N4-(3-bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d] pyrimidines as potent multiple receptor tyrosine kinase inhibitors: Design, synthesis, and in vivo evaluation

Article abstract of DOI:10.1016/j.bmc.2012.01.029

With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibition against PDGFRβ and VEGFR-2 we designed and synthesized eleven N⁴-(3-bromophenyl)-7- (substitutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a-19

Full text of DOI:10.1016/j.bmc.2012.01.029

Bioorganic & Medicinal Chemistry 20 (2012) 2444–2454
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N4-(3-Bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d]pyrimidines
as potent multiple receptor tyrosine kinase inhibitors: Design, synthesis,
and in vivo evaluation
Aleem Gangjee a, , Nilesh Zaware a, Sudhir Raghavan a, Jie Yang a, Jessica E. Thorpe b, Michael A. Ihnat b
⇑
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
b Department of Cell Biology, School of Medicine, University of Oklahoma Health Sciences 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
Article history:
With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibi-
tion against PDGFRb and VEGFR-2 we designed and synthesized eleven N4-(3-bromophenyl)-7-(substi-
tutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a–19a. These compounds were obtained from the key
intermediate N4-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 29. Various arylmethyl
groups were regiospecifically attached at the N7 of 29 via sodium hydride induced alkylation with substi-
tuted arylmethyl halides. Compounds 11a and 19a were potent dual inhibitors of PDGFRb and VEGFR-2.
In a COLO-205, in vivo tumor mouse model 11a demonstrated inhibition of tumor growth, metastasis,
and tumor angiogenesis that was better than or comparable to the standard compound TSU-68
(SU6668, 8).
Received 16 September 2011
Revised 11 January 2012
Accepted 19 January 2012
Available online 4 February 2012
Keywords:
Multiple receptor
Tyrosine kinase inhibitors
Antiangiogenic agents
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
triggers the release of growth factors, particularly, vascular endo-
thelial growth factor (VEGF), epidermal growth factor (EGF), and
Protein kinases are enzymes that transfer a phosphate group
from ATP to the hydroxyl group of serine, threonine, or tyrosine
of specific proteins inside cells.1 The phosphorylation by these en-
zymes achieves an important function of signal transduction in
eukaryotic cells and controls the processes of cell proliferation,
metabolism, survival, and apoptosis. A mis-regulation of these
tightly controlled processes results in the overexpression of ki-
nases, which is associated with a variety of disease states including
cancer.2
Angiogenesis is the process of formation of new blood vessels
from existing vascular bed. Angiogenesis is crucial for a tumor to
grow beyond 1–2 mm and for tumor invasion and metastasis.3,4
As a tumor grows in size, it becomes increasingly hypoxic and
platelet derived growth factor (PDGF) among others. The growth
factors activate adjacent blood vessels leading to angiogenesis.5,6
Following angiogenesis, the tumor can grow at an increased rate.7
For angiogenesis to occur, the pro-angiogenic growth factors
have to bind to members of the protein kinase family identified
as receptor tyrosine kinases (RTKs). Following binding, the RTKs
dimerize and undergo autophosphorylation, initiating a series of
downstream events leading to proliferation migration and cell sur-
vival.8 The growth factor VEGF triggers angiogenesis by binding to
a family of VEGF receptors, including VEGFR-2 (KDR), VEGFR-1
(Flt-1), and VEGFR-3 (Flt-4). Similarly PDGF binds to the family
of PDGF receptors, Flt-3 (FMS-like tyrosine kinase-3), PDGFRb, c-
Kit (a stem cell factor receptor), and CSF1R (colony-stimulating fac-
tor 1 receptor).9
Abrogation of angiogenesis by inhibition of RTK signaling path-
ways is an important mechanism for the development of novel
anticancer agents.8,10,11 Early work in the area of RTK inhibitor dis-
covery afforded small molecule inhibitors gefitinib 1 (Fig. 1) (spe-
cific EGFR inhibitor, approved for limited use for the treatment of
non-small cell lung cancer)12 and erlotinib 2 (specific EGFR inhib-
itor, approved for the treatment of non-small cell lung cancer).12
Recent data, however, shows that tumors treated with specific
RTK inhibitors usually develop resistance through the upregulation
of alternate kinase mediated pathways.13 In addition, cross-talk
has been implicated between EGFR and other growth factor
receptors involved in tumor development.14
Abbreviations: ATP, adenosine 50triphosphate; VEGF, vascular endothelial
growth factor; EGF, epidermal growth factor; PDGF, platelet derived growth factor;
RTK, receptor tyrosine kinase; KDR, kinase insert domain receptor; VEGFR-2,
vascular endothelial growth factor receptor-2; Flt-1, fms-related tyrosine kinase 4;
VEGFR-1, vascular endothelial growth factor receptor-1; Flt-4, fms-related tyrosine
kinase 4; VEGFR-3, vascular endothelial growth factor receptor-3; Flt-3, FMS-like
tyrosine kinase-3; PDGFRb, platelet derived growth factor receptor-b; CSF1R, colony
stimulating factor 1 receptor; CML, chronic myelogenous leukemia; GIST, gastro-
intestinal stromal tumor; Abl, abelson tyrosine kinase; ErbB-2, erythroblastic
leukemia viral oncogene homolog-2; TLC, thin layer chromatography; CAM,
chorioallantoic membrane.
⇑
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 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.029

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
2445
N
N
N
F
H
Cl
N
N
O
O
O
N
N
N
HN
N
HN
N
N
O
O
O
O
O
F
H
HN
N
N
O
.CH3SO3H
N
H
O
1
2
3
4
O
S
O
O
Cl
N
O
R
N
O
H
HN
CF3
N
H
O
HN
N
Cl
O
O
O
7
R = H
N
F
N
H
8 R =
N
H
N
OH
H
5
6
R
Ar
N
HN
N
9
H
N
Cl
Cl
NH3
NH3
10
2'-Me
O
O
Pt
11 2'-Cl
12
HN
Br
N
4'-Cl
4
5
13 2',5'-diOMe
14 2',4'-diCl
3
N
22
23
Ar
6
15
20 3'-BrPh
3',4'-diCl
N
H2N 2 N
1
7
H
21
2'-F,4'-ClPh
16 2',3'-C4H4
17
R
3',4'-C4H4
18 4'-Ph
19 3',4',5'-triOMe
9-19
Figure 1. RTK Inhibitors.
