N2-Trimethylacetyl substituted and unsubstituted-N 4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d] pyrimidine-2,4-diamines: Design, cellular receptor tyrosine kinase inhibitory activities and in vivo evaluation as antiangiogenic, antimetastatic and antitumor agents

Article abstract of DOI:10.1016/j.bmc.2012.12.045

Six novel N⁴-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H- pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N²-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

Full text of DOI:10.1016/j.bmc.2012.12.045

Bioorganic & Medicinal Chemistry 21 (2013) 1312–1323
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N2-Trimethylacetyl substituted and unsubstituted-N4-phenylsubsti-
tuted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mines: Design, cellular receptor tyrosine kinase inhibitory activities
and in vivo evaluation as antiangiogenic, antimetastatic and antitu-
mor agents
a
a
b
Aleem Gangjee a, , Ojas A. Namjoshi , Jianming Yu , Michael A. Ihnat ,
⇑
Jessica E. Thorpe b, Lora C. Bailey-Downs b
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States
b Department of Pharmaceutical Sciences, The University of Oklahoma College of Pharmacy, Oklahoma City, OK 73117, United States
a r t i c l e i n f o
a b s t r a c t
Six novel N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and
their N2-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibi-
tors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these com-
pounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory
activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture.
The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of
these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study
also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic
site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo
in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate,
in tumor vascular density and in metastases to the lung compared to the control.
Article history:
Received 21 August 2012
Revised 6 December 2012
Accepted 14 December 2012
Available online 10 January 2013
Keywords:
Antiangiogenic
Antimetastatic
In vivo
Sonogashira coupling
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
predominant mediator of angiogenesis.3,4 The expression of VEGF
is efficiently controlled under physiologic conditions. However,
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 over-
expressed in several tumors. These growth factors and their recep-
tors are directly as well as indirectly involved in cancer.1
Angiogenesis, the formation of new blood vessels from existing
vasculature, in solid tumors is essential for both physiologic and
pathologic processes. It is a complex cascade that is tightly regu-
lated by proangiogenic and antiangiogenic factors.2 VEGF is the
VEGF is secreted by a large number of tumor cells, leading to
angiogenesis that promotes tumor growth and metastases. In
addition to VEGF, several external factors are also involved in
stimulating tumor angiogenesis. Some of the principal proangio-
genic regulators include growth factors EGF, PDGF, fibroblast
growth factor (FGF), insulin-like growth factor-1 (IGF-1), trans-
forming growth factors (TGF
a and b) and tumor necrosis factor
a
(TNF-a
) among others.5 The newly sprouted blood vessels pro-
vide an additional supply of nutrients to the tumor to grow be-
yond 1–2 mm.3 Angiogenesis 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.6 In addi-
tion, angiogenesis contributes to the development of hematologic
malignancies, particularly multiple myeloma, leukemia, and lym-
phoma. However, the role of angiogenesis has not been clearly de-
fined in hematologic malignancies.7–9 Angiogenesis has been
described as one of the hallmarks of cancer.10 Thus inhibition of
tumor angiogenesis affords attractive targets for the development
of antitumor agents.
Abbreviations: RTK, receptor tyrosine kinase; EGFR, epithelial growth factor
receptor; VEGFR, vascular endothelial growth factor receptor; PDGFR, platelate
derived growth factor receptor; CAM, chorioallantoic membrane; Abl, abelson
tyrosine kinase; CML, chronic myelogenous leukemia; GIST, gastrointestinal
stromal tumors; FLT3, FMS-like tyrosine kinase 3; MTX, methotrexate; ELISA,
enzyme-linked immunosorbent assay.
⇑
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 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.045

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
1313
There has been considerable discussion in the literature regard-
ing the use of RTK inhibitors as monotherapy for cancer or the
combination of multiple RTK inhibitors either as single agents or
in combination with other chemotherapeutic agents. The majority
of earlier reports underlined the development of small molecule
targeted therapy against single RTK (Fig. 1), some of which affor-
ded clinical agents such as gefitinib (1)11 and erlotinib (2).11 How-
ever, tumors have redundant signaling pathways for angiogenesis
and often develop resistance to agents that target single specific
pathways.12 Recently, crosstalk has been implicated between EGFR
and other growth factor receptors involved in tumorigenesis.5
Hence to arrest angiogenesis, a multifaceted approach that targets
multiple signaling pathways has been shown to be more effective
than the inhibition of a single target. The most important conse-
quence of inhibiting multiple RTKs is to retard tumor resistance
by also blocking potential ‘escape routes’. Since RTKs are present
in endothelial cells (VEGFR, PDGFR), tumor cells (FGFR, PDGFR),
and pericytes/smooth muscle cells (FGFR, PDGFR), inhibition of
more than one RTKs provides synergistic inhibitory effects against
solid tumors.12 Recently, VEGFR-2 and PDGFR-b have been impli-
cated 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 produces a syner-
gistic effect in early stage as well as late stage pancreatic islet
cancer in mouse models by attacking the angiogenic process at
two different sites.13,14
pyrrolo[2,3-d]pyrimidine scaffold in the ATP binding pocket of
RTKs in at least three different binding modes (Fig. 2). In these dif-
ferent modes, the side chain substituents are oriented in different
sites in the ATP binding domain, the hydrophobic site I, sugar bind-
ing pocket or the phosphate binding region (Fig. 2).
The presence of a 2-pyridyl side chain makes these compounds
of further interest. These analogs are slightly more polar (clogP
ꢀ3.5) than the corresponding phenyl substituted analogs (clogP
ꢀ4.5) reported earlier.22 This may help in cellular transport of
the molecules by altering the lipid/water solubility ratio. The com-
pounds are also amenable to acid salt formation (pyridyl ring pKa
ꢀ5.2–5.4) and provide increased water solubility as acid salts for
ease of administration.
Various aniline substituents at the 4-position that were re-
ported in the literature to provide potent RTK inhibitors11,24–30
were incorporated in the design of 7–12 to determine their effects
on potency and/or selectivity against RTKs. Compound 7 was de-
signed with a 3-Br substituted aniline at the 4-position as similar
substituents have shown26,27 good EGFR inhibition in quinazolines.
Compound 8 was designed with a 3-Br, 4-F substituted aniline at
the 4-position as a logical step from 7 and as a prior analog to com-
pound 9 with a 3-Cl, 4-F substituted aniline moiety. This aniline
has been shown to improve EGFR inhibition by Wissner et al.28
in the case of quinazolines. Most importantly, gefitinib (1, Fig. 1)
also has a 3-Cl, 4-F substituted aniline. In a recent crystal structure
report29 of gefitinib (1) with EGFR it was found that the 3-Cl group
fits snuggly in a pocket formed by the side chains of residues
Lys745, Leu788 and Thr790. The 4-F substituent extends toward
the side chains of Leu788, Met766 and Glu762. Thus 3-Cl, 4-F
substituted aniline was included in our design to determine its ef-
fect on EGFR inhibition and to possibly extend the spectrum of RTK
inhibition of these compounds. The 3-ethynylaniline substituent of
10 was similar to that of erlotinib (2, Fig. 1). As reported in the
crystal structure of erlotinib (2) bound with EGFR, the acetylene
moiety is within 4 Å distance from side chains of Thr766, Lys721,
and Leu764 thus possibly forming hydrophobic interactions lead-
ing to improved binding of erlotinib with EGFR. The 3-trifluoro-
methyl group of 11 was included because electronegative groups
at the meta-position of the aniline moiety are better for EGFR
inhibitory activity as previously noted in the structure–activity
relationship studies of quinazoline compounds by Rewcastle
et al.26 and Bridges et al.27 Additionally Manley et al.30 has shown,
A flood of reports on the design of multikinase inhibitors have
appeared in the past few years,1,5,13–16 some of which have led to
clinically approved agents (Fig. 1) such as imatinib (3),17 sunitinib
(4),18 sorafenib (5),19 and lapatinib (6) (Fig. 1).20
2. Results and discussion
2.1. Design of inhibitors
The aim of this study was to design multikinase inhibitors, thus
a general RTK pharmacophore model was utilized rather than the
X-ray crystal structures of specific RTKs.21,22 As shown in this gen-
eral pharmacophore model depicted in Figure 2, the 2-amino group
is strategically incorporated in our pyrrolo[2,3-d]pyrimidine scaf-
fold to afford an additional hydrogen-bond contact with the back-
bones of the hinge region amino acids.23 This places the 2-amino
CH3
N
CH3
H
F
N
N
O
O
N
N
N
N
HN
N
HN
N
Cl
O
O
O
O
O
HN
N
N
O
CH3SO3H
2 (erlotinib)
3 (imatinib)
1 (gefitinib)
H3C
O
O
H3C
H3C
S
O
N
Cl
H3C
N
N
O
NH
O
NH
H
H3C
O
CF3
O
HN
N
Cl
O
CH3
F
N
F
O
N
H
N
H
N
H
4 (sunitinib)
5 (sorafenib)
Figure 1. Structures of RTK inhibitors in market.
