N4-Aryl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2, 4-diamines as receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.058

Six novel N⁴-substitutedphenyl-6-substitutedphenylmethyl-7H- pyrrolo[2,3-d]pyrimidine-2,4-diamines were synthesized as multiple receptor tyrosine kinase (RTK) inhibitors and antitumor agents. An improvement in the inhibitory potency against epi

Full text of DOI:10.1016/j.bmc.2011.11.058

Bioorganic & Medicinal Chemistry 20 (2012) 910–914
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N4-Aryl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines
as receptor tyrosine kinase inhibitors
Aleem Gangjee a, , Sonali Kurup a, Michael A. Ihnat b, Jessica E. Thorpe b, Bryan Disch b
⇑
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
b Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, College of Pharmacy, Oklahoma City, OK 73104, USA
a r t i c l e i n f o
a b s t r a c t
Six novel N4-substitutedphenyl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines
were synthesized as multiple receptor tyrosine kinase (RTK) inhibitors and antitumor agents. An improve-
ment in the inhibitory potency against epidermal growth factor receptor (EGFR), vascular endothelial
growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) assays
and in the A431 cellular proliferation assay was observed for compounds 8–13 over the previously reported
5–7. Three compounds (8, 9 and 13) demonstrated potent, multiple RTK inhibition and were more potent or
equipotent compared to the lead compounds 5 and 7 and the standard compounds. Compounds 10 and 12
showed potent inhibition of VEGFR-2 over EGFR, platelet-derived growth factor receptor-b (PDGFR-b) and
VEGFR-1. The results indicate that the RTK inhibitory profile could be modulated with slight variations to
the N4-aryl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamino scaffold.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 21 June 2011
Revised 18 November 2011
Accepted 25 November 2011
Available online 8 December 2011
Keywords:
Pyrrolo[2,3-d]pyrimidines
Receptor tyrosine kinase inhibitors
1. Introduction
for angiogenesis and often develop resistance to agents that target
one specific pathway.9,10 A multitargeted approach that inhibits
Dysfunctional receptor tyrosine kinases (RTKs) have been asso-
ciated with several cancers where they play a pivotal role in tumor
angiogenesis.1,2 Angiogenesis requires the transduction of signals
from the extracellular domain of endothelial cells to the nucleus
which is mediated by RTKs.3 Solid tumors require angiogenesis
to grow beyond 1–2 mm in size and metastasis requires the pres-
ence of blood vessels to allow transport of tumor cells to sites
distal to the primary tumor.3,4 Inhibition of tumor angiogenesis
prevents the growth and metastasis of several types of solid
tumors. Thus, inhibition of angiogenesis via RTK inhibition pro-
vides an attractive target for the treatment of cancer.1,5 Among
the RTKs implicated in tumor progression and angiogenesis are
members of the VEGFR family namely VEGFR-1 and VEGFR-2,
members of the EGFR family and members of the PDGFR family,
multiple signaling pathways has shown to be more effective than
the inhibition of a single target.10,11 Sorafenib, 3 an inhibitor of
VEGFR, PDGFR and Raf-1 kinase and sunitinib, 4 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 (CSF-1)
have been approved for renal cell carcinoma, and sunitinib most
recently for pancreatic cancer.12,13
Gangjee et. al.14 previously reported compounds 5–7 (Fig. 2) as
multiple RTK inhibitors in
a
series of N4-(3-bromophenyl)-
6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mines. It was demonstrated that variation of the phenyl
substituents in the 6-benzyl moiety determined both the potency
and specificity of inhibitory activity against various RTKs. To further
develop the structure–activity relationship, it was of interest to
determine if variation in the anilino moiety could similarly
influence potency and specificity for RTK inhibition. Thus, com-
pounds 8–13 (Fig. 2) were synthesized as analogs of 5–7 with two
different 4-anilino moieties in combination with the 6-benzyl
sidechains of 5–7. We elected the 4-chloro anilino and 4-chloro-
2-fluoroanilino substitutions in compounds 8–13 on the basis of
the potent multiple RTK inhibition seen for these anilines in 6–6
fused systems such as quinazolines, pthalazines and pyrido[2,
3-d]pyrimidines.15–19 Vatalinib bearing the 4-chloro anilino substi-
tution, has shown potent VEGFR-1 and VEGFR-2 inhibition,15,16
while quinazolines bearing the 4-chloro-2-fluoroanilino substitu-
tion have shown potent, dual VEGFR-2 and EGFR inhibition.17,18
The 2-NH2 moiety in 8–13 was maintained to provide additional
namely PDGFR-a
and PDGFR-b.5,6
Small molecule RTK inhibitors targeting the ATP binding site of
tyrosine kinases are currently in clinical use while others are in
clinical trials as antitumor agents.5,7,8 Initial strategies for RTK inhi-
bition focused on single RTK inhibitors such as erlotinib, 1 and gef-
itinib, 2 that were approved for non small cell lung cancer
(Fig. 1).7,8 However, tumors have redundant signaling pathways
Abbreviations: RTK, receptor tyrosine kinase; EGFR, epidermal growth factor
receptor; VEGFR, vascular endothelial growth factor receptor; CSF-1, colony
stimulating factor; PDGFR, platelet-derived growth factor receptors.
