Design, synthesis, and biological evaluation of novel 3-aryl-4-(1H-indole- 3yl)-1,5-dihydro-2H-pyrrole-2-ones as vascular endothelial growth factor receptor (VEGF-R) inhibitors

Article abstract of DOI:10.1021/jm8001185

In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly potent VEGF-R2/3 inhibitor with IC ₅₀ of 31/37 nM. The novel 3,4-diaryl-2H-pyrrole-2-ones were designed on the basis of the modeled binding mode of the corresponding 1H-pyrrole-2,5-dione (maleimide) VEGF-R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2//-pyrrole-2-ones and structure-activity relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC₅₀ values) are presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.

Full text of DOI:10.1021/jm8001185

3814
J. Med. Chem. 2008, 51, 3814–3824
Design, Synthesis, and Biological Evaluation of Novel
3-Aryl-4-(1H-indole-3yl)-1,5-dihydro-2H-pyrrole-2-ones as Vascular Endothelial Growth Factor
Receptor (VEGF-R) Inhibitors
Christian Peifer,*,§ Roland Selig,§ Katrin Kinkel,§ Dimitri Ott,§ Frank Totzke,‡ Christoph Scha¨chtele,‡ Regina Heidenreich,†
Martin Ro¨cken,† Dieter Schollmeyer,| and Stefan Laufer§
Department of Pharmacy, Eberhard-Karls UniVersity, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany, ProQinase GmbH,
Breisacherstrasse 117, D-79106 Freiburg, Germany, Department of Dermatology, Eberhard-Karls UniVersity, Liebermeisterstrasse 25,
D-72076 Tu¨bingen, Germany, and Department of Organic Chemistry, Johannes Gutenberg UniVersity Mainz, Duesbergweg 10-14,
D-55099 Mainz, Germany
ReceiVed December 12, 2007
In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly
potent VEGF-R2/3 inhibitor with IC50 of 31/37 nM. The novel 3,4-diaryl-2H-pyrrole-2-ones were designed
on the basis of the modeled binding mode of the corresponding 1H-pyrrole-2,5-dione (maleimide) VEGF-
R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but
not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2H-pyrrole-2-ones and structure-activity
relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC50 values) are
presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent
antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in
HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was
dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.
Introduction
diversity, VEGF-R2 seems to mediate at modulated high levels
the majority of pathological angiogenic effects including mi-
crovascular permeability, endothelial cell differentiation, pro-
liferation, invasion, and migration.5 The importance of VEGF-
R2 for the regulation of angiogenesis has been highlighted by
the fact that homozygous knockout mice die at an early
embryonic level, whereas the specific activation of VEGF-R2
alone by VEGF-E results in potent endothelial cell activity and
angiogenesis.6 At a molecular level of signal transduction,
extracellular binding of VEGF-A or VEGF-E causes dimeriza-
tion of the transmembrane VEGF-R2. Subsequently, the intra-
cellular tyrosine kinase domains of VEGF-R2 autophosphorylate
each other at different sites (Tyr951, Tyr1175, Tyr1214). Activated
VEGF-R2 through these cascades triggers multiple intracellular
downstream signaling pathways involving activation of phos-
pholipase C γ-protein kinase C-Raf-MAP kinase pathways7
resulting in proliferation of endothelial cells, whereas survival
and migration are mediated via activation of PI3K/Akt and Src/
focal adhesion kinase (FAK) pathways, respectively.8 Taken
together, VEGF-R2 situated at the top level of these cascades
is working as a significant integrative “biochemical amplifier”
of VEGF-A and VEGF-E signals and is considered to be a
validated drug target in angiogenesis related diseases. Hence, a
plethora of clinically effective strategies for its inhibition have
been developed. These include neutralizing antibodies to VEGF
or VEGF-Rs and small molecule tyrosine kinase inhibitors.6
Vascular endothelial growth factor receptors (VEGF-Rsa) play
pivotal roles in mediating the effects of hypoxia induced VEGFs
not only on physiological angiogenesis but also on pathophysi-
ological angiogenesis such as in pancreatic carcinomas, gastric
carcinomas, colorectal carcinomas, breast cancer, lung cancer,
prostate cancer, and melanoma.1 The VEGF-R family consists
of VEGF-R1 (also referred to as fms-like tyrosine kinase 1, Flt-
1), VEGF-R2 (KDR), and VEGF-R3 (Flt-4), which have differ-
ent binding specificities for VEGF members (VEGF-A to
VEGF-E). VEGF-R1 is critical for physiological and develop-
mental angiogenesis.2 VEGF-R3 is restricted mainly to lym-
phatic endothelial cells,3,4 whereas a low basic VEGF-R2
expression occurs on almost all endothelial cells. Despite this
* To whom correspondence should be addressed. Phone: 0049 7071 29
75278. Fax: 0049 7071 29 5037. E-mail: Christian.Peifer@uni-tuebingen.de.
§
Department of Pharmacy, Eberhard-Karls University.
ProQinase GmbH.
Department of Dermatology, Eberhard-Karls University.
Johannes Gutenberg University Mainz.
‡
†
|
a Abbreviations: AKT1, v-akt murine thymoma viral oncogene homo-
logue 1 (PKB); ARK5, AMPK-related protein kinase 5 (NUAK1); Aurora,
aurora kinase; B-RAF, v-raf murine sarcoma viral oncogene homologue
B1; CDI, carbonyldiimidazole; CDK, cyclin-dependent kinase; CK, casein
kinase; COT, mitogen-activated protein kinase kinase kinase 8 (MAP3K8);
DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; EGF-R, epidermal growth
factor receptor; EPHB, EPH receptor B; FAK, focal adhesion kinase; FLT3,
fms-related tyrosine kinase 3; HDMEC, human dermal microvascular
endothelial cells; HLMEC, human lung-derived microvascular endothelial
cells; HMDS, hexamethyldisilazide; HOBt, hydroxybenzotriazole; HPI,
hydrophobic pocket; HRII, hydrophobic region II; IGF1-R, insulin-like
growth factor 1 receptor; INS-R, insulin receptor; MET, met proto-oncogene
(hepatocyte growth factor receptor); PDGF-R, platelet-derived growth factor
receptor; PK, protein kinase; PLK1, polo-like kinase 1; PPA, polyphosporic
acid; SAK, serine/threonine-protein kinase (PLK4); SEM, 2-(trimethylsi-
lyl)ethoxymethyl; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral onco-
Small Molecule VEGF-R2 Inhibitors
Among protein kinases (PKs) as drug targets, tyrosine kinases
such as VEGFR-2 play an outstanding role for the development
of clinically effective inhibitors in oncology.5 Since VEGF-R2
and the other PKs of the kinome use ATP as cofactor for the
phosphorylation of proteins, they share a highly conserved ATP
binding pocket that is the molecular binding site of most
inhibitors. Many compounds that have been developed to
t
gene homologue; TBAF, tetra-n-butylammonium fluoride; BuOK, potas-
sium tert-butoxide; TIE2, tunica interna endothelial cell kinase; TIPS,
triisopropylsilyl; TMSCL, trimethylsilylchloride; VEGF-R, vascular endot-
helial growth factor receptor.
