Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents

Article abstract of DOI:10.1016/j.bmc.2008.04.019

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N⁴-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.¹ with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-β (PDGFR-β), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N⁴-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N⁴-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC₅₀ value of 30 nM in the CAM angiogenesis inhibition assay.

Full text of DOI:10.1016/j.bmc.2008.04.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5514–5528
Design, synthesis and biological evaluation of substituted
pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine
kinase inhibitors and antiangiogenic agents
Aleem Gangjee,a,* Ojas A. Namjoshi,a Jianming Yu,a Michael A. Ihnat,b
Jessica E. Thorpeb and Linda A. Warnkeb
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
bDepartment of Cell Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Received 18 March 2008; revised 4 April 2008; accepted 4 April 2008
Available online 14 April 2008
Abstract—Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal
targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently
demonstrated. We designed and synthesized eight N4-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side
chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthe-
sized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and
substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee
et al.1 with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs
are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable
inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1),
platelet-derived growth factor receptor-b (PDGFR-b), the growth of A431 cells in culture, and in the chicken embryo chorioallan-
toic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the
6-ethylaryl substituents as well as the N4-phenyl substituents of these analogs does indeed control both the potency and specificity
of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl
increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were
identified with the best compound, N4-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15),
with an IC50 value of 30 nM in the CAM angiogenesis inhibition assay.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
as indirectly involved in the growth of tumors and
metastases.2
Receptor tyrosine kinases (RTKs) are a subfamily of
protein tyrosine kinases, which play key roles in tumor
growth, survival, and dissemination. A variety of
growth factors particularly vascular endothelial growth
factor (VEGF), epithelial growth factor (EGF), platelet
derived growth factor (PDGF), and their receptors are
overexpressed in several tumors. These growth factors
and their receptors are thought to be directly as well
Angiogenesis, the formation of new blood vessels from
existing vasculature, is essential for both physiological
and pathological processes. It is a complex cascade that
is tightly regulated by proangiogenic and antiangiogenic
factors.3 VEGF is the predominant stimulator of angio-
genesis.4,5 The expression of VEGF is efficiently con-
trolled under physiologic conditions. However, VEGF
is secreted by a large number of tumor cells, leading to
angiogenesis that allows the tumor to grow and metasta-
size. In addition to VEGF, several external factors are
also involved in stimulating tumor angiogenesis. Some
of the principal proangiogenic regulators include growth
factors EGF, PDGF, fibroblast growth factor (FGF),
insulin-like growth factor-1 (IGF-1), transforming
Keywords: Multiple receptor; Tyrosine kinase inhibitors; Antiangio-
genic agents.
*
Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396
5593; e-mail: gangjee@duq.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.019

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5515
growth factors (TGFa and b), and tumor necrosis factor
a (TNF-a) among others.6 The newly sprouted blood
vessels provide an additional supply of nutrients to the
tumor to grow beyond a certain size (1–2 mm3). Angi-
ogenesis is a pivotal step in the transition of some solid
tumors from a dormant state to a malignant state; it also
provides metastatic pathways for solid tumors.7 In addi-
tion, angiogenesis contributes to the development of
hematologic malignancies, particularly multiple myelo-
ma, leukemia, and lymphoma. However, the role of
angiogenesis has not been clearly defined in hematologic
malignancies.8–10 Angiogenesis has been described as
one of the hallmarks of cancer.11 Thus inhibition of tu-
mor angiogenesis affords attractive targets for the devel-
opment of novel antitumor agents. Antiangiogenic
therapy is targeted to non-tumor cells (endothelial cells)
which are expected to have less ability to mutate in order
to produce resistance compared with tumor cells. Thus,
antiangiogenic agents have afforded a new paradigm for
the treatment of cancer.11
cies], sunitinib 4 [inhibits VEGFR-2, PDGFR-b, c-kit,
and FLT3 (FMS-like tyrosine kinase 3); approved for
the treatment of renal cell carcinoma and imatinib-resis-
tant gastrointestinal stromal tumor)],26 sorafenib 5
[inhibits Raf kinase and VEGFR-2, PDGFR-b and c-
kit; approved for the treatment of advanced renal cell
carcinoma],27 and lapatinib 6 [inhibits EGFR and
ErbB-2 (erythroblastic leukemia viral oncogene homo-
log 2); approved for advanced metastatic breast cancer
in conjunction with the chemotherapy].28 In 2003,
Gangjee et al. 29 demonstrated that fine-tuning of the
molecular substitution pattern on a pyrrolo[2,3-d]pyrim-
idine scaffold affords multikinase inhibitors with poten-
cies equivalent to or better than standard agents. Since
substitutions on the phenyl ring of the 6-arylmethyl
group in 7 dictated both the inhibitory potencies as well
as the selectivity against RTKs, it was of interest to
determine the effect of side chain homologation of the
previously reported compounds of general structure 7,
on the potency and spectrum of RTK inhibitory
activity.
There has been considerable discussion in the literature
regarding the use of RTK inhibitors as monotherapy for
cancer or the combination of multiple RTK inhibitors
either as single agents12–15 or in combination with other
chemotherapeutic agents.6–18 The majority of earlier re-
ports underlined the development of targeted therapy
against single RTK by small molecules, some of which
afforded clinical agents such as gefitinib 1 (specific
EGFR inhibitor; approved for limited use for the treat-
ment of non-small cell lung cancer)19 (Fig. 1) and erloti-
nib 2 (specific EGFR inhibitor; approved for the
treatment of non-small cell lung cancer).19 However, tu-
mors have redundant signaling pathways for angiogene-
sis and often develop resistance to agents that target one
specific pathway.20 Recently, crosstalk has been impli-
cated between EGFR and other growth factor receptors
involved in tumorigenesis.6 Hence, a multifaceted ap-
proach that targets multiple signaling pathways has
been shown to be more effective than the inhibition of
a single target.6,16 The most important consequence of
inhibiting multiple RTKs would be to retard tumor
resistance by blocking potential ‘escape routes.’21 Since
RTKs are present in endothelial cells (VEGFR,
PDGFR), tumor cells (FGFR, PDGFR), and peri-
cytes/smooth muscle cells (FGFR, PDGFR), inhibition
of more than one RTK does provide synergistic inhibi-
tory effects against solid tumors.20 Recently, VEGFR-
2 and PDGFR-b have been implicated in controlling
angiogenesis at two different stages of the angiogenic
process, and it has been shown that inhibition of VEG-
FR-2 and PDGFR-b with two separate inhibitors pro-
duces a synergistic effect in early stage as well as late
stage pancreatic islet cancer in mouse models by attack-
ing the angiogenic process at two different sites.17,22
2. Design of inhibitors
Since the aim of this study was to design multikinase
inhibitors, a general RTK pharmacophore model was
utilized rather than X-ray crystal structures of specific
RTKs. On the basis of this general pharmacophore
model depicted in Figure 2, we reasoned that the addi-
tion of a second carbon in the bridge at the 6-position
of the pyrrolo[2,3-d]pyrimidine 7 would afford greater
conformational flexibility and allow the phenyl ring to
adopt multiple low energy-binding orientations such
that it could perhaps bind to multiple RTKs providing
multi-RTK inhibitors. Such agents could expand the
spectrum of RTK inhibition compared to 7. Thus we
embarked on the homologation of our previously
reported RTK inhibitors using the pyrrolo[2,3-d]pyrim-
idine scaffold with a 2-NH2 moiety as in compounds
8–12 (Fig. 4). The 2-NH2 group in our compounds
provides a third H-bonding moiety in the Hinge Region
of RTKs, as shown in the pharmacophore model in
Figure 1, and should increase binding and consequently
potency of our scaffold, as compared to the majority of
the pyrimidine-containing RTK inhibitors which do not
contain the 2-NH2 group.30 The 4-anilino ring with a 3-
bromo substitution has provided potent inhibition of
RTKs and is accommodated either in the Hydrophobic
Region I, in the Sugar Pocket, or in the Phosphate Bind-
ing Region (Fig. 2) depending on the mode of attach-
ment for our proposed pyrrolo[2,3-d]pyrimidines.29 As
the first iteration of our previous series of RTK inhibi-
tors,29 we elected to keep these two substitutions [i.e.,
the 2-NH2 group and the 4-amino-(30-bromophenyl)]
constant. The purpose being to determine if variations
in the phenyl ring of a 6-phenylethyl moiety appended
to the pyrrolo[2,3-d]pyrimidine scaffold would allow
for inhibition of multiple RTKs and also dictate the
potency and selectivity as reported for 7.
A flood of reports on the design of multikinase inhibi-
tors have appeared in the past few years,2,6,17,22–24 some
of which have led to clinically approved agents such as
imatinib 3 [inhibits Abelson Tyrosine Kinase (Abl), c-
kit protein (CD117), and PDGFR; approved for chronic
myelogenous leukemia (CML),25 gastrointestinal stro-
mal tumors (GISTs), and a number of other malignan-
Molecular modeling, using the SEARCH option in
SYBYL 7.3,31 of the homologated ethyl side chain

