Synthesis and biological activity of N4-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents

Article abstract of DOI:10.1016/j.bmc.2010.03.052

A series of eight N⁴-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8-15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N⁴-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16-20 were synthesized to evaluate the importance of the 2-NH₂ moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of α-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of α-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40-42, respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8-20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.

Full text of DOI:10.1016/j.bmc.2010.03.052

Bioorganic & Medicinal Chemistry 18 (2010) 3575–3587
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of N4-phenylsubstituted-6-(2,4-dichloro
phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as vascular
endothelial growth factor receptor-2 inhibitors and antiangiogenic
and antitumor agents
a
b
b
b
Aleem Gangjee a, , Sonali Kurup , Michael A Ihnat , Jessica E. Thorpe , Satyendra S. Shenoy
*
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
b Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA
a r t i c l e i n f o
a b s t r a c t
A series of eight N4-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-
diamines 8–15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors
with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N4-phenylsubstitut-
ed-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16–20 were synthesized to evalu-
ate the importance of the 2-NH2 moiety for multiple receptor tyrosine kinase (RTK) inhibition.
Article history:
Received 30 November 2009
Revised 18 March 2010
Accepted 23 March 2010
Available online 27 March 2010
Cyclocondensation of
amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction
of -bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with form-
amide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40–42,
respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the tar-
get compounds 8–20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-
fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple
RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a
mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis
and metastasis.
a-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-
Keywords:
Pyrrolo[2,3-d]pyrimidines
Receptor tyrosine kinase inhibitors
Antiangiogenic agents
Antitumor agents
a
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Angiogenesis is the growth of new blood vessels from existing
vasculature.3 Physiological angiogenesis occurs during wound
Receptor tyrosine kinases (RTKs) such as epidermal growth fac-
tor receptor (EGFR), vascular endothelial growth factor receptor
(VEGFR) and platelet-derived growth factor receptor (PDGFR) have
important functions in signal transduction pathways that regulate
cell proliferation, differentiation and growth under normal cell
function, as well as under abnormal conditions.1,2 RTKs are overex-
pressed in several tumors and are associated with aberrant signal-
ing leading to increased angiogenesis, tumor cell proliferation and
metastasis.1,2
healing, the menstrual cycle and pregnancy, and produces well or-
dered vascular structures with mature, functional blood vessels.3,4
Pathological angiogenesis is associated with tumor growth and
several diseases such as psoriasis, diabetic retinopathies and endo-
metriosis.3 Angiogenesis is crucial for the sustained growth of tu-
mors. The new blood vessels provide nutrients and oxygen for
tumor proliferation, and also a route for metastasis. In addition
to providing tumors with oxygen and nutrients through angiogen-
esis, activated endothelial cells release growth factors such as
platelet-derived growth factor (PDGF), and insulin-like growth fac-
tor-1 (IGF-1) that on binding to the growth factor receptors acti-
vate RTKs that both maintain the endothelial cell activation and
stimulate tumor cell growth.3–6
Abbreviations: RTK, receptor tyrosine kinase; EGFR, epidermal growth factor
receptor; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endo-
thelial growth factor receptor; IGF-1, insulin-like growth factor-1; Flt-3, fms-like
tyrosine kinase 3; c-Kit, stem cell factor receptor; cFMS, colony stimulating factor
receptor; FGFR-1, fibroblast derived growth factor receptor-1; ATP, adenosine
triphospate.
Thus, inhibition of tumor angiogenesis offers a therapeutic
strategy for treating a variety of cancers. Antiangiogenic agents
are usually cytostatic and prevent the growth of the tumor but
are not usually tumoricidal and are much more successful in can-
cer chemotherapy when combined with cytotoxic agents.4–6
* Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396 5593.
E-mail address: gangjee@duq.edu (A. Gangjee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.052

3576
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
Among the RTKs implicated in tumor progression are members
series, the 6-(2,4-dichlorophenyl)methyl substituted compound
7, was a potent EGFR inhibitor, with poor VEGFR-2 inhibition.27
Since VEGFR-2 has been implicated as the principal mediator of
angiogenesis5,9–11 and VEGFR-2 stimulation alone is sufficient to
induce tumor growth and metastases,11 it was of interest to struc-
turally design VEGFR-2 inhibition in the 6-(2,4-dichloro-
phenyl)methyl pyrrolo[2,3-d]pyrimidine scaffold of compound 7,
to afford either a dual EGFR/VEGFR-2 inhibitor or perhaps a selec-
tive VEGFR-2 inhibitor.
of the VEGFR family that include VEGFR-1 and VEGFR-2, members
of the EGFR family and members of the PDGFR family namely
PDGFR-a
and PDGFR-b.7,8
Angiogenesis is facilitated by a number of growth factor recep-
tors of which VEGFR-2 is the key.5,9–11 VEGF stimulation is suffi-
cient to induce tumor growth and metastases. Inhibition of
VEGFR-2 leads to an inhibition of angiogenesis, decreased vascular
permeability and decreased endothelial cell survival.11
Several small molecule inhibitors of RTKs that target the aden-
osine triphosphate (ATP) binding site of tyrosine kinases are in
clinical use and several others are in clinical trials as antitumor
agents.1,2,12 Initial strategies for RTK inhibition focused on single
RTK inhibitors such as erlotinib, 1 and gefitinib, 2 (Fig. 1) that were
approved for non small cell lung cancer.12,13 However, tumors have
redundant signaling pathways for angiogenesis and often develop
resistance to agents that target one specific pathway.14,15 A multi-
faceted approach that targets multiple signaling pathways has
shown to be more effective than the inhibition of a single target
due to the probable synergism/potentiation/additive effects that
could lower the drug dose required.16–20 Multiple RTK inhibitors,
sorafenib, 3 (Fig. 1) an inhibitor of VEGFR, PDGFR and Raf-1 kinase
and sunitinib, 4 (Fig. 1) an inhibitor of VEGFR-1, VEGFR-2, fms-like
tyrosine kinase-3 (Flt-3), PDGFR, stem cell factor receptor (c-Kit)
and colony stimulating factor receptor (cFMS) have been approved
for renal cell carcinoma.21–23 These two RTK inhibitors are in clin-
ical evaluation alone and in combination with other agents against
a variety of cancers.
However, multiple RTK inhibitors are in some instances more
prone to yield side effects than selective RTK inhibitors.24–26 Pro-
tein kinases share very similar structural features, which are often
responsible for cross-reactivities in various kinases, producing the
undesirable side efects.24 For example, the approved multiple RTK
inhibitors sorafenib and sunitinib have been reported to be cardio-
toxic.25,26 Thus, specific inhibitors of VEGFR-2 or other RTKs would
also be of considerable interest.
In order to design VEGFR-2 inhibitory activity into compound 7,
various anilino substitutions were explored in compounds 8–15.
These anilino substitutions were selected on the basis of the potent
VEGFR-228–31 and EGFR32,33 inhibition reported for similar anilino
substitutions in 6-6 fused scaffolds such as quinazolines, pthala-
zines and pyrido[2,3-d]pyrimidines. Small, lipophilic, electron-
deficient substitutions at the ortho-position of the 4-anilino ring
combined with a larger, lipophilic, electron withdrawing moiety
at the para-position such as chlorine or bromine have provided po-
tent VEGFR-2 inhibition in quinazolines.30,31 Thus, compounds 8,
with the 2-chloro, 4-bromo anilino moiety and 9, with the 2-fluoro,
4-chloro aniline moiety were synthesized.
Small substitutions at the meta-position of the anilino ring such
as a 3-hydroxy and a 3-trifluoromethyl have also been reported to
afford potent VEGFR-2 and EGFR inhibition,28–31 while larger, elec-
tron withdrawing substitutions such as 3-bromo and 3-chloro are
preferred for EGFR inhibition in bicyclic 6-6 fused scaffolds.32,33 In
order to improve the potency of VEGFR-2 inhibition and perhaps
provide an optimal combination for VEGFR-2 and EGFR inhibition
in the target compounds, we elected to substitute the phenyl ring
of the anilino moiety at the 3-position with small, lipophilic, elec-
tron withdrawing substituents such as the 3-fluoro in 10, 3-trifluo-
romethyl in 11 and 3-ethynyl in 12.
Disubstitution at the 2,4- and 3,4-position of the anilino deriv-
atives have also shown good inhibitory activities against VEGFR-2,
while retaining moderate EGFR inhibition.30,31 In addition, these
substitutions showed a preference for VEGFR-2 and EGFR over
PDGFR and other kinases has been reported (VEG-
FR > EGFR > PDGFR, FGFR, IGF-1R).30,31 Thus, in addition to the
2,4-disubstituted compounds 8 and 9, the 3,4-disubstituted deriv-
ative 13 with a 3-fluoro, 4-trifluoromethyl anilino was also
synthesized.
Gangjee et al.27 previously reported compounds 5–7 (Fig. 2) as
multiple RTK inhibitors in a series of N4-(3-bromophenyl)-6-phe-
nylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines.
Compound 5 was a potent VEGFR-2 inhibitor and demonstrated
A431 cytotoxicity and moderate EGFR inhibition in the cellular as-
says.27 Compound 6 was a potent VEGFR-2 inhibitor and a moder-
ate PDGFRb and VEGFR-1 inhibitor.27 In the previously reported
Bulky groups at the para-position which include electron donat-
ing alkyl groups and electron withdrawing substitutions such as
F
Cl
NH
N
O
NH
O
O
N
N
N
O
O
N
O
O
1
2
, Gefitinib
, Erlotinib
O
N
Cl
N
H
HN
O
NH
O
CF3
N
H
F
N
O
O
N
H
N
H
3
4
, Sunitinib
, Sorafenib
Figure 1.