10.1021/jm8001185 CCC: $40.75
2008 American Chemical Society
Published on Web 06/05/2008

Pyrrole-2-ones as VEGFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3815
Figure 2. Retrosynthetic concepts for the preparation of 3,4-diaryl-
2H-pyrrole-2-ones from the adequate precursors by (a) regioselective
reduction of maleimides, (b) McMurry coupling, (c) aldol condensation
(Knoevenagel-reaction), (d) intramolecular Wittig reaction.
Figure 3. Modified Luche reduction of 1 yielded 4 as racemic mixture
(I, ethanol, CeCl3, NaBH4). McMurry reaction of 7 resulted in a racemic
mixture of 8 (besides undefined polymers) (II, THF, Ti (TiCl3), TMSCl,
reflux).
Figure 1. Crystallographically determined ATP-binding site of VEGF-
R2 (pdb code 1Y6A)17 and modelled binding modes of 1 and 2. Key
residues and hydrogen bonds are labeled. For compound 3 no rational
binding mode in the ATP binding pocket of VEGF-R2 could be
calculated.
also inhibit VEGF-R2/3. Accordingly, molecular modeling
studies revealed a rational binding mode in the ATP pocket of
VEGF-R2 comparable to 1 for 4-(1H-indole-3-yl)-3-(3,4,5-tri-
methoxyphenyl)-1,5-dihydro-2H-pyrrole-2-one (compound 2)
but not for the isomer 3-(1H-indole-3-yl)-4-(3,4,5-trimeth-
oxyphenyl)-1,5-dihydro-2H-pyrrole-2-one (compound 3, Figure
1).
To prove this hypothesis, 2 and 3 possessing analogue
substitution as compound 1 were synthesized and evaluated for
their potency against VEGF-R2/3.
selectively inhibit VEGF-R2 have also significant affinity for
VEGF-R1 and VEGF-R3 as well as for other tyrosine kinase
receptors such as the fibroblast growth factor receptor (FGF-
R),9 epidermal growth factor receptors (EGF-Rs),10,11 platelet
derived growth factor receptors (PDGFRR/ꢀ),12 c-kit, and Flt-3
and for various other PKs.13 Thus, the specificity of protein
kinase inhibitors within the kinome is a key question and a
robust evaluation of potency and selectivity is an essential part
of drug discovery programs.14,15
In this paper we report on the design, synthesis, and biological
evaluation of the 3-aryl-4-indolyl-2H-pyrrole-2-one 2 as a potent
VEGF-R2 inhibitor with an IC50 of 31 nM and strong in vivo
activity in the HLMEC sprouting assay. We designed the novel
pyrrole-2-ones derivatives on the basis of the rational binding
mode for the highly active and selective inhibitor 1 (3-(1H-indole-
3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione) modeled in
the ATP binding pocket of VEGF-R2 (Figure 1).16 Herein, central
hydrogen bonds to the hinge backbone carbonyl of Glu915 and to
the backbone amine of Cys917 are formed. The indole ring is
located in the hydrophobic region I with π-π stacking interactions
to Phe1045, whereas the trimethoxyphenyl moiety is situated in
the hydrophobic region II.
Chemistry
Although many synthetic approaches toward symmetrically
or asymmetrically substituted pyrrole-2-ones,18,19 pyrrole-2,5-
diones (maleimides),20–22 and related carbazoles23–26 exist, no
adequate procedures for the flexible preparation of 3,4-aryl-
(indole-3-yl)-2H-pyrrole-2-one have been reported. However,
a retrosynthetic analysis of 2 and 3 revealed different possibili-
ties for the flexible preparation of 3,4-diaryl-2H-pyrrole-2-one
including a regioselective reduction of maleimides, strategies
based on a McMurry coupling, an aldol reaction, and an
intramolecular Wittig reaction (Figure 2).
According to these concepts a modified Luche reduction27
of 1 yielded 4, but all further attempts to reduce the alcohol to
the methylene moiety (2) failed.25,28,29 Concerning the McMurry
reaction,30 we prepared precursor 7, which was reacted under
various coupling conditions,31,32 but the ring closure for the
pyrrole-2-one moiety did not perform and we obtained com-
pound 8 as the main product in combination with polymeric
tars (Figure 3).
Since, besides the NH-Glu915 in this calculated binding
mode, only the 5′ carbonyl oxygen and not the 2′ oxygen of
the maleimide moiety is involved in ligand-protein interactions
to Cys917, we hypothesized that the corresponding 3,4-diaryl-
2H-pyrrole-2-one scaffold was suitable as a pharmacophore to
The aldol condensation of 10 by use of the strong base tBuOK
under reflux (thermodynamic conditions)33,34 yielded the desired

3816 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Peifer et al.
In order to avoid the indole NH deprotonation according to
the tautomeric situation in 13 and to regain the required carbonyl
reactivity, we introduced a variety of indole NH protecting
groups to this molecule (Figure 6). Interestingly, for the
triisopropylsilyl (TIPS) protected derivative we obtained com-
pound 15 (Figure 6; structure of 15 has been proven by X-ray
analysis38), which was not stable for further reactions. Herein,
the tautomerization and unexpected subsequent protection of
the indole 3′ side chain oxygen in 15 confirm the deactiviation
of the indole 3′ side chain carbonyl reactivity as discussed above.
Furthermore, the phenylsulfonyl-protected compound 16 was
immediately oxidized under the alkaline conditions to 17. Albeit
the mechanism for this reaction is still under investigation, we
assume that the strong electron withdrawal effect of the phe-
nylsulfonyl group facilitates the oxidation of the pyrrole-2-one
methylene group. However, ring closure to the desired pyrrole-
2-one derivative 19 under the thermodynamic Knoevenagel
reaction conditions was successful for the 2-(trimethylsi-
lyl)ethoxymethyl (SEM) protected species 18 (Figure 6).33
Figure 4. Aldol condensation of 10 under thermodynamic Knoevenagel
conditions (III, THF, tBuOK, reflux) yielded 3, whereas kinetic
conditions (IV, dichloromethane, 4 equiv of pyridine, 1 equiv of K2CO3,
room temperature) yielded 11.
After successful achievement of the ring closure to pyrrole-
2-one 19, the subsequent removal of the SEM protecting group
to generate 2 unexpectedly turned out to be quite difficult. Even
if SEM deprotection of related indole derivatives commonly
achieved by TBAF (Bu4NF) has been reported to proceed
without further complications25,34 in this case, the removal was
a challenge. The mechanism of SEM cleavage usually involves
a hemiaminal that eliminates formaldehyde (commonly seques-
tered by bases such as ethylenediamine39) to yield the unsub-
stituted indole NH. Unfortunately in our case all attempts
involving fluoride induced cleavage of SEM in 19 using various
solvents and conditions failed.40 Instead of SEM cleavage by
TBAF under alkaline conditions, an oxidation of 19 to 19a
(possessing the pyrrole-2,5-dione moiety) occurred, as also seen
in related compounds.25 In particular, the use of bases such as
ethylenediamine typically resulted in oxidation and destruction
of 19 and gave complex mixtures of red tars. Thus, as already
seen for compound 17 the methylene bridge in the 3,4-diaryl-
1,5-dihydro-2H-pyrrole-2-one moiety of 19 was found to be
susceptible for oxidation particularly under basic conditions.