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.058

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 910–914
911
F
Cl
NH
N
O
NH
N
O
N
O
N
N
O
O
O
O
1
2
, Gefitinib
, Erlotinib
O
N
Cl
N
H
HN
O
O
CF3
NH
N
H
F
N
O
O
N
H
N
H
3
4
, Sunitinib
, Sorafenib
Figure 1. Reported RTK inhibitors.
R
Ar
2-MePh
2,3-C4H4Ph
2,5-diOMePh
R
Cl
R
5
6
7
8
9
3-Br
3-Br
3-Br
H2N
HN
Ar
17
a
N
R
Ar
R
N
HN
N
65-70%
H2N
N
2-F, 4-Cl 2-MePh
4-Cl 2-MePh
H
N
H
H2N
N
N
14 Ar = 2-MePh
10 2-F, 4-Cl 2,3-C4H4Ph
15 Ar = 1-naphthyl
8-11
N
H
Ar
11
H2N
N
4-Cl
12 2-F, 4-Cl 2,5-diOMePh
13
2,3-C4H4Ph
Cl
4-Cl
2,5-diOMePh
R
H2N
Ar
N
a, b
HN
17
Figure 2. Target compounds 8–13 as analogs of 5–7.
N
H
Ar
HN
Piv
N
N
61-86%
N
H2N
N
hydrogen bonding in the Hinge region of RTKs compared to other
known RTK inhibitors that lack this 2-NH2 moiety. The flexible 6-
benzyl substitutions were incorporated to allow for multiple con-
formations of this side chain and to perhaps afford interactions with
multiple RTKs.
H
16 Ar =2,5-diOMePh
12-13
Scheme 1. Reagents and conditions: (a) 17, iPrOH, 2–3 drops concd HCl, 4 h, reflux;
(b) KOH, 1,4-dioxane, reflux.
2. Chemistry
evaluations. The standard compounds used were semaxanib, 18
for VEGFR-220; 19, (4-chloro-2-fluorophenyl)-6,7-dimethoxy qui-
nazolin-4-yl-amine (CB676475) for VEGFR-117; 20, 4-[(3-bromo-
The synthesis of compounds 8–13 is shown in Scheme 1. The
4-chloro-6-substituted pyrrolo[2,3-d]pyrimidines, 14–1514 were
synthesized in five steps from the corresponding phenylacetic
acids. Treatment with the appropriate aniline, 17 in isopropanol
and a few drops of concd HCl at reflux afforded compounds
8–11. Reaction of 1614 (synthesized in six steps from 2,5-dime-
thoxyphenylacetic acid) with the appropriate aniline 17 in isopro-
panol and a few drops of concd HCl, followed by depivaloylation
with base, at reflux afforded compounds 12–13.
phenyl)amino]-6,7-dimethoxyquinazoline (PD153035) for EGFR21
;
and 21, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI)
for PDGFR-b.22 Cisplatin, 22 was used as the standard for the A431
cytoxicity assay. For a better demonstration of the kinase inhibitory
potential of the target compounds described in this study, two clin-
ically approved RTK inhibitors, erlotinib, 1 and sunitinib, 4 were
incorporated for comparison.7,13 The inhibitory potencies (IC50
values) of 8–13 are also compared with the previously synthesized
compounds 5–7 and standard compounds 1, 4, 18–22 in Table 1.