6 (lapatinib)

1314
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
HYDROPHOBIC SITE I
HYDROPHOBIC SITE I
N
Br
PHOSPHATE
BINDING
REGION
PHOSPHATE
O
HN
N
HN
O
HINGE
BINDING
REGION
HINGE
REGION
REGION
NH
O
NH
O
N
NH
NH
N
N
HN
HN
N
R1
Br
R1
HYDROPHOBIC
SITE II
HYDROPHOBIC
SITE II
SUGAR BINDING
POCKET
SUGAR BINDING
POCKET
Binding mode 1
Binding mode 2
HYDROPHOBIC SITE I
Br
PHOSPHATE
BINDING
REGION
O
HN
N
HINGE
REGION
NH
O
NH
N
N
HN
R1
HYDROPHOBIC
SITE II
SUGAR BINDING
POCKET
Binding mode 3
Figure 2. General pharmacophore model of pyrrolo[2,3-d]pyrimidines.
in the anthranilic acid amide series of compounds, that the 3-tri-
fluoromethyl substituted compound shows excellent inhibition of
VEGFR and was a potent antiangiogenic agent. Thus 11 was de-
signed with the idea that it may inhibit both EGFR as well as VEGFR
thus affording dual RTK inhibition. Compound 12 is an analog of 11
with a small electronegative fluoro substituent at the 4-position of
the aniline to further improve EGFR inhibitory activity.27
In addition to the 2-NH2 moiety providing additional H-bonding
with the hinge region,23 substituents on the 2-amino group could
afford access to the hydrophobic site II and perhaps improve the
potency against RTKs. As shown in Figure 2, compound 7 makes
three H-bond contacts with the hinge region of the ATP binding
pocket. In this binding mode, substituents on the 2-amino group
could access the hydrophobic site 2 and perhaps improve the po-
tency against RTKs. In EGFR the hydrophobic site 2 is formed by
Leu694 and Gly772. Thus, in this study, a trimethylacetyl group
on the 2-amino moiety was incorporated as an initial attempt to
explore this hypothesis in compounds 14–18 (Fig. 3). The substit-
uents on the 4-anilino ring for 14–18 were kept the same as in
8–12, respectively for direct comparison.
at room temperature for 72 h and afforded poor yields (49%).31
Thus it was necessary to optimize the reaction conditions for the
synthesis of 21.
As shown in Table 1 the reaction was very sluggish at room
temperature (entry 1). The reaction did not proceed in less polar
solvent - THF (entry 2), probably due to inferior solubility. In ace-
tonitrile the reaction did not go to completion even after stirring
for 72 h at room temperature (entry 3). Prolonged heating at
100 °C in DMF led to inferior results (entry 4). Hence a microwave
mediated reaction approach was utilized. Under microwave irradi-
ation maintaining the temperature at 100 °C for 1 h a spot corre-
sponding to the product was observed on TLC (entry 5). However
the reaction did not reach completion and unreacted starting
material was observed on TLC. At higher temperature (180 °C)
some decomposition products (baseline spot on TLC) were
observed on TLC. Slightly better results were obtained when the
catalyst was generated in situ instead of the preformed catalyst
and the reaction was run at 100 °C for 1 h (entry 7). Higher temper-
ature (150 °C) improved the conversion, but also led to some
decomposition products (baseline spot on TLC). Decreasing the
reaction time to 10 min improved the yield considerably (entry
9). Tweaking the reaction time further led to the optimized condi-
tion in which the reaction proceeded smoothly under microwave
irradiation at 150 °C in 3.5 min (entry 10). Additionally, the use
of in situ generated catalyst proved to be more efficient instead
of a pre-formed catalyst such as tetrakistriphenylphosphinepalla-
dium (0) (compare entry 11 with entry 10) (Table 1).
2.2. Chemistry
The syntheses of compounds 13–18 and 7–12 are shown in
Scheme 1. Intermediate 21 can be obtained by a Sonogashira cou-
pling of 19 with 2-ethynylpyridine 20.31 However the reported
conditions for this conversion required the reaction to be stirred

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
1315
R
R1
R
R1
R
7
3'-Br
H
H
H
H
H
H
14
15
16
17
18
3'-Br, 4'-F
3'-Cl, 4'-F
3'-C2H
COC(CH3)3
COC(CH3)3
COC(CH3)3
COC(CH3)3
HN
8
3'-Br, 4'-F
3'-Cl, 4'-F
3'-C2H
N
9
R1
10
11
12
3'-CF3
N
H
N
H
N
3'-CF3
3'-CF3, 4'-F
3'-CF3, 4'-F COC(CH3)3
N
7-12 and 14-18
Figure 3. Designed compounds.
O
O
O
N
20
N
b
HN
HN
PivHN
HN
PivHN
I
a
N
N
H
N
H
PivHN
N
N
N
H
N
21
19
22
NH
2
R
Cl
24a-f
HN
c
N
dR
e
N
N
N
H
PivHN
N
PivHN
N
H
N
N
23
13-18
Comp. #
R
R
HN
13; 7
14; 8
15; 9
16; 10
17; 11
18; 12
3'-Br
3'-Br, 4'-F
3'-Cl, 4'-F
3'-C2H
N
N
H
H2N
N
3'-CF3
3'-CF3, 4'-F
N
7-12
Scheme 1. Synthesis of target compounds.
Compound 21 was obtained from 19 via a microwave-mediated
Sonogashira reaction. Catalytic reduction of the triple bond of 21
with 5% Palladium on charcoal afforded 22 in 75% yield. Chlorina-
tion of 22 with phosphorus oxychloride afforded 23 in 70% yield.
Compound 23 was reacted with appropriately substituted anilines
24a–f in isopropanol at reflux in the presence of 2 drops of concd
HCl for 3 h to afford 13–18 in 80–95% yields. Deprotection of the
2-amino groups of 13–18 with 1 N NaOH in methanol followed
by chromatographic purification afforded 7–12.
shown to give a linear approximation of cell number.35 Finally,
the effect of selected compounds on angiogenesis was assessed
using the chicken embryo chorioallantoic membrane (CAM) assay,
a standard test for angiogenesis.36 Since the IC50 values of RTK
inhibitors vary under different assay conditions, we used a stan-
dard (control) compound in each of the evaluations (Fig. 4). For
EGFR the standards were clinically used agents erlotinib (2), suni-
tinib (4) and experimental PD153035 (25); for VEGFR-1 the stan-
dard was CB676475 (26); for VEGFR-2 the standards were
clinically used agents erlotinib (2), sunitinib (4) and semaxanib
(27); for PDGFR-b the standard was AG1295 (28); for the cytotox-
icity study against the growth of A431 cells in culture the standard
was cisplatin (29). Semaxanib (27) was also used as the standard
for the antiangiogenic CAM assay.