Interestingly, SEM cleavage at the pyrrole-2,5-dione core
proceeded successfully by refluxing 19a in TBAF, DMF, and
ethylenediamine to yield 1 (yield 70%).
This prompted us to develop a method for SEM cleavage
under acidic conditions for the deprotection of molecule 19.
However, refluxing SEM-protected 19 in methanol with catalytic
amounts of HCl quantitatively resulted in 21 formally possessing
the MOM protection (Figure 7). The mechanism for this reaction
can be explained via conventional elimination of ethene from
19 yielding the hemiaminal 20 as intermediate, which reacts,
consecutively and via proton catalysis, with an excess of alco-
holic solvent to generate the methoxyaminal. Accordingly, the
reaction of 19 in various alcohols gave the corresponding
derivatives 22-26 (Figure 7). Interestingly, reaction of HCl with
19 in tert-butyl alcohol as solvent gave a mixture of three main
products that were separated by flash chromatography to yield
2 (10%), 20 (46%), and 24 (16%, Figure 7). Thus, it is
noteworthy that in this study compound 2 could be generated
indirectly from 19 under acidic conditions. Furthermore, 2 was
also generated from 20, eliminating formaldehyde by the use
of sodium methanolate in dry methanol.
Table 1. Substitution patterns of compounds 3, 31, 33, and 35
compd
R1
R2
R3
R4
3
H
CH
H
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
31
33
35
CH3
H
H
product 3 in good amounts. The reaction mechanism herein
involves deprotonation of the benzylic position in 10 to generate
a carbanion, which in turn attacks the carbonyl function to form
the pyrrole-2-on ring by an E1cB mechanism. It is noteworthy
that the use of the weaker bases pyridine and potassium carbo-
nate at room temperature (kinetic conditions) yielded oxazole
11 by an aldol reaction of 10 (Figure 4).35,36
According to the straightforward strategy for the preparation
of 3, the analogue compounds 31, 33, and 35 were obtained
from adequate precursors (Table 1). Thus, because the aldol
strategy was successful, in this study the Wittig approach (Figure
2) was abandoned.
Interestingly, for the preparation of 2 the aldol condensation
methodology via reaction of precursor 13 was initially not
successful. In light of the established aldol mechanism for the
reaction of 10 (to generate 3) the complete loss of reactivity of
isomeric 13 (to generate 2) can be explained by the different
pKa values in these molecules affecting the deprotonation of
13 compared to the isomer 10 (Figure 5). Because of the car-
bonyl function in 3′ side chain position of the indole moiety,
compound 13 is able to form a vinylogue amide system in which
under the strong basic conditions the indole NH (pKaNH ≈ 13)37
is deprotonated rather than the benzylic position (pKaCH ≈ 16).
As a consequence of the indole NH deprotonation, the subse-
quently formed anion is delocalized over the vinylogue system,
thereby deactivating the carbonyl reactivity that is necessary
for the aldol condensation. However, reaction of 13 in poly-
phosporic acid (PPA) let to compound 14 (Figure 5), the isomer
of oxazole 11.35
Compound 29 was synthesized via the precursors 27 and 28
(Figure 8), wherein the 4-ethyl-2,2-dimethyl-1,3-dioxolane side
chain was used elegantly as a “protected protecting group” for

Pyrrole-2-ones as VEGFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3817
t
Figure 5. Aldol condensation of 13 under thermdynamic Knoevenagel conditions (V, THF, BuOK, reflux) was not successful because of the
deprotonation of indole NH. The tautomeric situation completely deactivates the carbonyl reactivity necessary for ring closure. Under acidic conditions
(VI, PPA, 80 °C), the oxazole 14 was formed.
Figure 6. Strategies for the introduction of protecting groups to compound 13 (VII, THF, NaH, TIPSCl; VIII, EtOH, KOH, benzoylsulfonylchloride;
t
IX, THF, NaHMDS, SEMCl; III, THF, BuOK, reflux).
the indole NH accordingly to the requirements for the Knoev-
enagel condensation (as discussed above).
of VEGF-R2 (IC50 ) 11 µM) and VEGF-R3 (IC50 ) 9.4 µM).
Hence, these data strongly confirm the calculated ligand-protein
interactions of 2 in particular for the lactame moiety, which
was supposed to address the amino acids Glu915 and Cys917.
However, the lactame 2 was determined to inhibit VEGF-R2/3
(and the other kinases of this panel) approximately 10-fold less
than maleimide 1. This moderate loss of activity may be due to
the lower polarization of the NH in amide 2 compared to imide
1 and a subsequently weaker H-bond to the carbonyl of Glu915
in VEGF-R2/3.
Biological Evaluation and Discussion
The pyrrole-2-ones of this study and maleimide 1 have been
evaluated in vitro for their biological activity in a panel of 24
therapeutically relevant PKs including VEGF-R2/3 (Table 2).
Consistent with the molecular modeling in the ATP pocket of
VEGF-R2 (Figure 1), compounds 2 and 116 were found to
potently inhibit these receptor tyrosine kinases (2 VEGF-R2 IC50
) 0.031 µM; VEGF-R3 IC50 ) 0.037 µM). In contrast to
compound 2 the isomer 3 showed a significant weaker inhibition
On the other hand, although 3 (as well as 31, 33, and 35)
compared with the isomer 2 showed the expected reduction of

3818 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Peifer et al.
Figure 7. Reaction of 19 in different alcohols and catalytic amount of HCl generated the corresponding compounds 21-26 (X, particular alcohol
as solvent, catalytic amounts of HCl, reflux). Because of the tert-butylcarbocation as a leaving group, hemiaminal 20 was found as an unstable
t
intermediate in the reaction of 19 with BuOH, and 2 was subsequently obtained from 20 by the elimination of formaldehyde.