In the 2-methylphenylsubstituted compounds, 9 with the 4-
chloro anilino substitution showed potent EGFR inhibition, being
equipotent to the standard compound, PD153035, fivefold more
potent than erlotinib and 38-fold better than compound 5. EGFR
inhibition decreased for the 2-fluoro-4-chloro anilino derivative 8
compared to 5. In the 6-(1-naphthyl)substituted compounds, vari-
ation of the 3-bromo anilino moiety to the 2-fluoro-4-chloro anili-
no in 10 and the 4-chloro anilino substitution in 11 did not
improve EGFR inhibition. For the 2,5-dimethoxyphenylsubstituted
pyrrolo[2,3-d]pyrimidines, EGFR inhibition further decreased on
variation to the 2-fluoro-4-chloro anilino substitution in 12 and
the 4-chloro anilino substitution in 13.
3. Results and discussions
The RTK inhibitory activities of compounds 8–13 were evaluated
in human tumor cells known to express high levels of EGFR, VEGFR-
2, VEGFR-1 and PDFGR-b using a phosphotyrosine ELISA assay.19 The
effect of compounds 8–13 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.19 Cellular evaluations
of RTK inhibitory activities afford more meaningful results for trans-
lation to in vivo studies than direct enzymatic assays. Since the IC50
valuesofcompoundsvaryunderdifferentassayconditions, standard
compounds 18–22 ( Fig. 3) were used as controls in each of the

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A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 910–914
decreased VEGFR-1 inhibition compared to 6 and the standard
Br
CB676475. Among the 2,5-dimethoxyphenylsubstituted com-
pounds, VEGFR-1 inhibition significantly decreased for the 2-flu-
oro-4-chloro anilino substituted compound 12 compared to 7 and
CB676475, and improved for the 4-chloro anilino compound 13.
Compound 13 showed a twofold better and almost equipotent VEG-
FR-1 inhibition compared to 7 and CB676475, respectively.
In the PDGFR-b assay, variation of the 3-bromo anilino moiety to
the 2-fluoro-4-chloro anilino and the 4-chloro anilino substitution
did not provide PDGFR-b inhibition in the 6-(2-methylphenyl)
substituted or 6-(1-naphthyl)substituted compounds 8–11. Among
the 2,5-dimethoxyphenylsubstituted compounds, the PDGFR-b
inhibitory activity decreased with variation in the anilino moiety
in 12 and 13 compared to 7.
A431 cytotoxicity significantly decreased for the 2-fluoro-4-
chloro anilino substituted compounds 8 and 10, while moderate
cytoxicity in the A431 proliferation assay was seen for the 4-chloro
anilino substituted compounds 9 and 11. Compound 9 was twofold
more potent than cisplatin and approximately fivefold less potent
than 5. Compound 11 showed a fourfold better inhibition compared
to 6, and was almost equipotent to cisplatin. In the 2,5-dimethoxy-
phenylsubstituted compounds, an improvement in A431 cytotoxic-
ity was observedon variationin the anilino moiety. Both compounds
12 and 13 were better or equivalent to the standard cisplatin, and
fourfold and eightfold better than 7, respectively.
Multiple kinase inhibitory profiles were observed for com-
pounds 8, 9 and 13. Compound 8 was a dual VEGFR-1 and VEGFR-
2 inhibitor. Compound 9 demonstrated potent EGFR, moderate
VEGFR-1 and VEGFR-2 inhibition and potent A431 cytotoxicity.
Compound 13 demonstrated potent VEGFR-1 inhibition, A431 cyto-
toxicity and moderate VEGFR-2 inhibition. Submicromolar, specific
inhibition of VEGFR-2 over EGFR, VEGFR-1 and PDGFR-b was ob-
served for compounds 10 and 12. Interestingly, compounds 11–13
showed potent A431 cytoxicities although they did not show signif-
icant EGFR inhibition. The A431 cell lines depend on EGFR for sur-
vival; perhaps these compounds do not directly inhibit EGFR but
influence the downstream signaling of EGFR and crosstalk with
other kinases which may be necessary for the functioning of
EGFR.14 There have been several literature reports in which the
EGFR inhibitory activity does not translate into the A431 cytotoxic-
ity.23,24 It will be of interest to determine whether another kinase
(other than EGFR) also plays a role in A431 cell survival. Since the
inhibitory activities are determined in cells, it is not possible to
make a definite conclusion on the structure–activity relationship
for compounds 8–13.