In the EGFR inhibition assay, the most potent compound was 7
(with a meta-Br aniline at the 4-position) which was more potent
than clinically used erlotinib (2) as well as sunitinib (4) and was
equipotent to the standard EGFR inhibitor PD153035 (25). An addi-
tional electron withdrawing group such as fluorine at the para po-
sition on the aniline moiety (8) was detrimental for EGFR
inhibitory activity. Replacing the meta-Br in 8 with a slightly smal-
ler meta-Cl in 9 was not conducive for EGFR inhibition either. Com-
pound 10 (with a meta-ethynyl aniline at the 4-position like
erlotinib) exhibited lower inhibitory activity against EGFR; indicat-
2.3. In vitro evaluation for kinase inhibition, A431 cytotoxicity
and CAM angiogenesis inhibition
RTK inhibitory activity of compounds 7–12 and intermediates
14–18 were evaluated using human tumor cells known to express
high levels of EGFR, VEGFR-1, VEGFR-2, and PDFGR-b using a phos-
photyrosine ELISA cytoblot (Table. 2).32,33 Compounds known to
inhibit a particular RTK were used as positive controls for these as-
says. Whole cell assays were used for RTK inhibitory activity with
the synthesized compounds and controls since these assays afford
more meaningful results for translation to in vivo studies. The ef-
fect of compounds on cell proliferation was measured using A431
cancer cells, known to overexpress EGFR.34 Cell proliferation was
assessed using CYQUANTÒ, a DNA intercalating dye that has been

1316
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
Table 1
Optimization of the Sonogashira coupling reaction
O
O
N
N
20
N
HN
HN
PivHN
I
N
H
N
H
Pd source,
ligand,
PivHN
N
solvent,
21
19
conditions
#
Pd source
Ligand
Solvent
Conditions
Yielda/Results (%)
1
2
3
4
5
6
7
8
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd (powder)
Pd (powder)
Pd (powder)
Pd (powder)
Pd(PPh3)4
—
—
—
—
—
—
PPh3
PPh3
PPh3
PPh3
—
DMF
THF
rt, 72 h
rt, 72 h
rt, 72 h
100 °C, 24 h
4928
No reaction
20b
MeCN
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
15c
l
l
l
l
l
l
l
wave, 100 °C, 1 h
wave, 180 °C, 1 h
wave, 100 °C, 1 h
wave, 150 °C, 1 h
wave, 150 °C, 10 min
wave, 150 °C, 3.5 min
wave, 150 °C, 3.5 min
25b
22c
35
65c
9
10
11
81
88
72
a
b
c
Isolated yield.
Unreacted starting material was observed on TLC.
Considerable amount of baseline spot was observed on TLC. Attempts to analyze this material were not made.
Table 2
IC50 values (lM) for kinase inhibition, A431 cytotoxicity and CAM angiogenesis inhibition assay
Compd
R
EGFR inhibitiona
VEGFR-1 inhibitiona
VEGFR-2 inhibitiona
PDGFR-b inhibitiona
A431 cytotoxicitya
CAM angiogenesis inhibitiona
2
4
7
8
—
—
30-Br
1.2 0.2
—
—
124.7 18.2
18.9 2.7
49.9 9.8
24.1 3.4
>200
43.6 6.1
16.7 3.1
124.5 21.1
>200
89.3 9.7
125.6 19.8
16.7 2.5
150.9
12.2 1.9
83.1 10.1
13.4 3.9
>500
>500
331.5 48.2
>500
>500
89.7 10.2
>500
430.1 50.1
>500
>500
—
—
—
—
172.1 19.4
0.3 0.04
85.9 5.1
110.6 0.19
19.9 2.1
19.3 1.5
15.6 2.3
5.4 0.31
16.3 2.8
>200
>50
93.6 10.2
>200
103.1 17.1
>200
>200
>200
>200
156.3 18.2
129.6 17.2
189.6 23.1
19.6 5.0
12.2 2.0
27.0 3.5
0.9 0.12
3.4 0.4
3.2 0.34
1.0 0.17
2.6 0.3
29.9 3.1
2.9 0.4
1.9 0.2
12.6 3.1
7.23 0.9
0.07 0.005
11.3 1.9
13.9 1.4
1.09 0.15
14.0 1.5
0.9 0.12
2.0 0.31
3.2 0.41
1.6 0.2
8.6 0.9
30-Br, 40-F
30-Cl, 40-F
30-C2H
30-CF3
30-CF3, 40-F
30-Br, 40-F
30-Cl, 40-F
30-C2H
30-CF3
30-CF3, 40-F
—
9
10
11
12
14
15
16
17
18
25
26
27
28
29
12.8 1.7
26.9 4.1
0.23 0.04
—
—
—
—
14.1 2.8
12.0 2.7
0.085 0.003
6.2 1.3
10.6 2.9
a
Standard deviation for three experiments.
ing a possible different binding mode. Similarly, substitution of a
larger electron withdrawing meta-CF3 group on the 4-anilino moi-
ety in 11 led to diminished EGFR inhibitory activity as compared to
7 and PD153035 (25). An additional electron withdrawing fluoro
group at the para-position in 12 did not improve the IC50 value
against EGFR as compared with 11. The substitution requirement
(meta-Br) for EGFR inhibition was very subtle. A bulkier or smaller
substituent at the meta-position on the 4-anilino ring was not con-
ducive for potent EGFR inhibition.
For compounds with a trimethylacetyl group on the 2-amino
moiety (14–18), EGFR inhibition was improved for 14 (16-fold
compared to 8), 15 (6.5-fold compared to 9) and 17 (1.4-fold com-
pared to 11). This improvement may be attributed to the additional
interaction of these analogs with hydrophobic site II as hypothe-
sized and/or due to improved cell permeability of the compounds.
However, the same trend was not observed for 16 (inactive) and 18
(1.7-fold less active than 12) in the EGFR inhibitory assay and per-
haps could be attributed to a different mode of binding of 16 and
18.
Compounds 8, 10, 16–18 inhibited VEGFR-1 with IC50 values in
the three-digit micromolar range and were about 5- to 11-fold less
potent than the standard CB676475 (26). The trimethylacetyl
group on the 2-amino moiety exhibited a detrimental effect on
the inhibitory potency against VEGFR-1 (compare 8 with 14 and
10 with 16). However this trend was not observed for 11 and 17.
Against VEGFR-2, compounds 11 and 17 (both with meta-CF3
substitution on the 4-aniline) were the most potent compounds
of the series and were much more potent than clinically used erl-
otinib (2) and were equipotent with clinically used sunitinib (4)
and only 1.5-fold less potent as compared to the standard semaxa-
nib (27) that is in clinical trials. In both compounds an additional
electron withdrawing fluoro group at the para-position of 4-anilino
substituent led to an 8- to 9-fold loss of activity (compounds 12
and 18, respectively), underlining the stringent requirement of

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
1317
R
R
HN
HN
HN
N
R
N
H
O
O
N
N
N
O
N
H
N
H
H2N
N
PivHN
N
N
H
N
N
25 (PD153035): R = 3'-Br
27 (semaxanib)
7-12
14-18
26 (CB676475): R = 2'-F,4'-Cl
O
COOH
COOH
NH2
N
N
H
NH3
NH3
Cl
Cl
N
N
N
CH3
Pt
H2N
N
N
N
MTX (methotrexate)
29 (cisplatin)
28 (AG1295)
Figure 4. Designed compounds and standard compounds used in the biological assays.
electronics on the 4-anilino substituent for VEGFR-2 inhibitory
activity. Compound 7 with a meta-Br anilino moiety exhibited
two-digit micromolar IC50 value and was more potent than erloti-
nib (2) but only about half as potent as sunitinib (4) and 4.7-fold
less potent than semaxanib (27). An additional electron withdraw-
ing fluoro group at the para-position of anilino moiety resulted in 8
which afforded a 2-fold improvement in activity and was much
more potent than clinically used erlotinib (2); about equipotent
with sunitinib (4) and only 2.4-fold less potent than semaxanib
(27). Compound 17 with a meta-CF3 group was equipotent with
sunitinib (4) and somewhat less active compared to semaxanib
(27). Replacement of the meta-Br in 8 to a slightly smaller meta-
Cl in 9 led to loss of activity. Substitution on the 2-amino group
did not affect VEGFR-2 inhibition. A bulkier, electron withdrawing
substituent at the meta-position on the 4-anilino ring was neces-
sary for potent VEGFR-2 inhibition. Substitution on the 2-amino
group did not improve inhibitory activity against VEGFR-2.