Figure 8. Synthesis of 29 via protected 27, ring closure to 28, and deprotection to yield 29 (XI, DMF, K2CO3, 4-(bromomethyl)-2,2-dimethyl-
t
1,3-dioxolane, reflux; III, THF, BuOK, reflux; XII, dichloromethane, catalytic amount of HCl).
activity against VEGF-R2/3, we found considerable inhibition
of ARK5, AuroraA/B, and SAK in the single digit micromolar
range for compound 3 and for the derivatives of this isomer,
depending on the aryl substitution. Interestingly, FLT3 was
inhibited by 1 with an IC50 of 270 nM, but the two isomers 2
and 3 were found to have both a similar IC50 in the low
micromolar range (2 IC50 ) 6.1 µM; 3 IC50 ) 2.2 µM). These
data indicate that the maleimide moiety in 1 presumably interacts
with the hinge region of FLT3 by the NH as well as both
oxygens, thereby accounting for the nanomolar potency. Com-
pared to the nanomolar inhibitors 1 and 2, the 2′-hydroxyl
function in 4 significantly reduced the biological activity for
VEGF-R2/3 to the micromolar range (4 IC50 ) 5.9 µM/3.4 µM)
and abolished the activity against many of the other kinases in
the panel. Since in this study compound 4 had been tested in
the kinase assays as a 2′-racemic mixture, it is not possible to
describe a clear stereochemical SAR for this position.
ing a benzyloxymethylene substitution at the indole nitrogen,
showed promising specificity for VEGF-R2/3 in the kinase
panel.
Consistent with the proposed binding mode of compound 2
in the ATP pocket of VEGF-R2 (Figure 1), we designed
compound 29 possessing a dihydroxy-functionalized side chain
that was intended to interact with Arg840 of the glycine-rich
loop (Figure 9). In light of the chiral 2′-hydroxyl function of
29, molecular modeling showed suitable ligand-protein interac-
tions for the propylene glycol S-enantiomer but not for the
corresponding R-enantiomer (not shown). However, the in vitro
inhibitory activity for racemic 29 at VEGF-R2/3 (IC50 ) 4.2
µM/2.5 µM) was determined to be in the micromolar range, as
also seen for the other indole N-substituted derivatives 19-26
(and 28). Although the defined enantiomers of 29 have not been
tested, the weak IC50 values of racemic 29 for VEGF-R2/3
comparable to protected 28 indicate that no additional H-bond
to the protein was formed by the hydroxy moieties of 29. Thus,
in contrast to the calculated binding mode of 29 in the ATP
pocket of VEGF-R2, this optimization strategy was not suc-
cessful and may be due to different reasons. First, protein kinase
domains such as the glycin-rich loop and the DFG motif are
highly flexible while the crystal structure used for modeling
reflects only one specific protein conformation,41 which was
presumably not realistic for the binding of 29 in VEGF-R2.
Second, the relatively rigid alkyl side chain of 29 bearing the
hydroxy functions may not provide the molecule with the
The unsubstituted indole-NH in compound 2 was found to
be essential for potent inhibition of VEGF- R2/3. This result
goes parallel with the SAR of 1 at VEGF-R2/3 where the free
indole-NH was found to be relevant for high affinity too.20 Thus,
as previously described,16 we suppose a similar water mediated
ligand-protein interaction for the indole NH of 2 to the ribose
pocket (Arg1030 and Asn1031) of VEGF-R2/3. Accordingly,
the substituted derivatives 19 and 21-26 showed IC50 values
only in the micromolar range (Table 2). However, within this
series compound 26 (VEGF-R2/3 IC50 ) 1.6/1.7 µM), possess-

Pyrrole-2-ones as VEGFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3819
Table 2. IC50 Values (µM) up to 50 µM Test Compounds in This Study in an Assay Panel of 24 Therapeutically Relevant PKs of the Kinomea
a None of the compounds siginificantly inhibited AKT1, CK2-A1, and PLK1 up to 50 µM. As a parameter for assay quality, the Z′-factor for the low and
high controls of each assay plate (n ) 8) was used (for details, see Supporting Information).
capability of adopting the optimal conformation necessary for
additional H-bond interactions to Arg840 in the glycin-rich loop
of VEGF-R2.42 Taken together, accurate and actual docking with
protein kinases and inhibitors remains challenging.43
Cellular in Vitro Characterization
Recent data show that multikinase inhibitors are clinically
more effective for tumor therapy than single targeting com-
pounds.44 Even though the data of 2 determined so far show
potent in vitro efficiency against VEGF-R2/3 and specificity
over the other 22 PKs involved in the panel (Table 2), cellular
in vitro studies are needed to analyze the efficacy of such
compounds on receptor functioning. In light of the nanomolar
potency of 1 and 2 and the significantly reduced activity of 3
against isolated VEGF-R2/3, these compounds have been
assayed in an endothelial cell sprouting assay using human lung
Figure 9. Crystallographically determined ATP-binding site of VEGF-
R2 (PDB code 1Y6A)17 and modelled binding mode of the S-
enantiomer of 29. Key residues and hydrogen bonds are labeled.
derived microvascular endothelial cells (HLMEC) in order to
reveal their antiangiogenic activity (Figures 10 and 11). In line
with the in vitro SAR data for VEGF-R, compounds 1 (81%
inhibition) and 2 (88% inhibition) actually showed strong

3820 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Peifer et al.
Figure 10. Antiangiogenic activity of compounds 1-3 at a concentration of 1.3 µM in the HLMEC sprouting assay presented as sprouts/bead (
SD (for each experiment n ) 18). Cn ) control negative without VEGF stimulation: 0.69 ( 0.22 sprouts/bead. Cp ) control positive with 20
ng/mL VEGF stimulation: 4.54 ( 0.45 sprouts/bead. Compound 1: 0.86 ( 0.2 sprouts/bead. Compound 2: 0.52 ( 0.36 sprouts/bead. Compound
3: 2.98 ( 0.41 sprouts/bead. Statistical analysis was performed by using GraphPad InStat3 software. ANOVA, Tukey-Kramer test, and Bonferroni
test showed the results of compounds 1-3 to be highly significant compared to Cp (///, p < 0.001). Compound 1 vs 2 was not significant (p >
0.05). Compounds 1 and 2 vs 3 were highly significant (///, p < 0.001).
presented in Supporting Information). Furthermore, at 1.3 µM
the derivatives 19 (30% ( 13% inhibition), 21 (42% ( 7%
inhibition), and 25 (41% ( 4% inhibition) blocked the HLMEC
sprouting less efficiently than highly active 2. These results are
also in accordance with their reduced in vitro VEGF-R activity.
Interestingly, although 2 compared to 1 has been determined
to be approximately 10-fold less active in vitro against VEGF-
R2/3 (Table 2), compound 2 reduced the sprouting of HLMECs
down to the negative control level slightly more potent than 1,
and yet this difference was not significant (Figures 10 and 11).
This effect may be due to different reasons. First, since
maleimides have been reported to possess both poor water
solubility and poor cellular bioavailability,45 these parameters
are presumably limiting for maleimide 1 but less for the lactame
2 to reach their cellular target(s). Second, further PK(s) involved
in the angiogenic signaling process may be blocked more
potently by 2 than by 1, even though these PK(s) were not
detected in our 24 kinase profiles. An enhanced panel of PKs
to profile the compounds will be used in new experiments to
address this question.
Since VEGF-R inhibition may result in considerable apop-
tosis46 of endothelial cells (EC), we investigated if compounds
1-3 could induce apoptosis in EC such as human dermal
microvascular endothelial cells (HDMEC). Herein, the com-
pounds were tested at the antiangiogenic effective concentrations
of 1.3 and 2.6 µM compared to staurosporine47 as a positive
control (Figure 12).
