, %)
401 (M+, 7). Anal. (C24H22N2O4‚0.75H2O) C, H, N.
3-{5-[6-(2-Met h oxy-p h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-
ylid en em eth yl]-4-m eth yl-1H-p yr r ol-3-yl}p r op ion ic Acid
(11g). This compound was prepared using the same procedure
as for preparation of 11c with a yield of 86%: 1H NMR (360
MHz, DMSO-d6) δ 13.29 (s, br, 1H, NH-1′), 11.59 (s, br, 1H,
CH2CH2COOH-4′), 10.78 (s, br, 1H, NH-1), 7.76 (d, J ) 8.13
Hz, 1H, H-4), 7.62 (s, 1H, H-vinyl), 7.29-7.34 (m, 2H), 6.99-
7.15 (m, 6H), 3.76 (s, 3H, OCH3-2′′), 2.66 (t, J ) 7.46 Hz, 2H,
CH2CH2COOH-4′), 2.45 (t, J ) 7.46 Hz, 2H, CH2CH2COOH-
4′), 2.27 (s, 3H, CH3-3′); MS m/z (relative intensity, %) 401
([M - 1]+, 100). Anal. (C24H22N2O4‚H2O) C, H, N.
3-{5-[6-(4-Met h oxyp h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-
ylid en em eth yl]-4-m eth yl-1H-p yr r ol-3-yl}p r op ion ic Acid
(11h ). This compound was prepared using the same procedure
as for preparation of 11c with a yield of 59%: 1H NMR (360
MHz, DMSO-d6) δ 13.26 (s, br, 1H, NH-1′), 10.83 (s, br, 1H,
NH-1), 7.78 (d, J ) 8.07 Hz, 1H, H-4), 7.61 (s, 1H, H-vinyl),
7.57 (d, J ) 8.95 Hz, 2H, H-2′′,6′′), 7.22 (dd, J ) 1.44, 8.07 Hz,
1H, H-5), 7.13 (d, J ) 3.09 Hz, 1H, H-5′), 7.04 (d, J ) 1.44 Hz,
1H, H-7), 7.00 (d, J ) 8.95 Hz, 2H, H-3′′,5′′), 3.79 (s, 3H, OCH3-
4′′), 2.65 (t, J ) 7.65 Hz, 2H, CH2CH2COOH-4′), 2.44 (t, J )
7.65 Hz, 2H, CH2CH2COOH-4′), 2.27 (s, 3H, CH3-3′); MS m/z
(relative intensity, %) 403 ([M + 1]+, 100). Anal. (C24H22N2O4‚
0.5H2O) C, H, N.
3-[2,4-Dim et h yl-5-(2-oxo-1,2-d ih yd r oin d ol-3-ylid en e-
m eth yl)-1H-p yr r ol-3-yl]p r op ion ic Acid (16a ). 4-(2-Meth-
oxycarbonylethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid
benzyl ester (2.0 g, 6.3 mmol) was hydrogenated over 0.2 g of
10% palladium on carbon in 40 mL of methanol for 2 h at room
temperature. The catalyst was removed by filtration and
washed with 40 mL of methanol. The filtrate was evaporated
to dryness and dried overnight in a vacuum oven to give 1.3 g
(92% yield) of 4-(2-methoxycarbonylethyl)-3,5-dimethyl-1H-
pyrrole-2-carboxylic acid (12).
Compound 12 (1.3 g, 5.8 mmol) was ground with 0.5 g of
anhydrous sodium acetate and then heated at 100 °C for 3
days. The mixture was dissolved in water, extracted with ethyl
acetate, and chromatographed on a column of silica gel in ethyl
acetate-hexane to give 0.4 g (38% yield) of 3-(2,4-dimethyl-
1H-pyrrol-3-yl)propionic acid methyl ester (13) as a thick, pale
yellow oil.
Compound 13 (0.35 g, 1.9 mmol) and 0.5 g (3.9 mmol) of
chloromethylenedimethylammonium chloride in 10 mL of
dichloroethane were stirred at room temperature under ni-
trogen for 2 h. The reaction mixture was concentrated and
treated with 10 mL of 6 M sodium hydroxide solution and then
extracted three times with 10 mL of ethyl acetate. The ethyl
acetate extracts were combined, dried over anhydrous sodium
sulfate, and evaporated to dryness to give 0.24 g (60% yield)
of 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid meth-
yl ester (14) as a brown oil.