F
Cl
HN
HN
N
H
OMe
OMe
OMe
OMe
N
N
O
N
H
N
N
18, semaxinib (SU5416)
20 (PD153035)
19 (CB676475)
N
Cl
H2N Pt Cl
NH2
O
N
H
22, cisplatin
21(DMBI)
Figure 3. Reported RTK Inhibitors used as standard compounds for comparison in
the cellular evaluation of 8–13.
In the VEGFR-2 assay, the variation of the 3-bromo anilino
substitution to the 2-fluoro-4-chloro anilino in compound 8 and
the 4-chloro anilino substitution in 9 showed a decrease in activity
compared to 5 among the 6-(2-methylphenyl)substituted pyrrol-
o[2,3-d]pyrimidines. In the 6-(1-naphthyl)substituted compounds,
the VEGFR-2 inhibitory activity improved in the 2-fluoro-4-chloro
anilino substituted compound 10 compared to 6 and was 25-fold
more potent than semaxanib, and 39-fold more potent than suni-
tinib, 4. Compound 11 with the 4-chloro anilino substitution
showed moderate VEGFR-2 inhibition and was approximately
threefold and twofold less potent compared to semaxanib and sun-
itinib, respectively. In the 2,5-dimethoxyphenylsubstituted com-
pounds, the VEGFR-2 inhibition improved with variation of the
2-fluoro-4-chloro anilino moiety in compound 12, but decreased
on variation to the 4-chloro anilino moiety in 13. Compound 12
was remarkably 120-fold and 189-fold more potent against VEG-
FR-2 compared to semaxanib and sunitinib, respectively.
In the VEGFR-1 assay, inhibitory potency improved with varia-
tion to the 2-fluoro-4-chloro anilino moiety in the 2-methylphenyl-
substituted compound 8 compared to 5 and was almost equipotent
to the standard CB676475. The 4-chloro anilino substituted com-
pound 9 was also almost equipotent to 5 and showed a fourfold
lower inhibition of VEGFR-1 compared to CB676475. In the 6-(1-
naphthyl)substituted compounds, variation to the 2-fluoro-4-
chloro anilino in 10 and the 4-chloro anilino substitution in 11
Table 1
IC50 values (lM) of kinase inhibition and A431 cytotoxicity for compounds 8–13
Cpd
EGFR
VEGFR-2
VEGFR-1
PDGFR-b
A431 cytotoxicity
5a
8
9
9.19 1.8
>200
0.24 0.031
>50
0.25 0.04
29.8 5.0
45.4 6.2
5.08 0.83
0.48 0.06
32.1 5.2
0.62 0.21
0.1 0.021
56.7 5.1
12.0 2.7
>50
>50
>300
>300
>50
193.2 20.1
>300
8.92 1.6
145.9 23.8
44.3 5.2
1.21 0.42
>200
5.6 0.08
>50
46.7 6.2
8.5 0.93
>50
13.4 0.21
5.8 0.62
16 1.9
45.9 4.3
19.2 4.3
>200
6a
10
11
7a
12
13
>200
112.1 18.2
12.62 3.3
112.7 20.2
>200
>200
31.1 5.8
185.6 27.5
15.2 0.26
Semaxanib
CB676475
PD153035
DMBI
14.1 2.8
0.23 0.04
3.75 0.06
Cisplatin
Erlotinib
Sunitinib
10.6 2.9
1.2 0.2
172.1 19.4
124.7 18.2
18.9 2.7
83.1 10.1
12.2 1.9
a
IC50 values from Ref. 14.