There are a number of RTK mediated processes that can pro-
mote angiogenesis, thus inhibitors targeting a broader range of
RTKs may lead to a more robust antitumor response and prevent
resistance by targeting two or more angiogenic pathways.15,16
The FDA approval of several compounds targeting a broader-spec-
trum of RTKs lends credence to the development of single agents
that target multiple RTKs. These include imatinib 3, sunitinib 4,
sorafenib 5 and lapatinib 6 (Fig. 1). Compound 3 inhibits Abelson
Tyrosine Kinase (Abl), c-kit protein (CD117), and PDGFR, and has
been approved for chronic myelogenous leukemia (CML),17 gastro-
intestinal stromal tumors (GISTs), and other malignancies. Com-
pound 4 inhibits VEGFR-2, PDGFRb, c-kit, and FMS-like tyrosine
kinase 3 (FLT3)18 and has been approved for the treatment of renal
cell carcinoma and imatinib-resistant GIST. Compound 5 inhibits
Raf kinase, VEGFR-2, PDGFRb, and c-kit19 and has been approved
for advanced renal cell carcinoma. Compound 6 inhibits EGFR
and erythroblastic leukemia viral oncogene homolog-2 (ErbB-2)20
and has been approved for advanced metastatic breast cancer in
conjunction with chemotherapy.
In angiogenesis, VEGFs cause a significant increase in the forma-
tion of blood vessels, but these vessels are immature and leaky. The
formation of thicker, more stable vessels requires an encapsulation
by pericytes. This process of encapsulation is driven by PDGFRb
signaling. Recent reports indicate that the inhibition of VEGFR-2
and PDGFRb with two separate inhibitors produces a synergistic ef-
fect in early stage as well as late stage pancreatic islet cancer in
mouse models.21 Timke et al.22 have investigated the therapeutic
potential of a VEGFR inhibitor SU5416 (7) (Fig. 1) and PDGFR inhib-
itor 823,24 in combination with radiotherapy in vitro and in vivo. It
was observed that dual inhibition of VEGFR and PDGFR signaling
was a more efficient antitumor mechanism than single pathway
inhibition. In addition radiation markedly enhanced the therapeu-
tic efficacy in vitro and in human glioblastoma and human pros-
trate carcinoma in mice.
We25 previously reported a series of eleven N4-(3-bromophe-
nyl)-6-(substituted benzyl) pyrrolo[2,3-d] pyrimidines 9–19
(
Fig. 1). These compounds were evaluated in human tumor cells
known to over express high levels of specific RTKs. Compound 10
with a 20-Me benzyl substitution exhibited toxicity against A431
cells (cell lines overtly dependent on EGFR for survival)26 at values
10-fold better than the standard agent PD153035 (20) used in this
assay (Table 1). In addition, 10 also demonstrated VEGFR-2 inhibi-
tion 40-fold better than the standard agent 7. EGFR and A431 cell
lines were inhibited by 14 (with a 20,40-diCl benzyl substitution)
at concentrations that were comparable to and fivefold better than
the standard 20, respectively. Compound 13 with a 20,50-diOMe
benzyl substitution inhibited VEGFR-2 and PDGFRb at values 17-
fold better than, and comparable to, standard agents 23 and
AG1295 (22), respectively. Thus the N4-(3-bromophenyl)-6-
(substituted benzyl) pyrrolo[2,3-d]pyrimidine scaffold with varia-
tions in the phenyl ring affords multi-kinase inhibition with poten-
cies equivalent to or better than standard agents. In an attempt to
develop compounds with multikinase inhibition potential it was of
interest to explore the effect of moving the 6-position substituent
in the parent compounds to the 7-position. Hence a series of N4-(3-
bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d]pyrimidines—
9a–19a (Fig. 2) was designed. The selection of the substituents
on the N7-position is the same as that at N6 in the parent series
(9–19), and is rationalized below.
Compound 9a (Fig. 2) with a benzyl at N7 served as a compar-
ison with compounds 10a to 19a (Fig. 2) that possess varied sub-
stituents at the N7 position. The basis for including the 20-Me

2446
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
Table 1
IC50 values (lM) of kinase inhibition and the A431 cytotoxicity assay
Compd #
PDGFR-b inhibition
VEGFR-2 inhibition
VEGFR-1 inhibition
EGFR inhibition
A431 cytotoxicity
9a
>50
>50
>50
>50
8.92 1.6
17.0 5.6
>50
>50
>50
>50
14.7 3.4
>500
159.6 26.3
1.5 0.21
>500
>50
>50
>50
26.8 4.1
42.7 6.1
31.1 5.8
>50
1.67 0.3
9.19 1.8
4.31 1.75
17.42 3.9
12.62 3.3
0.23 0.06
19.77 5.6
>50
1.24 0.21
6.16 1.2
>50
166.4 20.6
113.3 18.9
>200
31.8 6.3
1.21 0.42
>50
28.6 5.1
>50
2.8 1.1
33.5 6.2
>50
33.2 5.9
23.5 5.2
42.1 18.5
50.4 5.9
15.7 2.8
88.4 10.2
16.1 2.2
40.6
15.3 1.9
19.2 3.0
36.3 4.9
20.4 3.5
13.3 20.5
39.0 6.8
12.6 2.9
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
9a
0.25 0.04
5.58 0.69
8.28 0.69
0.62 0.21
28.11 9.9
>50
>50
5.08 0.83
>50
19.2 4.3
15.2 2.9
>50
5.97 0.78
9.42 1.9
23.8 3.0
113.4 17
17.9 2.4
65.3 7.9
>200
>50
99.3 10.3
129.3 20.4
126.3 19.1
79.9 8.4
>200
138.1 24.2
118.6 11.4
150 22.1
50.8 6.2
>200
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20
99.9 18.6
69.2
6
34.2 4.4
>200
>200
>200
>200
>200
>200
>200
229.6 37.1
129.3 21.1
212.4 16.2
129.1 20.5
1.8 0.29
64.5 7.8
14.9 2.1
22.9 2.9
22.9 10.7
25.7 4.6
156.5 25
0.2 0.04
21
17.7 5.5
7
22
23
2.43 0.32
10.6 3.5
19.2 1.1
6.2 1.3
a
IC50 values taken from Ref. 25.