Compound 14 (0.23 g, 1.1 mmol) in 6 N sodium hydroxide
(10 mL) was heated at 100 °C for 2 h. The reaction mixture
was cooled to room temperature, acidified with 6 N hydro-
chloric acid, and extracted three times with 10 mL of ethyl
acetate. The combined organic layers were washed with 10 mL
of water and 5 mL of brine, dried over anhydrous sodium
sulfate, and evaporated to dryness to give 230 mg (100%) of
crude 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid
(15) as a brown oil.
3-[5-(6-Met h oxy-2-oxo-1,2-d ih yd r oin d ol-3-ylid en em e-
th yl)-2,4-d im eth yl-1H-p yr r ol-3-yl]p r op ion ic Acid (16e).
This compound was prepared using the same procedure as for
preparation of 16b with a yield of 76%: 1H NMR (360 MHz,
DMSO-d6) δ 13.15 (s, br, 1H, NH-1′), 11.70 (s, br, 1H, CH2-
CH2COOH-4′), 10.65 (s, br, 1H, NH-1), 7.58 (d, J ) 8.27 Hz,
1H, H-4), 7.39 (s, 1H, H-vinyl), 6.54 (dd, J ) 2.26, 8.27 Hz,
1H, H-5), 6.43 (d, J ) 2.26 Hz, 1H, H-7), 3.74 (s, 3H, OCH3-6),
2.62 (t, J ) 7.67 Hz, 2H, CH2CH2COOH-4′), 2.30 (t, J ) 7.67
Hz, 2H, CH2CH2COOH-4′), 2.26 (s, 3H, CH3), and 2.22 (s, 3H,
CH3); MS m/z (relative intensity, %) 341 (M+, 100). Anal.
(C19H20N2O4‚0.5H2O) C, H, N.
3-[2,4-Dim eth yl-5-(2-oxo-6-p h en yl-1,2-d ih yd r oin d ol-3-
ylid en em eth yl)-1H-p yr r ol-3-yl]p r op ion ic Acid (16f). This
compound was prepared using the same procedure as for
1
preparation of 16b with a yield of 71%: H NMR (360 MHz,
DMSO-d6) δ 13.38 (s, br, 1H, NH-1′), 12.05 (s, br, 1H, CH2-
CH2COOH-4′), 10.81 (s, br, 1H, NH-1), 7.79 (d, J ) 7.84 Hz,
1H, H-4), 7.62 (d, J ) 7.51 Hz, 2H, H-2′′,6′′), 7.58 (s, 1H,
H-vinyl), 7.44 (t, J ) 7.51 Hz, 2H, H-3′′,5′′), 7.32 (t, br, J )
Compound 15 (0.22 g, 1.1 mmol), 0.15 g (1.1 mmol) of

Substituted Indolin-2-ones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5129
7.51 Hz, 1H, H-4′′), 7.26 (dd, J ) 1.13, 7.84 Hz, 1H, H-5), 7.09
(d, J ) 1.13 Hz, 1H, H-7), 2.64 (t, J ) 7.71 Hz, 2H, CH2CH2-
COOH-4′), 2.35 (t, J ) 7.71 Hz, 2H, CH2CH2COOH-4′), 2.30
(s, 3H, CH3), 2.26 (s, 3H, CH3); MS m/z (relative intensity, %)
387 ([M + 1]+, 100). Anal. (C24H22N2O3‚0.5H2O) C, H, N.
for PDGF-Râ and 5 min for EGF-R at room temperature and
then was stopped by addition of EDTA. The amount of
phosphotyrosine present on the receptors in the individual
wells was determined by incubating the immunolocalized
receptor with a biotinylated monoclonal antibody directed
against phosphotyrosine. After removal of the unbound anti-
phosphotyrosine antibody, avidin-conjugated horseradish per-
oxidase H was added to the wells. A stabilized form of 3,3′,5,5′-
tetramethylbenzidine dihydrochloride (TMB) and hydrogen
peroxide (H2O2) was added to the wells. The color readout of
the assay was allowed to develop for approximately 30 min
and the reaction was stopped with sulfuric acid (H2SO4).