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 910–914
913
The crude product was purified by flash chromatography on silica
gel (gradient, CHCl3 to 2% MeOH/CHCl3) to afford 80 mg (70%) of 8
as a white solid; TLC Rf 0.54 (CHCl3/CH3OH, 10:1); mp 210 °C;
1H NMR (DMSO-d6) d 2.29 (s, 3H, CH3), 3.89 (s, 2H, CH2), 5.60 (s,
2H, NH2), 5.94 (s, 1H, C5–H), 7.16–7.92 (m, 7H, Ar–H), 8.62 (s,
1H, NH), 10.89 (s, 1H, NH). Anal. (C20H17ClFN5Á0.08 CHCl3) C, H,
N, F, Cl.
4. Conclusion
We synthesized six novel RTK inhibitors to determine the effect
of substitution in the 4-anilino ring along with variations in the 6-
substituent of the pyrrolo[2,3-d]pyrimidine scaffold. Compound 12
emerged as the most viable candidate for future evaluation with its
remarkable 120-fold and 189-fold increased potency against VEG-
FR-2 compared to semaxinib and sunitinib, respectively. Several
analogs (8, 9 and 13) demonstrated multiple RTK inhibition while
others (10 and 12) were specific for VEGFR-2. The cytotoxicity
against A431 cells in culture was somewhat better for some com-
pounds (9, 11 and 13) compared with cisplatin, although 9, 11 and
13 did not show improved EGFR inhibition. Compounds 9, 11 and
13 are interesting candidates for further evaluation against addi-
tional kinases.
Our results indicate that the potency and selectivity of cellular
inhibition of different RTKs does indeed vary with different anilino
substitutions and that an optimal combination of the substitutions
in the 4-anilino ring and the 6-benzyl substituent is essential for
RTK inhibition of the N4-aryl-6-substituted phenylmethyl-7H-pyr-
rolo[2,3-d]pyrimidine-2,4-diamines.
5.1.2. N4-(4-Chlorophenyl)-6-(2-methylbenzyl)-7H-pyrrolo[2,3-
d]pyrimidine-2,4-diamine (9)
Compound 9 was synthesized as described for 8 with 4-chloro-
aniline and was obtained as an off white solid (65%); TLC Rf 0.52
(CHCl3/CH3OH, 10:1); mp 212 °C; 1H NMR (DMSO-d6) d 2.27 (s,
3H, CH3), 3.89 (s, 2H, CH2), 5.99 (s, 1H, C5–H), 5.70 (s, 2H, NH2),
7.17–7.23 (m, 4H, Ar–H), 7.26(d, 2H, Ar–H), 7.96 (d, 2H, Ar–H),
8.87 (s, 1H, NH), 10.88 (s, 1H, NH). Anal. (C20H18ClN5Á0.23 H2O) C,
H, N, Cl.
5.1.3. N4-(4-Chloro-2-fluorophenyl)-6-(1-naphthylmethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (10)
Compound 10 was synthesized as described for 8 with 15 and
was obtained as an off white solid (70%); TLC Rf 0.58 (CHCl3/
CH3OH, 10:1); mp 212 °C; 1H NMR (DMSO-d6) d 4.37 (s, 2H, CH2),
5.91(s, 1H, C5–H), 5.57 (s, 2H, NH2), 6.55–8.07 (m, 10H, Ar–H),
9.3 (s, 1H, NH), 10.96 (s, 1H, NH). Anal. (C23H18ClFN5) C, H, N, Cl, F.
5. Experimental section
5.1. Synthesis
5.1.4. N4-(4-Chlorophenyl)-6-(1-naphthylmethyl)-7H-pyrrolo[2,
3-d]pyrimidine-2,4-diamine (11)
Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-
DRY drying apparatus over P2O5 at 80 °C. Melting points were
determined on a MEL-TEMP II melting point apparatus with FLUKE
51 K/J electronic thermometer and are uncorrected. Nuclear mag-
netic resonance spectra for proton (1H NMR) were recorded on a
Compound 11 was synthesized as described for 10 with 4-chlo-
roaniline and was obtained as a white solid (68%); TLC Rf 0.55
(CHCl3/CH3OH, 10:1); mp 209 °C; 1H NMR (DMSO-d6) d 4.38 (s,
2H, CH2), 5.95 (s, 1H, C5–H), 5.69 (s, 2H, NH2), 7.45–8.04 (m, 9H,
Ar–H), 7.23 (m, 2H, Ar–H), 8.77 (s, 1H, NH), 10.99 (s, 1H, NH). Anal.