5516
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
H3C
H3C
CH3
N
N
CH3
H
NH
F
N
N
H3C
O
O
O
N
N
N
N
HN
N
HN
N
Cl
CH3
O
O
O
F
O
O
HN
N
N
O
O
N
CH3SO3H
H
Gefitinib (1)
Erlotinib (2)
H3C
Imatinib (3)
Sunitinib (4)
O
S
O
O
Cl
H3C
N
O
N
O
NH
H
HN
Br
CF3
O
HN
N
N
Cl
O
N
F
N
H
H2N
N
N
N
H
H
R2
Sorafenib (5)
Lapatinib (6)
7
HN
NH3
NH3
R
Cl
N
H
O
O
N
N
Pt
N
Cl
N
O
N
H
PD153035: R= 3'-Br
CB676475: R = 2'-F,4'-Cl
Semaxanib (SU5416)
AG1295
Cisplatin
Figure 1. Structures of RTK inhibitors.
analog 8 using the five rotatable bonds through 30ꢁ
increments afforded 97875 conformations (compared
to 3174 for 7). The lowest energy conformation and
conformations within 2 kcal/mol generate side chain
orientations of the anilino and the 6-phenylethyl side
chain that place these side chains in multiple positions
that include the Hydrophobic Region 1, the Sugar
Pocket, and the Phosphate Binding Region for the 6-
phenylethyl moiety when in Mode 1 (Fig. 2). The ani-
lino moiety can be oriented in the Hydrophobic Region
1 in Mode 1 and in the Sugar Pocket in Mode 2. The
conformations within 1 kcal/mol of the least energy
conformation also show multiple orientations of both
side chains on the pyrrolo[2,3-d]pyrimidine scaffold.
Three low energy conformations of 8 (within 0.5 kcal/
mol of the lowest energy conformation) are depicted
in Figure 3 and show the wide variations of the orien-
tations of the 4-anilino group (green) and the 6-phenyl-
ethyl substituents (yellow). Thus molecular modeling
lends credence to the idea that adding an additional
carbon in the side chain significantly increases the flex-
ibility and allows for multiple low energy orientations
of the 6-side chain phenyl ring to perhaps allow for
multiple low energy binding orientations to target dif-
ferent RTKs. Thus we synthesized compounds 8–12
which contain a 2-amino group, a 4-meta-bromo ani-
line and a phenylethyl substitution at the 6-position.
Compounds 8–11 were previously disclosed without
synthetic details, and were found inactive against
IGF-1R.1 The synthesis, characterization, and activities
of 8–11 against EGFR, VEGFR-1, VEGFR-2, PDGFR-
b, A431 cells in culture, and the CAM assay are re-
ported for the first time in this study.
In addition, as proposed in the pharmacophore model in
Figure 2, the 4-anilino ring could bind in the Hydropho-
bic Pocket 1 (Binding mode 1 and Binding mode 3) or in
the Sugar Binding Pocket (Binding mode 2). Molecular
modeling indicates that the anilino moiety adopts a vari-
ety of low energy conformations similar to the 6-substi-
tuent. Hence, variations on the 4-anilino ring (13–20)
were also synthesized to probe these binding areas and
to determine whether selectivity and/or potency can be
influenced by substitutions on the 4-anilino moiety with
the 6-phenylethyl substituent constant. Compound 8,
the phenyl unsubstituted analog, was the most potent
and this side chain at position-6 was maintained in com-
pounds 13–20. Several substituents on the 4-position
that were reported in the literature to provide potent
RTK inhibitors19,32,33 were incorporated in target com-
pounds 13–20 . Compound 20 was designed to influence
and perhaps improve the cellular activity of the com-
pound as reported for a quinazoline series with similar
bicyclic 4-substituents.34,35

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5517
HYDROPHOBIC REGION I
HYDROPHOBIC REGION I
R
Br
PHOSPHATE
BINDING
REGION
PHOSPHATE
BINDING
REGION
O
HN
N
HN
O
HINGE
REGION
HINGE
REGION
NH
O
NH
O
N
NH
NH
N
N
H2N
H2N
R
Br
HYDROPHOBIC
REGION II
HYDROPHOBIC
REGION II
SUGAR BINDING
POCKET
SUGAR BINDING
POCKET
Binding mode 1
Binding mode 2
HYDROPHOBIC REGION I
Br
PHOSPHATE
BINDING
REGION
O
HN
N
R
HINGE
REGION
NH
O
NH
N
H2N
HYDROPHOBIC
REGION II
SUGAR BINDING
POCKET
Binding mode 3
Figure 2. General pharmacophore model of pyrrolo[2,3-d]pyrimidines.
3. Chemistry
moketone 28. Stirring 28 with 2,4-diamino-6-hydroxy-
pyrimidine 29 in DMF in the presence of molecular
sieves (13·; 16–2.5 mm beads) afforded 30.29 The pres-
The synthesis of compounds 8–12 is shown in Scheme 1.
Gangjee et al.36 previously reported 21a–e via the palla-
dium-catalyzed Sonogashira coupling followed by cata-
lytic reduction of the triple bond with 5% Pd on
charcoal. Chlorination of 21a–e with phosphorus oxy-
chloride afforded 22a–e. Compounds 22a–e were reacted
with 3-bromoaniline 23 in isopropanol at reflux in the
presence of two drops of concd HCl for 1–3 h afforded
24a–e. Depivalation of 24a–e with 1 N NaOH in meth-
anol followed by chromatographic purification afforded
8–12.
1
ence of a singlet at d5.85 in H NMR corresponding to
C5-CH of the pyrrolo[2,3-d]pyrimidine confirmed the
cyclization to 30. Chlorination of 30 with phosphorous
oxychloride at reflux afforded 31. Nucleophilic displace-
ment of the 4-chloro group in 31 with various substi-
tuted anilines gave the target compounds 13–20.
4. Results and discussion
RTK inhibitory activity of the compounds 8–20 was
evaluated using human tumor cells known to express
high levels of EGFR, VEGFR-1, VEGFR-2, PDFGR-
b, and FGFR1 using a phosphotyrosine ELISA cyto-
blot.38,39 Compounds known to inhibit a particular
RTK were used as positive controls for these assays.
Whole cell assays were used for RTK inhibitory activity
since these assays afford more meaningful results for
translation to in vivo studies. The effect of compounds
on cell proliferation was measured using inhibition of
the growth of A431 cancer cells known to overexpress
EGFR. EGFR has been shown to play a role in the
The synthesis of target compounds 13–20 is shown in
Scheme 2. 3-Phenylpropanoic acid 25 was converted to
3-phenylpropanoyl chloride 26 with thionyl chloride in
benzene at reflux. Compound 26 in diethyl ether was
added dropwise to a solution (in diethyl ether) of freshly
generated diazomethane37 (from N-methyl-N-nitrosou-
rea) in an ice-salt bath; maintaining the temperature be-
tween 0 and 5 ꢁC. The resulting yellow solution was
warmed to room temperature and stirred for an addi-
tional 30 min. The diazo intermediate 27 was treated
with 48.5% HBr solution and refluxed to form the a-bro-

5518
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
cell number.41 Finally, the effect of selected compounds
on blood vessel formation was assessed using the chick-
en embryo chorioallantoic membrane (CAM) assay, a
standard test for angiogenesis.42 In this assay, purified
angiogenic growth factors are placed locally on a vascu-
larized membrane of a developing chicken embryo to-
gether with possible inhibitors. Digitized images of the
vasculature are taken at 48 h after growth factor admin-
istration and the number of vessels per unit area evalu-
ated as a measure of vascular density.
Since the IC50 values of RTK inhibitors vary under dif-
ferent assay conditions, we used a standard (control)
compound in each of the evaluations. For VEGFR-2
the standard was SU5416; for VEGFR-1 the standard
was CB676475 (4-chloro-2-fluoro-phenyl)-(6,7-dime-
thoxy-quinazolin-4-yl)-amine; for EGFR the standard
was PD153035; for PDGFR-b the standard was
AG1295; for the cytotoxicity study against the growth
of A431 cells in culture the standard was cisplatin.
SU5416 was also used as the standard for the antiangio-
genic CAM assay.
4.1. EGFR inhibition
Compound 8 with an unsubstituted phenyl side chain
was the most potent compound and was similar in po-
tency to the standard analog PD153035. Compounds
9–12 showed single-digit micromolar inhibition of
EGFR, and were about 10- to 20-fold less than the stan-
dard PD153035. Electron-donating group (OCH3, com-
pound 9) in the 40-position and withdrawing groups (Cl,
compound 10) in the 20-position on the phenyl ring were
detrimental to activity. Both compounds 11 and 12 with
2,3- and 3,4-di substitution with hydrophobic carbon
atoms and bulkier side chains also showed a decrease
in potency by about 16-fold as compared with the 6-phe-
nyl compound 8. Thus any substitution of a 40-OCH3,
20-Cl, 2,3-, and 3,4-carbon substitution is detrimental
to EGFR inhibition. Homologation from one to two
carbon chain improved EGFR inhibitory activity for
Figure 3. Stereoview of low energy conformations of 8 using SYBYL
7.3.31 4-Anilino group (green); 6-phenylethyl substituent (yellow). The
conformations within 1 kcal/mol of the least energy conformation
show multiple orientations of both side chains on the pyrrolo[2,3-
d]pyrimidine scaffold.
overall survival of the cells.40 Cell proliferation was as-
sessed using CYQUANTꢂ, a DNA intercalating dye
that has been shown to give a linear approximation of
R2
Ar
R2
13
14
15
16
17
18
19
3'-Br, 4'-F
3'-Cl, 4'-F
3'-CF3
3'-C
2'-F, 4'-Cl
4'-Cl
8
9
Phenyl
HN
Br
HN
4'-methoxyphenyl
10 2'-chlorophenyl
11 1'-naphthyl
12 2'-napthyl
N
N
CH
N
N
H
H2N
N
H2N
N
H
Ar
3'-CF3, 4'-F
NH
HN
N
N
H
H2N
N
20
Figure 4. Homologated analogs.

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5519
NH2
23
Br
Cl
HN
Br
HN
Br
O
N
a
N
N
N
c
b
HN
PivHN
N
N
H
N
H
N
PivHN
N
PivHN
N
H2N
N
H
H
Ar
Ar
Ar
Ar
21a-e
Ar
22a-e
24a-e
8-12
21a Phenyl
21b 4'-methoxyphenyl
21c 2'-chlorophenyl
21d 1'-naphthyl
21e 2'-napthyl
Scheme 1. Synthesis of 8–12. Reagents and conditions: (a) POCl3, reflux, 2 h; (b) 3-bromoaniline, i-PrOH, 2 drops of concd HCl, reflux, 1–3 h; (c)
1 N NaOH, methanol, 70 ꢁC, 10 h.
a
c
b
Cl
N+
Br
COOH
N
O
O
O
25
26
27
28
R2
O
N
NH2
O
N
Cl
N
HN
HN
H2N
29
32a-h
R2
NH2
e
HN
H2N
N
N
f
d
N
N
N
H
H2N
H2N
N
H
H
30
31
13-20
Scheme 2. Synthesis of 13–20. Reagents and conditions: (a) Thionyl chloride, benzene, reflux, 1.5 h; (b) diazomethane, ether, 0–5 ꢁC 15 min; rt
30 min.; (c) 47.5% aq. HBr, ether, reflux, 1 h; (d) dimethyl formamide, MS 13·, rt, 3d; (e) phosphorus oxychloride, N,N0-dimethylaniline, reflux, 4 h;
(f) isopropanol, 2–3 drops concd HCl, reflux 4 h (for 17, no concd HCl).
8, 10, and 11 and only in 12 was there a decrease in
activity compared to the single atom bridge. For the
four analogs for which direct comparison is available
there was an increase in inhibitory activity in three of
the four.
4.3. VEGFR-2 inhibition
Compound 12 was the most potent inhibitor of VEGFR-
2 and was only 1.5-fold less than the standard SU5416.
Compounds 8 and 9 were inactive at 50 lM and com-
pound 10 was about one fourth as potent as SU5416.
Homologation improved the activity in 12 but decreased
the activity for 10. Thus no clear-cut trends were apperent
for VEGFR-2 inhibition for this series.
4.2. VEGFR-1 inhibition
Compounds 8–10 exhibited two-digit micromolar inhi-
bition of VEGFR-1, which is about 2- to 2.5-fold less
than the standard CB676475. Compounds 11 and 12,
the naphthyl analogs, showed better inhibitory activity
than the standard compound against VEGFR-1. Elec-
tron-withdrawing or electron-donating groups did not
have much influence on VEGFR-1 inhibition. Against
VEGFR-1, the bulk on the phenyl ring affords better
inhibition compared to the phenyl unsubstituted analog
8. In this regard the activities against VEGFR-1 are in
contrast to the inhibitory activity against EGFR, where
bulk was detrimental to activity. Once again, homologa-
tion from one to two carbon atoms in the chain im-
proved inhibitory activity (VEGFR-1) in three (8, 11,
and 12) of the four compounds.
4.4. PDGFR-b inhibition
Compounds 8, 11, and 12 showed 2-digit micromolar
inhibition of PDGFR-b. Compound 11 with a 1-naph-
thyl side chain was the best in this series and was only
1.7-fold less potent than the standard AG1295. Homol-
ogation from a one to a two-carbon chain provides for
PDGFR-b inhibition in three (8, 11, and 12) of the five
analogs synthesized. The corresponding single atom-
bridged analogs were all devoid of PDGFR-b inhibitory
activity. These results suggest that increasing the chain
length of the bridge from one to two-carbon atoms does
provide for additional RTK inhibitory activity. It is