At an antiangiogenic effective concentration of 1.3 µM,
compounds 1 (22.4 ( 10.8%) and 2 (25.0 ( 7.6%) both slightly
increased the apoptosis rate compared to the negative control.
At a concentration of 2.6 µM, compound 2 significantly induced
apoptosis (42.1% ( 8.8%), although this compound was not as
potent as staurosporine, a wildly unselective PK inhibitor.15 As
expected, compound 3 showed no significant induction of
apoptosis at both concentrations. Taken together, in light of the
Figure 11. Antiangiogenic activity of compounds 1-3 in the HLMEC
sprouting assay: (A) negative control, no VEGF stimulation; (B) positive
control, stimulation with 20 ng/mL VEGF; (C) 1.3 µM 1 and 20 ng/
mL VEGF; (D) 2.6 µM 1 and 20 ng/mL VEGF; (E) 1.3 µM 2 and 20
ng/mL VEGF; (F) 2.6 µM 2 and 20 ng/mL VEGF; (G) 1.3 µM 3 and
20 ng/mL VEGF; (H) 2.6 µM 3 and 20 ng/mL VEGF.
considerable antiangiogenic effect of 2 in the sprouting assay
at 1.3 µM, it is remarkable that this concentration does not
significantly induce apoptosis in endothelial cells.
Conclusion
antiangiogenic potency at 1.3 µM whereas compound 3 (34%
inhibition) was significantly less active. The concentration range
of the compounds for effective inhibition of HLMEC sprouting
was dose dependent, as determined by titration experiments in
this assay (data not shown; the results of 1-3 at 2.6 µM are
Novel regioselective substituted 3,4-diaryl-2H-pyrrole-2-ones
were rationally designed, synthesized and subsequently evalu-
ated as VEGF-R2/3 inhibitors. As a highly active and selective
VEGF-R2/3 inhibitor with IC50 values of 31/37 nM against the
isolated enzymes compound 2 was also determined to potently

Pyrrole-2-ones as VEGFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3821
Figure 12. Dose dependent induction of apoptosis of compounds 1-3 in the HDMEC assay after 24 h of incubation represented as percent
apoptotic cells ( SD (for each experiment n ) 3). Cn ) control negative, 14.4 ( 6.0%. Cp ) control positive, 2.1 µM staurosporine after 5 h, 63.9
( 13.8%.
once. The mixture immediately changed to deep-red and was stirred
under reflux for 15 min (reaction progress monitored by TLC) when
the starting material disappeared. The mixture was cooled in an
ice bath and quenched with 1 mL of 10% HCl. The mixture was
extracted by ethyl acetate and washed with brine. The organic phase
was dried over Na2SO4 and evaporated. The product 19 was purified
by flash chromatography on silica gel using a gradient of ethyl
acetate/ethanol (chromatogram see Supporting Information for
details).
block angiogenesis in the HLMEC sprouting assay at a
concentration of 1.3 µM and to slightly induced apoptosis in
the HDMEC assay at a concentration of 2.6 µM. Thus, in light
of the high in vitro activity and selectivity and also being
bioavailable in the cellular models compound 2 may have po-
tential for clinical development as an antiangiogenic drug.
Experimental Section
3-(1H-Indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-
dione (1). The compound was prepared as described previously.20
HRMS m/z ) 378.120 88, calculated 378.121 541; purity HPLC
98.7%, tR ) 7.882 min; mp 245.8 °C.
Chemistry. Infrared spectra were recorded on a “Perkin Elmer
Spectrum one” infrared spectrophotometer. H (200 MHz, digital
1
resolution of 0.3768 Hz) and 13C (50 MHz, digital resolution of
1.1299 Hz) NMR were recorded on a Bruker AC 200. The data
are reported as follows: chemical shift in ppm from Me4Si as
external standard, multiplicity, and coupling constant (Hz). GC/
MS was performed on a HP6890 series system. EI mass spectra
were recorded on a Varian MAT 311A (70 eV). HRMS and FD
mass spectra were recorded on a MAT-95 (Finnigan). For clarity
only the highest measured signal is given for FD mass spectra.
Melting points/decomposition temperatures were determined on a
Bu¨chi apparatus according to Dr. Tottoli and are uncorrected. X-ray
structure determination was performed on a CAD4-Enraf-Nonius
using Cu KR radiation with graphite monochromator. Where
appropriate, column chromatography was performed for crude
precursors with Merck silica gel 60 (0.063-0.200 mm) or Acros
organics silica gel (0.060-0.200 mm, pore diameter of ∼60 nm).
Column chromatography for test compounds was performed using
a La-flash-System (VWR) with Merck silica gel 60 (0.015-0.040
mm) or RP8 columns. The progress of the reactions was monitored
by thin-layer chromatography (TLC) performed with Merck silica
gel 60 F-245 plates. Where necessary, reactions were carried out
in a nitrogen atmosphere using 4 Å molecular sieves. All reagents
and solvents were obtained from commercial sources and used as
received (THF was used after distillation over K/benzophenone).
Reagents were purchased from Sigma-Aldrich Chemie, Steinheim,
Germany; Lancaster Synthesis, Mu¨hlheim, Germany; or Acros,
Nidderau, Germany.
4-(1H-Indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-1,5-dihydro-2H-
pyrrole-2-one (2). The compound was obtained after purification
by flash chromatography from reaction mixture described for
compound 20, yielding 10% (70 mg) of 2 as a pale-white solid.
Alternative preparation of 2 is as follows. An amount of 400
mg (1 mmol) of 20 was dissolved under argon in 10 mL of dry
methanol. To the mixture 100 µL of a 0.5 M NaMeOH solution in
methanol was added and stirred for 20 min. The mixture was cooled
in an ice bath when 1 mL 10% HCl was added. The mixture was
extracted by ethyl acetate and washed with brine. The organic phase
dried over Na2SO4 and evaporated. The product was purified by
flash chromatography on silica gel using ethyl acetate and 10%
ethanol to yield 47% (170 mg, 0.47 mmol) of 2. LC-MS for
C21H20N2O4, 365.1 [M + H+], 363.0 [M - H+]; mp 131.8 °C;
HRMS m/z ) 364.140 69, calculated 364.142 281; purity HPLC
97.9%, tR ) 5.199 min. The crystal structure of 2 has been proven
by X-ray analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-
97, CCDC No. 680816. Further details of the crystal structure
analysis are available in Supporting Information.
3-(1H-Indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1,5-dihydro-2H-
pyrrole-2-one (3). The compound was prepared from 0.78 mmol
of 10 following a general procedure. Yield after flash chromatog-
raphy purification was 63% (0.49 mmol) as a fawn solid. FAB-
MS for C21H20N2O4, 364.9 [M+•]; mp 217.0 °C; HRMS m/z )
364.141 00, calculated 364.142 281; purity HPLC 97.5%, tR
5.285 min.