Against PDGFR-b, the most potent compound was again 7 (with
a meta-Br aniline) and was equipotent with erlotinib (2) and about
6-fold more potent than sunitinib (4) and only about 2-fold less po-
tent than AG1295 (28). Additional substitution of a small electron
withdrawing fluro group at the para-position on the aniline moiety
(in 8) resulted in significantly diminished inhibition of PDGFR-b.
Similarly 9 showed diminished inhibitory activity. Substitution of
a meta-Br with a larger ethynyl group (in 11) led to a 25-fold loss
of activity. Thus bulky groups at meta-position (11, 12) were not
tolerated for PDGFR-b inhibitory activity. For PDGFR-b inhibition,
substitution on the 2-amino group improved activity for 14 (com-
pared with 8); and was equipotent as sunitinib (4) while for 16
(compared with 10) activity decreased.
pound 14 was only 10-fold less potent than semaxanib (27). In
addition compounds 11 and 15–18 exhibited IC50 values in the
one-digit micromolar range in the CAM angiogenesis assay. In most
cases the trimethylacetyl group on the 2-amino moiety improved
CAM inhibition (except for 14 and 17). Compounds exhibiting po-
tent inhibition of angiogenesis (8, 11, 14 and 17) have shown inhi-
bition of at least two RTKs.
From this study compound 8, was found to be a potent inhibitor
of angiogenesis (IC50 = 0.07 lM in the CAM angiogenesis inhibition
assay) and it also inhibited VEGFR-2, EGFR and VEGFR-1. Though
the potency of 8 against these three RTKs was not the best (as com-
pared to the corresponding standards used), the overall effect of
simultaneous inhibition of three RTKs may account for the potent
inhibition of angiogenesis (CAM assay). In general, from this
in vitro study, compounds that exhibit relatively good inhibition
of angiogenesis (CAM assay) also exhibited inhibition of at least
two of the RTKs evaluated. This underscores the fact that angiogen-
esis is a multifaceted, multicellular, multikinase, and multiprotein
activated process and that perhaps RTKs other than those evalu-
ated, may also be inhibited, providing a synergistic effect on the
overall angiogenesis process.
On the basis of the in vitro activity of compound 8 it was se-
lected for in vivo evaluation in a mouse tumor model in order to
determine its effect on tumor growth rates, metastasis, and tumor
angiogenesis.
2.4. In vivo evaluation of compound 8 in a B16-F10 syngeneic
tumor model
To examine whether compound 8 was effective in vivo in reduc-
ing tumor volume and metastasis, a syngeneic mouse tumor model
was used. This model is a widely accepted model for testing tumor
growth and metastases.37 The B16 model of tumor growth pro-
duces almost quantitative (100%) tumor take in a very short time
after administration,38,39 so animals are not wasted. The B16 tumor
cells are skin-derived, so subcutaneous implantation is adequate
and resulting tumors are superficial, and easily measured. The
B16-F10 variant used in these studies is highly metastatic, and
most if not all metastases go to the lung, making metastasis eval-
uation very facile.38–41 Unlike most human tumor xenograft mod-
els, the B16 model produces highly vascularized tumors so that the
effect of tumor-mediated angiogenesis can be evaluated.38–41
The maximal tolerated dose for 8 was 12.5 mg/kg twice weekly
as determined in the whole animal toxicity assay (Section 4). This
same dose was used for the in vivo evaluation. Compound 8 was
injected intraperitoneally (IP) every Monday (AM) and Thursday
Most of the compounds in this series exhibited good potency in
the A431 cytotoxicity assay compared to the standard cisplatin
(29). Compound 7 showed cytotoxicity against A431 cells at IC50
of 19.6 lM. Substitution of an electron withdrawing fluoro group
led to 1.6-fold improvement in cytotoxicity. Substitution of meta-
Br with slightly smaller chloro group resulted in about 2.2-fold loss
of activity. Compound 10 (meta-ethynyl) was the most potent
compound in A431 cytotoxicity assay, and was 8-fold more potent
than cisplatin (29). Substitution at the 2-amino group improved
cytotoxicity in most cases (14, 15, 17 and 18) except one (16).
Activity of 14 against EGFR appears to be translated into A431
cytotoxicity. Overall, compounds with bulkier substituents at the
meta-position of the 4-anilino ring exhibited improved cytotoxicity
against A431 cells.
In the CAM angiogenesis assay compound 8 was the most po-
tent compound and was 1.2-fold better than semaxanib (27). Com-

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A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
(PM) at 12.5 mg/kg dose and was compared with the antifolate
methotrexate (MTX) injected IP at 10 mg/kg. Tumor volume was
calculated using the formula length ꢁ width ꢁ depth. Tumor
growth rate was calculated using a linear regression analysis algo-
rithm using the software GraphPad Prism 4.0.c. At the experiment’s
end, animals were humanly euthanized using carbon dioxide, tu-
mors and lungs excised, fixed in 20% neutral buffered formalin
for 8–10 h, embedded into paraffin, and hematoxylin–eosin
(H&E) stain of three separate tissue sections completed to span
the tumor/lung. Metastases per lung lobe counted using the H&E
stained sections. Blood vessels per unit area were counted in 5
fields at 100ꢁ magnification and averaged. Tumor growth rates
were compared statistically using two-way ANOVA with a Re-
peated Measures post-test and tumor vascularity and metastases
were compared using one-way ANOVA and a Neuman–Keuls post
test with the null hypothesis rejected when P <0.05.
The results of compound 8 on the B16-F10 syngeneic tumor
model are shown in Figure 5A–D.
It was found that 8 at 12.5 mg/kg given twice weekly resulted in
a significant decrease in B16-F10 tumor growth rate as compared
to solvent treated animals (Fig. 5A). On the other hand, MTX at
10 mg/kg given twice weekly was slightly better at inhibiting pri-
mary tumor growth as compared to 8.
There was also significantly less increase in tumor volume
(Fig. 5B) in animals treated with compound 8 or MTX as compared
to untreated animals. Compound 8 (12.5 mg/Kg twice weekly dose)
was as effective as MTX (10 mg/kg twice weekly dose) in prevent-
ing increase in tumor volume over the entire course of the exper-
iment (Fig. 5B).
With respect to the effect of these agents on the vascular den-
sity (Fig. 5C) of the resulting tumors, compound 8 led to slightly
more vessels per unit area as compared to treatment with MTX.
A
B
Tumor growth rates
1500
1000
500
0
*
**
***
Treatment
* P<0.05 and ** P<0.01 by one-way ANOVA and
Neuman-Keuls post-test. n=7-10.
B16-F10 lung metastases
C
D
B16-F10 vascular density
5000
200
4000
150
**
100
***
50
*
3000
**
2000
1000
0
0
Treatment
Vascular density
** P<0.01 by one-way ANOVA and Neuman-Keuls
post-test. n=7-10. Average metastases in
untreated group - 2.5/lobe.
*** P<0.001 and ** P<0.01 by one-way ANOVA and
Neuman-Keuls post-test. n=7-10.