5520
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
interesting to note that only the 6-phenyl and 6-naphthyl
analogs (hydrocarbons) had PDGFR-b activity. The
electron-donating 40-methoxy and electron withdrawing
20-chloro analogs 9 and 10, respectively, were not active
against PDGFR-b.
4.5. A431 cytotoxicity
Compound 11 was equipotent with the standard cis-
platin but was more than 2-fold better than SU5416,
and was also better than the other standard
PD153035. Compound 10 was a reasonably potent
inhibitor comparable to PD153035. However, the corre-
lation with EGFR inhibitory potency did not always
translate to high A431 inhibitory activity. This may re-
flect transport and/or other pharmacokinetic differences
of analogs between the two cell systems. The EGFR
inhibitory activity of 10 and 11 does translate to A431
cytotoxicity. However, for 9 and 12 the EGFR inhibi-
tory activity is similar to 10 and 11 but the A431 inhibi-
tion is not. Similar discrepancies have been noted in
several literature reports43–50 and may reflect differences
in transport or other factors.
4.6. CAM angiogenesis
All the compounds 8–12 showed IC50s at two-digit
micromolar concentration in the CAM assay. The best
compound is 10. However, this is about 118-fold less po-
tent than the standard SU5416, but could reflect differ-
ences in transport or other pharmacokinetic differences
of the analogs.
Taken together the inhibitory activities against RTKs
(Table 1) show that the spectrum of RTK inhibition
for three of the four homologated analogs 8, 11, and
12 includes PDGFR-b which was distinctly absent for
the corresponding 6-methylphenyl compounds29, and
lends support to our hypothesis that homologation
and greater conformational flexibility do indeed provide
for the inhibition of an additional RTK and depend on
the nature of the substitution on the 6-phenyl side chain.
Compounds 13–20 were synthesized to determine the ef-
fect of the different substitutions on the aniline phenyl
ring and were chosen, as mentioned above, on the basis
of the activities these substitutions provided against
RTK with other scaffolds.19,32,33 These analogs were also
evaluated against RTKs, A431 cells in culture, and in the
CAM assay and the results are listed in Table 2.
4.7. EGFR inhibition
The best compound in this series against EGFR is 17,
with a 20-fluoro, 40-chloroaniline substituents. It is, how-
ever, 6.6-fold less active than the standard PD153035.
Similarly, 18 with a 40-chloro substituent on the aniline
exhibited the next best inhibitory activity against
EGFR. However, both compounds were less potent
than the meta-Br analog 8. Thus substitution on the ani-
line ring affords significant variability in EGFR inhibi-
tion. Compounds 13, 15, 16, and 20 were inactive
against EGFR.

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5521
Table 2. IC50 values (lM) of kinase inhibition, A431 cytotoxicity, and inhibition of the CAM assay
Compound
EGFR inhibition
VEGFR-1
inhibition
VEGFR-2
inhibition
PDGFR-b
inhibition
A431
cytotoxicity
CAM angiogenesis
inhibition
8
13
0.3 0.042
>200
45.1 8.9
>200
>200
>50
11.8 3.7
>500
>500
28.6 8.2
3.4 0.41
6.8 0.71
4.6 0.67
8.5 1.1
19.5 5.1
6.1 0.83
5.9 0.7
198.3 24.3
132.1 16.7
>200
14
15
22.8 4.7
>200
>200
>200
>200
>500
0.03 0.004
1.1 0.07
8.6 0.76
3.3 0.82
0.12 0.012
50.6 7.1
16
17
>200
>200
100.4 22.1
>500
>500
1.32 0.09
3.18 0.41
122 30.1
>200
>200
>200
1.4 0.12
1.6 0.06
9.8 1.0
18
19
>200
>200
24.9 4.1
>200
>200
90.0 17.8
77.4 8.9
20
5.5 0.92
12.6 2.9
PD153035
CB676475
SU5416
AG1295
Cisplatin
0.2 0.04
17.7 5.5
10.6 2.7
19.2 1.1
7.65 1.4
0.085 0.0031
6.2 1.3
4.8. VEGFR-1 inhibition
4.12. CAM angiogenesis
All the compounds 13–20 in this series were inactive
against VEGFR-1 indicating a lack of tolerance for
VEGFR-1 inhibitory activity with substituents other
than a meta-Br.
Compounds 13–19 showed improved inhibition of angi-
ogenesis as compared with 8. Compound 15 with 30-tri-
fluoromethylaniline group was the best and was about
2.8 times more potent than the standard SU5416 in
the inhibition of angiogenesis. Similarly 19, with 30-tri-
fluoromethyl, 40-fluoroaniline substitution, showed sig-
nificant antiangiogenic effects. However, for both these
compounds, the excellent potency against angiogenesis
could not be explained with the limited number of RTKs
evaluated. This raises the possibility of alternate antian-
giogenic mechanisms for these compounds and is cur-
rently under investigation.
4.9. VEGFR-2 inhibition
Compound 20, with 5-aminoindole substituents, is the
best inhibitor of VEGFR-2 and is only 2.3-fold less po-
tent than the standard SU5416.
4.10. PDGFR-b inhibition
All the compounds showed diminished activity as com-
pared to 8. Only compounds 19 and 20 exhibited 2-digit
micromolar IC50 values, however, this was more than
12-fold less potent than the standard AG1295 and about
7-fold less potent than 8. Clearly the 30-Br substituents
(8) are the most potent, among the substituents evalu-
ated, for PDGFR-b inhibition. This indicates that, as
for the other RTKs, both the aniline and the 6-substitu-
ent determines the inhibitory activity.
5. Summary
Thirteen N4-phenylsubstituted-6-(2-phenylethylsubsti-
tuted)-7H-pyrrolo[2,3-d]pyrimidine 2,4-diamines 8–20
were designed and synthesized as RTK inhibitors. The
biological evaluation showed several analogs had
remarkable inhibitory activity against EGFR, VEG-
FR-1, A431 proliferation, and in the CAM angiogenesis
assay. Of the analogs evaluated, 11 compounds (com-
pounds 8 and 11–20) were equipotent or more potent
as compared to the standard compounds tested. Eight
analogs, 9, 13–18, and 20, showed potent cytotoxic ef-
fects against the growth of A431 cells in culture and
two analogs, 15 and 19, demonstrated high antiangio-
genic activity in the CAM assay. In addition, one analog
(compound 8) showed dual inhibitory activity against
EGFR and PDGFR-b; while another analog (com-
pound 12) inhibits three of the kinases (VEGFR-1,
VEGFR-2, and PDGFR-b) evaluated. The inhibitory
data against the RTKs in this study demonstrate that
variation of the 6-ethylaryl substituents as well as the
N4-phenyl substituents of these N4-phenylsubstituted-
6-(2-phenylethylsubstituted)-7H- pyrrolo[2,3-d]pyrimi-
dine-2,4-diamines does indeed control both the potency
and specificity of inhibitory activity against RTKs. In
addition, homologation of the 6-chain length does in-
crease the spectrum of RTK inhibition to include
PDGFR-b that was absent for the unhomologated ana-
logs. Using the analogs described in this study as leads, a
4.11. A431 cytotoxicity
Interestingly, all the compounds 13–20 showed good
inhibition (single-digit micromolar) as compared to 8
and the standards PD153035 and cisplatin (except 16
and 19). Compound 17 is the most potent and is 9-
and 14-fold more potent than PD153035 and SU5416,
respectively, and about 5.5-fold more potent than cis-
platin. Compound 18 showed similar activity as 17.
Compounds 17 and 18 were the most potent EGFR
inhibitors and this rank order translates into A431 cyto-
toxicity. None of the other analogs 13–16 and 19–20
showed any significant EGFR inhibitory activity and
yet were reasonably potent against the growth of A431
cells in culture. This could be attributed to differences
in the transport and/or inhibition of other targets. As
mentioned for compounds 8–12 (Table 1), the litera-
ture43–50 contains several reports of similar activities
where EGFR inhibitory activity does not translate to
A431 cell inhibitory activity.