)
HPLC analysis was performed on a Hewlett-Packard HP 1090
series II using a Thermo Betasil C8 (150 µm × 4.6 5 µm) column
(mobile phase flow 1.5 mL/min, gradient KH2PO4 buffer, pH 2.3/
methanol, UV detection at 230/254 nm). A table summarizing the
purity of key target compounds can be found in Supporting Infor-
mation.
5-Hydroxy-4-(1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-1,5-
dihydro-2H-pyrrole-2-one (4). LC-MS for C21H20N2O5, **382
[M + H]+; mp >250 °C dec; HRMS m/z ) 380.138 19, calculated
380.137 191; purity HPLC 98.0%, tR ) 5.356 min. Crystal structure
of 4 has been proven by X-ray analysis: CAD4 Enraf Nonius, Cu
KR, SIR-92, SHELXL-97, CCDC No. 680817. Further details of
the crystal structure analysis are available in Supporting Information.
Oxo(3,4,5-trimethoxyphenyl)acetic Acid (5).48 LC-MS for
C11H12O6, 239 [M - H]+; mp 148.7 °C.
N-[2-(1H-Indol-3-yl)ethyl]-2-oxo-2-(3,4,5-trimethoxyphenyl)-
acetamide (6). GC-MS for C21H22N2O5, 382 [M]+; mp 211.9 °C.
Caution! The mixture must be absolutely free of water! If water
was involved in this reaction, the salt 2-(1H-indol-3-yl)etha-
naminium-oxo(3,4,5-trimethoxyphenyl)acetate (6a) was obtained
Routine experimental and NMR data are given in Supporting
Information.
General Method for Preparing Substituted Pyrrole-2-one
Derivatives by Thermodynamic Knoevenagel Condensation. A
typical procedure (exemplified by the preparation of compound 19)
is as follows. A dry three-necked round-bottom flask was charged
through a septum under argon with 1 mmol of a 20% (w/w) solution
t
of BuOK in THF and diluted with an additional 10 mL of dry
THF. This mixture was heated to 80 °C at which temperature a
solution of 1 mmol of 18 in 10 mL of dry THF was added all at

3822 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Peifer et al.
in our study. Crystal structure of 6a has been proven by X-ray
analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-97,
CCDC No. 680818. Further details of the crystal structure analysis
are available in Supporting Information.
N-[2-(1H-Indol-3-yl)-2-oxoethyl]-2-oxo-2-(3,4,5-trimethox-
yphenyl)acetamide (7). EI-MS for C21H20N2O6, 396 [M]+; mp
194.3 °C.
N-[2-(1H-Indol-3-yl)-2-oxoethyl]-2-hydroxy-2-(3,4,5-trimethox-
yphenyl)acetamide (8). LC-MS for C21H22N2O6, 397 [M - H]+;
mp 175.0 °C.
65 mg (0.17 mmol) of 1 as an orange solid (which was identical to
a sample of 1 from our earlier study20).
4-[1-(Hydroxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethoxyphe-
nyl)-1,5-dihydro-2H-pyrrole-2-one (20). LC-MS for C22H22N2O5
(m/z 394.4), 395 [M + H]+; mp 176.3 °C.
4-[1-(Methoxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethoxyphe-
nyl)-1,5-dihydro-2H-pyrrole-2-one (21). LC-MS for C23H24N2O5
(m/z 408.1), 409.1 [M + H+]; mp 194.6 °C; HRMS m/z )
408.167 41, calculated 408.168 491; purity HPLC 100%, tR ) 7.763
min.
1-Bromo-2-oxo-2-(3,4,5-trimethoxyphenyl)ethanaminium Bro-
mide (9). The compound was prepared according to the literature.49
FAB-MS for C11H16NO4, 226.1 [M+•] (-bromide); mp 236.0 °C.
2-(1H-Indol-3-yl)-N-[2-(3,4,5-trimethoxyphenyl)2-oxoethyl]ac-
etamide (10). FAB-MS for C21H22N2O5, 383.2 [M + H+.]; mp
125.7 °C.
4-[1-(Ethoxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethoxyphe-
nyl)-1,5-dihydro-2H-pyrrole-2-one (22). LC-MS for C24H26N2O5
(m/z 422.5), 423.1 [M + H+]; mp 167.1 °C; HRMS m/z )
422.183 11, calculated 422.184 141; purity HPLC 97.2%, tR
)
8.428 min.
4-[1-(Isopropoxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethox-
yphenyl)-1,5-dihydro-2H-pyrrole-2-one (23). LC-MS for
C25H28N2O5 (m/z 436.5), 437.1 [M + H+]; mp 172.7 °C; HRMS
m/z ) 436.200 59, calculated 436.199 791; purity HPLC 98.5%,
tR ) 9.036 min. Crystal structure of 23 has been proven by X-ray
analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-97,
CCDC No. 680822. Further details of the crystal structure analysis
are available in Supporting Information.
4-[1-(tert-Butoxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethox-
yphenyl)-1,5-dihydro-2H-pyrrole-2-one (24). The compound was
obtained in a procedure described for 20. It was purified by flash
chromatography to yield 16% of compound 24 as a brownish solid
(for chromatogram, see Supporting Information). LC-MS for
C26H30N2O5 (m/z 450.5), 451.1 [M + H+]; mp 193.0 °C; HRMS
m/z ) 450.213 02, calculated 450.215 441; purity HPLC 98.8%,
tR ) 9.50 min. Crystal structure of 24 has been proven by X-ray
analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-97,
CCDC No. 680823. Further details of the crystal structure analysis
are available in Supporting Information.
3-{[5-(3,4,5-Trimethoxyphenyl)-1,3-oxazol-2-yl]methyl}-1H-
indole (11). The compound was obtained following the procedure
of method B for the preparation of 10. FAB-MS for C21H20N2O4,
364.1 [M+•]; mp 138.0 °C; HRMS m/z ) 364.142 05, calculated
364.142 281; purity HPLC 98.8%, tR ) 9.100 min. Crystal structure
of 11 has been proven by X-ray analysis: CAD4 Enraf Nonius, Cu
KR, SIR-92, SHELXL-97, CCDC No. 680819. Further details of
the crystal structure analysis are available in Supporting Information.
N-[2-(1H-Indol-3-yl)ethyl]-2-(3,4,5-trimethoxyphenyl)aceta-
mide (12). GC-MS for C21H24N2O4, 368 [M+.]; mp 120.1 °C.
N-2-(1H-Indol-3-yl)-2-oxoethyl-2-(3,4,5-trimethoxyphenyl)ac-
etamide (13). Oxidation was done with dichlorodicyanoquinone
(DDQ).50 GC-MS for C21H22N2O5, 381.2 [M - H+]; mp 191.1 °C.
3-[2-(3,4,5-Trimethoxybenzyl)-1,3-oxazol-5-yl]-1H-indole (14).