Figure 5. (A) Effect of compound 8 and MTX on the growth of B16-F10 primary tumors. Mice bearing B16-F10 GFP-tagged tumors were treated biweekly with 12.5 mg/kg of
compound 8 or 10 mg/kg of MTX and tumors measured biweekly. Tumor growth rates were calculated using linear regression analysis in GraphPad Prism 4.0 software. ⁄P
<0.05, ⁄⁄P <0.01 as compared to untreated animals; using two-way ANOVA with Repeated Measures post-test, n = 7–10. (B) Effect of compound 8 and MTX on the tumor
volume (mm3) of B16-F10 primary tumors. Mice bearing B16-F10 GFP-tagged tumors were treated biweekly with 12.5 mg/kg of compound 8 or 10 mg/kg of MTX and tumors
measured biweekly. Tumor volume was calculated using the formula length ꢁ width ꢁ depth. (C) Effect of compound 8 and MTX on the vascular density of B16-F10 primary
tumors. Blood vessels per unit area were counted in 5 fields at 100ꢁ magnification and averaged. ⁄⁄⁄P <0.001, ⁄⁄P <0.01 as compared to untreated animals; using one-way
ANOVA and Neuman-Keuls post-test, n = 7–10. (D) Effect of compound 8 and MTX on B16-F10 lung metastases. Metastases per lung lobe lobe counted using the H&E stained
sections. ⁄⁄P <0.01 as compared to untreated animals; using one-way ANOVA and Neuman-Keuls post-test, n = 7–10. Average metastases in untreated group ꢂ2.5/lobe.

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
1319
However this decrease in vascular density of B16-F10 tumors by
4.2. 2,2-Dimethyl-N-[4-oxo-6-(pyridin-2-ylethynyl)-4,7-
dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl]propanamide (21)31
compound 8 (as well as MTX) was statistically significant as com-
pared to solvent treated (untreated) animals. These results support
the antiangiogenic activity of compound 8.
To
a
5 mL microwave vial were added 1931 (100 mg,
Treatment with 8 also resulted in significantly decreased num-
bers of B16-F10 lung metastases (Fig. 5D) as compared to treat-
ment with MTX and untreated control.
Compound 8 exhibited excellent activity in the in vivo antitu-
mor evaluation, compared to MTX. This could be the result of a
synergistic effect of multiple RTK inhibition. Decrease in number
of lung metastases along with a decrease in vascular density of tu-
mor in animals treated with 8 corroborates our hypothesis.
0.28 mmol), 2-ethynylpyridine 20 (30 mg, 0.29 mmol), palladium
(14 mg, 0.13 mmol), CuI (10 mg, 0.05 mmol), triphenyl phosphine
(53.2 mg, 0.2 mmol) followed by the addition of DMF (4 mL) and
triethylamine (0.5 mL). The mixture was irradiated in a Smith Cre-
ator (personal chemistry) microwaves or a Initiator (Biotage)
microwave for 3.5 min at 150 °C. Silica gel (500 mg) was added
to the mixture and the solvents were removed under reduced pres-
sure to obtain a silica gel plug which was loaded on a silica gel col-
umn (2.5 cm ꢁ 15 cm) and chromatographed with 1.5% MeOH in
CHCl3. Fractions containing the product (TLC) were pooled and
evaporated to afford 21 (83 mg, 88%) as a pale yellow solid: TLC
Rf 0.25 (MeOH/CHCl3, 1:19); mp >270 °C dec. (lit.31 mp >270 °C
dec.); 1H NMR (300 MHz) (DMSO-d6) d 1.24 (s, 9H, C(CH3)3), 6.89
(s, 1H, C5–H), 7.43 (d, 1H, J = 4.8 Hz, C5H4N), 7.61 (d, 1H,
J = 4.8 Hz, C5H4N), 7.86 (t, 1H, J = 4.8 Hz, C5H4N), 8.61 (br, 1H,
C5H4N), 10.99 (s, 1H, 2-NHPiv, exch), 11.97 (br, 1H, 3-NH, exch),
12.36 (br, 1H, 7-NH, exch).
3. Summary
In summary six novel N4-phenylsubstituted-6-(2-pyridin-2-
ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N2-
trimethylacetyl substituted intermediate analogs were synthesized
using a microwave-assisted Sonogashira reaction as a key step. The
optimized microwave-mediated reaction resulted in considerably
higher yields compared to the previously described bench-top
reaction. These compounds were evaluated in cell-based assay
for their inhibitory activity against EGFR, VEGFR-1, VEGFR-2, and
PDFGR-b. Minor variations in the substitution on the 4-anilino ring
led to significant differences in inhibition of the various RTKs. In
addition, in some cases, the substitution on the 2-amino group re-
sulted in improved inhibitory activity against particular RTKs.
However this trend was not observed in all cases. These variations
in activity could be attributed to the stringent requirements for
binding in individual RTKs and/or due to differences in cell perme-
ability of these compounds. Compound 8 was evaluated in vivo at
12.5 mg/kg dose in B16-F10 syngeneic mouse tumor model. In this
study a significant reduction in tumor growth rate, in tumor vascu-
lar density and in metastases of the primary tumor to the lung was
observed for compound 8.
4.3. 2,2-Dimethyl-N-[4-oxo-6-(2-pyridin-2-ylethyl)-4,7-dihydro-
3H-pyrrolo[2,3-d]pyrimidin-2-yl]propanamide (22)31
To a Parr hydrogenation bottle was added 5% Pd/C (200 mg) fol-
lowed by 21 (200 mg, 0.59 mmol) dissolved in a mixture of DMF
and THF (1:5) (24 mL) and 2–3 drops of concd ammonium hydrox-
ide. The reaction mixture was shaken under 50 psi hydrogen pres-
sure for 24 h. At the end of the reaction, the slurry was filtered
through a celite pad which was washed with hot THF–DMF
(25 mL ꢁ 2). The filtrate was concentrated under reduced pressure.
Silica gel (1 g) was added and the solvent was evaporated to form a
plug which was loaded on top of a silica gel column (2.5 x 10 cm)
and eluted with 1.5% MeOH in CHCl3. Fractions containing the
product (TLC) were pooled and evaporated to afford 22 (161 mg,
75%) as an off-white solid: Rf 0.20 (MeOH/CHCl3, 1:19); mp
216.5–217.8 °C (lit.31 mp 217–218 °C) 1H NMR (300 MHz)
(DMSO-d6): d 1.24 (s, 9H, C(CH3)3), 3.01–3.05 (m, 4H, CH2CH2),
6.08 (s, 1H, C5–H), 7.19–7.23 (m, 1H, C5H4N), 7.27 (d, 1H,
J = 4.8 Hz, C5H4N), 7.68 (t, 1H, J = 4.8 Hz, C5H4N), 7.27 (d, 1H,
J = 4.8 Hz, C5H4N), 10.76 (br, 1H, 2-NHPiv, exch), 11.45 (br, 1H, 3-
NH, exch), 11.79 (br, 1H, 7-NH, exch).
4. Experimental
4.1. General methods for synthesis
All evaporations were carried out in vacuo with a rotary evapo-
rator. 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. Nuclear
magnetic resonance spectra for proton (1H NMR) were recorded on
a Bruker WH-300 (300 MHz) spectrometer. The chemical shift val-
ues are expressed in ppm (parts per million) relative to tetrameth-
ylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad singlet. The relative integrals 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 visualized under 254 and/or 365 nm illumination. Propor-
tions of solvents used for TLC are by volume. Column chromatogra-
phy was performed on a 230–400 mesh silica gel purchased from
Fisher Scietific. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. The results of elemental analyses for
the compounds that were biologically evaluated were in agree-
ment with calculated values within 0.4% range. Fractional moles
of water or organic solvents sometimes found in some analytical
samples could not be prevented in spite of 24–48 h of drying in va-
cuo and were confirmed where possible by their presence in the 1H
NMR spectra. All solvents and chemicals were purchased from Al-
drich Chemical Co. or Thermo Fisher Scientific and were used as
received.