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3577
R
Br
8
9
4-Br, 2-Cl
4-Cl, 2-F
R
Ar
5
6
7
2-MePh
HN
10 3-F
11
12 3- C CH
HN
2,5-diOMePh
2,4-diClPh
3-CF3
N
Cl
Cl
N
N
H
13
14
H2N
N
3-F, 4-CF3
4-i Pr
15 2-i Pr
N
H
Ar
H2N
N
Figure 2.
halogens and aryl substitutions have also been reported to be con-
ducive for potent VEGFR-2 inhibition,28,29,34,35 thus the 4-isopropyl
aniline derivative 14 was synthesized. Its regioisomer, 15 was syn-
thesized to restrict the conformation of the anilino ring with re-
spect to the pyrrolo[2,3-d]pyrimidine scaffold and thereby
determine its effect on RTK inhibition.
molecule to VEGFR-2 and appropriately orient the other parts of
the molecule in the ATP binding site.
Compound 5 in Mode 1 in VEGFR-2 (PDB code: 1YWN) showing
the key interactions. Hydrogen bonding in the Hinge region be-
tween the 2-NH2 and the backbone carbonyl of Cys917, N3 and
NH of Cys917 and 4-anilino NH and backbone carbonyl of
Glu915. Hydrophobic interactions between the 4-anilino phenyl
ring and Val897 and Cys1043 are indicated. Hydrophobic interac-
tions between the 6-benzyl ring and Val846 and carbon chain of
Lys866 (shown in dotted van der Waals surface for VEGFR-2).
Compound 5 in Mode 2 in VEGFR-2 (PDB code: 1YWN) showing
hydrogen bonding in the Hinge region between the 2-NH2 and the
backbone carbonyl of Cys917, N3 and NH of Cys917 and pyrrolo
NH and backbone carbonyl of Glu915.
An alternate mode of binding or Mode 2 (Fig. 4), is obtained
from Mode 1 by rotating the C2–NH2 bond by 180°. Mode 2 has
the 4-anilino moiety binding in the Sugar pocket rather than in
the hydrophobic region as in Mode 1. In Mode 2, the 7-position
pyrrolo NH mimics the anilino NH of Mode 1 and forms one of
the H-bonds with the Hinge region, substituting for the 4-NH of
ATP; the N1-nitrogen forms the second H-bond and mimics the
N3-nitrogen of Mode 1 and ATP.
The 2-NH2 moiety of compound 727 was maintained in com-
pounds 8–15 to provide an additional hydrogen bond in the Hinge
region of RTKs compared to other known RTK inhibitors that lack
this 2-NH2 moiety. The flexible 6-(2,4-dichlorobenzyl) substitution
was maintained to allow for multiple conformations of the side
chain and as a result, perhaps afford interactions with two or more
RTKs.
Molecular modeling using SYBYL 7.034 showed two possible
modes of binding of 5 to VEGFR-2. The first or Mode 1 is shown
in Figure 3. Compound 5 was energy minimized (minimize and
search option in SYBYL 7.0) and superimposed onto the crystal struc-
ture of a furo[2,3-d]pyrimidine in VEGFR-2 (PDB: 1YWN)35,36
(Fig. 3). In this model, the pyrrolo[2,3-d]pyrimidine occupies the
adenine region of the ATP binding site. The N3 of the pyrimidine
in 5 is hydrogen bonded to the backbone NH of Cys917 and the ani-
line NH is hydrogen bonded to the backbone carbonyl of Glu915.
An additional hydrogen bond between the backbone carbonyl of
Cys917 and the 2-NH2 on the pyrimidine ring of 5 is not observed
in ATP or the reported furo[2,3-d]pyrimidine.35,36 These hydrogen
bond interactions serve to anchor the heterocyclic portion of the
Thus, molecular modeling provided credence to our hypothesis
of the possible binding modes of our target compounds 8–15 to
VEGFR-2, similar to that shown for 5.
An additional aspect of our work was to evaluate the role of the
2-NH2 moiety for RTK inhibition. The 2-NH2 moiety was originally
incorporated in our design27 of pyrrolo[2,3-d]pyrimidines on the
basis of molecular modeling as shown in Figures 3 and 4 to afford
an additional hydrogen bond in the Hinge region of RTKs compared
Figure 3. Compound 5 in Mode 1 in VEGFR-2 (PDB code: 1YWN) showing the key
interactions. Hydrogen bonding in the Hinge region between the 2-NH2 and the
backbone carbonyl of Cys917, N3 and NH of Cys917 and 4-anilino NH and backbone
carbonyl of Glu915. Hydrophobic interactions between the 4-anilino phenyl ring
and Val897 and Cys1043 are indicated. Hydrophobic interactions between the 6-
benzyl ring and Val846 and carbon chain of Lys866 (shown in dotted van der Waals
surface for VEGFR-2).
Figure 4. Compound 5 in Mode 2 in VEGFR-2 (PDB code: 1YWN) showing hydrogen
bonding in the Hinge region between the 2-NH2 and the backbone carbonyl of
Cys917, N3 and NH of Cys917 and pyrrolo NH and backbone carbonyl of Glu915.

3578
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
to most of the known 6-6 and 6-5 bicyclic pyrido[2,3-d]pyrimi-
dines, quinazolines and furo[2,3-d]pyrimidines that lack the 2-
NH2 moiety.28–34
We repeated the reported methodology to obtain 23 as de-
scribed previously.27 Careful scrutiny of the TLC for the reaction
of
a-bromomethyl-(2,4-dichlorobenzyl)ketone, 21 with 22 re-
In order to specifically demonstrate that the 2-NH2 moiety does
indeed contribute to an increase in the potency of the 6-phenylm-
ethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-2,4-diamines, it was
necessary to synthesize pairs of analogs with and without the 2-
NH2 group. Thus, N4-phenylsubstituted-6-phenylmethylsubstitut-
ed-7H-pyrrolo[2,3-d]pyrimidin-4-amines, 16–20 (Fig. 5) were syn-
thesized as the 2-des NH2 analogs of 5–7, 11 and 12, respectively.
vealed three spots [TLC Rf 0.57, 0.60, 0.65 (CHCl3/CH3OH, 5:1)].
Two of the products, 23 and 24 were separated by column chroma-
tography, however the third product, 25 (<5% yield) could not be
separated from 24. The 6-(2,4-dichlorobenzyl)-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one, 23 (Rf 0.57) was the major product,
isolated in 44% yield. The Rf and the 1H NMR for the minor products
were distinct from the starting materials 21 and 22 and the prod-
uct 23. 1H NMR for one of the minor products, 24 (Rf 0.60), showed
two D2O exchangeable peaks at 5.91 ppm and 6.12 ppm that inte-
grated for two protons each and were assigned to the 2,4-diNH2 of
24. A singlet at 5.13 ppm that integrated for one proton was as-
signed to the C5–H of the pyrimidine. In addition, two singlets that
integrated for two protons each at 4.85 ppm and 4.00 ppm and aro-
matic peaks at 7.37–7.58 ppm were also observed. These proton
positions and their integration along with the HRMS confirmed
the structure to be the monocyclic 2,4-diamino-6-substituted
pyrimidine 24 (Scheme 2). Compound 24 is most likely formed
by the attack of the 4-hydroxy group of 22 on the halogen of the
2. Chemistry
The key intermediate in the synthesis of 8–15 was the 6-(2,4-
dichlorophenyl)methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
4-one 23 (Scheme 1). Several methods have been published for the
synthesis of 6-substituted pyrrolo[2,3-d]pyrimidines.27,37–43 We27
previously synthesized 23 by utilizing the method reported by Sec-
rist and Liu37 that involved the reaction of
a-bro-
momethylbenzylketones with 2,6-diamino-4-oxopyrimidine, 22.
Secrist and Liu37 reported the regiospecific formation of 6-substi-
a-bromomethylbenzylketone, 21 and could be an intermediate in
tuted pyrrolo[2,3-d]pyrimidines in the reaction of
a-bromometh-
the pathway toward the 2,4-diamino-5-substituted furo[2,3-
ylketones with 22 in DMF. These authors also reported a mixture
of the 6-substituted-pyrrolo[2,3-d]pyrimidines and the 5-substi-
d]pyrimidine, a by product isolated in previous reports.37
The above method for 23 involved the formation of additional
side products, moderate yields, prolonged reaction times and re-
quired purification by column chromatography. We attempted an
alternate method for the synthesis of 23. The regiospecific synthe-
tuted-furo[2,3-d]pyrimidines by the reaction of a-chloromethylke-
tones with 22 in DMF. In subsequent reports, Gangjee et al.38
reported the reaction of 3-bromo-4-piperidone hydrobromide with
22 to afford only the tricyclic tetrahydropyrido annulated-2,4-
diaminofuro[2,3-d]pyrimidine. Secrist and Liu37 had also reported
sis of 6-substituted pyrrolo[2,3-d]pyrimidines by the reaction of a-
chloromethylbenzylketones with 22 in sodium acetate and water
at reflux has been previously reported by Gangjee et al.39–42 and
other groups.43 We attempted this method (Scheme 2) for the syn-
thesis of 6-(2,4-dichlorophenyl)methyl-3,7-dihydro-4H-pyrrol-
o[2,3-d]pyrimidin-4-one 23 based on the shorter reaction time
(6–12 h vs 72 h) and significantly improved yields for the pyrrol-
o[2,3-d]pyrimidines, without the formation of any furo[2,3-
d]pyrimidines or byproducts.39–43
The appropriate chloroketones 28 and 29 were synthesized by
treating the appropriate diazoketones 2627 and 2727 with concd
HCl at reflux (Scheme 2). The chloroketones 28 and 29 were trea-
ted with 22, NaOAc and water at reflux to afford 23 and 30, respec-
tively, in 65–70% yield. Chlorination of 23 proceeded in poor yields,
the formation of furo[2,3-d]pyrimidines exclusively with cyclic a-
chloromethylketones.
R1
3-Br
Ar
2-MePh
16
R1
17 3-Br
2,5-diOMePh
2,4-diClPh
2,4-diClPh
HN
18
19
20
3-Br
3 - CF3
3- C CH 2,4-diClPh
N
N
H
Ar
H
N
Figure 5.
Cl
O
HN
H2N
Cl
N
H
N
O
Cl
23
(44%)
a
HN
H2N
Br
O
N
NH2
Cl
NH2
22
21
N
Cl
H2N
N
O
O
Cl
24 (10%)
25
(~ 5)%
Scheme 1. Reagents and conditions: (a) DMF, rt, 3 days.