displacement of the 4-chloro group in 27 with 3-bromoaniline 28
furnished the common intermediate 29. The target compounds
9a–19a were obtained in yields ranging from 5% to 47% by a so-
dium hydride induced regiospecific alkylation of 29 at the 7-posi-
tion with the corresponding substituted arylmethyl halides 30a–k.
The purification of the target compounds was tedious since the by-
products had Rf values (TLC) close to the desired compounds 9a–
19a. This necessitated the exploration of alternate synthetic proce-
dures, particularly for large scale synthesis of the compounds for
in vivo evaluation.
A gram scale synthesis of 11a was accomplished by an im-
proved method28 as shown in Scheme 2. A sodium hydride induced
alkylation of the N7-position of 27 furnished 31. This was followed
by a nucleophilic displacement of the 4-chloro group in 31 with 28
to afford 11a. This modified protocol provided a remarkable
improvement in the overall reaction yield of 27 to 11a from a mere
5% (Scheme 1) to 39% (Scheme 2).
R
H
9a
10a 2'-Me
2'-Cl
12a 4'-Cl
2',5'-diOMe
14a 2',4'-diCl
HN
N
Br
11a
13a
N
H2N
N
15a
3',4'-diCl
16a 2',3'-C4H4
17a
R
3',4'-C4H4
18a 4'-Ph
19a 3',4',5'-triOMe
Figure 2. Target compounds.
benzyl group in 10a, 20,50-diOMe benzyl group in 13a, and 20,40-diCl
benzyl group in 14a, was the potent RTK inhibition produced by
the lead compounds 10, 13, and 14 (Fig. 1) which have a similar
substitution pattern at the 6-position.
3. Biological evaluation and discussion
The 20-Cl phenyl group in 11a, 40-Cl benzyl group in 12a, and
30,40-diCl benzyl in group in 15a would afford information on activ-
ity obtained on having an electron withdrawing group (or groups)
at the respective positions. The 30,40,50-triOMe benzyl group in 19a
would afford information on activity obtained on having electron
donating groups at these positions.
The RTK inhibitory activities of compounds 9a–19a (Table 1)
were evaluated using a phosphotyrosine ELISA in human tumor
cells known to express high levels of EGFR, VEGFR-1, VEGFR-2,
and PDGFRb.29,30 Compounds known to inhibit a specific RTK were
used as a positive control for these assays. Whole cell assays were
used for measuring RTK inhibition as these assays afford more
meaningful results for translation to in vivo studies.25,31–34 To
study the effect of compounds on cell proliferation, A431 cancer
cells known to over express EGFR were used. EGFR has been shown
to be a factor in the overall survival of A431 cells.26 Cell-prolifera-
tion was measured using CYQUANTÒ, a DNA intercalating dye
shown to provide a linear approximation of cell number.35 In this
assay, purified angiogenic growth factors are positioned locally
on a vascularized membrane of a developing chicken embryo along
with possible inhibitors. Digitized images of the vasculature are ta-
ken 48 h after growth factor administration and the number of
vessels per unit area is quantified to measure vascular density.
The 1-naphthyl moiety in 16a, 2-naphthyl in 17a, and 40-phenyl
benzyl group in 18a were included to obtain information about
bulk tolerance in this position in the RTK active site.
2. Chemistry
The synthesis of compounds 9a–19a is shown in Scheme 1.
Compound 26 was obtained by cyclocondensation of chloroacetal-
dehyde 25 with 24 at reflux for 4 h as previously reported.27
Chlorination of 26 with phosphorus oxychloride, in the presence
of N,N0-dimethylaniline as a catalyst afforded 27. Nucleophilic

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
2447
O
O
Cl
O
N
Cl
N
HN
Br
H2N
Br
H
25
a
28
c
HN
H2N
HN
H2N
N
N
b
N
H
N
N
H
N
NH2
H2N
H2N
N
H
27
24
26
29
R
X
30a
H
Br
30b 2'-Me
Br
HN
Br
30c
30d
2'-Cl
4'-Cl
Br
d
Cl
N
30e 2',5'-diOMe
Cl
N
H2N
N
30f
2',4'-diCl
Cl
X
30g 3',4'-diCl
Cl
30h
30i
R
2',3'-C4H4
3',4'-C4H4
Cl
30a-k
Cl
Cl
R
30j 4'-Ph
9a-19a
30k
3',4',5'-triOMe Cl
Scheme 1. Reagents and conditions: (a) sodium acetate, water, reflux, 4 h; (b) phosphorus oxychloride, N,N0-dimethylaniline, reflux, 4 h; (c) isopropanol, three drops concd
HCl, reflux, 4 h; (d) sodium hydride, dimethylformamide, 0 °C–1 h, rt–10 min-4 h.
Br
In the VEGFR-2 assay, compounds 9a with an unsubstituted
phenyl was twofold less potent than the standard 23. The 2-naph-
30c
Cl
N
H2N
Br
HN
Br
thyl substituted 17a was also twofold less active than 23, while the
1-naphthyl substituted 16a was 1.4-fold less active than 23 and
was the most active compound in this series, in the VEGFR-2 assay.