4.4. N-[4-chloro-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (23)
To a round-bottom flask was added 22 (300 mg, 0.96 mmol;
pre-dried over phosphorous pentoxide using high vacuum for
8 h) and phosphorous oxychloride (10 mL). The slurry was heated
to form a light brown solution. Reaction was continued at reflux
for 3 h; at the end of which, the mixture was allowed to cool to
room temperature. Unreacted phosphorous oxychloride was re-
moved under reduced pressure to afford a gummy residue. Tritur-
ation of this residue with ice-water (approx 50 ml); followed by
the addition of concd NH4OH to adjust the pH to 4. The light brown
precipitate thus formed was filtered, air-dried and then dissolved
in CHCl3–MeOH (1:1, 50 mL). Silica gel (1 g) was added and sol-
vents evaporated to form a silica gel plug which was loaded on
top of silica gel column (2.5 ꢁ 15 cm) and eluted with 1% MeOH
in CHCl3. Fractions containing the product (TLC) were pooled and
evaporated under reduced pressure to afford 23 (240 mg, 70%) as
a white solid: TLC Rf 0.3 (MeOH/CHCl3, 1:19); mp 157–158.5 °C;
1H NMR (300 MHz) (DMSO-d6): d 1.22 (s, 9H, C(CH3)3), 3.16 (s,
4H, –CH2–CH2–), 6.22 (s, 1H, C5–CH), 7.25–7.28 (m, 1H, C5H4N),
7.29 (d, 1H, J = 5.1 Hz, C5H4N), 7.96 (t, 1H, J = 4.8 Hz, C5H4N), 8.51

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A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
(d, 1H, J = 4.8 Hz, C5H4N), 9.97 (s, 1H, 2-NHPiv, exch), 12.30 (s, 1H,
7-NH, exch). HRMS (EI) calcd for C18H20ClN5O: 357.1356, found
357.1392.
gave 16 (130 mg; 85%) as a off-white solid: TLC Rf 0.46 (MeOH/
CHCl3, 1:10); mp 120–125 °C; 1H NMR (300 MHz) (DMSO-d6): d
1.23 (s, 9H, C(CH3)3), 3.12–3.14 (m, 4H, –CH2CH2–), 4.10 (s, 1H,
C2H), 6.42 (s, 1H, C5–CH), 7.24–7.28 (m, 3H, ArH), 7.69 (t, 1H,
J = 6.0 Hz, ArH), 8.12 (s, 1H, ArH), 8.24 (d, 1H, J = 6.0 Hz, ArH),
8.51 (s, 1H ArH), 9.22 (s, 1H, 2-NHPiv or 4-NH, exch), 9.36 (s, 1H,
2-NHPiv or 4-NH, exch), 11.51 (br, 1H, 7-NH, exch); HRMS (ESI)
calcd for C26H27N6O (M+H)+: 439.2267, found 439.2246.
4.5. General procedure for the synthesis of compounds 13–18
To a round-bottom flask was added 23 and the appropriate
substituted anilines 24a–e in isopropanol (10 mL), followed by
the addition of two drops of concd HCl. The reaction was continued
at reflux for 3 h at the end of which the solvent was evaporated un-
der reduced 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 ob-
tained was loaded onto a silica gel column and eluted with 1.5%
methanol in chloroform. Fractions corresponding to the product
(TLC) were pooled and evaporated to dryness under reduced pres-
sure to afford the product.
4.5.5. N-{4-[(3-Trifluoromethylphenyl)amino]-6-(2-pyridin-2-
yl-ethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-
dimethylpropanamide (17)
Reaction of 20 (125 mg, 0.35 mmol) and 3-trifluoromethylani-
line 21e (225 mg, 1.4 mmol), using the general procedure de-
scribed above, gave 17 (151 mg; 89%) as a white fluffy solid: TLC
Rf 0.47 (MeOH/CHCl3, 1:10); mp 155.9–158.2 °C; 1H NMR
(300 MHz) (DMSO-d6): d 1.24 (s, 9H, C(CH3)3), 3.13–3.16 (m, 4H,
–CH2CH2–), 6.45 (s, 1H, C5–CH), 7.23–7.26 (m, 3H, ArH), 7.49 (t,
1H, J = 7.2 Hz, ArH), 7.69 (t, 1H, J = 7.2 Hz, ArH), 8.30 (d, 1H,
J = 7.2 Hz, ArH), 8.51 (s, 1H, ArH), 8.65 (s, 1H, ArH), 9.42 (s, 1H, 2-
NHPiv or 4-NH, exch), 9.48 (s, 1H, 2-NHPiv or 4-NH, exch), 11.55
(br, 1H, 7-NH, exch). Anal. (C25H25N6F3O 0.3H2O) CHNF.
4.5.1. N-{4-[(3-Bromophenyl)amino]-6-(2-pyridin-2-yl-ethyl)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide
(13)
Reaction of 20 (80 mg, 0.24 mmol) and 3-bromoaniline 21a
(160 mg, 0.96 mmol), using the general procedure described above,
gave 13 (110 mg; 98%) as yellow foam: TLC Rf 0.15 (MeOH/CHCl3,
1:19); 1H NMR (300 MHz) (DMSO-d6): d 1.24 (s, 9H, C(CH3)3),
3.07–3.19 (m, 4H, –CH2CH2–), 6.43 (s, 1H, C5–CH), 7.08–7.27 (m,
5H, ArH), 7.66–7.71 (m, 1H, ArH), 8.02 (d, 1H, J = 8.0 Hz, ArH),
8.50 (d, 1H, J = 4.9 Hz, ArH), 9.29 (s, 1H, 2-NHPiv or 4-NH, exch),
9.38 (s, 1H, 2-NHPiv or 4-NH, exch), 11.51 (s, 1H, 7-NH, exch);
HRMS (EI) calcd for C24H25N6BrO: 492.1273, found 492.1275.
4.5.6. N-{4-[(3-Trifluoromethyl,4-fluorophenyl)amino]-6-(2-
pyridin-2-yl-ethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-
dimethylpropanamide (18)
Reaction of 20 (150 mg, 0.42 mmol) and 4-fluoro-3-trifluoro-
methylaniline 21f (300 mg, 1.67 mmol), using the general proce-
dure described above, gave 18 (200 mg; 95%) as a white fluffy
solid: TLC Rf 0.46 (MeOH/CHCl3, 1:10); mp 167.5–169 °C; 1H
NMR (300 MHz) (DMSO-d6): d 1.23 (s, 9H, C(CH3)3), 3.13–3.15
(m, 4H, –CH2CH2–), 6.41 (s, 1H, C5–CH), 7.21–7.24 (m, 2H, ArH),
7.39 (t, 1H, J = 7.5 Hz, ArH), 7.69 (t, 1H, J = 7.5 Hz, ArH), 8.34 (br,
1H, ArH), 8.51 (d, 1H, J = 4.2 Hz, ArH), 8.67 (d, 1H, J = 5.4 Hz, ArH),
9.41 (s, 1H, 2-NHPiv or 4-NH, exch), 9.48 (s, 1H, 2-NHPiv or 4-
NH, exch), 11.53 (s, 1H, 7-NH, exch). Anal. (C25H24N6F4O 0.8H2O)
CHNF.
4.5.2. N-{4-[(3-Bromo,4-fluorophenyl)amino]-6-(2-pyridin-2-yl-
ethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl}the-2,2-
dimethylpropanamide (14)
Reaction of 20 (143 mg, 0.4 mmol) and 3-bromo, 4-fluoroaniline
21b (300 mg, 1.6 mmol), using a general procedure described
above, gave 14 (200 mg; 96%) as off-white fluffy crystals: TLC Rf
0.49 (MeOH/CHCl3, 1:10); mp 149.5–151.5 °C; 1H NMR
(300 MHz) (DMSO-d6):
d 1.24 (s, 9H, C(CH3)3), 3.14 (t, 2H,
J = 6.3 Hz, –CH2–), 3.26 (t, 2H, J = 6.3 Hz, –CH2–), 6.40 (s, 1H, C5–
CH), 7.25–7.27 (m, 3H, ArH), 7.69 (s, 1H, ArH), 7.98 (br, 1H, ArH),
8.52 (d, 1H, J = 7.4 Hz, ArH), 8.72 (br, 1H, ArH), 9.31 (s, 1H, 4-NH
or 2-NHPiv, exch), 9.39 (s, 1H, 2-NHPiv or 4-NH, exch), 11.49 (s,
1H, 7-NH, exch). Anal. (C24H24N6BrFO) CHNBrF.