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3579
O
O
N
HN
Ar
H2N
N
NH2
HN
H2N
a
22
ArCH2COCHN2
ArCH2COCH2Cl
b
N
H
26 Ar = 2,4-diClPh
28 Ar = 2,4-diClPh
29 Ar = 2-MePh
65- 70%
27
Ar = 2-MePh
23 Ar = 2,4-diClPh
30
Ar = 2-MePh
Cl
N
O
N
Ar
Ar
d
HN
HN
c
75-85%
N
H
N
H
HN
Piv
N
45-58%
Piv
31 Ar = 2,4-diClPh
33 Ar = 2,4-diClPh
34
32
Ar = 2-MePh
Ar = 2-MePh
R
R
H2N
HN
e or f
35
Ar
N
61-86%
N
H
H2N
N
5,8-15
Scheme 2. Reagents and conditions: (a) concd HCl, 1 h, 70–80 °C; (b) 21, NaOAc, H2O, 12 h, 100 °C; (c) PiV2O, 2 h, 120 °C; (d) POCl3, 3 h, reflux; (e) 35, iPrOH, 2–3 drops concd
HCl, 16–48 h, reflux; (f) 35, iPrOH, 2–3 drops concd HCl, microwave, 45 min.
hence, 23 was first pivaloylated at the 2-NH2 moiety followed by
chlorination and treatment with appropriate aniline to the target
compounds 8–15 as described in Scheme 2. Prolonged reflux
(16–48 h) of the 4-chloro intermediates 33 and 34 with the appro-
priate anilines in isopropanol and a few drops of concd HCl re-
sulted in displacement of the 4-chloro with the aniline and
simultaneous depivaloylation to afford the target compounds 8,
9, 12 and 14. Reaction with the anilines in isopropanol and 2–3
drops of concd HCl in the microwave reactor (Emrys Liberator
microwave synthesizer, Biotage Inc.) for 45 min at 120 °C afforded
compounds 10, 11 and 13, respectively.
say as described in Scheme 2. The 2-NH2 moiety in 30 was first
pivaloylated to 32 and then chlorinated with POCl3 to afford N-
[4-chloro-6-(2-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-
2,2-dimethylpropanamide, 34 in 55% yield compared to the 4-
chloro-6-(2-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidine-2-
amine27 that was previously reported in 28% yield. Nucleophilic
displacement of the 4-chloro in 34 with 3-bromoaniline and simul-
taneous depivaloylation on prolonged reflux afforded 5.
The 2-des NH2 compounds 16–20 (Fig. 5) were synthesized
(Scheme 3), somewhat differently from 8–15. Reaction of bromoac-
etone with ethylamidinoacetate, 3645 to afford the corresponding
pyrroles, followed by cyclization with formamide to the corre-
sponding pyrrolo[2,3-d]pyrimidines, has been reported in the liter-
Based on the potent RTK inhibition seen for the previously re-
ported 5, we resynthesized 5 in larger quantities for an in vivo as-
O
EtOOC
H2N
Br
Ar
EtOOC
H2N
Ar
R
HN
O
b
a
N
H
N
H
H
N
NH.HCl
45-70%
40-45%
36
37
38
Ar = 2-Me
40 Ar = 2-Me
41
42
Ar = 2,5-diOMe
Ar = 2,5-diOMe
Ar = 2,4-diCl
39 Ar = 2,4-diCl
NH2
R
R
Cl
N
HN
Ar
35
d
c
N
Ar
N
55-76%
55-84%
N
H
H
N
H
H
N
43 Ar = 2-Me
44
45
16-20
Ar = 2,5-diOMe
Ar = 2,4-diCl
Scheme 3. Reagents and conditions: (a)
a-bromobenzylketones, EtOAc, NEt3, N2, 15 min, rt then 30 min, reflux; (b) formamide, Na/MeOH, 8 h, 120 °C; (c) POCl3, 3 h, reflux;
(d) 35, iPrOH, 2–3 drops concd HCl, 4–6 h, reflux.

3580
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
ature.44–46 We modified this procedure for the synthesis of 16–20.
The appropriate -bromomethylbenzylketones with 36 at reflux
(Fig. 6) were used as controls in each of the evaluations. The stan-
dard compounds used were semaxanib (SU5416), 46 for VEGFR-
2;48 cisplatin, 47 for A431 cytotoxicity, (4-chloro-2-fluoro-
phenyl)-6,7-dimethoxyquinazolin-4-yl-amine (CB676475), 48 for
VEGFR-1;32 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline
(PD153035), 49 for EGFR;31 and 3-(4-dimethylamino-benzylide-
nyl)-2-indolinone (DMBI), 50 for PDGFR-b.49 In addition, FDA ap-
a
afforded the appropriately substituted pyrroles 37–39 which were
heated with formamide to afford the 6-substituted phenylmethyl-
3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40–42. Com-
pounds 40–42 were chlorinated with POCl3 to 43–45 and subse-
quent treatment with the appropriate anilines, 35 afforded the
target compounds 16–20.
proved receptor tyrosine kinase inhibitors erlotinib,
1 and
sunitinib, 4 were also incorporated for a comparison in the
in vitro study against EGFR, VEGFR-2 and PDGFR-b kinases. Suniti-
nib, 4 displayed two-digit micromolar inhibitions of VEGFR-2 and
PDGFR-b while erlotinib displayed single-digit micromolar inhibi-
tion of EGFR. The inhibitory potencies (IC50 values) of 8–15 are
compared with the previously synthesized compound 727 (Fig. 1)
and the standard compounds 1, 4 and 46–50 in Table 1.
2.1. Biological evaluation and discussion
Compounds 8–20 were evaluated as RTK inhibitors using hu-
man tumor cells known to express high levels of EGFR, VEGFR-2,
VEGFR-1 and PDFGR-b using a phosphotyrosine ELISA cytoblot (Ta-
ble 1).47 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 A431 cancer cells, known to overexpress
EGFR. EGFR is known to play a role in the overall survival of
A431 cells.47
2.1.1. VEGFR-2
Previously synthesized compound 7 did not show significant
VEGFR-2 inhibition and was less potent than semaxanib in the cel-
lular assay. Compounds 8, 10 and 14 of this series showed inhibi-
tion of VEGFR-2 that was more potent than the standards,
semaxanib 46 and sunitinib 4. In the disubstituted anilino deriva-
Since the IC50 values of compounds vary under different assay
conditions (e.g., ATP concentrations), standard compounds
Table 1
IC50 values (lM) of kinase inhibition and A431 cytotoxicity for compounds 7–15
VEGFR-2
EGFR
VEGFR-1
PDGFR-b
A431 cytotoxicity
7a
8
9
28.1 9.9
0.1 0.3
45.7 6.4
0.3 0.04
>300
85.6 12.2
113.1 21.1
1.4 0.1
>300
0.2 0.06
>300
>50
>300
70.9 9.1
162.9
156.1 29.1
>300
>300
157 22.3
163.1 27.9
17.0 5.6
>300
24.6 4.1
183.1 30.2
>300
>300
>300
174.9 27.3
43.0 9.4
2.8 1.1
16.2 2.5
4.9 0.51
45.2 7.2
195.2 28.8
9.5 0.09
13.8 2.5
26.2 4.0
160.2 25.6
2.5 0.09
161.6 25.3
27.2 3.1
55.2 7.1
91.7 29.3
45.6 13.2
96.4 14.9
10
11
12
13
14
15
46
47
48
49
50
1
12.0 2.7
10.6 2.9
14.1 2.8
0.2 0.04
3.7 0.06
83.1 10.1
12.2 1.9
124.7 18.2
18.9 2.7
1.2 0.2
172.1 19.4
4
a
IC50 values from Ref. 27.
F
Cl
HN
Cl
H2N Pt
N
H
OMe
OMe
Cl
N
O
NH2
N
H
N
46
, Semaxanib (SU5416)
47
, Cisplatin
48, CB676475
Br
COOH
N
HN
N
H
OMe
OMe
N
O
O
N
H
N
H
N
51, SU6668
50, DMBI
49, PD153035
Figure 6.