The electron withdrawing 20-Cl benzyl substituted 11a was the
next most active compound and was 1.6-fold less active than 23;
however the electron donating 20-Me benzyl substituted 10a was
11-fold less potent than 23. Comparison of 11a and 10a, suggests
that an electron withdrawing substituent at the 20-position is ben-
eficial for activity relative to an electron donating group. The 40-Cl
benzyl substituted 12a was sixfold less active than 23, while the
20,40-diCl benzyl substituted 14a was inactive. The 30,40-diCl benzyl
substituted 15a was also sixfold less active than 23. The 4-phenyl
benzyl substituted 18a was only twofold less active than 23 indi-
cating that bulk is tolerated in this position. The 30,40,50-triOMe
benzyl substituted 19a was also twofold less active than 23. The
Cl
28
b
N
N
a
27
N
N
H2N
H2N
N
Cl
Cl
31
11a
Scheme 2. Reagents and conditions: (a) sodium hydride, dimethylformamide, 0 °C–
30 min, rt–2 h; (b) isopropanol, three drops concd HCl, reflux, 4 h.
The IC50 values of RTK inhibition vary under different assay con-
ditions. Hence, we used a standard (control) compound in each of
the evaluations. For EGFR, the standard was 20 (Fig. 1); for VEGFR-1
the standard was 21; for VEGFR-2 the standard was 7; for PDGFRb
the standard was 22; for the cytotoxicity study against the growth
of A431 cells in culture the standards were 20, 7 and 23.
20,50-diOMe benzyl substituted 13a was inactive even at 200
lM.
The most active compound in the parent series, the 20-Me benzyl
substituted 10, is 450-fold more active relative to its regioisomer
10a. The most active compound in the N7 substituted series, the
1-naphthyl substituted 16a is threefold less active than its parent
regioisomer 16.
Since the inhibitory activities are determined in cells, a definite
structure–activity relationship cannot be determined for 9a–19a
and RTK inhibition. Compounds 11a with a 20-Cl benzyl substitu-
tion and 19a with a 30,40,50-triOMe benzyl substitution inhibited
PDGFRb at single digit micromolar concentrations. Compounds
11a and 19a inhibited PDGFRb fourfold and threefold better
respectively than the standard 22. The 20,40-diCl benzyl substituted
14a, inhibited PDGFRb at an IC50 5.5-fold lower than 22. Com-
pounds 16a (1-naphthyl substituted) and 18a (4-phenyl benzyl
substituted) had similar potencies and were 20-fold less active
than 22. The 2-naphthyl substituted 17a was 34-fold less potent
than 22. Hence bulky 7-substituents were not tolerated (16a–
18a). An electron withdrawing group at the 20-position (20-Cl ben-
zyl in 11a) is favorable for activity, but an electron donating group
in this position (20-Me benzyl in 10a), or an unsubstituted phenyl
(9a) is not favored. Moving the 20-chloro group to the 40-position
(compare 11a to 12a), or disubstitution with chloro groups (14a,
15a) is detrimental to activity. The most active compound in the
parent series in this assay is the 20,50-diOMe benzyl substituted
13. The most active compound in the N7 substituted series, the
20-Cl benzyl substituted 11a is >33-fold more active than its parent
regioisomer 11.
In the VEGFR-1 assay, compound 9a, with an unsubstituted
benzyl group was 5.6-fold less active than the standard 21. Com-
pound 10a has a methyl group in the 20 position of the benzyl sub-
stituent (compared to 9a). This compound is sevenfold less active
than standard 21. Compound 11a bears a 20-chloro benzyl substi-
tuent and is also sevenfold less active than 21. The 20-substituted
compounds 10a and 11a were less active than the unsubstituted
9a. Compound 16a with a (naphthalene-1-yl)methyl substitution,
and, bulk at the 20 position, is 8.4-fold less active than 21. Con-
versely the (naphthalene-2-yl)methyl substitution in compound
17a is beneficial for activity, with this compound being only 2.8-
fold less active than 21 and the most potent compound in this ser-
ies against VEGFR-1. Compound 12a with a 40-Cl benzyl is 4.5-fold
less active than 21; while 11a with a 20-Cl benzyl is sevenfold less
active than 19. Excessive bulk in the 40-position is not tolerated as
exemplified by the (biphenyl-4-yl)methyl substituted 18a which is
inactive at a concentration of 200 lM. Dichloro benzyl substituted
compounds 14a and 15a were eight- and sevenfold less active than
21 respectively. The 20,50-dimethoxy benzyl substituted compound

2448
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
is inactive at 200
l
M. The 30,40,50-triOMe benzyl substituted com-
the Hinge region. Additional hydrophobic interactions of the pyr-
rolo[2,3-d]pyrimidine ring with Leu1033 (not labeled) can stabilize
the docked pose. In this pose, the N7 benzylic substitution extends
towards Hydrophobic region I and is involved in interactions with
Val846, Ala864, Val897 and Val914. The N7 benzylic substitution
also interacts with the side chain carbon atoms of Glu883 and
Cys1043. The N4-(3-bromophenyl) is extended towards Hydropho-
bic region II and interacts with the side chains of Phe916, Leu838
(not shown) and Leu1033 (not shown).