4.6. General procedure for the synthesis of compounds 7–12
To a round-bottom flask was added 13–18 in methanol (10 mL),
followed by the addition of 1 N NaOH (2 mL). The reaction was
completed after heating at 70 °C for 10 h. Methanol was evapo-
rated under reduced pressure to afford a residue, which was fil-
tered, washed with water (10 mL) and air-dried. 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 thus obtained was loaded on top of a silica gel
column (1.5 ꢁ 15 cm) 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.
4.5.3. N-{4-[(3-Chloro,4-fluorophenyl)amino]-6-(2-pyridin-2-yl-
ethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-
dimethylpropanamide (15)
Reaction of 20 (143 mg, 0.4 mmol) and 3-chloro,4-fluoroaniline
21c (235 mg, 1.6 mmol), using the general procedure described
above, afforded 15 (150 g; 80%) as a white fluffy solid: TLC Rf 0.5
(MeOH/CHCl3, 1:10); mp 157–159 °C; 1H NMR (300 MHz)
(DMSO-d6): d 1.24 (s, 9H, C(CH3)3), 3.13 (t, 2H, J = 6.0 Hz, –CH2–),
3.27 (t, 2H, J = 6.0 Hz, –CH2–), 6.40 (s, 1H, C5–CH), 7.27–7.29 (m,
3H, ArH), 7.68 (s, 1H, ArH), 7.95 (br, 1H, ArH), 8.47 (d, 1H,
J = 7.8 Hz, ArH), 8.66 (d, 1H, J = 4.8 Hz, ArH), 9.31 (s, 1H, 4-NH or
2-NHPiv, exch), 9.39 (s, 1H, 2-NHPiv or 4-NH, exch), 11.50 (s, 1H,
7-NH, exch). HRMS (EI) calcd for C24H24N6ClFO: 466.1678, found
466.1684.
4.6.1. N4-(3-Bromophenyl)-6-(2-pyridin-2-yl-ethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (7)
Compound 7 was obtained from 13 (0.09 g, 0.19 mmol) using
the general procedure described above to afford after purification
40 mg (54%) as a pale yellow solid: TLC Rf 0.31 (MeOH/CHCl3,
1:9); mp 200–202 °C; 1H NMR (300 MHz) (DMSO-d6): d 2.97–
3.01 (m, 4H, –CH2CH2–), 5.83 (s, 2H, 2-NH2, exch), 6.24 (s, 1H,
C5–CH), 7.08–7.28 (m, 4H, ArH), 7.69 (br, 1H, ArH), 8.00 (d, 1H,
J = 8.0 Hz, ArH), 8.11 (m, 1H, ArH), 8.50 (d, 1H, J = 4.8 Hz, ArH),
8.97 (s, 1H, 4-NH, exch), 10.96 (s, 1H, 7-NH, exch). HRMS (EI) calcd
for C19H17N5Br: 408.0698, found 408.0684.
4.5.4. N-{4-[(3-Ethynylphenyl)amino]-6-(2-pyridin-2-yl-ethyl)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl}-2,2-dimethylpropanamide
(16)
Reaction of 20 (125 mg, 0.35 mmol) and 3-ethynylaniline 21d
(164 mg, 1.4 mmol), using the general procedure described above,

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
1321
4.6.2. N4-(3-Bromo,4-fluorophenyl)-6-(2-pyridin-2-yl-ethyl)-
7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (8)
4.7. General methods for biological evaluation
4.7.1. 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 (Manas-
sas, VA).
Compound 8 was obtained from 14 (130 mg, 0.25 mmol) using
the general procedure described above to afford a gummy residue
which was stirred with hexanes and filtered to obtain 65 mg (61%)
of 8 as a off-white solid: TLC Rf 0.23 (MeOH/CHCl3, 1:10); mp 223–
226 °C; 1H NMR (300 MHz) (DMSO-d6): d 3.00 (t, 2H, J = 6.9 Hz, –
CH2–), 3.15 (t, 2H, J = 6.9 Hz, –CH2–), 5.74 (br, 2H, 2-NH2, exch),
6.19 (s, 1H, C5–CH), 7.21–7.24 (m, 3H, ArH), 7.69 (t, 1H,
J = 7.2 Hz, ArH), 7.85 (br, 1H, ArH), 8.22 (d, 1H, J = 6.3 Hz, ArH),
8.49 (s, 1H, ArH), 8.90 (s, 1H, 4-NH, exch), 10.89 (br, 1H, 7-NH,
exch). Anal. (C19H16N6BrFꢃ 0.1 C6H14) CHNBr. HRMS (EI) calcd for
4.7.2. Chemicals
All growth factors (bFGF, VEGF, EGF, and PDGF-b) were pur-
chased from Peprotech (Rocky Hill, NJ). PD153035 (25),
CB676475 (26), semaxanib (27), AG1295 (28), and cisplatin (29)
were purchased from Calbiochem (San Diego, CA). The CYQUANT
cells proliferation assay was from Molecular Probes (Eugene, OR).
All other chemicals were from Sigma Chemical Co. unless other-
wise noted.
C19H16N6BrF: 426.0598, found 426.0603.
4.6.3. N4-(3-Chloro,4-fluorophenyl)-6-(2-pyridin-2-yl-ethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (9)
Compound 9 was obtained from 15 (100 mg, 0.21 mmol) using
the general procedure described above to afford after purification
51 mg (62%) as a light brown solid: TLC Rf 0.25 (MeOH/CHCl3,
1:10); mp >200 °C (decomposed); 1H NMR (300 MHz) (DMSO-
d6): d 3.07 (t, 2H, J = 6.9 Hz, –CH2–), 3.20 (t, 2H, J = 6.9 Hz, –CH2–
), 5.74 (br, 2H, 2-NH2, exch), 6.19 (s, 1H, C5–CH), 7.23 (m, 3H,
ArH), 7.69 (t, 1H, J = 7.2 Hz, ArH), 7.85 (br, 1H, ArH), 8.22 (d, 1H,
J = 6.3 Hz, ArH), 8.49 (s, 1H, ArH), 8.91 (s, 1H, 4-NH, exch), 10.88
(s, 1H, 7-NH, exch). Anal. (C19H16N6ClF) CHNClF.
4.7.3. Antibodies
The PY-HRP antibody was from BD Transduction Laboratories
(Franklin Lakes, NJ). Antibodies against EGFR, PDGFR-b, FGFR-1,
Flk-1, and Flt-1 were purchased from Upstate Biotech (Framing-
ham, MA).
4.7.4. Phosphotyrosine ELISA
Cells used were tumor cell lines naturally expressing high levels
of EGFR (A431), Flk-1 (U251), Flt-1 (A498), and PDGFR-b (SF-539).