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3581
tives, compound 8 with the 2-chloro, 4-bromo anilino substitution
was 100-fold more potent than semaxanib. However, the 2-fluoro,
4-chloro anilino derivative 9 and the 3-fluoro, 4-trifluoromethyl
anilino derivative 13 did not inhibit VEGFR-2. VEGFR-2 inhibition
also improved in the 3-substituted anilino derivatives, when the
3-bromo in 7 was replaced with a 3-fluoro in 10. Compound 10
was 40-fold better than semaxanib, 46. VEGFR-2 inhibition de-
creased significantly with variations to the 3-ethynyl in 12 and
was abolished on variation to the 3-trifluoromethyl in 11. The 4-
isopropyl substitution in 14, however provided potent VEGFR-2
inhibition that was 8-fold more potent than semaxanib, 46 while
the 2-isopropyl substitution in 15 abrogated VEGFR-2 inhibition.
against VEGFR-2 and 10-fold less potent than the standard com-
pound 49 (PD153035) against EGFR. Compound 9 also showed
moderate VEGFR-1 inhibition and was 5-fold less potent than the
standard 48 (CB676475). The 3-ethynyl substitution present in
12 was derived from the quinazoline-derived EGFR inhibitor, erl-
otinib, 1.12,32,33 Compound 12 however, did not show EGFR inhibi-
tion. This indicates that the scaffold that holds the anilino moiety is
also important for specificity of inhibition of an RTK.
Since the inhibitory activities are determined in cells, it is not
possible to make a definite structure–activity relationship for com-
pounds 8–15 and RTK inhibition. However, the cellular assays dis-
cussed above clearly indicate that variation in the phenyl
substitution of the anilino moiety does indeed control the potency
and specificity of RTK inhibition. Since some anilino substitutions
in 8–15 do not follow RTK inhibitory profiles observed for the 6-
6 fused scaffolds, the optimal substitution pattern on the anilino
moiety needs to be determined individually for each of the various
scaffolds showing RTK inhibition.
2.1.2. EGFR
In the 6-(2,4-dichlorophenylmethyl)substituted pyrrolo[2,3-
d]pyrimidines, compound 7 was a potent EGFR inhibitor. EGFR
inhibition significantly decreased or was abolished on variation
of the 3-bromo anilino substitution in 7 to other anilino moieties
in 8–15. Compound 9 with the 2-fluoro, 4-chloro anilino substitu-
tion was the most potent EGFR inhibitor in this series of com-
pounds, although EGFR inhibition decreased by 10-fold in 9
compared to 7 and the standard 49 (PD153035).
A comparison of the RTK inhibitory activities and A431 cytotox-
icity for the 2-NH2 substituted compounds 5–7 and 14–15 with
their 2-des NH2 analogs 16–20, respectively, is provided in Table 2,
along with the standards.
2.1.3. VEGFR-1
2.2.1. VEGFR-2
Compounds 8–15 and the lead compound 7 were inactive
against VEGFR-1. Compound 9 with the 2-fluoro, 4-chloro anilino
substitution was the best and was fivefold less active than the
standard 48 (CB676475).
The 2-NH2 substituted compounds 5 and 6 showed potent sub-
micromolar inhibition of VEGFR-2. The corresponding 2-des NH2
analogs 16 and 17 were 108-fold and >300-fold less potent than
5 and 6, respectively, and were 2-fold and >16-fold less potent than
the standard, semaxanib 46. The 2-NH2 substituted compounds 7,
11 and 12 did not show potent inhibition of VEGFR-2. The VEGFR-2
inhibition further decreased for the corresponding 2-des NH2 ana-
logs 18, 19 and 20, respectively.
2.1.4. PDGFR-b
Compound
7 showed two-digit micromolar inhibition of
PDGFR-b and was less potent than the standard 50 (DMBI) and
sunitinib, 4. Analogs 8–15 also showed diminished activity com-
pared to 50 however compounds 9 and 15 retained two-digit
micromolar inhibition as seen in the lead compound 7 and were
6.5-fold and 11.5-fold less potent than 50, respectively. Com-
pounds 9 and 15 were approximately 2-fold and 4-fold less potent
than the standard compound 4.
2.2.2. EGFR
The 2-des NH2 compounds 16–20 showed poor inhibitory
potencies against EGFR, compared to the 2-NH2 substituted com-
pounds 5–7 and 11 and 12 and the standard compound 49
(PD1530305).
2.1.5. A431 cytotoxicity
2.2.3. VEGFR-1
Compound 7 showed potent inhibition in the A431 cytotoxicity
assay and was more potent than the standard, cisplatin. All three
disubstituted anilino compounds 8, 9 and 13 showed potent
A431 cytotoxicity, more potent or equipotent with cisplatin, but
slightly less potent than compound 7. The IC50 value for compound
9 was 1.7-fold less potent than the lead compound 7. The 3-ethy-
nylsubstituted anilino derivative, 12 also showed single-digit
micromolar inhibitory activities in the A431 cytotoxicity assay
and was almost equipotent with cisplatin and approximately
threefold less potent than 7. Interestingly, compounds 8, 12 and
13 showed reasonably potent A431 cytotoxicities although they
did not show significant EGFR inhibition. The A431 cell lines de-
pend on EGFR for survival; perhaps these compounds do not di-
rectly inhibit EGFR but influence the downstream signaling of
EGFR and crosstalk with other kinases which may be necessary
for EGFR function. The literature contains several similar reports
in which the EGFR inhibitory activity does not necessarily translate
into A431 cytotoxicity.31,34
The 2-NH2 substituted compound 6 showed moderate VEGFR-1
inhibition, approximately 2-fold less potent than the standard 48.
The corresponding 2-des NH2 analog 17 was >6-fold less potent
than 6 and >14-fold less potent than the standard 48. VEGFR-1
inhibition did not improve for the 2-des NH2 analogs 16, 18, 19
and 20 compared to the 2-NH2 substituted compounds 5, 7, 11
and 12, respectively, and also compared to the standard, 48
(CB676475).
2.2.4. PDGFR-b
The PDGFR-b inhibition did not improve for the 2-des NH2 ana-
logs 16–20 compared to the 2-NH2 substituted compounds 5–7, 11
and 12, respectively.
2.2.5. A431 cytotoxicity
The 2-NH2 substituted compounds 5, 7 and 12 showed potent
A431 cytotoxicity being more potent or equipotent to the standard,
cisplatin. The A431 cytotoxicity significantly decreased for the cor-
responding 2-des NH2 analogs 16, 18 and 20 compared to 5, 7 and
12, respectively, and compared to cisplatin. The A431 cytotoxicity
improved for the 2-des NH2 analog 17 compared to 6 and cisplatin,
47.
A consistent decrease in RTK inhibition in whole cells was ob-
served for the 2-des NH2 compounds, 16–20, with the exception
of the potent inhibition seen in the A431 cytotoxicity assay for
17. The study of the 2-NH2 substituted compounds and their corre-
2.2. Multiple RTK inhibition
Compounds 8, 10 and 14 were the most potent VEGFR-2 inhib-
itors of this study. The improvement in VEGFR-2 inhibition for 8,
10 and 14 was obtained with a simultaneous decline in EGFR inhi-
bition. Compound 9 retained moderate, dual EGFR and VEGFR-2
inhibition, and was 3.8-fold less potent than the semaxanib, 46

3582
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
Table 2
IC50 values (
l
M) of kinase inhibition and A431 cytotoxicity for compounds 5–7 and 11–12 and 16–20
VEGFR-2
EGFR
VEGFR-1
PDGFR-b
A431 cytotoxicity
5a
6a
0.2 0.04
0.6 0.2
28.1 9.9
>300
85.6
27.2 4.2
>300
>300
>300
>300
12.0 2.7
9.1 1.8
12.6 3.3
0.2 0.06
27.2 3.1
55.2 7.1
>300
>300
>300
33.1 5.3
>300
>50
31.1 5.8
>50
156.1 29.1
>300
>300
>300
150.9 27.4
>300
>50
8.9 1.6
17.0 5.6
>300
>300
182.0 31.3
171.3 31.6
220.4 33.4
110.1 31.1
142.2 34.1
1.2 0.4
>50
7a
2.8 1.1
195.2 28.8
9.5 0.09
191.3 30.2
4.5 1.7
>300
11
12
16
17
18
19
20
46
47
48
49
50
1
>300
198.1 31.2
>300
10.6 2.9
14.1 2.8
0.2 0.04
3.7 0.06
83.1 10.1
12.2 1.9
124.7 18.2
18.9 2.7
1.2 0.2
172.1 19.4
4
a
IC50 values from Ref. 27.
sponding 2-des NH2 analogs confirms our original hypothesis that a
2-NH2 should provide additional hydrogen bonding interactions
that translates into improved inhibition for RTK and A431 cytotox-
icity for the 2-NH2 substituted compounds compared to their 2-des
NH2 analogs.
2.3. In vivo evaluation
Two compounds, compound 8 of this study and previously syn-
thesized analog 5 were selected on the basis of their cellular RTK
inhibitory activities for in vivo evaluation of inhibition of tumor
growth, vascularity and metastasis. The compounds were evalu-
ated in a B16-F10 murine metastatic melanoma model. This model
is widely accepted for evaluating tumor growth and metastases,
with highly vascularized tumors so that tumor-mediated angio-
genesis can also be evaluated. Compound 5 showed potent VEG-
FR-2 inhibition, A431 cytotoxicity and moderate EGFR inhibition
in the cellular assays, while 8 showed potent VEGFR-2 inhibition
and A431 cytotoxicity. Compounds 5 and 8 were dosed intraperito-
neally, three times weekly at 35 mg/kg. SU6668, 51,21 (Fig. 6), an
analog of the approved drug sunitinib and a potent inhibitor of c-
Kit, VEGFR-2, PDGFR-b and fibroblast derived growth factor recep-
tor-1 (FGFR-1) was used as a standard in this study and was dosed
three times weekly at 10 mg/kg.
Figure 7. Inhibitory activity of compounds 5, 8 and 51 (SU6668) on primary tumor
growth in the B16-F10 melanoma model in mice. Values are mean SEM,
***P <0.001, **P <0.01.
The results of the inhibitory activity of compounds 5, 8 and 51
on primary tumor growth are shown in Figure 7. Compounds 5 and
8 showed an inhibition in tumor growth compared to the un-
treated (sham) animals. Both 5 and 8 were effective antitumor
agents compared to untreated animals, however they were some-
what less effective than 51.
The effect of compounds 5, 8 and 51 on primary tumor vascular-
ity is shown in Figure 8. All three agents (5, 8 and 51) induced a sig-
nificant decrease in tumor vascularity compared to tumors from
untreated (sham) animals. Compounds 5 and 8 were both some-
what more effective than 51 in the inhibition of tumor vascularity.
The effect of compounds 5, 8 and 51 on tumor metastasis is
shown in Figure 9. The F10 variant of the B16-F10 tumor was se-
lected for lung metastasis and one month after implantation, 90%
of the animals showed lung metastases. These studies revealed that
lung metastasis was inhibited with all three compounds 5, 8 and 51
compared with the untreated (sham) animals. Compound 5 was
more effective than 51 in the inhibition of lung metastasis.
Compound 8 also showed some inhibition of lung metastasis
compared to the untreated animals but was less effective than either
5 or 51.
Figure 8. The effect of compounds 5, 8 and 51 (SU6668) on tumor vascularity in the
B16-F10 melanoma model in mice. Values are mean SEM, **P <0.01.
The in vivo antitumor evaluation of 5 and 8 suggest that both
compounds cause an inhibition in tumor growth, angiogenesis
and metastasis. Compound 5 shows a better inhibition of tumor
angiogenesis and metastasis compared to 8. Compound 8 shows