In contrast, the pyrrolo[2,3-d]pyrimidine scaffold of 11 docks in
a flipped conformation compared to 11a described above that en-
ables the formation of three hydrogen bonds with the hinge region
(Fig. 3). The 2-NH2, N1 and pyrrole NH of 11 form hydrogen bonds
with the backbone of Glu915 and Cys917. Additionally, the pyrrol-
o[2,3-d]pyrimidine scaffold forms hydrophobic interactions with
Leu838 (not shown), Val846, Ala864, Val897 and Leu1033 (not
shown). This mode places the N4-(3-bromophenyl) moiety in
Hydrophobic region I where it forms hydrophobic interactions
with Val846, Ala864, Leu887, Val897, Val912 and Val914. Addi-
tional hydrophobic interactions with the side chain carbon atoms
of Lys866 and Glu883 stabilize this docked pose. The 6-benzylic
substitution extends towards Hydrophobic region II and forms
interactions with Leu838 (not shown), side chain atoms of
Phe916, Cys917, Lys918 and Gly920 (not shown). Thus, molecular
modeling and docking studies suggests that the 7-benzylic substi-
tution forces 11a to adopt a binding mode different from that
docked for the 6-benzylic compound 11 in VEGFR2.
pound 19a, which lacks the 20-substitution is about 8.8-fold less
active than 21. Compared to the parent 6-benzyl substituted series,
9a–19a showed a decrease in VEGFR-1 inhibition.
In the EGFR assay, the 20,50-dimethoxy benzyl substituted com-
pound 13a exhibited 2-digit micromolar inhibition. Though this
compound was the most potent compound of the series in this as-
say, its activity was 346-fold lower than the standard 20. Com-
pounds 10a with a 20-Me benzyl and 12a with a 40-Cl benzyl
substitution were about 500-fold less potent than 20. Compound
9a with an unsubstituted phenyl group was about 800-fold less ac-
tive than 20. EGFR inhibition was abolished on variations with a
bulky substitution (1-naphthyl methyl in 16a, 2-naphthyl methyl
in 17a, and 4-phenyl benzyl in 18a). In addition, alternative di-sub-
stitution (20,40-diCl benzyl in 14a, 30,40-diCl benzyl in 15a) and tri-
substitution (30,40,50-triOMe benzyl in 19a) also lead to inactivity in
the EGFR assay. Relative to the parent 6-benzyl substituted series
(9–19), 9a–19a showed a decrease in activity in the EGFR assay.
The most potent compound, in the A431 cytotoxicity assay, the
4-phenyl benzyl substituted 18a was equipotent to standard 20,
1.4-fold better than 7 and 1.7-fold less active than 23. The 20-Me
benzyl substituted 10a, 40-Cl benzyl substituted 12a, and 20,40-diCl
benzyl substituted 14a were equipotent to the standard 20, and
1.2-fold more potent than 7. Every compound had an improved
A431 cytotoxicity value relative to their corresponding EGFR ki-
nase IC50’s. This is presumably due to the varied transport and/or
pharmacokinetic differences of analogs between the two cell
systems.
There is no reported crystal structure of PDGFRb bound to a li-
gand. Hence a homology model of PDGFRb was built using the
structure of c-KIT kinase complex (pdb code 1PKG) as a template.30
Docking studies were performed with 11 and 11a as described
above for VEGFR-2. Compound 11a binds to the ATP binding site
of PDGFRb with the pyrrolo[2,3-d]pyrimidine portion occupying
the adenine binding site ( Fig. 4). Critical hydrogen bonds with
the Hinge region are maintained in this binding mode: the aniline
NH hydrogen bonds with the backbone of Cys684 while the N3 and
2-NH2 moieties form hydrogen bonds with Glu682. This binding
mode places the N4-(3-bromophenyl) moiety in Hydrophobic re-
gion I and forms hydrophobic interactions with Val614, Ala632
(not shown), Val665 and Leu833 (not shown) and with the side
chain carbon atoms of Lys634 andThr681. The N7-benzyl substitu-
ent lies in the Sugar binding pocket where it can interact with
Leu606 (not shown), Val614, Val615 (not shown) and Ala848. A
similar binding mode was seen with the best scored pose of 11
in PDGFRb. The 6-benzylic substituent of 11 accesses the same re-
gion accessed by the 7-benzylic substituent of 11a however in a
different conformation as shown in Figure 4. It was interesting to
note that alternate binding modes of 11a that scored 1-2 kcal/
mol higher than the best docked pose indicate different bound con-
formations of the 7-benzylic substituent (Fig. 5). In the alternate
binding mode, the 7-benzylic substituent accesses Hydrophobic re-
gion II instead of the sugar binding pocket as is seen in the bound
conformation in Fig. 5 where it interacts with Phe916 and Leu938
(not shown). Compound 11, which has a 6-benzylic substituent
cannot access Hydrophobic region II in the poses seen in Figures
3 and 4. Similar poses were observed from the docking studies
on other compounds. These binding modes suggested that the
presence of multiple docked poses could translate into differences
in the activity and selectivity of these compounds against different
kinases as compared to the parent series.
Taken together, transposing the 6-benzyl substituent in the par-
ent compounds to the 7-position produced significant differences
in the inhibition of the various RTKs. Although the EGFR inhibition
present in the parent series was lost in 9a–19a, some of these com-
pounds (11a, 19a) were much more potent PDGFRb inhibitors. The
variation in activity of analogs 9a–19a may either be due to the
stringent requirements for binding in individual RTKs and/or due
to differences in cell permeability of these compounds.
From this study, compounds 11a and 19a were identified as
inhibitors of multiple RTKs. The discovery of these compounds is
significant because they are potent inhibitors of VEGFR-2. In addi-
tion, 11a and 19a inhibit PDGFRb at concentrations better than the
standard agents. As stated above VEGFR-2 and PDGFRb are the two
principal mediators of angiogenesis. Compared to the most potent
multikinase inhibitor 11a, the most potent multikinase inhibitor in
the parent series—compound 13—had a 20,50-diOMe benzyl group
at N6 and exhibited inhibition against PDGFRb at values 1.5-fold
less than the standard 22 and in addition, demonstrated VEGFR-2
inhibition 17-fold better than the standard 23 (Table 1).