3-{5-[6-(3-Met h oxy-p h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-
ylid en em et h yl]-2,4-d im et h yl-1H -p yr r ol-3-yl}p r op ion ic
Acid (16g). This compound was prepared using the same
procedure as for preparation of 16b with a yield of 91%: 1H
NMR (360 MHz, DMSO-d6) δ 13.38 (s, br, 1H, NH-1′), 12.04
(s, br, 1H, CH2CH2COOH-4′), 10.79 (s, br, 1H, NH-1), 7.77 (d,
J ) 8.05, 1H, H-4), 7.58 (s, 1H, H-vinyl), 7.35 (t, J ) 8.11 Hz,
1H, H-5′′), 7.27 (dd, J ) 1.49, 8.05 Hz, 1H, H-5), 7.19 (d, br, J
) 8.11 Hz, 1H, H-6′′), 7.14 (t, J ) 2.31 Hz, 1H, H-2′′), 7.09 (d,
J ) 1.49 Hz, 1H, H-7), 6.90 (dd, J ) 2.31, 8.11 Hz, 1H, H-4′′),
3.81 (s, 3H, OCH3-3′′), 2.65 (t, J ) 7.62 Hz, 2H, CH2CH2COOH-
4′), 2.35 (t, J ) 7.62 Hz, 2H, CH2CH2COOH-4′), 2.30 (s, 3H,
CH3), 2.27 (s, 3H, CH3); MS m/z (relative intensity, %) 417 ([M
+ 1]+, 72). Anal. (C25H24N2O4‚0.25H2O) C, H, N.
3-{5-[6-(2-Met h oxyp h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-
ylid en em et h yl]-2,4-d im et h yl-1H -p yr r ol-3-yl}p r op ion ic
Acid (16h ). This compound was prepared using the same
procedure as for preparation of 16b with a yield of 44%: 1H
NMR (360 MHz, DMSO-d6) δ 13.38 (s, br, 1H, NH-1′), 12.00
(s, br, 1H, CH2CH2COOH-4′), 10.70 (s, br, 1H, NH-1), 7.71 (d,
J ) 7.81 Hz, 1H, H-4), 7.55 (s, 1H, H-vinyl), 7.28-7.33 (m,
2H, H-4′′,6′′), 7.09 (d, J ) 7.60 Hz, 1H, H-3′′), 7.05 (dd, J )
1.27, 7.81 Hz, 1H, H-5), 7.01 (t, J ) 7.60 Hz, 1H, H-5′′), 7.00
(d, J ) 1.27 Hz, 1H, H-7), 3.76 (s, 3H, OCH3-2′′), 2.66 (t, J )
7.60 Hz, 2H, CH2CH2COOH-4′), 2.35 (t, J ) 7.60 Hz, 2H,
CH2CH2COOH-4′), 2.30 (s, 3H, CH3), 2.26 (s, 3H, CH3); MS
m/z (relative intensity, %) 415 ([M - 1]+, 100). Anal.
(C25H24N2O4‚0.2H2O) C, H, N.
3-{5-[6-(4-Met h oxyp h en yl)-2-oxo-1,2-d ih yd r oin d ol-3-
ylid en em et h yl]-2,4-d im et h yl-1H -p yr r ol-3-yl}p r op ion ic
Acid (16i). This compound was prepared using the same
procedure as for preparation of 16b with a yield of 57%: 1H
NMR (360 MHz, DMSO-d6) δ 13.35 (s, br, 1H, NH-1′), 12.02
(s, br, 1H, CH2CH2COOH-4′), 10.75 (s, br, 1H, NH-1), 7.74 (d,
J ) 6.75 Hz, 1H, H-4), 7.56 (d, J ) 9.01 Hz, 2H, H-2′′,6′′), 7.54
(s, 1H, H-vinyl), 7.21 (dd, J ) 1.57, 6.75 Hz, 1H, H-5), 7.04 (d,
J ) 1.57 Hz, 1H, H-7), 7.00 (d, J ) 9.01 Hz, 2H, H-3′′,5′′), 3.79
(s, 3H, OCH3-4′′), 2.65 (t, J ) 7.54 Hz, 2H, CH2CH2COOH-4′),
2.35 (t, J ) 7.54 Hz, 2H, CH2CH2COOH-4′), 2.29 (s, 3H, CH3),
2.26 (s, 3H, CH3); MS m/z (relative intensity, %) 417 ([M +
1]+, 100). Anal. (C25H24N2O4‚0.25H2O) C, H, N.