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3583
mined on a MEL-TEMP II melting point apparatus with FLUKE 51 K/J
electronic thermometer and are uncorrected. Nuclear magnetic res-
onance spectra for proton (1H NMR) were recorded on a Bruker WH-
300 (300 MHz) or a Bruker 400 MHz/52 MM (400 MHz) spectrome-
ter. The chemical shift values are expressed in ppm (parts per
million) relative to tetramethylsilane as an internal standard: s, sin-
glet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet.
Mass spectra were recorded on a VG-7070 double-focusing mass
spectrometer or in a LKB-9000 instrument in the electron ionization
(EI) or electron spray (ESI) mode. Chemical names follow IUPAC
nomenclature. Thin-layer chromatography (TLC) was performed
on Whatman Sil G/UV254 silica gel plates with a fluorescent indica-
tor, and the spots were visualized under 254 and 366 nm illumina-
tion. Proportions of solvents used for TLC are by volume. Column
chromatography was performed on a 230–400 mesh silica gel (Fish-
er, Somerville, NJ) column. Elemental analyses were performed by
Atlantic Microlab, Inc., Norcross, GA. Element compositions are
within 0.4% of the calculated values. Fractional moles of water or or-
ganic solvents frequently found in some analytical samples 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.
Microwave-assisted synthesis was performed utilizing an Emrys
Liberator microwave synthesizer (Biotage) utilizing capped reaction
vials. All microwave reactions were performed with temperature
control. All solvents and chemicals were purchased from Aldrich
Chemical Co. or Fisher Scientific and were used as received.
Figure 9. The effect of compounds 5, 8 and 51 (SU6668) on tumor metastasis in the
B16-F10 melanoma model in mice. Values are mean SEM, ***P <0.001, **P <0.01.
significant inhibition of angiogenesis but is less effective in inhibi-
tion of tumor metastasis.
3. Summary
In summary, compounds 8–15 were synthesized to improve the
VEGFR-2 inhibition by variation of the phenyl substitutions of the 4-
anilino moiety in compound 7. Three compounds 8, 10 and 14
showed potent cellular VEGFR-2 inhibitions and were much more
potent or equipotent to the standard compound semaxanib. In fact,
compounds 8, 10 and 14 were selective VEGFR-2 inhibitors and were
devoid of any inhibitory activity against the other RTKs evaluated.
Two compounds, 9 and 12 showed potent cytotoxic effects against
A431 cells in culture, equipotent or more potent than the standard.
The cellular inhibition assays demonstrated that the potency and
selectivity of the inhibition of different RTKs varies with different
anilino substitutions, and that a combination of the substitutions
in the 4-anilino ring and the 6-benzyl substituent dictates both spec-
ificity and potency of RTK inhibition in the 6-benzyl substituted pyr-
rolo[2,3-d]pyrimidines. The different RTK inhibitory profiles
observed for compounds 8–15 in this study suggest that individual
substituent optimization of the anilino moiety will be necessary
for each of the various scaffolds for different RTK inhibitions.
The lack of inhibitory activity of the 2-des NH2 analogs, 16–20
supports our design strategy of incorporating a 2-NH2 moiety in
the 6-benzyl substituted pyrrolo[2,3-d]pyrimidines for improved
potency of RTK inhibition by providing an additional hydrogen
bond with the Hinge region of RTKs. The in vivo antitumor activi-
ties of 5 and 8 indicate that both compounds cause an inhibition in
tumor growth, angiogenesis and metastasis. Compound 5 was
more effective in the inhibition of tumor angiogenesis and metas-
tasis than the standard 51. Compound 8 was also more effective in
the inhibition of tumor angiogenesis than the standard 51, but less
effective than both 5 and 51 in the inhibition of tumor growth and
metastasis. The demonstrated VEGFR-2 selectivity of 8 provides
antiangiogenic activity in vivo as predicted. The in vivo antitumor
activity of 5 and 8 indicates that whole cell RTK inhibitory activity
does translate into successful in vivo activity and warrants the fur-
ther development of 5 and 8 as antitumor agents.
4.1.1. 2-Amino-6-(2,4-dichlorobenzyl)-3,7-dihydro-4H-pyrrolo-
[2,3-d]pyrimidin-4-one (23)
To a 250-mL round-bottom flask was added a solution of 26
(2.3 g, 10 mmol) in diethylether (50 mL). Aqueous HCl (15 mL)
was added dropwise and the yellow mixture was refluxed for
1 h. The reaction mixture was cooled and the ether layer was sep-
arated, washed with water (50 mL) and dried over Na2SO4. The or-
ganic layer was evaporated to afford 2 g (84%) of 28 as a yellow
solid. To a 250-mL round-bottom flask was added 22 (1 equiv),
NaOAc (1.3 equiv) and water (180 mL). The reaction mixture was
refluxed for 20 min, and then a solution of 28 (2 g, 8.4 mmol) in
MeOH (2 mL) was added. The reaction mixture was refluxed for an-
other 12 h. The reaction mixture was cooled to 0 °C and filtered.
The solid collected was washed with MeOH and diethyl ether to af-
ford 1.96 g (75%) of 23; mp 265 °C (lit. mp 265 °C).27 The com-
pound was identical in all respects to the mp, Rf, 1H NMR as that
reported.27
4.1.2. 2-Amino-6-(2-methylbenzyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (30)
Compound 30 was synthesized as described for 23 with 27 and
was obtained as an off white solid (65%); mp 290 °C (lit. mp
290 °C).27 The compound was identical in all respects to the mp,
Rf, 1H NMR as that reported.27
4.1.3. 1-(2,6-Diamino-pyrimidin-4-yloxy)-3-(2,4-dichlorophenyl)-
propan-2-one (24)
To
a
250-mL round-bottom flask was added 1727 (2 g,
7.1 mmol), 2,6-diaminopyrimidin-4-one and dry DMF (10 mL).
The mixture was stirred at room temperature for 3 days. The reac-
tion mixture was dried in vacuo and the residue was purified by
flash chromatography on silica gel (gradient, 1–5% MeOH in CHCl3)
to afford 970 mg (44%) of 23 as a yellow solid (mp, Rf, 1H NMR as
that reported)27 and 23 mg (10%) of 24 as an off-white solid; TLC
Rf 0.60 (CHCl3/CH3OH, 5:1); mp 152–153 °C; 1H NMR (DMSO-d6)
d 4.01 (s, 2H, CH2), 4.85 (s, 2H, CH2), 5.12 (s, 1H, CH), 5.92 (s, 2H,
NH2), 6.12 (s, 2H, NH2), 7.35–7.60 (m, 3H, C6H3). HRMS (EI): calcd
for C13H12N4O2Cl2 m/z = 326.0337, found m/z = 326.0337.
4. Experimental
4.1. Synthesis
Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-
DRY drying apparatus over P2O5 at 80 °C. Melting points were deter-