4. Molecular modeling studies
Molecular modeling studies were carried out using Flexx 3.1.236
and MOE 2008.1037 in VEGFR2 (pdb code 1YWN)38 and a homology
model of PDGFRb.39 Figures 3–5 depict the docked poses of 11 and
its regioisomer 11a as a representative study in VEGFR2 and the
homology model of PDGFRb. Figure 3 shows the superimposition
of the best scored docked poses of 11 and 11a in the ATP binding
site of VEGFR-2. The binding site of ATP competitive inhibitors in
RTKs consists of a Hinge region, two hydrophobic binding sites
(Hydrophobic region
I and II) and a Sugar binding pocket
(Fig. 3).25,40–42 Compounds 11 and 11a can be seen to adopt differ-
ent docked conformations in the active site. The pyrrolo[2,3-
d]pyrimidine ring of 11a occupies the adenine binding portion of
the ATP binding site. The 2-NH2 moiety is involved in a hydrogen
bond with Glu915 in the Hinge region while the N3 and 4-anilino
NH are involved in hydrogen bonds with the backbone of Cys917 in
On the basis of its in vitro activity, compound 11a was selected
for in vivo evaluation in a mouse model to determine its effect on
tumor growth, metastasis, and angiogenesis.
To examine whether compound 11a was effective in vivo in
reducing tumor volume and metastasis, a syngeneic mouse tumor
model was used. Athymic mice implanted with COLO-205 meta-

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
2449
Figure 3. Stereo view. Superimposition of docked poses of 11 (red) and 11a (white) in the ATP binding site of VEGFR2 (PDB: 1YWN).
Figure 4. Stereo view of the docked pose of 11 (red) and 11a (white) in the putative binding site of PDGFRb. The putative binding site of PDGFRb was generated by homology
modeling.30
static colon cancer cells, shown to overexpress PDGFRb were
used.43 In a six week toxicity determination study, it was found
that 35 mg/kg of 11a and 10 mg/kg (MTD) 844 caused no overt tox-
icity in mice (data not shown). At day 10 after implantation, the
multitargeted inhibitor 8 at the MTD of 10 mg/kg three times
weekly and 11a at 35 mg/kg three times weekly were administered
to mice (Fig. 6A). Tumor growth was measured using calipers. It
was found that 8 and 11a resulted in significant decreases in pri-
mary tumor growth rate (Fig. 6B) compared to the control. At the
end of the experiment, tumors were taken and stained for blood
vessels as described in the methods section. It was found that both
8 and 11a resulted in a significant decrease in primary tumor vas-
cularity as compared to control animals (Fig. 6C). Finally, livers
were stained for the presence of metastases and it was found that
11a, but not 8, resulted in significantly reduced numbers of COLO-
205 metastases to the liver (Fig. 6D). Thus 11a demonstrated
significant tumor inhibitory activity, antiangiogenic activity and
antimetastatic activity in a COLO-205 mouse tumor xenograft
model and is being considered for further preclinical development.
In summary, eleven novel N4-(3-bromophenyl)-7-(substitut-
edbenzyl) pyrrolo[2,3-d]pyrimidines 9a–19a were designed and
synthesized as RTK inhibitors. The biological evaluation showed
that several analogs afforded potent inhibition against PDGFRb,
VEGFR-2 and the A431 cytotoxicity assay. Of the analogs evaluated,
11a and 19a were better than or comparable to the standard com-
pounds used in these assays against two principal RTKs responsible
for angiogenesis in tumors—PDGFRb and VEGFR-2. Compound 11a
was evaluated in a COLO-205 xenograft mouse model, where it
exhibited tumor growth inhibition, antimetastatic and antiangio-
genic effects better than or comparable to standard 8. Using the
analogs described in this study as leads, structure based design
and synthesis of novel multi-RTK inhibitors are currently in pro-
gress. In addition, selected compounds from this study are being
evaluated in preclinical studies and in other tumor xenograft as-

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A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
Figure 5. Stereo view. Superimposition of the best scored pose (white) and alternate docked pose (blue) of 11a in the putative binding site of PDGFRb. The putative binding
site of PDGFRb was generated by homology modeling.39
Figure 6. Tumor growth (A), primary tumor growth rate (B), primary tumor vascularity (C), and liver metastasis of COLO-205 tumor xenografts after various treatments (D).
COLO-205 cells were implanted into male athymic mice as in the research design and methods section. Compounds were administered at the listed doses three times weekly
and tumor growth measured using calipers. At the experiment end, tumors were stained for vascularity and livers stained for metastases as in research design and methods
section. Data represent the average SD of 6–9 animals. ⁄ P > 0.05, ⁄⁄ P < 0.01, ⁄⁄⁄ P < 0.001 by one way ANOVA and Repeated Measures post test. The average tumor growth
rate for control treated animals (B) is 100.2 mm3/day, average number of tumor vessels in control-treated animals are 14.3/field (C) and average metastases in control treated
animals are 1.2/lobe.
says. One of the observed differences on transposing the 6-benzyl
substituent in the parent compounds to the 7-position was that—
although the EGFR inhibition present in the parent series was lost
in 9a–19a, some of these compounds (11a, 19a) were much more
potent PDGFRb inhibitors. Molecular modeling studies indicate
that access of Hydrophobic region II by the 7-benzyl substituent

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
2451
in this series could explain, in part, the observed differences in
activities as compared to the 6-substituted lead series of
compounds.
eluted with 2% methanol in chloroform. Fractions corresponding to
the product (TLC) were pooled and evaporated to dryness under re-
duced pressure to afford 29 (70%) as a green solid: mp 210–
212.5 °C; TLC Rf 0.56 (CHCl3/CH3OH, 5:1 with two drops of concen-
trated NH4OH); 1H NMR (DMSO-d6): d 5.81 (br s, 2H, NH2, exch),
6.55 (s, 1H, C5–CH), 6.79 (s, 1H, C6–CH), 7.11 (s, 1H, Ar–H), 7.22
(d, J = 12 Hz, 1H, Ar–H), 8.05 (d, J = 12 Hz, 1H, Ar–H), 8.14 (s, 1H,
Ar–H), 9.04 (s, 1H, NH, exch), 10.91 (s, 1H, NH, exch). Anal.