Expression levels at the RNA level were derived from the NCI
Developmental Therapeutics Program (NCI-DTP) web site public
molecular target information (http://www.dtp.nci.nih.gov/mtar-
gets/mt_index.html). Briefly, cells at 60–75% confluence are placed
in serum-free medium for 18 h to reduce the background of phos-
phorylation. Cells were always >98% viable by Trypan blue exclu-
sion. Cells are then pretreated for 60 min with 10, 3.33, 1.11,
4.6.4. N4-(3-Ethynylphenyl)-6-(2-pyridin-2-yl-ethyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (10)
Compound 10 was obtained from 16 (100 mg, 0.23 mmol) using
the general procedure described above to afford after purification
70 mg (86%) as a off-white solid: TLC Rf 0.2 (MeOH/CHCl3, 1:10);
mp 229.5–231.5 °C; 1H NMR (300 MHz) (DMSO-d6): d 2.98 (t, 2H,
J = 6.9 Hz, –CH2–), 3.09 (t, 2H, J = 6.9 Hz, –CH2–), 4.12 (s, 1H, C2H),
5.68 (br, 2H, 2-NH2, exch), 6.22 (s, 1H, C5–CH), 7.01 (d, 1H,
J = 7.5 Hz, ArH), 7.20–7.23 (m, 3H, ArH), 7.68 (t, 1H, J = 7.4 Hz,
ArH), 7.92 (s, 1H, ArH), 8.08 (d, 1H, J = 8.1 Hz, ArH), 8.50 (d, 1H,
J = 3.5 Hz, ArH), 8.81 (s, 1H, 4-NH, exch), 10.86 (br, 1H, 7-NH, exch).
Anal. (C21H18N6) CHN.
0.37, and 0.12 lM compound followed by 100 ng/ml EGF, VEGF,
PDGF-BB, or bFGF for 10 min. The reaction is stopped and cells per-
meabilized by quickly removing the media from the cells and add-
ing ice-cold Tris-buffered saline (TBS) containing 0.05% Triton X-
100, protease inhibitor cocktail and tyrosine phosphatase inhibitor
cocktail. The TBS solution is 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 are further exposed to block (TBS
with 1% BSA) for 1 h, washed, and then a horseradish peroxidase
(HRP)-conjugated phosphotyrosine (PY) antibody added overnight.
The antibody is removed, cells are washed again in TBS, exposed to
an enhanced luminal ELISA substrate (Pierce Chemical, Rockford,
IL) and light emission measured using a UV products (Upland,
CA) BioChemi digital darkroom. Data were graphed as a percent
of cells receiving growth factor alone and IC50 values were esti-
mated from two to three separate experiments (n = 8–24) using
hand drawn probit plots. In each case, the activity of a positive con-
trol inhibitor did not deviate more than 10% from the IC50 values
listed in the text.
4.6.5. N4-(3-Trifluoromethylphenyl)-6-(2-pyridin-2-yl-ethyl)-
7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (11)
Compound 11 was obtained from 17 (125 mg, 0.26 mmol) using
the general procedure described above to afford after purification
85 mg (84%) as a light yellow solid: TLC Rf 0.19 (MeOH/CHCl3,
1:10); mp 226.5–227.6 °C; 1H NMR (300 MHz) (DMSO-d6): d 3.00
(t, 2H, J = 6.1 Hz, –CH2–), 3.09 (t, 2H, J = 6.1 Hz, –CH2–), 5.73 (br,
2H, 2-NH2, exch), 6.24 (s, 1H, C5–CH), 7.22–7.25 (m, 3H, ArH),
7.47 (t, 1H, J = 7.4 Hz, ArH), 7.67 (d, 1H, J = 6.7 Hz, ArH), 8.15 (s,
1H, ArH), 8.44 (d, 1H, J = 7.8 Hz, ArH), 8.50 (s, 1H, ArH), 9.08 (s,
1H, 4-NH, exch), 10.92 (br, 1H, 7-NH, exch). Anal. (C20H17N6F3)
CHNF.
4.7.5. CYQUANT cell proliferation assay
4.6.6. N4-(3-Trifluoromethl,4-fluorophenyl)-6-(2-pyridin-2-yl-
ethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (12)
As a measure of cell proliferation, the CYQUANT cell counting/
proliferation assay was used as previously described.35 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 an UV prod-
ucts BioChemi digital darkroom. A positive control used for cyto-
toxicity in each experiment was cisplatin, with an apparent
Compound 12 was obtained from 18 (100 mg, 0.2 mmol) using
the general procedure described above to afford after purification
65 mg (78%) as a white lustrous crystals: TLC Rf 0.26 (MeOH/CHCl3,
1:10); mp 233.8–234.7 °C; 1H NMR (300 MHz) (DMSO-d6): d 3.00
(t, 2H, J = 6.6 Hz, –CH2–), 3.09 (t, 2H, J = 6.6 Hz, –CH2–), 5.70 (br,
2H, 2-NH2, exch), 6.19 (s, 1H, C5–CH), 7.22–7.24 (m, 2H, ArH),
7.37 (t, 1H, J = 7.8 Hz, ArH), 7.68 (t, 1H, J = 7.7 Hz, ArH), 8.19 (s,
1H, ArH), 8.44 (d, 1H, J = 4.5 Hz, ArH), 8.50 (s, 1H, ArH), 9.07 (s,
1H, 4-NH, exch), 10.90 (br, 1H, 7-NH, exch). Anal.
(C20H16N6F4ꢃ0.2H2O) CHNF.
average IC50 value of 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.

1322
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1312–1323
4.7.6. CAM assay of angiogenesis
embedded into paraffin, and hematoxylin–eosin (H&E) stain of
three separate tissue sections completed to span the tumor/lung.
Metastases per lung lobe counted using the H&E stained sections.
The metastases can be seen as purple clusters of disorganized cells
on the highly organized largely pink lung. Together with the
OUHSC Department of Pathology core, blood vessels per unit area
were counted in 5 fields at 100ꢁ magnification and averaged. Tu-
mor growth rates were compared statistically using two-way AN-
OVA with a Repeated Measures post-test and tumor vascularity
and metastases were compared using one-way ANOVA and a Neu-
man–Keuls post test with the null hypothesis rejected when P
<0.05.
The chorioallantoic membrane (CAM) assay is a standard assay
for testing antiangiogenic agents.36 The CAM assay used in these
studies was modified from a procedure by Sheu42 and Brooks43
and as published previously.44 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 saturation to a 3 mm microbial test-
ing disk (BBL, Cockeysville, MD) and placed onto the CAM by
breaking a small hole in the superior surface of the egg. Antiangio-
genic compounds are added 8 h after the VEGF/bFGF at saturation
to the same microbial testing disk and embryos allowed to incu-
bate for an additional 40 h. After 48 h, the CAMs are perfused with
2% paraformaldehyde/3% glutaraldehyde containing 0.025% Triton
X-100 for 20 s, excised around the area of treatment, fixed again
in 2% paraformaldehyde/3% glutaraldehyde for 30 min, placed on
petri dishes, and a digitized image taken using a dissecting micro-
scope (Wild M400; Bannockburn, IL) at 7.5X and Retiga enhanced
digital imaging system (QImaging, 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 vascularity.
Semaxanib (27) was 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 exper-
iments (n = 5–11) using nonlinear regression analysis and a Sig-
moidal Dose–response analysis using Prism 4.0 (GraphPad
Software, San Diego, CA).
4.7.9. Statistics
All analysis was done using Prism 4.0. (GraphPad Software, San
Diego, CA.)
Acknowledgments
This work was supported, in part, by the National Institutes of
Health, National Cancer Institute Grant CA98850 (A.G.) and the Du-
quesne University Adrian Van Kaam Chair in Scholarly Excellence
(AG).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.045.
4.7.7. Whole animal toxicity assay
To first determine the maximal tolerated dose (MTD) for a twice
weekly dose of 8, male NCr nu/nu mice (4 animals per group) at
8 weeks of age without tumors were injected with 5, 10, 12.5, 15,
17.5 and 20 mg/kg 8 two times weekly, on Monday AM and Thurs-
day PM. Weights were taken and animals observed for acute dis-
tress during the first 24 h after injection and animals observed
and weighed throughout the 6 week study. It was found that 8 at
20, 17, and 15 mg/kg twice weekly resulted in a significant (P
<0.05) weight loss at the 6 week time point. At 12.5 mg/kg twice
weekly, a small (1%) but statistically insignificant weight loss oc-
curred and the doses of either 7.5 or 10 mg/kg twice weekly
showed no weight loss as compared to their untreated counter-
parts. The dose of 12.5 mg/kg twice weekly was chosen for the
study.
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