3584
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
4.1.4. N-[4-Chloro-6-(2,4-dichlorobenzyl)-7H-pyrrolo[2,3-d]pyr-
imidin-2-yl]-2,2-dimethylpropanamide (33)
4.1.9. N4-(4-Isopropylphenyl)-6-(2,4-dichlorobenzyl)-7H-pyr-
rolo[2,3-d]pyrimidine-2,4-diamine (14)
To a 50-mL round-bottom flask, under nitrogen, was added
compound 23 (800 mg, 2.59 mmol) and excess Piv2O (4 equiv).
The mixture was stirred at 100–120 °C for 2 h. The reaction mix-
ture was cooled and hexane (40 mL) was added. The suspension
was filtered to afford 850 mg (85%) of 31 as a light brown solid;
TLC Rf 0.56 (CHCl3/MeOH, 10:1). To a 100-mL round-bottom flask
was added 31 (300 mg, 1.3 mmol) and POCl3 (12 mL). The mixture
was refluxed for 3 h. The reaction mixture was evaporated and
quenched with water (15 mL). The resulting solution was cooled
in an ice bath, and the pH was adjusted to 8–9 with dropwise addi-
tion of NH4OH. The mixture was diluted with CHCl3 (120 mL). The
organic layer was separated and dried over Na2SO4, filtered, and
concentrated under reduced pressure to afford a yellow solid.
The crude product was purified by flash chromatography on silica
gel (isocratic, CHCl3) to afford 180 mg (58%) of 33 as a yellow fluffy
solid; TLC Rf 0.81 (CHCl3/MeOH, 10:1); mp 122–124 °C; 1H NMR
(DMSO-d6) d 1.21 (s, 9H, C(CH3)3), 4.17 (s, 2H, CH2), 6.07 (s, 1H,
CH), 7.39–7.84 (m, 3H, C6H3), 10.01 (s, 1H, NH), 12.40 (s, 1H,
NH). HRMS (ESI) [M+Na]+: calcd for C19H21N4Cl3 m/z = 433.0729,
found m/z = 433.0744.
Compound 14 was synthesized as described for 8 with 4-isopro-
pylaniline and was obtained as an off-white solid (122 mg, 63%);
mp 218 °C; TLC Rf 0.54 (CHCl3/CH3OH, 10:1); 1H NMR (DMSO-d6)
d 1.19 (d, 6H, CH3, J = 6 Hz), 2.8 (m, 1H, CH, J = 6 Hz), 4.00 (s, 2H,
CH2), 5.63 (s, 2H, NH2), 6.02 (s, 1H, CH), 7.09 (d, 2H, C6H4,
J = 9 Hz), 7.77 (d, 2H, C6H4, J = 9 Hz), 7.37–7.64 (m, 3H, C6H3),
8.69 (s, 1H, NH), 10.86 (s, 1H, NH). Anal. (C22H21Cl2N5) C, H, N, Cl.
4.1.10. N4-(2-Isopropylphenyl)-6-(2,4-dichlorobenzyl)-7H-pyr-
rolo[2,3-d]pyrimidine-2,4-diamine (15)
Compound 15 was synthesized as described for 8 with 2-isopro-
pylaniline and was obtained as a white solid (102 mg, 72%); TLC Rf
0.54 (CHCl3/CH3OH, 10:1); mp 216 °C; 1H NMR (DMSO-d6) d 1.09
(d, 6H, CH3, J = 6 Hz), 3.17 (m, 1H, CH, J = 6 Hz), 3.90 (s, 2H, CH2),
5.40 (s, 2H, NH2), 5.44 (s, 1H, CH), 7.15–7.59 (m, 7H, C6H4 and
C6H3), 8.33 (s, 1H, NH), 10.74 (s, 1H, NH). Anal. (C22H21Cl2N5Á0.05
CHCl3) C, H, N, Cl.
4.1.11. N4-(3-Bromophenyl)-6-(2-methylbenzyl)-7H-pyrrolo-
[2,3-d]pyrimidine-2,4-diamine (5)
Compound 5 was synthesized as described for 8 with 34 and 3-
bromoaniline and was obtained as an off white solid (315 mg,
70%); mp 226 °C (lit. mp 225–228 °C).27 The compound was iden-
tical in all respects to the mp, Rf, 1H NMR as that reported.27 Anal.
(C20H18BrN5Á0.04 CHCl3) C, H, N, Br.
4.1.5. N-[4-Chloro-6-(2-methylbenzyl)-7H-pyrrolo[2,3-d]pyrim-
idin-2-yl]-2,2-dimethylpropanamide (34)
Compound 34 was synthesized as described for 33 with 30 and
was obtained as a yellow fluffy solid (55%); Rf = 0.76 (CHCl3/MeOH
10:1); mp 117–119 °C; 1H NMR (DMSO-d6) d 1.22 (s, 9H, C(CH3)3),
2.27 (s, 3H, CH3), 4.05 (s, 2H, CH2), 6.07 (s, 1H, CH), 7.15–7.19 (m,
4H, C6H4), 9.99 (s, 1H, NH), 12.36 (s, 1H, NH). HRMS (ESI) [M+H]+:
calcd for C19H22N4OCl m/z = 357.1482, found m/z = 357.1470.
4.1.12. N4-(3-Fluorophenyl)-6-(2,4-dichlorobenzyl)-7H-pyrrolo-
[2,3-d]pyrimidine-2,4-diamine (10)
To a 2–5 mL microwave reaction vial was added 33 (300 mg,
0.73 mmol), 3-fluoroaniline (1.5 equiv), iPrOH (4 mL), DMF (1 mL)
and four drops of concd HCl. The reaction mixture was irradiated
in a microwave apparatus at 120 °C for 45 min. After being cooled,
the reaction mixture was dried in vacuo. The residue was neutral-
ized with NH4OH (1 mL) and extracted with CHCl3 (40 mL). The or-
ganic layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford a yellow solid. The crude product was
purified by flash chromatography on silica gel (gradient, CHCl3 to
2% MeOH/CHCl3) and washed with diethyl ether to afford 180 mg
(61%) of 6 as a light yellow solid; TLC Rf 0.56 (CHCl3/CH3OH,
10:1); mp 221 °C; 1H NMR (DMSO-d6) d 4.01 (s, 2H, CH2), 5.82 (s,
2H, NH2), 6.05 (s, 1H, CH), 6.69–8.08 (m, 7H, C6H4 and C6H3),
8.94 (s, 1H, NH), 10.96 (s, 1H, NH). Anal. (C19H14Cl2FN5Á0.11 CHCl3)
C, H, N, F, Cl.
4.1.6. N4-(2-Bromo-4-chlorophenyl)-6-(2,4-dichlorobenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (8)
To a 100-mL round-bottom flask was added 33 (200 mg,
0.48 mmol), 2-bromo-4-chloroaniline (1.5 equiv), iPrOH (20 mL)
and six drops of concd HCl. The mixture was refluxed for 12 h. After
being cooled, the reaction mixture was dried in vacuo. The residue
was neutralized with NH4OH (1 mL) and extracted with CHCl3
(30 mL). The organic layer was dried over Na2SO4, filtered, and con-
centrated under reduced pressure to afford a yellow solid. The
crude product was purified by flash chromatography on silica gel
(gradient, CHCl3 to 2% MeOH/CHCl3) and washed with diethyl
ether to afford 180 mg (74%) of 8 as a white solid; TLC Rf 0.58
(CHCl3/CH3OH, 10:1); mp 203 °C; 1H NMR (DMSO-d6) d 3.99 (s,
2H, CH2), 5.60 (s, 2H, NH2), 5.91 (s, 1H, CH), 7.36–7.72 (m, 6H,
C6H3 and C6H3), 8.45 (s, 1H, NH), 10.91 (s, 1H, NH). Anal.
(C19H13Cl3BrN5Á0.35H2O) C, H, N, Br, Cl.
4.1.13. N4-(3-Trifluoromethylphenyl)-6-(2,4-dichlorobenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (11)
4.1.7. N4-(2-Fluoro-4-chlorophenyl)-6-(2,4-dichlorobenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (9)
Compound 11 was synthesized as described for 10 with 3-tri-
fluoromethylaniline and was obtained as an off white solid
(65%); TLC Rf 0.56 (CHCl3/CH3OH, 10:1); mp 216 °C; 1H NMR
(DMSO-d6) d 4.02 (s, 2H, CH2), 6.04 (s,1H, CH), 5.78 (s, 2H, NH2),
6.69 (t, 1H, C6H4), 7.20–8.46 (m, 7H, C6H4 and C6H3), 9.07 (s,1H,
NH), 11.00 (s, 1H, NH). Anal. (C20H14Cl2F3N5Á0.03 CHCl3) C, H, N,
F, Cl.
Compound 9 was synthesized as described for 8 with 4-chloro-
2-fluoroaniline and was obtained as a light yellow solid (86%); TLC
Rf 0.57 (CHCl3/CH3OH, 10:1); mp 214 °C; 1H NMR (DMSO-d6) d 4.00
(s, 2H, CH2), 5.62 (s, 2H, NH2), 5.99 (s, 1H, CH), 7.19–7.90 (m, 6H,
C6H3 and C6H3), 8.95 (s, 1H, NH), 10.92 (s, 1H, NH). Anal.
(C19H13Cl3FN5Á0.3H2O) C, H, N, F, Cl.
4.1.14. N4-(3-Fluoro-4-trifluoromethylphenyl)-6-(2,4-dichloro-
benzyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (13)
4.1.8. N4-(3-Ethynylphenyl)-6-(2,4-dichlorobenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (12)
Compound 13 was synthesized as described for 10 with 3-flu-
oro-4-trifluoromethylaniline and was obtained as an off white so-
lid (57%); TLC Rf 0.58 (CHCl3/CH3OH, 10:1); mp 220–222 °C; 1H
NMR (DMSO-d6) d 4.00 (s, 2H, CH2), 6.01 (s, 1H, C5-H), 5.79 (s,
2H, NH2), 7.26–8.19 (m, 6H, Ar-H), 8.91(s,1H, NH), 10.97 (s, 1H,
NH). Anal. (C20H13Cl2F4N5Á0.44 CH3OH) C, H, N, F, Cl.
Compound 12 was synthesized as described for 8 with 3-ethy-
nylaniline and was obtained as a yellow solid (52 mg, 53%); TLC Rf
0.55 (CHCl3/CH3OH, 10:1); mp 219 °C; 1H NMR (DMSO-d6) d 4.01
(s, 2H, CH2), 4.1 (s, 1H, CH), 5.72 (s, 2H, NH2), 6.04 (s,1H, CH),
6.90–8.06 (m, 7H, C6H4 and C6H3), 8.81 (s, 1H, NH), 10.92 (s,1H,
NH). Anal. (C21H15Cl2N5Á0.21H2O) C, H, N, Cl.