(C12H10BrN5ꢀ0.1H2O) C, H, N, Br.
5. Experimental section
Analytical samples were dried in vacuo (0.2 mm Hg) 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
5.3. General procedure for the synthesis of compounds 9a–19a
Bruker WH-300 (300 MHz) or
a Bruker 400 MHz/52 MM
To a round bottomed flask was added 29 and dissolved in 5 ml
of DMF. The solution was cooled to 0 °C and sodium hydride was
added. After stirring at 0 °C for 1 hour, the appropriate (halometh-
yl)arene was added. The reaction was continued at rt till the
appearance of a new spot (TLC) after which the reaction was
quenched with water. The water phase was extracted with chloro-
form. The organic phase was dried over sodium sulfate and evapo-
rated under reduced pressure. The residue was dissolved in
methylene chloride, 250 mg silica gel was added to the solution
which was then evaporated to dryness to form a plug. The silica
gel plug obtained was loaded onto a silica gel column and eluted
with 1% methanol in chloroform. Fractions corresponding to the
product (TLC) were pooled and evaporated to dryness under re-
duced pressure to afford the product.
(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. Thin-layer chromatography (TLC) was
performed on Whatman Sil G/UV254 silica gel plates with a fluo-
rescent 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 anal-
yses were performed by Atlantic Microlab, Inc., Norcross, GA. Ele-
ment compositions are within (0.4% of the calculated values and
indicate >95% purity. Fractional moles of water or organic solvents
frequently found in some analytical samples could not be pre-
vented in spite of 24–48 h of drying in vacuo and were confirmed
where possible by their presence in the 1H NMR spectra. All sol-
vents and chemicals were purchased from Aldrich Chemical Co.
or Fisher Scientific and were used as received. High resolution
mass spectra (HRMS) were obtained for compounds 13a and 18a
and TLC of these compounds produced a single spot in three differ-
ent solvent systems.
5.4. 7-Benzyl-N4-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrim
idine-2,4-diamine (9a)
Reaction of 29 (210 mg, 0.69 mmol), 30a (236 mg, 1.38 mmol)
and sodium hydride (16 mg, 0.65 mmol), using the general proce-
dure described above, gave 11a: yield 36%, mp >200 °C; TLC Rf
0.87 (CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6): d 5.30 (s, 2H, CH2), 6.03 (br s, 2H, NH2, exch),
6.60 (d, J = 9 Hz, 1H, Ar–H), 6.71 (s, 1H, C5–CH), 6.91 (s, 1H, C6–
CH), 7.12–7.15 (s, 1H, Ar–H), 7.22–7.31 (m, 4H, Ar–H), 7.49–7.51
(m, 2H, Ar–H), 8.07–8.13 (m, 2H, Ar–H), 9.17 (s, 1H, NH, exch).
Anal. (C19H16N5Brꢀ5CHCl3) C, H, N, Br.
5.1. 4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (27)
To a round-bottomed flask were added 26 (3 g, 19.9 mmol) of
and three drops of N,N0-dimethyl aniline. The mixture was sus-
pended on phosphorus oxychloride (250 mL). The reaction was
continued at reflux for 4 h. After cooling to room temperature,
the solvent was evaporated under reduced pressure and the resi-
due was treated with ice water (20 mL) in an ice bath. The pH of
the suspension was adjusted to 7 with concentrated ammonium
hydroxide solution. The precipitate was filtered, air-dried and then
dissolved in methanol. Silica gel (6 g) was added to the solution
which was then evaporated to dryness to form a plug. The silica
gel plug obtained was loaded onto a silica gel column and eluted
with 2% methanol in chloroform. Fractions corresponding to the
product (TLC) were pooled and evaporated to dryness under re-
duced pressure to afford 27 (10%) as a sparkling white solid: mp
215–216 °C; TLC Rf 0.56 (CHCl3/CH3OH, 5:1 with two drops of con-
centrated NH4OH); 1H NMR (DMSO-d6): d 6.24 (s, 1H, C5–CH), 6.49
(br s, 2H, NH2, exch), 7.08 (s, 1H, C6–CH), 11.46 (s, 1H, NH, exch).
Anal. (C6H5ClN4ꢀ0.25H2O) C, H, N, Cl.
5.5. N4-(3-Bromophenyl)-7-(2-methylbenzyl)-7H-pyrrolo[2,3-d]
pyrimidine-2,4-diamine (10a)
Reaction of 29 (500 mg, 1.64 mmol), 30b (607 mg, 3.28 mmol)
and sodium hydride (39 mg, 1.64 mmol), using the general proce-
dure described above, gave 10a: yield 34%, mp 177 °C; TLC Rf 0.85
(CHCl3/CH3OH, 5:1 with two drops concentrated NH4OH); 1H NMR
(DMSO-d6): d 2.30 (s, 3H, CH3), 5.19 (s, 2H, CH2), 5.97 (br s, 2H,
NH2, exch), 6.61 (m, 1H, Ar–H), 6.65 (s, 1H, C5–CH), 6.77 (s, 1H,
C6–CH), 7.05–7.27 (m, 5H, Ar–H), 7.95–8.13 (m, 2H, Ar–H), 9.12
(s, 1H, NH, exch). Anal. (C20H18N5Br) C, H, N, Br.
5.6. N4-(3-Bromophenyl)-7-(2-chlorobenzyl)-7H-pyrrolo[2,3-d]
pyrimidine-2,4-diamine (11a)
5.2. N4-(3-Bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-
Method A: Reaction of 29 (200 mg, 0.65 mmol), 30c (270 mg,
1.31 mmol) and sodium hydride (15 mg, 0.62 mmol), using the
general procedure described above, gave 11a: yield 7%.
Method B: To a round bottomed flask were added 31 (320 mg,
1.1 mmol)and 3-bromoaniline 28 (1.42 g, 8.25 mmol) in isopropa-
nol (10 mL), followed by the addition of three drops of concd HCl.