5522
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
structure-based design and synthesis of further RTK
inhibitors are currently in progress and will be the sub-
ject of future reports.
CH2CH2), 6.23(s, 1H, C5-CH), 7.24 (m, 5H, C6H5),
9.99 (s, 1H, 2-NHPiv, exch), and 12.32 (s, 1H, 7-NH,
exch). Anal. Calcd for C19H21N4OCl: C, 63.95; H,
5.93; N, 15.70; Cl, 9.93. Found C, 63.84; H, 5.96; N,
15.64; Cl, 10.04.
6. Experimental
6.1.2. N-{4-Chloro-6-[2-(4-methoxyphenyl)ethyl]-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide
(22b ). Compound 21b (0.3 g, 0.80 mmol) using the gen-
eral procedure described above, afforded 22 b (0.25 g;
79%) as a white solid: TLC Rf 0.5 (MeOH/CHCl3,
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo
(0.2 mm Hg) in a CHEM-DRY drying apparatus over
P2O5 at 70 ꢁC. Melting points were determined on a
MEL-TEMP II melting point apparatus with FLUKE
51 K/J electronic thermometer and are uncorrected. Nu-
clear magnetic resonance spectra for proton (1H NMR)
were recorded on a Bruker WH-300 (300 MHz) spec-
trometer. The chemical shift values are expressed in
ppm (parts per million) relative to tetramethylsilane as
an internal standard: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad singlet. The relative inte-
grals of peak areas agreed with those expected for the
assigned structures. Thin-layer chromatography (TLC)
was performed on WHATMAN UV254 silica gel plates
with a fluorescent indicator, and the spots were visual-
ized under 254 and/or 365 nm illumination. Proportions
of solvents used for TLC are by volume. Column chro-
matography was performed on a 230–400 mesh silica gel
purchased from Fisher Scientific. Elemental analyses
were performed by Atlantic Microlab, Inc., Norcross,
GA. Element compositions are within 0.4% of the calcu-
lated values. Fractional moles of water or organic sol-
vents frequently found in some analytical samples of
compounds in this report related to the ones like antifo-
lates,1,29,51 could not be prevented in spite of 24–48 h of
drying in vacuo and were confirmed where possible by
their presence in the 1H NMR spectra. All solvents
and chemicals were purchased from Aldrich Chemical
Co. or Fisher Scientific and were used as received.
1
1:19); mp: 204.5–206 ꢁC; H NMR (DMSO-d6): d 1.2
(s, 9H, C(CH3)3), 2.97 (s, 4 H, CH2CH2), 3.7 (s, 3H,
OCH3), 6.22 (s, 1 H, C5-CH), 6.82 (d, 2H, C6H4,
J = 8.5 Hz), 7.13 (d, 2H, C6H4, J = 8.5 Hz), 9.97 (s,
1H, 2-NHPiv, exch), and 12.29 (s, 1H, 7-NH, exch).
Anal. Calcd for C20H23N4O2Cl: C, 62.09; H, 5.99; N,
14.48; Cl, 9.16. Found C, 61.91; H, 6.01; N, 14.54; Cl,
9.33.
6.1.3. N-{4-Chloro-6-[2-(2-chlorophenyl)ethyl]-7H-pyrrol-
o[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (22c).
Compound 21c (0.23 g, 0.6 mmol) using the general pro-
cedure described above, gave 22c (0.16 g; 64%) as a pale
yellow solid: TLC Rf 0.45 (MeOH/CHCl3, 1:19); mp
195–197 ꢁC; 1H NMR (DMSO-d6): d 1.22 (s, 9H,
C(CH3)3), 3.03 (t, 2H, CH2CH2, J = 7.2 Hz), 3.16 (t,
2H, CH2CH2, J = 7.2 Hz), 6.24 (s, 1H, C5-CH), 7.26
(m, 4H, C6H4), 9.98 (s, 1H, 2-NHPiv), and 12.14 (s,
1H, 7-NH, exch).
6.1.4 . N-{4-[Chloro]-6-[2-(1-naphthyl)ethyl]-7H-pyrrolo-
(22d).
[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide
Compound 21d (0.5 g, 1.29 mmol) using the general pro-
cedure described above, gave 22d (0.27 g; 52%) as a pale
yellow foam: TLC Rf 0.52 (MeOH/CHCl3, 1:19); 1H
NMR (DMSO-d6): d 1.24 (s, 9H, C(CH3)3), 3.1(t, 2H,
CH2CH2), 3.52 (t, 2H, CH2CH2), 6.33 (s, 1H, C5-CH),
7.36–7.85 (m, 4H, C10H7), 7.95 (d, 1H, C10H7,
J = 9 Hz), 8.24 (d, 1H, C10H7, J = 9 Hz), 10.23 (s, 1H,
2-NHPiv), and 12.44 (s, 1H, 7-NH, exch).
6.1. General procedure for the synthesis of compounds
22a–e
To a round-bottomed flask were added 21a–e suspended
in phosphorous oxychloride (20 mL). The reaction was
continued at reflux for 4 h. After cooling to room tem-
perature, the solvent was evaporated under reduced
pressure and the residue was treated with ice water
(20 mL) in ice bath. The pH of the suspension was ad-
justed to 4 with ammonium hydroxide. The precipitate
was filtered, air-dried and then dissolved in chloro-
form/methanol (1:1, 25 mL). Silica gel (1 g) was added
to the solution which was then evaporated to dryness
to form a plug. The silica gel plug obtained was loaded
onto a silica gel column and eluted with 20% ethyl ace-
tate in hexanes. Fractions corresponding to the product
(TLC) were pooled and evaporated to dryness under re-
duced pressure to afford the product.
6.1.5. N-{4-[Chloro]-6-[2-(2-naphthyl)ethyl]-7H-pyrrol-
o[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide (22e).
Compound 21e (0.12 g, 0.31 mmol) using the general
procedure described above, gave 22 d (0.08 g; 64%) as
a pale yellow solid: TLC Rf 0.45 (MeOH/CHCl3, 1:19);
1
mp 217.5–220 ꢁC; H NMR (DMSO-d6): d 1.2 (s, 9H,
C(CH3)3), 3.22 (m, 4H, CH2CH2), 6.27 (s, 1H, C5-
CH), 7.47 (m, 3H, C10H7), 7.5 (s, 1H, C10H7), 7.85 (s,
1H, C10H7), 9.99 (s, 1H, 2-NHPiv), and 12.36 (s, 1H,
7-NH, exch). Anal. Calcd for C23H23N4OCl Æ 0.2 H2O:
C, 67.29; H, 5.75; N, 13.65; Cl, 8.64. Found C, 67.22;
H, 5.65; N, 13.38; Cl, 8.92.
6.2. General procedure for the synthesis of compounds
24a–e
6.1.1. N-[4-Chloro-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyr-
imidin-2-yl]-2,2-dimethylpropanamide (22a). Compound
21a (0.3 g, 1 mmol) using a general procedure described
above, gave 22a (0.25 g; 68%) as a white solid: TLC Rf
0.60 (MeOH/CHCl3, 1:19); mp: 230–231 ꢁC; H NMR
(DMSO-d6): d 1.22 (s, 9H, C(CH3)3), 3.02 (s, 4H,
To a round-bottomed flask were added 22a–e and 3-bro-
moaniline 23 in isopropanol (10 mL), followed by the
addition of two drops of concd HCl. The reaction was
continued at reflux for 1–3 h at the end of which the
solvent was evaporated under reduced pressure. The
1