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3585
4.1.15. Ethyl-2-amino-5-(2-methylbenzyl)-1H-pyrrole-3-carboxy-
late (37)
To a 250-mL round-bottom flask, under nitrogen was added car-
bethoxyacetamidine hydrochloride 36,44 NEt3 (20 mL) and ethyl
acetate (40 mL). The reaction mixture was stirred for at room tem-
separated and dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford 150 mg (71%) of 44 as a white solid; TLC
Rf 0.61 (CHCl3/MeOH, 10:1); mp 172–176 °C; 1H NMR (DMSO-d6) d
3.70 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 4.05 (s, 2H, CH2), 6.12 (s, 1H,
CH), 6.82–6.83 (m, 3H, C6H3), 8.52 (s, 1H, CH), 12.53 (s, 1H, NH).
perature for 20 min, and then
a-bromomethyl-(2-methylben-
zyl)ketone (2 g, 9 mmol),27 was added and the mixture was
refluxed for 1 h. After being cooled, the reaction mixture was dried
in vacuo. The crude product was purified by flash chromatography
on silica gel (gradient, 10–50%. EtOAc/hexane) to afford 1.3 g (60%)
of 37 as a red semisolid that degraded on storage and was directly
used in the next step; TLC Rf 0.46 (EtOAc/hexane, 1:4); 1H NMR
(DMSO-d6) d 1.17 (t, 3H, CH3, J = 6 Hz), 2.23 (s, 3H, CH3), 3.65 (s,
2H, CH2), 4.01 (q, 2H, CH2, J = 6 Hz), 5.48 (s, 2H, NH2), 7.11–7.13
(m, 4H, C6H4), 9.96 (s, 1H, NH).
4.1.21. N-4-Chloro-6-(2,4-dichlorobenzyl)-3,7-dihydro-4H-pyrrolo-
[2,3-d]pyrimidine (45)
To a 250-mL round-bottom flask, under nitrogen was added 38
(1.0 g, 5 mmol), NaOMe (0.3 g, 50 mmol), and formamide (100 mL).
The reaction mixture was heated to 160 °C for 12 h. The reaction
mixture was cooled and 41 was precipitated with ice. Compound
41 was collected by filtration and directly used for the synthesis
of 45. To a 100-mL round-bottom flask was added 41 (200 mg,
0.7 mmol) and POCl3 (12 mL). The mixture was refluxed for 3 h.
The reaction mixture was evaporated and quenched with water
(15 mL). The resulting solution was cooled in an ice bath, and the
pH was adjusted to 8–9 with dropwise addition of NH4OH. The
mixture was diluted with CHCl3 (120 mL). The organic layer was
separated and dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford compound 45 as a white solid (76%);
TLC Rf 0.65 (CHCl3/MeOH, 10:1); 1H NMR (DMSO-d6) 4.23 (s, 2H,
CH2), 6.13 (s, 1H, CH), 7.39–7.65 (m, 3H, C6H3), 8.5 (s, 1H, CH),
12.6 (s, 1H, NH). Anal. (C13H8Cl3N3) C, H, N, Cl.
4.1.16. Ethyl-2-amino-5-(2,4-dichlorobenzyl)-1H-pyrrole-3-carb-
oxylate (38)
Compound 38 was synthesized as described for 37 with a-bro-
momethyl-(2,4-dichlorobenzyl)ketone and was obtained as an or-
ange semisolid (70%) that degraded on storage and was directly
used in the next step; TLC Rf 0.5 (hexane/ethylacetate,1:4); 1H
NMR (DMSO-d6) d 1.20 (t, 3H, CH3), 3.49(s, 1H, C5-H), 4.02 (s,
2H, CH2), 4.23 (q, 2H, CH2), 5.59 (s, 2H, NH2), 7.24–7.58 (m, 3H,
C6H4), 10.08 (s, 1H, NH).
4.1.22. N-4-(3-Bromophenyl)-6-(2-methylbenzyl)-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-amine (16)
4.1.17. Ethyl-2-amino-(2,5-dimethoxybenzyl)-1H-pyrrole-3-carb-
oxylate (39)
To a 100-mL round-bottom flask was added 40 (200 mg,
0.7 mmol) and POCl3 (12 mL). The mixture was refluxed for 3 h.
The reaction mixture was evaporated and quenched with water
(15 mL). The resulting solution was cooled in an ice bath, and the
pH was adjusted to 8–9 with dropwise addition of NH4OH. The mix-
ture was diluted with CHCl3 (120 mL). The organic layer was sepa-
rated and dried over Na2SO4, filtered, and concentrated under
reduced pressure to afford 133 mg of 43 as a residue that was treated
with 3-bromoaniline (1.5 equiv), iPrOH (20 mL) and 2–3 drops of
concd HCl. The mixture was refluxed for 4 h. After being cooled,
the reaction mixture was dried in vacuo. The residue was neutral-
ized with NH4OH (1 mL) and extracted with CHCl3 (30 mL). The or-
ganic layer was dried over Na2SO4, filtered, and dried in vacuo to
afford 16 as a yellow solid. The crude product was purified by flash
chromatography on silica gel (gradient, CHCl3 to 2% MeOH/CHCl3)
and washed with diethyl ether to afford 170 mg (84%) of 16 as a
white solid; TLC Rf 0.56 (CHCl3/CH3OH, 10:1); mp 242–244 °C; 1H
NMR (DMSO-d6): d 2.26 (s, 3H, CH3), 4.03 (s, 2H, CH2), 6.24 (s, 1H,
CH), 7.11–7.79 (m, 8H, C6H4 and C6H4), 8.26 (s, 1H, CH), 9.29 (s,
1H, NH), 11.85 (s, 1H, NH). Anal. (C20H17BrN4Á0.17H2O) C, H, N, Br.
Compound 39 was synthesized as described for 37 with a-bro-
momethyl(2,5-dimethoxybenzyl)ketone and was obtained as a red
semisolid (45%) that degraded on storage and was directly used in
the next step; TLC Rf 0.40 (EtOAc/hexane, 1:4); 1H NMR (DMSO-d6)
d 1.16 (t, 3H, CH3), 3.67 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.95 (s,
2H, CH2), 4.20 (q, 2H, CH2), 5.75 (s, 2H, NH2), 6.73–6.88 (m, 3H,
C6H3), 9.98 (s, 1H, NH).
4.1.18. 6-(2-Methylbenzyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrim-
idin-4-one (40)
To a 250-mL round-bottom flask, under nitrogen was added 37
(1.3 g, 5.4 mmol), NaOMe (0.3 g, 50 mmol), and formamide
(100 mL). The reaction mixture was heated to 160 °C for 12 h.
The reaction mixture was cooled and the product was precipitated
with ice. The precipitate was collected by filtration and dried in va-
cuo to afford 0.54 g (45%) of 40 as a pale brown solid; TLC Rf 0.55
(CHCl3/MeOH, 5:1); 1H NMR (DMSO-d6) 2.26 (s, 3H, CH3), 3.92 (s,
2H, CH2), 5.95 (s, 1H, CH), 7.12–7.15 (m, 4H, C6H4), 7.76 (s, 1H,
CH), 11.71 (s, 1H, NH), 11.80 (s, 1H, NH).
4.1.19. 6-(2,5-Dimethoxybenzyl)-3,7-dihydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (41)
4.1.23. N-4-(3-Bromophenyl)-6-(2,5-dimethoxybenzyl)-3,7-dihy-
dro-4H-pyrrolo[2,3-d]pyrimidin-4-amine (17)
Compound 41 was synthesized as described for 40 with 39 and
was obtained as a brown solid (40%); TLC Rf 0.51 (CHCl3/MeOH,
5:1); mp 220 °C; 1H NMR (DMSO-d6) d 3.67 (s, 3H, OCH3), 3.74 (s,
3H, OCH3), 3.86 (s, 2H, CH2), 6.02 (s, 1H, CH), 6.71–6.92 (m, 3H,
C6H3), 7.76 (s, 1H, CH), 11.71 (s, 1H, NH), 11.75 (s, 1H, NH). HRMS
(ESI) [M+Na]+: calcd for C15H15N3O3Na m/z = 308.1011, found m/
z = 308.1013.
To a 100-mL round-bottom flask was added 44, 3-bromoaniline
(1.5 equiv), iPrOH (20 mL) and 2–3 drops of concd HCl. The mixture
was refluxed for 4 h. After being cooled, the reaction mixture was
dried in vacuo. The residue was neutralized with NH4OH (1 mL)
and extracted with CHCl3 (30 mL). The organic layer was dried over
Na2SO4, filtered, and dried in vacuo to afford 17 as a white solid
(55%); TLC Rf 0.54 (CHCl3/CH3OH, 10:1); mp 208–210 °C; 1H NMR
(DMSO-d6) d 3.67 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.96 (s, 2H,
CH2), 6.29 (s, 1H, CH), 6.80–7.81 (m, 7H, C6H4 and C6H3), 8.26 (s,
1H, CH), 9.28 (s,1H, NH), 11.79 (s,1H, NH). Anal. (C21H19BrN4O2)
C, H, N, Br.
4.1.20. N-4-Chloro-6-(2,5-dimethoxybenzyl)-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidine (44)
To a 100-mL round-bottom flask was added 41 (200 mg,
0.7 mmol) and POCl3 (12 mL). The mixture was refluxed for 3 h.
The reaction mixture was evaporated and quenched with water
(15 mL). The resulting solution was cooled in an ice bath, and the
pH was adjusted to 8–9 with dropwise addition of NH4OH. The
mixture was diluted with CHCl3 (120 mL). The organic layer was
4.1.24. N-4-(3-Bromophenyl)-6-(2,4-dichlorobenzyl)-3,7-dihydro-
4H-pyrrolo[2,3-d]pyrimidin-4-amine (18)
Compound 18 was synthesized as described for 17 with 45 and
was obtained as a white solid (77%); TLC Rf 0.61 (CHCl3/CH3OH,