F GF -R1 a n d F lk -1/KDR Kin a se Assa ys. The catalytic
portion of FGF-R1 and Flk-1/KDR were expressed as GST
fusion proteins following infection of Spodoptera frugiperda
(sf9) cells with engineered baculoviruses. GST-FGFR1 and
GST-Flk1 were purified to homogeneity from infected sf9 cell
lysates by glutathione sepharose chromatography. The assays
were performed in 96-well microtiter plates that had been
coated overnight with 2.0 µg of a polyGlu-Tyr peptide (4:1)
(Sigma P-0275) in 0.1 mL of PBS per well. The purified kinases
were diluted in kinase assay buffer (100 mM Hepes pH 7.5,
100 mM NaCl, and 0.1 mM sodium orthovanadate) and added
to all test wells at 5 ng of GST fusion protein per 0.05 mL
volume buffer. Test compounds were diluted in 4% DMSO and
added to test wells (0.025 mL/well). The kinase reaction was
initiated by the addition of 0.025 mL of 40 µM ATP/40 mM
MnCl2, and plates were shaken for 10 min before stopping the
reactions with the addition of 0.025 mL of 0.5 M EDTA. The
final ATP concentration was 10 µM, which is twice the
experimentally determined Km value for ATP. Negative control
wells received MnCl2 alone without ATP. The plates were
washed three times with 10 mM Tris pH 7.4, 150 mM NaCl,
and 0.05% Tween-20 (TBST). Rabbit polyclonal anti-phospho-
tyrosine antiserum was added to the wells at a 1:10000
dilution in TBST for 1 h. The plates were then washed three
times with TBST. Goat anti-rabbit antiserum conjugated with
horseradish peroxidase was then added to all wells (Biosource
Cat. No. ALI0404; 1:10000 dilution in TBST) for 1 h. The plates
were washed three times with TBST, and the peroxidase
reaction was detected with the addition of 2,2′-azinobis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sigma A1888).
The color readout of the assay was allowed to develop for 20-
30 min and read on a Dynatech MR5000 ELISA plate reader
using a 410 nM test filter.
P DGF a n d EGF -Dep en d en t Br d U In cor p or a tion As-
sa ys. NIH 3T3 cells overexpressing human EGF receptors
(8000/well) were added to a 96-well microtiter plate in DMEM
containing 10% calf serum (Rockville, MD). The plates were
incubated overnight at 37 °C, and then deprived of serum for
24 h by incubation with DMEM containing 1% BSA, followed
by stimulation with PDGF-â (3.8 nM) or EGF (4 nM) in the
presence of various test compounds. After 20 h of incubation,
BrdU (Roche Molecular Biochemicals, Indianapolis, IN) was
added for a 2 h labeling period, and the cells were fixed with
Fix/Denat solution (Roche Molecular Biochemicals, Indianapo-
lis, IN). The amount of BrdU incorporation was then deter-
mined using anti-BrdU/POD and the horseradish peroxidase
substrate, ABTS (both from Roche Molecular Biochemicals,
Indianapolis, IN). All experiments were performed three times,
and the IC50 values were determined by nonlinear regression
using the data analysis program PRISM (Graphpad Inc., San
Diego, CA).
Molecu la r Mod elin g. Homology models for the catalytic
domains of VEGF-R2 (Flk-1/KDR) and PDGF-R were gener-
ated using the Modeler program in InsightII 95.5 released from
Molecular Simulations, Inc. The “open form” of FGF-R1
cocrystallized with SU4984 (PDB code: 1agw) was used as
reference, and t he sequence alignment was based on previous
study15 with slight modifications. Since the sequence homolo-
gies between FGF-R1 and VEGF-R2 (Flk-1/KDR) (62%) and
between FGF-R1 and PDGF-R (51%) are very high, the overall
structures were similar. The VEGF-R2 (Flk-1/KDR) and
PDGF-R models were superimposed with the FGF-R1 crystal
structures based on the CR trace. Docking of substituted
indolin-2-ones to these receptors were done manually based
on the FGF-R1/SU4984 crystal structure followed by simple
energy minimization.
P DGF -R a n d EGF -R Kin a se Assa ys. Solubilized mem-
branes derived from NIH3T3 mouse fibroblasts overexpressing
human PDGF-Râ or EGF-R were added to polystyrene 96-well
microtiter plates which had been coated with a monoclonal
antibody that recognized either the PDGF-R or EGF-R.14 After
a 30 min incubation with lysate, the plates were washed to
remove unbound material, and serial dilutions of chemical
inhibitors were added to the immunolocalized receptor. The
kinase reaction was started by the addition of ATP to the wells
at a final concentration of 20 µM for PDGF-Râ and 3 µM for
EGF-R, twice the experimentally determined Km values for
ATP. The kinase reaction was allowed to proceed for 30 min
VEGF a n d F GF Cell Br d U In cor p or a tion Assa ys. These
cellular assays were preformed as previously described.8
Ackn owledgm en t. The authors thank Robert Blake,
Danny Tam, J ames Rodda, and Kim Lach for providing
data on the enzymatic activities of the inhibitors,
Zhaoyang Wen for preparing compounds 16c and 16d ,
and Kenneth E. Lipson for organizing the BrdU data.

5130 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
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