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5523
residue was dissolved in methanol (10 mL), and silica gel
(10 g) was added to the solution which was then evapo-
rated to dryness to form a plug. The silica gel plug ob-
tained was loaded onto a silica gel column and eluted
with 1.5% methanol in chloroform. Fractions corre-
sponding to the product (TLC) were pooled and evapo-
rated to dryness under reduced pressure to afford the
product.
3-NH, exch), and 11.63 (s, 1H, 7-NH, exch). HRMS
(EI) calcd for C29H28N5OBr: 541.1477, found 541.1477.
6.2.5. N-{4-[(3-Bromophenyl)amino]-6-[2-(2-naphthyl)-
ethyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpro-
panamide (24e). Reaction of 22e (0.12 g, 0.28 mmol) and
3-bromoaniline 23 (0.19 g, 1.12 mmol), using the general
procedure described above, gave 24e (0.08 g; 53%) as a
yellow foam: TLC Rf 0.43 (MeOH/CHCl3, 1:19); 1H
NMR (DMSO-d6): d 1.25 (s, 9H, C(CH3)3), 3.16 (m,
4H, CH2CH2), 6.45 (s,1H, C5-CH), 7.08–8.00 (m, 9H,
C10H7 and C6H4), 8.02 (d, 1H, C6H4), 8.49 (s, 1H,
C6H4), 9.31 (s, 1H, 2-NHPiv or 4-NH, exch), 9.41 (s,
1H, 2-NHPiv or 4-NH, exch), and 11.56 (s, 1H, 7-NH,
exch). HRMS (EI) calcd for C29H28N5OBr: 541.1477,
found 541.1491.
6.2.1. N-{4-[(3-Bromophenyl)amino]-6-(2-phenylethyl)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropana-
mide (24a). Reaction of 22a (0.07 g, 0.19 mmol) and 3-
bromoaniline 23 (0.13 g, 0.76 mmol), using a general
procedure described above, gave 24a (0.1 g; 100%) as a
brown solid: TLC Rf 0.40 (MeOH/CHCl3, 1:19); mp:
93–95 ꢁC; 1H NMR (DMSO-d6): d 1.25 (s, 9H,
C(CH3)3), 2.99–3.15 (m, 4H, CH2CH2), 6.45(s, 1H,
C5-CH), 7.08–7.44 (m, 7 H, C6H5 and C6H4), 8.02 (d,
1H, C6H4), 8.51 (s, 1H, C6H4), 9.31 (s, 1H, 2-NHPiv
or 4-NH, exch), 9.42 (s, 1H, 2-NHPiv or 4-NH, exch),
and 11.55 (s, 1H, 7-NH, exch). HRMS (EI) calcd for
C25H26N5OBr: 491.1320, found 491.1301.
6.3. General procedure for the synthesis of compounds 8–
12
Into a round-bottomed flask was added 24a–e in meth-
anol (10 mL), followed by the addition of 1 N NaOH
(2 mL). The reaction was completed after heating at
70 ꢁC for 10 h. The solvent was evaporated under re-
duced pressure. The residue was dissolved in methanol
(10 mL), and silica gel (1 g) was added to the solution
which was then evaporated to dryness to form a plug.
The silica gel plug obtained was loaded onto a silica
gel column and eluted with 1.5% methanol in chloro-
form. Fractions corresponding to the product (TLC)
were pooled and evaporated to dryness under reduced
pressure to afford the product.
6.2.2.
N-{4-[(3-Bromophenyl)amino]-6-[2-(4-methoxy-
phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dim-
ethylpropanamide (24b). Reaction of 22 b (0.13 g,
0.32 mmol) and 3-bromoaniline 23 (0.22 g, 1.3 mmol),
using the general procedure described above, afforded
24 b (0.16 g; 92%) as a pale yellow solid: TLC Rf 0.30
(MeOH/CHCl3, 1:19); mp: 97.5–99.5 ꢁC; 1H NMR
(DMSO-d6): d 1.25 (s, 9H, C(CH3)3), 2.94 (m, 4H,
CH2CH2), 3.70 (s, 3H, OCH3), 6.42 (s, 1H, C5-CH),
6.82–7.25 (m, 6H, C6H4 and C6H4), 8.04 (d, 1H,
C6H4), 8.50 (s, 1H, C6H4), 9.29 (s, 1H, 2-NHPiv or 4-
NH, exch), 9.39 (s, 1H, 2-NHPiv or 4-NH, exch), and
11.49 (s, 1H, 7-NH, exch). HRMS (EI) calcd for
C26H28N5O2Br: 521.1426, found 521.1431.
6.3.1. N4-(3-Bromophenyl)-6-(2-phenylethyl)-7H-pyrrol-
o[2,3-d]pyrimidine-2,4-diamine (8). Using the general
procedure described above, reaction of 24a (0.08 g,
0.19 mmol) afforded 8 (0.04 g; 53%) as a pale yellow so-
lid: TLC Rf 0.20 (MeOH/CHCl3, 1:19); mp: 185–187
ꢁC; 1H NMR (DMSO-d6): d 2.88–3.07 (m, 4H,
CH2CH2), 5.74 (br, 2H, 2-NH2, exch), 6.27 (s, 1H, C5-
CH), 7.07–8.13 (m, 9H, C6H5 and C6H4), 8.91 (s, 1H,
4-NH, exch), and 10.92 (br, 1H, 7-NH, exch). HRMS
(EI) calcd for C20H18N5Br 407.0746, found 407.0732.
6.2.3. N-{4-[(3-Bromophenyl)amino]-6-[2-(2-chlorophenyl)-
ethyl]-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpro-
panamide (24c). Reaction of 22c (0.06 g, 0.15 mmol) and
3-bromoaniline 23 (0.10 g, 0.60 mmol), using the general
procedure described above, gave 24 c (0.07 g; 95%) as a
yellow foam: TLC Rf 0.46 (MeOH/CHCl3, 1:19); 1H
NMR (DMSO-d6): d 1.25 (s, 9H, C(CH3)3), 2.99–3.14
(m, 4H, CH2CH2), 6.45 (s, 1H, C5-CH), 7.09–7.45 (m,
6H, C6H4 and C6H4), 8.02 (d, 1H, C6H4), 8.50 (s, 1H,
C6H4), 9.29 (s, 1H, 2-NHPiv or 4-NH, exch), 9.39 (s,
1H, 2-NHPiv or 4-NH, exch), and 11.54 (s, 1H, 7-NH,
exch). HRMS (EI) calcd for C25H25N5OBrCl:
525.0931, found 525.0917.
6.3.2. N4-(3-Bromophenyl)-6-[2-(4-methoxyphenyl)ethyl]-7
H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (9). Using the
general procedure described above, reaction of 24b
(0.12 g, 0.23 mmol) afforded 9 (0.04 g; 35%) as a off-
white solid: TLC Rf 0.21 (MeOH/CHCl3, 1:19); mp:
1
196.5–198.5 ꢁC; H NMR (DMSO-d6) d 2.86 (m, 4H,
CH2CH2), 3.70 (s, 3H, OCH3), 5.79 (br, 2H, 2-NH2,
exch), 6.23 (s, 1H, C5-CH), 6.82–7.24 (m, 6H, C6H4
and C6H4), 8.00 (d, 1H, C6H4), 8.12 (s, 1H, C6H4),
8.95 (s, 1H, 4-NH, exch), 10.91 (br, 1H, 7-NH, exch).
HRMS (EI) calcd for C21H20N5BrO 437.0851, found
437.0837.
6.2.4. N-{4-[(3-Bromophenyl)amino]-6-[2-(1-naphthyl)-
ethyl]-7H-pyrrolo[2,3- d]pyrimidin-2-yl}-2,2-dimethylpro-
panamide (24d). Reaction of 22d (0.11 g, 0.27 mmol) and
3-bromoaniline 23 (0.19 g, 1.08 mmol), using the general
procedure described above, gave 24 d (0.13 g; 90%) as a
pale yellow foam: TLC Rf 0.42 (MeOH/CHCl3, 1:19);
1H NMR (DMSO-d6): d 1.25 (s, 9H, C(CH3)3), 3.07
(t, 2H, J = 9.0 Hz, CH2CH2), 3.48 (t, 2H, J = 9.0 Hz,
CH2CH2), 6.47 (s, 1H, C5-CH), 7.11–8.38 (m, 10H,
C10H7 and C6H4), 8.50 (s, 1H, C6H4), 9.29 (s, 1H,
2-NHPiv or 4-NH, exch), 9.39 (s, 1H, 2-NHPiv or
6.3.3. N4-(3-Bromophenyl)-6-[2-(2-chlorophenyl)ethyl]-
7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (10). Using the
general procedure described above, reaction of 24c
(0.06 g, 0.11 mmol) afforded 10 (0.03 g; 56%) as an or-
ange solid: TLC Rf 0.17 (MeOH/CHCl3, 1:19); mp:
178.5–180.5 ꢁC; 1H NMR (DMSO-d6) d 2.88–3.07

5524
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
(m, 4H, CH2CH2), 5.79 (br, 2H, 2-NH2, exch), 6.26 (s,
1H, C5-CH), 7.07–7.42 (m, 6H, C6H4 and C6H4), 8.01
(d, 1 H C6H4), 8.12 (s, 1H, C6H4), 8.94 (s, 1H, 4-NH,
exch), 10.95 (br, 1H, 7-NH, exch). HRMS (EI) calcd
for C20H17N5ClBr 441.0356, found 441.0356.
evaporated in vacuo to afford a dry plug. This plug
was placed on the top of silica gel column
(20 cm · 3 cm) and eluted with chloroform/methanol (a
gradient elution, 2% methanol in chloroform, and 5%
methanol in chloroform). Fractions containing the
product (TLC) were pooled and evaporated to obtain
a light pink residue, which was suspended in acetone
(10 mL) and filtered to afford the pure 2-amino-6-(2-
phenylethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one 30 (1.22 g, 45%) as a light pink solid. TLC Rf 0.56
6.3.4. N4-(3-Bromophenyl)-6-[2-(1-naphthyl)ethyl]-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (11). Using the gen-
eral procedure described above, reaction of 24d (0.10 g,
0.18 mmol) afforded 11 (0.02 g; 18%) as a pale yellow so-
lid: TLC Rf 0.23 (MeOH/CHCl3, 1:19); mp: 120–122
1
(MeOH/CHCl3, 1:5); mp: 145ꢁC; HNMR (DMSO-d6):
ꢁC; 1H NMR (DMSO-d6)
d
2.98–3.15 (m, 4H,
d 2.77 (t, 2H, CH2), 2.88 (t, 2H, CH2), 5.85 (s, 1H,
C5-CH), 5.96(s, 2H, NH2, exch), 7.22 (m, 5H, C6H5),
10.12( s, 1H, NH, exch), and 10.87( s, 1H, NH, exch).
Anal. Calcd for C14H14N4OÆ0. 2741 C3H6O: C, 65.88;
H, 5.83; N, 20.73. Found C, 66.23; H, 5.86; N, 20.67.
CH2CH2), 5.92 (br, 2H, 2-NH2, exch), 6.31 (s, 1H, C5-
CH), 7.10–8.20 (m, 11H, C10H7 and C6H4), 9.01 (s,
1H, 4-NH, exch), and 11.12 (br, 1H, 7-NH, exch).
HRMS (EI) calcd for C24H20N5Br 457.0902, found
457.0881.
6.5. Synthesis of 4-chloro-6-(2-phenylethyl)-7H-pyrrol-
o[2,3-d]pyrimidin-2-amine (31)
6.3.5. N4-(3-Bromophenyl)-6-[2-(2-naphthyl)ethyl]-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine ( 12). Using the
general procedure described above, reaction of 24e
(0.06 g, 0.11 mmol) afforded 12 (0.04 g; 75%) as a yel-
low solid: TLC Rf 0.22 (MeOH/CHCl3, 1:19); mp:
97.5–99.5 ꢁC; 1H NMR (DMSO-d6) d 2.97–3.14 (m,
4H, CH2CH2), 5.71 (br, 2H, 2-NH2, exch), 6.24 (s, 1H,
C5-CH), 7.07–8.11 (m, 11H, C10H7 and C6H4), 8.88 (s,
1H, 4-NH, exch), and 10.92 (br, 1H, 7-NH, exch).
HRMS (EI) calcd for C24H20N5Br 457.0902, found
457.0885.
A mixture of 30 (2.54 g; 10 mmol) and N,N-dimethylan-
iline (0.13 ml, 1 mmol) was kept at reflux with freshly
distilled phosphorous oxychloride (25 mL, excess) for
4 h. At the end of the reaction (monitored by TLC),
the solvent was evaporated under reduced pressure.
The resulting brown residue was cooled in ice-bath.
Crushed ice (10 g) was added to the residue followed
by dropwise addition of ammonium hydroxide to adjust
the pH to 7–8. The resulting olive green sticky residue
was extracted with chloroform (50 mL · 3). The extracts
were dried (anhydrous MgSO4), evaporated under re-
duced pressure followed by addition of silica gel (5 g)
to make a silica gel plug which was placed on top of a
silica gel column (15 cm · 3 cm) and eluted with 2%
methanol in chloroform. Fractions containing the prod-
uct were evaporated to obtain 31 (0.9 g; 33%) as a dull
white solid. TLC Rf 0.76 (MeOH/CHCl3, 1:5); mp
6.4. Synthesis of 2-amino-6-(2-phenylethyl)-3,7-dihydro-
4H-pyrrolo[2,3-d]pyrimidin-4-one (30)
3-Phenyl propanoic acid 25 (2 g, 13.3 mmol) in dry ben-
zene (10 mL) was reacted with thionyl chloride (10 mL,
excess) at reflux for 1 h. Evaporation of the reaction
mixture, followed by washings with toluene (thrice, to
remove excess thionyl chloride) gave 3-propanoyl chlo-
ride 26 (crude; 2 g). Compound 26 was dissolved in
anhydrous ether (15 mL) and added dropwise with stir-
ring to a solution of diazomethane in ether (50 mL;
about 30 mmol, prepared from 50 mmol of N-methyl-
N-nitroso urea)37 maintaining the temperature between
0 and 5 ꢁC. The resulting yellow solution was warmed
to room temperature, stirred for 1 h, then 47.5% aque-
ous HBr (15 mL) was added dropwise to the solution
and the resulting yellow mixture was heated at 70–
80 ꢁC (oil bath) for 1 h. The reaction was cooled to
room temperature and the ether layer was separated,
washed with water (25 mL · 1), saturated NaHCO3
aqueous solution (25 mL · 1), and brine (25 mL · 1)
and dried (anhydrous Na2SO4). The solution was evap-
orated to afford 1-bromo-4-phenylbutan-2-one 28
(2.43 g; crude) as a light yellow liquid, which was used
directly in the next step without purification. Compound
28 (2.43 g, 10.7 mmol) was placed in a 100-mL flask and
an equivalent amount of 2,4-diamino-6-hydroxy-pyrim-
idine 29 (1.35 g, 10.7 mmol), molecular sieves (13·; 16–
2.5 mm beads; 100 mg), and anhydrous DMF (15 mL)
was added. The slurry was stirred at room temperature
for 3 days. The color of the slurry changed from yellow
to orange on the second day. After 3 days, silica gel (5 g)
was added to the reaction mixture and the solvent was
1
212.5–213.5 ꢁC; HNMR (DMSO-d6): d 2.91 (t, 2H,
CH2), 2.94 (t, 2H, CH2), 5.95 (s, 1H, C5-CH), 6.38
(br, 2H, NH2, exch), 7.22–7.9 (m, 5H, C6H5), 11.41(br,
1H, 7-NH). Anal. Calcd for C14H13N4ClÆ0. 10H2O: C,
61.25; H, 4.85; N, 20.41; Cl, 12.91. Found: C, 61.05;
H, 4.79; N, 20.25; Cl, 12.7.
6.6. General method for synthesis of N4-phenylsubstitut-
ed-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mines (13–20)
Into a 50-mL round-bottomed flask was added 4-chloro-
6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine
31, substituted aniline, and isopropanol (10 mL) fol-
lowed by 2–3 drops of concd HCl. The mixture was
heated at reflux for 4 h. At the end of the reaction, mix-
ture was cooled to room temperature and pH was ad-
justed to 11–12 using ammonium hydroxide followed
by evaporation in vacuo. The residue was dissolved in
methanol and silica gel (0.5 g) was added followed by
evaporation in vacuo to afford a silica gel plug, which
was loaded on top of silica gel column (10 cm · 1 cm;
pre-treated with 1% ammonia–methanol in chloroform).
Column was eluted with 2% methanol in chloroform.
Fractions containing the product were pooled and evap-
orated under reduced pressure to afford a residue, which