3586
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
10:1); mp 264–265 °C; 1H NMR (DMSO-d6) d 4.15 (s, 2H, CH2), 6.27
(s, 1H, CH), 7.15–7.67 (m, 7H, C6H4 and C6H3), 8.29 (s, 1H, CH), 9.29
(s, 1H, NH), 11.92 (s, 1H, NH). Anal. (C19H13BrCl2N4) C, H, N, Br.
50 for PDGFR-b. Erlotinib, 1 and sunitinib, 4 were also evaluated
against VEGFR-2, EGFR and PDGFR-b in this assay. Data were
graphed as a percent of cells receiving growth factor alone and
IC50 values estimated from 2–3 separate experiments (n = 8–24)
using non-linear regression Sigmoidal dose–response analysis with
GraphPad Prism (San Diego, CA). In every case, the activity of a posi-
tive control inhibitor did not deviate more than 10% from the IC50
values listed in the text.
4.1.25. N-4-(3-Ethynylphenyl)-6-(2,4-dichlorobenzyl)-3,7-dihydro-
4H-pyrrolo[2,3-d]pyrimidin-4-amine (19)
Compound 19 was synthesized as described for 17 with 45 and
3-ethynylaniline and was obtained as a yellow solid (65%); TLC Rf
0.57 (CHCl3/CH3OH, 10:1); mp 243–245 °C; 1H NMR (DMSO-d6) d
4.11 (s, 2H, CH2), 4.11 (s, 1H, CH), 6.29 (s, 1H, CH), 7.06–8.10 (m,
7H, C6H4 and C6H3), 8.28 (s, 1H, CH), 9.28 (s, 1H, NH), 11.89 (s,
1H, NH). Anal. (C21H14Cl2N4) C, H, N, Cl.
4.4. Antiproliferative assay
The assay was performed as described previously.47 Briefly, cells
were first treated with compounds for 12 h and then allowed to
grow for an additional 36 h. The cells were then lysed and the CY-
QUANT dye, which intercalates into the DNA of cells, was added
and after 5 min the fluorescence of each well measured using an
UV Products BioChemi digital darkroom. Cisplatin, 47 was used
as the standard for cytotoxicity in each experiment. Data were
graphed as a percent of cells receiving growth factor alone and
IC50 values estimated from 2 to 3 separate experiments (n = 6–
15) using non-linear regression Sigmoidal dose–response analysis
with GraphPad Prism (San Diego, CA).
4.1.26. N-4-(3-Trifluoromethylphenyl)-6-(2,4-dichlorobenzyl)-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-amine (20)
Compound 20 was synthesized as described for 17 with 45 and
3-trifluoromethylaniline and was obtained as a white solid (77%);
TLC Rf 0.58 (CHCl3/CH3OH, 10:1); mp 244–246 °C; 1H NMR
(DMSO-d6) d 4.18 (s, 2H, CH2), 6.28 (s, 1H, CH), 7.28–8.17 (m, 7H,
C6H4 and C6H3), 8.30 (s, 1H, CH), 9.44 (s, 1H, NH), 11.92 (s, 1H,
NH). Anal. (C20H13Cl2F3N4Á0.3H2O) C, H, N, F, Cl.
4.2. Biological evaluation
4.5. In vivo antitumor activity
All cells were maintained at 37 °C in a humidified environment
containing 5% CO2 using media from Mediatech (Hemden, NJ, USA).
The A-431 cells were from the American Type Tissue Collection
(Manassas, VA, USA). All growth factors (bFGF, VEGF, EGF, PDGF-
BB) were purchased from Peprotech (Rocky Hill, NJ, USA). The
PY-HRP antibody was from BD Transduction Laboratories (Franklin
Lakes, NJ, USA). Antibodies against EGFR, PDGFRb, FGFR-1, Flk-1,
and Flt-1 were purchased from Upstate Biotech (Framingham,
MA, USA). The CYQUANT cell proliferation assay was from Molec-
ular Probes (Eugene, OR, USA).The standard compounds used for
comparison in the assays were purchased from Calbiochem (San
Diego, CA, USA).
In vivo antitumor activity of compounds was tested at a dose of
35 mg/kg for 5 and 8, and 10 mg/kg of standard compound 51
(SU6668) three times a week by intraperitoneal route against the
B16-F10 (lung colonizing) melanoma implanted in athymic NCr
Nu/Nu male mice. 50,000 B16-F10 mouse melanoma cells were in-
jected orthotopically SQ just behind the ear of the mice, 8 weeks in
age. Two experiments were done starting with 5–6 animals/group.
Animals were monitored every other day for the presence of tu-
mors. Drug treatment was started 9 days after tumor implantation,
the time at which most tumors were measurable by calipers.
DMSO stocks (30 mM) of drugs were dissolved into sterile water
for injection and 35 mg/kg was injected intraperitoneally (IP) every
Monday (AM), Wednesday (noon) and Friday (PM). Sham treated
animals received water only Monday, Wednesday and Friday. Val-
ues are mean SEM, ***P <0.001, **P <0.01 versus untreated ani-
mals using one-way ANOVA with Neuman–Keuls post-test.
4.3. Inhibition of cellular tyrosine phosphorylation
Inhibition of EGF, VEGF and PDGF-BB-stimulated total cellular
tyrosine phosphorylation in tumor cells naturally expressing high
levels of EGFR (A431), VEGFR-2 (U251), VEGFR-1 (A498) and
PDGFR-b (SF-539), respectively, were measured using the ELISA as-
say as previously reported.38 Briefly, cells at 60–75% confluence
were placed in serum-free medium for 18 h to reduce the back-
ground of phosphorylation. Cells were always >98% viable by Trypan
blue exclusion. Cells were then pre-treated for 60 min with 333, 100,
4.5.1. Tumor growth
The length (long side), width (short side) and depth of the tu-
mors were measured using digital Vernier Calipers each Monday,
Wednesday, and Friday. Tumor volume was calculated using the
formula: length  width  depth. Tumor growth rate was calcu-
lated using a linear regression analysis algorithm using the soft-
ware GraphPad Prism 4.0 as the slope of the curve from day 9
after tumor implantation, the date of first treatment, out to day
28 of the experiment. The tumor volumes at the first measurement
(day 9, day of first treatment) were untreated:12.3 1.8 mm3,
compound 5: 13.24 3.0 mm3, compound 8: 14.1 2.1 mm3 and
51 (SU6668): 11.9 1.2 mm3.
33.3, 10, 3.33, 1.00, 0.33 and 0.10 lM compound followed by 100 ng/
mL EGF, VEGF, PDGF-BB, or bFGF for 10 min. The reaction was
stopped and cells permeabilized by quickly removing the media
from the cells and adding ice-cold Tris-buffered saline (TBS) contain-
ing 0.05% triton X-100, protease inhibitor cocktail and tyrosine
phosphatase inhibitor cocktail. The TBS solution was then removed
and cells fixed to the plate by 30 min at 60 °C and further incubated
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 phosphotyrosine antibody was added
overnight. The antibody was removed, cells were washed again in
TBS, exposed to an enhanced luminol ELISA substrate (Pierce Chem-
ical, Rockford, IL, USA) and light emission measured using an UV
Products (Upland, CA, USA) BioChemi digital darkroom. Standard
compounds were used as controls in each of the evaluations. The
standard compounds used were semaxanib, 46 for VEGFR-2; (4-
chloro-2-fluorophenyl)-6,7-dimethoxy quinazolin-4-yl-amine, 48
for VEGFR-1; 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazo-
line, 49 for EGFR; 3-(4-dimethylamino-benzylidenyl)-2-indolinone,
4.5.2. Tumor metastases and vascularity
At the experiment’s end, day 28, the animals were humanely
euthanized using carbon dioxide, tumors and lungs excised, fixed
in 20% neutral buffered formalin for 8–10 h, embedded into paraf-
fin, and hematoxylin–eosin (H&E) stain of three separate tissue
sections completed to span the tumor/lung. Metastases per lung
lobe were counted using the H&E stained sections. Metastasis
was seen as purple clusters of disorganized cells on the highly or-
ganized, largely pink lung. Blood vessels per unit area were
counted in five fields at 100Â magnification and averaged. Values

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 3575–3587
3587
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Acknowledgements
This work was supported, in part, by Grant CA98850 (A.G.) from
the National Cancer Institute, National Institutes of Health, and an
equipment grant from the National Science Foundation (NMR: CHE
0614785).
Supplementary data
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