(C23H18ClN5Á0.2 H2O) C, H, N, Cl.
Bruker WH-300 (300 MHz) or
a Bruker 400 MHz/52 MM
(400 MHz) spectrometer. The chemical shift values are expressed
in ppm (parts per million) relative to tetramethylsilane as an inter-
nal standard: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; br, 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. Chemi-
cal names follow IUPAC nomenclature. Thin-layer chromatography
(TLC) was performed on Whatman Sil G/UV254 silica gel plates
with a fluorescent indicator, and the spots were visualized under
254 and 366 nm illumination. Proportions of solvents used for
TLC are by volume. Column chromatography was performed on a
230–400 mesh silica gel (Fisher, Somerville, NJ) column.
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 solvents fre-
quently 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-as-
sisted synthesis was performed utilizing an Emrys Liberator micro-
wave 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.
5.1.5. N4-(2-Fluoro-4-chlorophenyl)-6-(2,5-dimethoxybenzyl)-
7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (12)
To a 100 mL round-bottom flask was added 16 (200 mg,
0.49 mmol), 2-fluoro-4-chloroaniline (1.5 equiv), iPrOH (20 mL)
and 6 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. To this
was added 1,4-dioxane (10 mL) and 15% KOH (2 mL) and the mix-
ture was refluxed for 10 h. After removal of the solvent, water
(20 mL) was added, extracted with CHCl3 (30 mL), dried with anhy-
drous Na2SO4 and concentrated under reduced pressure. The crude
product was purified by flash chromatography on silica gel (gradi-
ent, CHCl3 to 2% MeOH/CHCl3) to afford 150 mg (86%) of 12 as a
light yellow solid; mp 186–187 °C; TLC Rf 0.51 (CHCl3/CH3OH,
10:1); 1H NMR (DMSO-d6) d 3.34 (s, 2H, CH2), 3.65 (s, 3H, OCH3),
3.72 (s, 3H, OCH3), 5.95 (s, 2H, NH2), 6.81 (s, 1H, CH), 6.94–7.38
(m, 6H, Ar–H), 8.89 (s, 1H, NH), 11.06 (s, 1H, NH). Anal.
(C21H19ClFN5O2Á0.9 H2O) C, H, N, F, Cl.
5.1.6. N4-(4-Chlorophenyl)-6-(2,5-dimethoxybenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (13)
5.1.1. N4-(4-Chloro-2-fluorophenyl)-6-(2-methylbenzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (8)
Compound 13 was synthesized as described for 12 with 4-chlo-
roaniline and was obtained as a white solid (61%); TLC Rf 0.48
(CHCl3/CH3OH, 10:1); mp 188 °C; 1H NMR (DMSO-d6) d 3.62 (s,
2H, CH2), 3.73 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 5.87 (s, 2H, NH2),
6.35 (s, 1H, CH), 6.74–7.15 (m, 3H, Ar–H), 7.22 (d, 2H, Ar–H),
7.44 (d, 2H, Ar–H), 11.03 (s, 1H, NH), 11.41 (s, 1H, NH). Anal.
(C21H20ClN5O2Á0.18 H2O) C, H, N, Cl.
To a 100 mL round-bottom flask was added 14 (100 mg,
0.44 mmol), 2-fluoro-4-chloroaniline (1.5 equiv), iPrOH (20 mL)
and 6 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
concentrated under reduced pressure to afford a yellow solid.

914
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 910–914
5.2. Biological evaluation
grow for an additional 36 h. The cells were then lysed and the
CYQUANT 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, 22 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).
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, PDGFR-b, 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).
Acknowledgments
This work was supported, in part, by the National Institutes of
Health, National Cancer Institute Grant CA 098850 (A.G.), the
Duquesne University Adrian Van Kaam Chair in Scholarly
Excellence (A.G.) and an equipment grant from the National
Science Foundation (NMR: CHE 0614785).
5.3. 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
assay as previously reported.19 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
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.058.
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The assay was performed as described previously.19 Briefly, cells
were first treated with compounds for 12 h and then allowed to