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5525
was analytically pure (13, 15–17). Compounds that
showed trace impurities were washed with acetone and
hexanes (14); triturated with diethyl ether (18); recrystal-
lized from hexanes-ethyl acetate (19) or from chloro-
form (20) to afford analytically pure N4-
phenylsubstituted-6-(2-phenylethyl)-7H-pyrrolo[2,3-
d]pyrimidine-2,4-diamines.
C22H19N5Æ0. 4H2O: C, 73.27; H, 5.53; N, 19.42. Found
C, 73.63; H, 5.77; N, 19.04.
6.6.5. N4-(4-chloro-2-fluorophenyl)-6-(2-phenylethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (17). Using the
general procedure described above, reaction of 31
(0.08 g, 0.29 mmol) and 4-chloro-2-fluoroaniline 32 e
(0.064 g, 0.43 mmol) gave 17 (0.095 g, 85%) as light
brown solid. TLC Rf 0.66 (MeOH/CHCl3, 1:5); mp:
160–164 ꢁC. 1HNMR (DMSO-d6): d 2.853–2.869 (t,
2H, CH2), 2.91–2.938 (t, 2H, CH2), 5.6 (br,2H, 2-NH2,
exch), 6.119 (s, 1H, C5-CH), 7.176–7.303 (m, 5H, C6
H5), 7.422–7.96 (m, 3H, C6 H3), 8.569 (br, 1H, 4-NH,
exch), 10.855 (br, 1H, 7-NH, exch). Anal. Calcd for
C20H17ClFN5Æ0. 1H2O: C, 62.62; H, 4.52; N, 18.25; Cl,
9.24; F,4.95. Found: C, 62.4; H, 4.58; N, 18.12; Cl,
9.44; F, 4.69.
6.6.1. N4-(3-bromo-4-fluorophenyl)-6-(2-phenylethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (13). Using the gen-
eral procedure described above, reaction of 31 (0.1 g,
0.36 mmol) and 3-bromo-4-fluoroaniline 32a (0.102 g,
0.54 mmol) afforded 13 (0.095 g, 61%) as an off-white
solid. TLC Rf 0.73 (MeOH/CHCl3, 1:5); mp: 222–224
ꢁC. 1HNMR (DMSO-d6):
d 2.89–3.00 (m, 4H,
CH2CH2), 5.70 (br,2H, 2-NH2, exch), 6.20 (s, 1H, C5-
CH), 7.17–7.30 (m, 5H, C6H5), 7.96–8.26 (m, 3H,
C6H3), 8.87 (br, 1H, 4-NH, exch), 10.86 (br, 1H, 7-
NH, exch). Anal. Calcd for C20 H17N5BrFÆ0.2H2O: C,
55.88; H, 4.08; N, 16.29; Br, 18.59; F, 4.42. Found: C,
55.68; H, 3.91; N, 16.08; Br, 18.21; F, 4.38.
6.6.6 . N4-(4-Chlorophenyl)-6-(2-phenylethyl)-7H-pyrrol-
o[2,3-d ]pyrimidine-2,4-diamine (18). Using the general
procedure described above, reaction of 31 (0.08 g,
0.29 mmol) and 4-chloroaniline 32f (0.055 g, 0.43 mmol)
gave a semisolid; which was triturated with diethyl ether
to afford a solid which was filtered and washed with
diethyl ether (5 mL) to get 18 (0.09 g, 85% yield) as a
light brown solid. TLC Rf 0.57 (MeOH/CHCl3, 1:5);
6.6.2.
N4-(3-Chloro-4-fluorophenyl)-6-(2-phenylethyl)-
7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (14). Using the
general procedure described above, reaction of 31 (0.1
g, 0.36 mmol) and 3-chloro-4-fluoroaniline 32 b
(0.078 g, 0.54 mmol) afforded a dark brown residue
which was warmed with acetone (1 mL) and filtered fol-
lowed by washing with hexanes (10 mL) to afford 14
(0.107 g, 80%) as light brown solid. TLC Rf 0.606
(MeOH/CHCl3, 1:5); mp: 214.5–216.5 ꢁC. 1HNMR
(DMSO-d6): d 2.89 (t, 2H, CH2), 2.93 (t, 2H, CH2),
5.80 (br, 2H, 2-NH2, exch), 6.21(s, 1H, C5-CH), 7.17–
8.23 (m, 8H, C6H5 & C6H3), 8.96 (br, 1H, 4-NH, exch),
10.91(br, 1H, 7-NH, exch). Anal. Calcd for
C20H17N5ClFÆ0.1299 C6H14: C, 63.50; H, 4.82; N,
17.81; Cl, 9.02; F, 4.83. Found: C, 63.18; H, 4.66; N,
17.48; Cl, 8.88; F, 4.54.
1
mp: 165–168 ꢁC. HNMR (DMSO-d6): d 2.88 (t, 2H,
CH2), 2.94 (t, 2H, CH2), 5.7 (bs, 2H, 2-NH2, exch),
6.23 (s, 1H, C5-CH), 7.24–7.28 (m, 7H, C6H5 & C
6H4), 7.96–7.99 (d, 2H, C6H4), 8.88 (br, 1H, 4-NH,
exch), and 10.85 (s, 1H, 7-NH, exch). Anal. Calcd for
C20H18ClN5Æ0.46 C4H10O: C, 65.91; H, 5.72; N,17.59;
Cl, 8.9. Found C, 66; H, 5.48; N, 17.52; Cl, 8.82.
6.6.7. N4-[4-Fluoro-3-(trifluoromethyl)phenyl]-6-(2-phen-
ylethyl)-7H -pyrrolo[2,3- d]pyrimidine-2,4-diamine (19).
Using the general procedure described above, reaction
of 31 (0.07 g, 0.255 mmol) and 3-ethynylaniline 32 g
(0.068 g, 0.38 mmol) afforded a pale yellow solid which
was recrystallized from hexanes/ethyl acetate (1:1) to
get 19 (0.087 g, 82%) as an off-white solid. TLC Rf
6.6.3. N4-(3-Trifluromethylphenyl)-6-(2-phenylethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (15). Using the
general procedure described above reaction of 31
(0.06 g, 0.21 mmol) and 3-trifluoromethylaniline 32c
(0.051 g, 0.32 mmol) afforded 15 (0.076 g, 89%) as a light
brown solid. TLC Rf 0.59 (MeOH/CHCl3, 1:5); mp:
1
0.61 (MeOH/CHCl3, 1:5); mp: 164–166 ꢁC. HNMR
(DMSO-d6): d 2.93 (t, 2H, CH2), 2.97 (t, 2H, CH2),
5.70 (br,2H, 2-NH2, exch), 6.20(s, 1H, C5-CH), 7.24–
8.42 (m, 8H, C6 H5 &C6H3), 9.07(s, 1H, 4-NH, exch),
and 10.89 (br, 1H, 7-NH, exch). Anal. Calcd for
C21H17N5F4Æ0.41 C6H14: C, 61.19; H, 4.37; N, 16.52;
F, 17.92. Found: C, 61.13; H, 4.20; N, 16.54; F, 17.97.
1
189–192 ꢁC. HNMR (DMSO-d6): d 2.93 (t, 2H, CH2),
2.98 (t, 2H, CH2), 5.72 (br, 2H, 2-NH2, exch), 6.24 (s,
1H, C5-CH), 7.24–8.44 (m, 9H, C6H5& C6H4), 9.08 (s,
1H, 4-NH, exch), and 10.90(s, 1H, 7-NH, exch). Anal.
Calcd for C21H18N5F3: C, 63.47; H, 4.56; N, 17.62; F,
14.34. Found: C, 63.22; H, 4.68; N, 17.43; F, 14.41.
6.6.8.
N4-1H-indol-5-yl-6-(2-phenylethyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (20). Initial attempts to
synthesize 20 using the general procedure described
above failed. The reaction mixture turned black almost
instantaneously. Use of excess 32h did not lead to any
improvement. However, avoiding the addition of concd
HCl and using 5-aminoindole 32h in excess (20-fold), the
reaction went to completion. Using the general proce-
dure described above for chromatographic purification
afforded light brown solid, which was recrystallized
from chloroform to obtain 20 (50 mg, 61% yield) as a
buff colored solid. TLC Rf 0.52 (MeOH/CHCl3,
6.6.4. N4-(3-Ethynylphenyl)-6-(2-phenylethyl)-7H-pyrrol-
o[2,3-d ]pyrimidine-2,4-diamine (16). Using the general
procedure described above, reaction of 31 (0.08 g,
0.29 mmol) and 3-ethynylaniline 32d (0.052 g,
0.43 mmol) afforded 16 (0.093 g, 90%) as a lustrous light
brown solid. TLC Rf 0.566 (MeOH/CHCl3, 1:5); mp:
163–167 ꢁC. 1HNMR (DMSO-d6): d 2.89 (t, 2H,
CH2), 2.93 (t, 2H, CH2), 4.12 (s, 1H, C2H), 5.69 (br,
2H, 2-NH2, exch), 6.23 (s, 1H, C5-CH), 7.03–8.09 (m,
9H, C6 H5& C6H4), 8.83 (s, 1H, 4-NH, exch), and
10.86 (s, 1H, 7-NH, exch). Anal. Calcd for
1
1:10 + 1 drop of NH4OH); mp: 154–156.6 ꢁC. HNMR
(DMSO-d6): d 2.84–2.9 (m, 4H, CH2CH2), 5.46 (s, 2H,