LC-MS for C21H20N2O4, 365.3 [M + H+]; mp 171.1 °C; HRMS
m/z ) 364.140 60, calculated 364.142 281; purity HPLC 100%, tR
) 8.767 min. Crystal structure of 14 has been proven by X-ray
analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-97,
CCDC No. 680820. Further details of the crystal structure analysis
are available in Supporting Information.
N-2-(N-[2-(1-{[2-(Trimethylsilyl)ethoxy]methyl}1H-indol-3-
yl)-2-oxoethyl-2-(3,4,5-trimethoxyphenyl)acetamide (15). LC-
MS for C39H62N2O5Si2 (m/z 695.1), 695.5 [M+•]; mp 131.5 °C.
Crystal structure of 15 has been proven by X-ray analysis.38 Further
details of the crystal structure analysis are available in Supporting
Information.
N-2-(1-Phenylsulfonyl-1H-indol-3-yl)-2-oxoethyl-2-(3,4,5-tri-
methoxyphenyl)acetamide (16). LC-MS for C27H26N2O7S, 523.0
[M + H+]; mp 151.6 °C.
4-[1-(Phenoxymethyl)-1H-indol-3-yl]-3-(3,4,5-trimethoxyphe-
nyl)-1,5-dihydro-2H-pyrrole-2-one (25). LC-MS for C28H26N2O5
(m/z 470.5), 471.1 [M + H+]; mp 226.7 °C; HRMS m/z )
470.183 43, calculated 470.184 141; purity HPLC 76.5%, tR
9.204 min.
)
4-{1-[(Benzyloxy)methyl]-1H-indol-3-yl}-3-(3,4,5-trimethox-
yphenyl)-1,5-dihydro-2H-pyrrole-2-one (26). LC-MS for C29H28-
N2O5 (m/z 484.5), 485.1 [M + H+]; mp 65.0 °C; HRMS m/z )
484.196 83, calculated 484.199 791; purity HPLC 98.2%, tR
9.772 min.
)
N-2-(N-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]-1H-indol-3-
yl)-2-oxoethyl-2-(3,4,5-trimethoxyphenyl)acetamide (27). LC-
MS for C27H32N2O7 (m/z 496.55), 497.0 [M + H+].
3-[1-(Phenylsulfonyl)-1H-indol-3-yl]-4-(3,4,5-trimethoxyphe-
nyl)-1H-pyrrole-2,5-dione (17). LC-MS for C27H22N2O7S (m/z
522.1), 522 [M•]+.
N-(2-Oxo-2-(1-(2-trimethylsilyl)ethoxy)methyl)-1H-indol-3-yl)-
ethyl-2-(3,4,5-trimethoxyphenyl)acetamide (18). LC-MS for C27-
H36N2O6Si (m/z 512), 513 [M + H]+; mp 126.7 °C.
4-{1-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]-1H-indol-3-yl}-
3-(3,4,5-trimethoxyphenyl)-1,5-dihydro-2H-pyrrole-2-one (28).
The compound was prepared from 700 mg of 27 following a general
procedure. Yield after flash chromatography purification (for
chromatogram, see Supporting Information) was 73% as yellow
solid. LC-MS for C27H30N2O6 (m/z 478.54), 479.2 [M + H+];
mp 118.8 °C; HRMS m/z ) 478.209 83, calculated 478.210 352;
purity HPLC 98.4%, tR ) 8.579 min.
4-[1-(2,3-Dihydroxypropyl)-1H-indol-3-yl]-3-(3,4,5-trimethox-
yphenyl)-1,5-dihydro-2H-pyrrole-2-one (29). LC-MS and FAB-
MS for C24H26N2O6 (m/z 438.47), 439.2 [M + H+]; mp 177.5 °C;
HRMS m/z ) 438.177 10, calculated 438.179 052; purity HPLC
97.1%, tR ) 5.790 min.
2-(N-Methyl-1H-indol-3-yl)-N-[2-(3,4,5-trimethoxyphenyl)2-
oxoethyl]acetamide (30). FAB-MS for C22H24N2O5, 397.2 [M +
H+]; mp 130.6 °C.
3-(N-Methyl-1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)-1,5-di-
hydro-2H-pyrrole-2-one (31). The compound was prepared from
500 mg (1.26 mmol) of 30 following general procedure. The product
precipitated from ethyl acetate to yield 73% 320 mg (0.85 mmol)
of 31 as yellowish solid. FAB-MS for C22H22N2O4, 378.2 [M+•];
mp 204.4 °C; HRMS m/z ) 378.155 40, calculated 378.157 931;
purity HPLC 97.4%, tR ) 7.908 min.
3-(3,4,5-Trimethoxyphenyl)-4-(1-{[2-(trimethylsilyl)ethoxy]-
methyl}-1H-indol-3-yl)-1,5-dihydro-2H-pyrrole-2-one (19). LC-
MS for C27H34N2O5Si (m/z 494), 495 [M + H]+; mp 115.8 °C;
HRMS m/z ) 494.222 30, calculated 494.223 672; purity HPLC
98.6%, tR ) 10.782 min. Crystal structure of 19 has been proven
by X-ray analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-
97, CCDC No. 680821. Further details of the crystal structure
analysis are available in Supporting Information.
3-(3,4,5-Trimethoxyphenyl)-4-(1-{[2-(trimethylsilyl)ethoxy]-
methyl}-1H-indol-3-yl)-1H-pyrrole-2,5-dione (19a). LC-MS for
C27H32N2O6Si (m/z 808.6), 507.1 [M - H+]; mp >250 °C dec.
4-(1H-Indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2(5H)-
one (1). Compound 19a (120 mg, 0.24 mmol) was dissolved in 10
mL of DMF and 0.5 mL of ethylenediamine and heated to reflux
when 1 mL of a 1 M TBAF solution in THF was added. After 2 h,
the mixture was cooled to room temperature and quenched with
saturated NH4Cl solution and extracted by ethyl acetate. The organic
phase separated, dried over Na2SO4, and evaporated. The product
was purified by flash chromatography on silica gel to yield 72%

Pyrrole-2-ones as VEGFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3823
2-(1H-Indol-3-yl)-N-[2-(4-methoxyphenyl)2-oxoethyl]aceta-
mide (32). FAB-MS for C19H18N2O3, 323.2 [M + H+]; mp 147.7
°C. Crystal structure of 32 has been proven by X-ray analysis:
CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-97, CCDC No.
680824. Further details of the crystal structure analysis are available
in Supporting Information.
3-(1H-Indol-3-yl)-4-(4-methoxyphenyl)-1,5-dihydro-2H-pyr-
role-2-one (33). FAB-MS for C19H16N2O2, 304.1 [M+]; mp 260.4
°C; HRMS m/z ) 304.118 63, calculated 304.121 161; purity HPLC
97.9%, tR ) 7.816 min. Crystal structure of 33 has been proven by
X-ray analysis: CAD4 Enraf Nonius, Cu KR, SIR-92, SHELXL-
97, CCDC No. 680825. Further details of the crystal structure
analysis are available in Supporting Information.
tion with the different test compounds. From a stem solution of the
compounds (concentration of 1 mM in DMSO), an amount of 2 µL
was dissolved in 1.5 mL of gel to give a final compound concentration
of 1.3 µM in the assay (the data in Supporting Information additionally
show the results for testing the compounds at a final assay concentration
of 2.6 µM). Fibrinogen gels without growth factors served as a negative
control. Polymerization of fibrin gels was started by adding 0.65 U/mL
thrombin. Afterward, gels were incubated in MCDB131 containing
the appropriate concentrations of factors, 5% FCS, 5% human serum,
and 200 U/mL Trasylol (Bayer Leverkusen, Germany). After 24 h
the gel was fixed in 1% PFA and the number of sprouts per 50 beads
was counted by microscope. The assay was performed in triplets per
condition (final number of different experiments n ) 9) and statistically
analyzed.
Apoptose Assay. Human dermal microvascular endothelial cells
(HDMECs) were incubated with appropriate concentrations of the
compounds for 24 h; adherent cells and the supernatant were
harvested for detection of apoptotic cells by cleaved caspase-3
staining with a specific antibody (Cell Signaling, Danvers, MA)
and subsequent FACS analysis. Staurosporine-treated HDMECs (5
h, 2.1 µM) served as a positive control. The experiment was
repeated three times.
Molecular Modeling. All modeling was performed on a Red
Hat Linux system. For visualization and building of the structures,
SYBYL 7.2.3 was used. The Connolly surface was calculated using
the MOLCAD module in Sybyl and colored according to the
lipophilicity (from very hydrophobic to hydrophilic corresponds
to brown to green to blue). Compound 2 was docked into the active
site of VEGFR-2 using the FlexX docking program.53 The FlexX
scoring function was applied during the placement and construction
phase of the ligands and DrugScore for the final ranking.54 The
3D coordinates of the VEGFR-2 catalytic core in complex with a
2-anilino-5-aryloxazole inhibitor were taken from the Brookhaven
Protein Databank (PDB code 1Y6A).17
2-(N-Methyl-1H-indol-3-yl)-N-[2-(4-methoxyphenyl)2-oxoet-
hyl]acetamide (34). FAB-MS for C20H20N2O3, 337.2 [M + H+];
mp 114.1 °C.
3-(N-Methyl-1H-indol-3-yl)-4-(4-methoxyphenyl)-1,5-dihydro-
2H-pyrrole-2-one (35). The compound was prepared from 170 mg
(0.5 mmol) of 34 following a general procedure. The product was
purified by flash chromatography to yield (56%) 90 mg (0.28 mmol)
of 35 as slightly ginger solid. FAB-MS for C20H18N2O2, 319.2 [M
+ H+]; mp 203.0 °C; HRMS m/z ) 318.138 50, calculated
318.136 811; purity HPLC 100%, tR ) 8.854 min. Crystal structure
of 35 has been proven by X-ray analysis: CAD4 Enraf Nonius, Cu
KR, SIR-92, SHELXL-97, CCDC No. 680826. Further details of
the crystal structure analysis are available in Supporting Information.
Selectivity Profiling of Compounds by IC50 Values Using 24
Protein Kinases. Recombinant Protein Kinases. The inhibitory
profile of compounds was determined using the following 24 protein
kinases (GenBankAcc.No. available on http://www.proqinase.com/
pages/science): AKT1, ARK5, Aurora-A, Aurora-B, B-Raf-VE, CDK2/
CycA, CDK4/CycD1, CK2-A1, EGF-R, EPHB4, ERBB2, FAK, IGF1-
R, SRC, VEGF-R2, VEGF-R3, FLT3, INS-R, MET, PDGFRbeta,
PLK1, SAK, TIE2, COT (name as listed in the literature51).
All protein kinases were expressed using human cDNAs in Sf9
insect cells as recombinat GST-fusion proteins or His-tagged pro-
teins by means of the baculovirus expression system. Kinases were
purified by affinity chromatography using either GSH-agarose
(Sigma) or Ni-NTH-agarose (Qiagen). The purity and identity of
each kinase were checked by SDS-PAGE/silver staining and by
Western blot analysis with specific antibodies.
Acknowledgment. We thank Susanne Weidemann, Depart-
ment of Dermatology, University of Tu¨bingen, for her helpful
assistance in performing the cellular assays. Financial support
by Merckle GmbH, Blaubeuren, is gratefully acknowledged.
Supporting Information Available: (1) IR data, (2) 1H and 13
C
NMR data, (3) purification data of compounds 2, 13, 19, 20, 23-28
by flashchromatography, (4) purity of key target compounds, (5)
X-ray analysis results for compounds 1, 2, 4, 6a, 11, 14, 15, 19,
23, 24, 32, 33, and 35, (6) quality parameters for the protein kinase
assay (IC50 values at 24 protein kinases), and (7) details of HLMEC
sprouting assay of compounds 1-3 at 2.6 µM. This material is
available free of charge via the Internet at http://pubs.acs.org.
Protein Kinase Assay. A proprietary protein kinase assay
(33PanQinase activity assay) was used for measuring the kinase
activity of the 24 protein kinases. All kinase assays were performed
in 96-well FlashPlates from Perkin-Elmer/NEN (Boston, MA) in a
50 µL reaction volume.
The assay for all enzymes contained 60 mM HEPES-NaOH,
pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM sodium orthovanadate,
1.2 mM DTT, 50 µg/mL PEG20000, 1 µM [γ-33P]ATP (ap-
proximately 5 × 105 cpm per well), and recombinant protein kinase
(50-400ng).Dependingonthekinase,thefollowingsubstrate-proteins
were used: AKT1 (GSK3/14-27), ARK5 (autophosphorylation),
Aurora-A, Aurora-B (Tetra(LRRWSLG)), B-Raf-VE (MEK1 KM),
CDK2/CycA (histone H1), CDK4/CycD1 (Rb-CTF), CK2-A1
(casein), EGF-R, EPHB4, ERBB2, FAK, IGF1-R, SRC, VEGF-
R2, VEGF-R3 (poly(Glu,Tyr) 4:1), FLT3, INS-R, MET, PDGFRꢀ
(poly(Ala,Glu,Lys,Tyr) 6:2:5:1), PLK1 (casein), SAK (autophos-
phorylation), TIE2 (poly(Glu,Tyr) 4:1), COT (autophosphorylation).
The IC50 values were measured by testing 10 concentrations of
compounds. The final DMSO concentration in the assay was 1%.
The reaction cocktails were incubated at 30 °C for 80 min. The
reaction was stopped with 50 µL of 2% (v/v) H3PO4. Plates were
aspirated and washed two times with 200 µL of 0.9% (w/v) NaCl.
Incorporation of 33Pi was determined with a microplate scintillation
counter (Microbeta Trilux, Wallac). All assays were performed with
a BeckmanCoulter/Sagian robotic system.
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