5526
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
2-NH2, exch), 6.05 (s, 1H, C5-CH), 6.35 (s, 1H, C8H6N),
7.23–7.34 (m, 7H, C6 H5 and C8H6N), 8.01 (s, 1H,
C8H6N), 8.5 (s, 1H, 4-NH, exch), 10.7 (br, 1H, NH,
exch), 10.9 (br, 1H, NH, exch). Anal. Calcd for
C22H20N6Æ0. 547CHCl3: C, 62.43; H, 4.77; N, 19.37.
Found: C, 62.52; H, 4.89; N, 19.12.
known RTK-specific kinase inhibitor, PD153035, was
used as a positive control compound for EGFR kinase
inhibition; SU5416 for Flk1 kinase inhibition; AG1295
for PDGFR-b kinase inhibition; and CB676475 (4-[(40-
chloro-20-fluoro)phenylamino]-6,7-dimethoxyquinazo-
line) was used as a positive control for both Flt1 and
Flk1 kinase inhibition. Data were graphed as a percent
of cells receiving growth factor alone and IC50 values
were estimated from two to three separate experiments
(n = 8–24) using hand-drawn probit plots. In each case,
the activity of a positive control inhibitor did not
deviate more than 10% from the IC50 values listed in
the text.
HRMS (ES) calcd for C22H21N6 (M+H)+: 369.1828,
found 369.1830.
6.7. Biological evaluation
6.7.1. Cells. All cells were maintained at 37 ꢁC in a
humidified environment containing 5% CO2 using media
from Mediatech (Hemden, NJ). A-431 cells were
from the American Type Tissue Collection (Manassas,
VA).
6.7.5. CYQUANT cell proliferation assay. As a measure
of cell proliferation, the CYQUANT cell counting/pro-
liferation assay was used as previously described.41
Briefly, cells are first treated with compounds for 12 h
and then allowed to grow for an additional 36 h. The
cells are then lysed and the CYQUANT dye, which
intercalates into the DNA of cells, is added and after
5 min the fluorescence of each well measured using a
UV product BioChemi digital darkroom. A positive
control used for cytotoxicity in each experiment was cis-
platin, with an apparent average IC50 value of
6.7.2. Chemicals. All growth factors (bFGF, VEGF,
EGF, and PDGF-b) were purchased from Peprotech
(Rocky Hill, NJ). PD153035, SU5416, AG1295, and
CB676475
(4-[(40-chloro-20-fluoro)phenylamino]-6,7-
dimethoxyquinazoline) were purchased from Calbio-
chem (San Diego, CA). The CYQUANT cell prolifera-
tion assay was from Molecular Probes (Eugene, OR).
All other chemicals were from Sigma Chemical unless
otherwise noted.
8.2
0.65 lM. Data are graphed as a percent of cells
receiving growth factor alone and IC50 values estimated
from two to three separate experiments (n = 6–15) using
probit plots.
6.7.3. Antibodies. The PY-HRP antibody was from BD
Transduction Laboratories (Franklin Lakes, NJ). Anti-
bodies against EGFR, PDGFR-b, FGFR-1, Flk-1,
and Flt-1 were purchased from Upstate Biotech (Fra-
mingham, MA).
6.7.6. Chorioallantoic membrane assay of angiogenesis.
The chorioallantoic membrane (CAM) assay is a stan-
dard assay for testing antiangiogenic agents.42 The
CAM assay used in these studies was modified from
a procedure by Sheu52 and Brooks53 and as published
previously.54 Briefly, fertile leghorn chicken eggs
(CBT Farms, Chestertown, MD) are allowed to grow
until 10 days of incubation. The proangiogenic factors,
human VEGF-165 and bFGF (100 ng each) are then
added at saturation to a 3-mm microbial testing disk
(BBL, Cockeysville, MD) and placed onto the CAM
by breaking a small hole in the superior surface of
the egg. Antiangiogenic compounds are added 8 h after
the VEGF/bFGF at saturation to the same microbial
testing disk and embryos allowed to incubate for an
additional 40 h. After 48 h, the CAMs are perfused
with 2% paraformaldehyde/3% glutaraldehyde contain-
ing 0.025% Triton X-100 for 20 s, excised around the
area of treatment, fixed again in 2% paraformalde-
hyde/3% glutaraldehyde for 30 min, placed on petri
dishes, and a digitized image taken using a dissecting
microscope (Wild M400; Bannockburn, IL) at 7.5·
and Retiga enhanced digital imaging system (QImag-
ing, Burnaby, BC, Canada). A grid is then added to
the digital CAM images and the average number of
vessels within 5–7 grids counted as a measure of vascu-
larity. AGM-1470 (a kind gift of the NIH Develop-
mental Therapeutics Program) and SU5416 are used
as a positive control for antiangiogenic activity. Data
are graphed as a percent of CAMs receiving bFGF/
VEGF and IC50 values estimated from two to three
separate experiments (n = 5–11) using nonlinear regres-
6.7.4. Phosphotyrosine ELISA. Cells used were tumor
cell lines naturally expressing high levels of EGFR
(A431), Flk-1 (U251), Flt-1 (A498), PDGFR-b (SF-
539), and FGFR-1 (NIH OVCAR-8). Expression levels
at the RNA level were derived from the NCI Develop-
mental Therapeutics Program (NCI-DTP) web site pub-
lic
molecular
target
information
(http://
www.dtp.nci.nih.gov/mtargets/mt_index.html). Briefly,
cells at 60–75% confluence were placed in serum-free
medium for 18 h to reduce the background of phosphor-
ylation. Cells were always >98% viable by Trypan blue
exclusion. Cells were then pretreated for 60 min with
10, 3.33, 1.11, 0.37, and 0.12 lM compounds followed
by 100 ng/ml EGF, VEGF, PDGF-BB, or bFGF for
10 min. The reaction was stopped and cells permeabili-
zed by quickly removing the media from the cells and
adding ice-cold Tris-buffered saline (TBS) containing
0.05% Triton X-100, protease inhibitor cocktail, and
tyrosine phosphatase inhibitor cocktail. The TBS solu-
tion was then removed and cells fixed to the plate for
30 min at 60 ꢁC and further incubation in 70% ethanol
for an additional 30 min. Cells were further exposed to
block (TBS with 1% BSA) for 1 h, washed, and then a
horseradish peroxidase (HRP)-conjugated phosphotyro-
sine (PY) antibody added overnight. The antibody was
removed, cells were washed again in TBS, exposed to
an enhanced luminal ELISA substrate (Pierce Chemical,
Rockford, IL), and light emission measured using a UV
product (Upland, CA) BioChemi digital darkroom. The

A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
5527
sion analysis and a Sigmoidal Dose–response analysis
using Prism 4.0 (GraphPad Software, San Diego, CA).
Acknowledgement
This work was supported, in part, by the National Insti-
tutes of Health and National Cancer Institute Grant
CA98850 (A.G.).
6.7.7. Statistics. All analysis was done using Prism 4.0.
(GraphPad Software, San Diego, CA).
Appendix A
Formula
Calculated (%)
Cl
Found (%)
C
H
N
Br
F
C
H
N
Cl
Br
F
22a C19H21N4OCl
22b C19H21N4O2Cl
22e C23H23N4OClÆ0.2H2O 67.29 5.75 13.65 8.64
30 C14H14N4OÆ
0.2741C3H6O
63.95 5.93 15.7 9.93
62.09 5.99 14.48 9.16
63.84 5.96 15.64 10.04
61.91 6.01 14.54 9.33
67.22 5.65 13.38 8.92
66.23 5.86 20.67
65.88 5.83 20.73
31 C14H13N4ClÆ0.10 H2O 61.25 4.85 20.41 12.91
13 C20 H17N5BrFÆ0.2 H2O 55.88 4.08 16.29
61.05 4.79 20.25 12.7
18.59 4.42 55.68 3.91 16.08
4.74 63.18 4.66 17.48 8.88
18.21 4.38
4.54
14 C20H17N5ClFÆ0.3
CH3COCH3
15 C21H18N5F3
62.87 4.76 17.49 8.85
63.47 4.56 17.62
73.27 5.53 19.42
14.34 63.22 4.68 17.43
73.63 5.77 19.04
4.95 62.4 4.58 18.12 9.44
66.0 5.48 17.52 8.82
14.41
4.69
16 C22H19N5Æ0.4 H2O
17 C20H17ClFN5Æ0.1 H2O 62.62 4.52 18.25 9.24
18 C20H18ClN5Æ0.6
CH3COCH3
65.67 5.46 17.55 8.89
19 C21H17N5F4Æ0.13
CH3COCH3
60.74 4.24 16.54
17.95 61.13 4.20 16.54
17.97
High-resolution mass spectra (HRMS) (EI)
Compound
Formula
Calcd. mass
Found mass
24a
24b
24c
24d
24e
8
9
10
11
12
C25H26N5OBr
C26H28N5O2Br
C25H25N5OBrCl
C29H28N5OBr
C29H28N5OBr
C20H18N5Br
C21H20N5BrO
C20H17N5ClBr
C24H20N5Br
491.1320
521.1426
525.0931
541.1477
541.1477
407.0746
437.0851
441.0356
457.0902
457.0902
369.1828
491.1301
521.1431
525.0917
541.1477
541.1491
407.0732
437.0837
441.0356
457.0881
457.0885
369.1830
C24H20N5Br
C22H21N6
20
5. Ferrara, N. Semin. Oncol. 2002, 29, 10–44.
6. Tabernero, J. Mol. Cancer Res. 2007, 5, 203–220.
7. Folkman, J. Annu Rev. Med. 2006, 57, 1–18.
8. Vacca, A.; Ribatti, D. Leukemia 2006, 20, 193–199.
9. Schuch, G.; Oliveira-Ferrer, L.; Loges, S.; Laack, E.;
Bokemeyer, C.; Hossfeld, D. K.; Fiedler, W.; Ergun, S.
Leukemia 2005, 19, 1312–1317.
10. Koster, A.; Raemaekers, J. M. Curr. Opin. Oncol. 2005,
17, 611–666.
11. Quesada, A. R.; Munoz-Chapuli, R.; Medina, M. A. Med.
Res. Rev. 2006, 26, 483–530.
12. Druker, B. J. Adv. Cancer Res. 2004, 91, 1–30.
References and notes
1. Li, W.; Favelyukis, S.; Yang, J.; Zeng, Y.; Yu, J.; Gangjee,
A.; Miller, W. T. Biochem. Pharmacol. 2004, 68, 145–154.
2. Herbst, R. S.; Johnson, D. H.; Mininberg, E.; Carbone, D.
P.; Henderson, T.; Kim, E. S.; Blumenschein, G., Jr.; Lee,
J. J.; Liu, D. D.; Truong, M. T.; Hong, W. K.; Tran, H.;
Tsao, A.; Xie, D.; Ramies, D. A.; Mass, R.; Seshagiri, S.;
Eberhard, D. A.; Kelley, S. K.; Sandler, A. J. Clin. Oncol.
2005, 23, 2544–2555.
3. Ellis, L. M. Hematol. Oncol. Clin. North America 2004,
18, 1007–1021, viii.
4. Jain, R. K. Semin. Oncol. 2002, 29, 3–9.

5528
A. Gangjee et al. / Bioorg. Med. Chem. 16 (2008) 5514–5528
13. Herbst, R. S.; Fukuoka, M.; Baselga, J. Nat. Rev. Cancer
2004, 4, 956–965.
W.; Ullrich, A.; Hirth, K. P.; McMahon, G. Cancer Res.
1999, 59, 99–106.
14. Slamon, D.; Pegram, M. Semin. Oncol. 2001, 28, 13–99.
15. Muhsin, M.; Graham, J.; Kirkpatrick, P. Nat. Rev. Drug
Discov. 2004, 3, 995–996.
16. Laird, A. D.; Cherrington, J. M. Expert Opin. Investig.
Drugs 2003, 12, 51–64.
39. Stockwell, B. R.; Haggarty, S. J.; Schreiber, S. L. Chem.
Biol. 1999, 6, 71–83.
40. Schilder, R. J.; Hall, L.; Monks, A.; Handel, L. M.;
Fornace, A. J., Jr.; Ozols, R. F.; Fojo, A. T.; Hamilton, T.
C. Int. J. Cancer 1990, 45, 416–422.
17. Bergers, G.; Song, S.; Meyer-Morse, N.; Bergsland, E.;
Hanahan, D. J. Clin. Invest. 2003, 111, 1287–1295.
18. Potapova, O.; Laird, A. D.; Nannini, M. A.; Barone, A.;
Li, G.; Moss, K. G.; Cherrington, J. M.; Mendel, D. B.
Mol. Cancer Ther. 2006, 5, 1280–1289.
19. Comis, R. L. Oncologist 2005, 10, 467–470.
20. Faivre, S.; Djelloul, S.; Raymond, E. Semin. Oncol. 2006,
33, 407–420.
41. Wilson, S. M.; Barsoum, M. J.; Wilson, B. W.; Pappone,
P. A. Cell Prolif. 1999, 32, 131–140.
42. Vu, M. T.; Smith, C. F.; Burger, P. C.; Klintworth, G. K.
Lab Invest. 1985, 53, 499–508.
43. Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.;
Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani,
C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.;
Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.
F.; Greenberger, L. M.; Tsou, H. R. J. Med. Chem. 2003,
46, 49–63.
21. de Jonge, M. J.; Verweij, J. Eur. J. Cancer 2006, 42, 1351–
1356.
22. Erber, R.; Thurnher, A.; Katsen, A. D.; Groth, G.;
Kerger, H.; Hammes, H. P.; Menger, M. D.; Ullrich, A.;
Vajkoczy, P. FASEB J. 2004, 18, 338–340.
23. Shen, J.; Vil, M. D.; Zhang, H.; Tonra, J. R.; Rong, L. L.;
Damoci, C.; Prewett, M.; Deevi, D. S.; Kearney, J.;
Surguladze, D.; Jimenez, X.; Iacolina, M.; Bassi, R.;
Zhou, K.; Balderes, P.; Mangalampalli, V. R.; Loizos, N.;
Ludwig, D. L.; Zhu, Z. Biochem. Biophys. Res. Commun.
2007, 357, 1142–1147.
24. Gille, J. Exp. Dermatol. 2006, 15, 175–186.
25. Marcucci, G.; Perrotti, D.; Caligiuri, M. A. Clin. Cancer
Res. 2003, 9, 1248–1252.
26. Chow, L. Q.; Eckhardt, S. G. J. Clin. Oncol. 2007, 25,
884–896.
44. Hunt, J. T.; Mitt, T.; Borzilleri, R.; Gullo-Brown, J.;
Fargnoli, J.; Fink, B.; Han, W. C.; Mortillo, S.; Vite, G.;
Wautlet, B.; Wong, T.; Yu, C.; Zheng, X.; Bhide, R. J.
Med. Chem. 2004, 47, 4054–4059.
45. Jin, Y.; Li, H. Y.; Lin, L. P.; Tan, J.; Ding, J.; Luo, X.;
Long, Y. Q. Bioorg. Med. Chem. 2005, 13, 5613–5622.
46. Tsou, H. R.; Overbeek-Klumpers, E. G.; Hallett, W. A.;
Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Michalak, R.
S.; Nilakantan, R.; Discafani, C.; Golas, J.; Rabindran, S.
K.; Shen, R.; Shi, X.; Wang, Y. F.; Upeslacis, J.; Wissner,
A. J. Med. Chem. 2005, 48, 1107–1131.
47. Klutchko, S. R.; Zhou, H.; Winters, R. T.; Tran, T. P.;
Bridges, A. J.; Althaus, I. W.; Amato, D. M.; Elliott, W.
L.; Ellis, P. A.; Meade, M. A.; Roberts, B. J.; Fry, D. W.;
Gonzales, A. J.; Harvey, P. J.; Nelson, J. M.; Sherwood,
V.; Han, H. K.; Pace, G.; Smaill, J. B.; Denny, W. A.;
Showalter, H. D. J. Med. Chem. 2006, 49, 1475–1485.
48. Antonello, A.; Tarozzi, A.; Morroni, F.; Cavalli, A.;
Rosini, M.; Hrelia, P.; Bolognesi, M. L.; Melchiorre, C. J.
Med. Chem. 2006, 49, 6642–6645.
49. Sun, L.; Cui, J.; Liang, C.; Zhou, Y.; Nematalla, A.;
Wang, X.; Chen, H.; Tang, C.; Wei, J. Bioorg. Med. Chem.
Lett. 2002, 12, 2153–2157.
50. Petrov, K. G.; Zhang, Y. M.; Carter, M.; Cockerill, G. S.;
Dickerson, S.; Gauthier, C. A.; Guo, Y.; Mook, R. A., Jr.;
Rusnak, D. W.; Walker, A. L.; Wood, E. R.; Lackey, K.
E. Bioorg. Med. Chem. Lett. 2006, 16, 4686–4691.
51. Gangjee, A.; Zeng, Y.; Ihnat, M.; Warnke, L. A.; Green,
D. W.; Kisliuk, R. L.; Lin, F. T. Bioorg. Med. Chem. 2005,
13, 5475–5491.
27. Gridelli, C.; Maione, P.; Del Gaizo, F.; Colantuoni, G.;
Guerriero, C.; Ferrara, C.; Nicolella, D.; Comunale, D.;
De Vita, A.; Rossi, A. Oncologist 2007, 12, 191–200.
28. Nelson, M. H.; Dolder, C. R. Ann. Pharmacother. 2006,
40, 261–269.
29. Gangjee, A.; Yang, J.; Ihnat, M. A.; Kamat, S. Bioorg.
Med. Chem. 2003, 11, 5155–5170.
30. Gangjee A.; Namjoshi, O. A.; Ihnat, M.A.; Kamat, S.,
97th Annual Meeting, American Association of Cancer
Research, April 1–5, 2006, Washington DC, 2006, Amer-
ican Association of Cancer Research, Philadelphia, PA.
31. Tripos Inc.: 1699 South Hanley Road, St. Louis, MO
63144–2319 USA.
32. Mazitschek, R.; Giannis, A. Curr. Opin. Chem. Biol. 2004,
8, 432–441.
33. Isanbor, C. O. H. D. J. Fluo Chem. 2006, 127, 303–319.
34. Bradbury, R. H. PCT Int. Appl. WO2006117521, 2006.
35. Bradbury, R. H. PCT Int. Appl. WO 2006092574, 2006.
36. Gangjee, A.; Yu, J.; Kisliuk, R. L. J. Het. Chem. 2002, 39,
833–840.
52. Sheu, J. R.; Fu, C. C.; Tsai, M. L.; Chung, W. J.
Anticancer Res. 1998, 18, 4435–4441.
53. Brooks, P. C.; Montgomery, A. M.; Cheresh, D. A.
Methods Mol. Biol. 1999, 129, 257–269.
37. Arndt, F. Organic Syntheses, Collective 1943, 2, 165.
38. Fong, T. A.; Shawver, L. K.; Sun, L.; Tang, C.; App, H.;
Powell, T. J.; Kim, Y. H.; Schreck, R.; Wang, X.; Risau,
54. Marks, M. G.; Shi, J.; Fry, M. O.; Xiao, Z.; Trzyna, M.;
Pokala, V.; Ihnat, M. A.; Li, P. K. Biol. Pharm. Bull. 2002,
25, 597–604.