The reaction was continued at reflux for 4 h at the end of which the
solvent was evaporated under reduced pressure. The residue was
dissolved in methanol (10 mL), and silica gel gel (3 g) was added
to the solution which was then evaporated to dryness to form a
diamine (29)
To a round bottomed flask were added 27 (111 mg, 0.65 mmo-
l)and 3-bromoaniline 28 (226 mg, 1.31 mmol) in isopropanol
(10 mL). Following this three drops of concd HCl were added. The
reaction was continued at reflux for 4 h at the end of which the sol-
vent was evaporated under reduced pressure. The residue was dis-
solved in methanol (10 mL), and silica gel (250 mg) was added to
the solution which was then evaporated to dryness to form a plug.
The silica gel plug obtained was loaded onto a silica gel column and

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A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
plug. The silica gel plug obtained was loaded onto a silica gel col-
umn and eluted with plain chloroform. Fractions corresponding
to the product (TLC) were pooled and evaporated to dryness under
reduced pressure to afford 11a (81%) as a white solid: mp 182.8 °C;
TLC Rf 0.87 (CHCl3/CH3OH, 5:1 with two drops concentrated
NH4OH); 1H NMR (DMSO-d6): d 5.28 (s, 2H, CH2), 6.02 (br s, 2H,
NH2, exch), 6.59 (d, J = 6 Hz, 1H, Ar–H), 6.68 (s, 1H, C5–CH), 6.89
(s, 1H, C6–CH), 7.12 (d, J = 6 Hz, 1H, Ar–H), 7.21–7.29 (m, 3H, Ar–
H), 7.49 (d, J = 9 Hz, 1H, Ar–H), 8.05–8.11 (m, 2H, Ar–H), 9.17 (s,
1H, NH, exch). Anal. (C19H15N5BrCl) C, H, N, Br, Cl.
dure described above, gave 16a: yield 18%, mp 237.5 °C; TLC Rf 0.92
(CHCl3/CH3OH, 5:1 with two drops concentrated NH4OH); 1H NMR
(DMSO-d6): d 5.67 (s, 2H, CH2), 6.01 (br s, 2H, NH2, exch), 6.64 (d,
J = 3 Hz, 1H, C5–CH), 6.82 (d, J = 3 Hz, 1H, C6–CH), 6.93–8.21 (m,
11H, Ar–H), 9.12 (s, 1H, NH, exch). Anal. (C23H18N5Br) C, H, N, Br.
5.12. N4-(3-Bromophenyl)-7-(2-naphthylmethyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (17a)
Reaction of 29 (200 mg, 0.65 mmol), 30i (290 mg, 1.31 mmol)
and sodium hydride (15 mg, 0.62 mmol), using the general proce-
dure described above, gave 17a: yield 19%, mp 130.2 °C; TLC Rf
0.73 (CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6): d 5.37 (s, 2H, CH2), 5.98 (br s, 2H, NH2, exch),
6.65 (d, J = 3 Hz, 1H, C5–CH), 6.92 (d, J = 3 Hz, 1H, C6–CH), 7.12–
8.12 (m, 11H, Ar–H), 9.11 (s, 1H, NH, exch). HRMS calcd for
5.7. N4-(3-Bromophenyl)-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]
pyrimidine-2,4-diamine (12a)
Reaction of 29 (200 mg, 0.65 mmol), 30d (211 mg, 1.31 mmol)
and sodium hydride (15 mg, 0.62 mmol), using the general proce-
dure described above, gave 12a: yield 47%, mp 127–128 °C; TLC
Rf 0.88 (CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6): d 5.19 (s, 2H, CH2), 6.04 (br s, 2H, NH2, exch),
6.63 (d, J = 3 Hz, 1H, C5–CH), 6.89 (d, J = 3 Hz, 1H, C6–CH), 7.14–
8.09 (m, 8H, Ar–H), 9.14 (s, 1H, NH, exch). Anal. (C19H15N5BrCl)
C, H, N, Br, Cl.
C23H18N5Br 443.0745, found 443.0792.
5.13. 7-Biphenyl-4-ylmethyl-N4-(3-bromo-phenyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (18a)
Reaction of 29 (500 mg, 1.64 mmol), 30j (666 mg, 3.28 mmol)
and sodium hydride (37 mg, 1.55 mmol), using the general proce-
dure described above, gave 18a: yield 10%, mp 86.2 °C; TLC Rf 0.82
(CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH); 1H
NMR (DMSO-d6): d 5.24 (s, 2H, CH2), 5.99 (br s, 2H, NH2, exch),
6.64 (s, 1H, C5–CH), 6.91 (s, 1H, C6–CH), 7.07–8.11 (m, 13H, Ar–
H), 9.12 (s, 1H, NH, exch). HRMS calcd for C25H20N5Br 469.0902,
found 469.1001.
5.8. N4-(3-Bromophenyl)-7-(2,5-dimethoxybenzyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (13a)
Reaction of 29 (300 mg, 0.98 mmol), 30e (368 mg, 1.97 mmol)
and sodium hydride (22 mg, 0.93 mmol), using the general proce-
dure described above, gave 13a: yield 10%, mp 150.9 °C; TLC Rf
0.71 (CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6): d 3.56 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 5.11
(s, 2H, CH2), 6.00 (br s, 2H, NH2, exch), 6.11 (s, 1H, Ar–H), 6.64
(d, J = 3 Hz, 1H, C5-CH), 6.77–6.79 (m, 1H, Ar–H), 6.85 (d, J = 3 Hz,
1H, C6-CH), 6.92–8.11 (m, 5H, Ar–H), 9.13 (s, 1H, NH, exch). HRMS
calcd for C21H20N5O2Br 453.0800, found 453.0788.
5.14. N4-(3-Bromo-phenyl)-7-(3,4,5-trimethoxy-benzyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (19a)
Reaction of 29 (400 mg, 1.31 mmol), 30k (570 mg, 2.63 mmol)
and sodium hydride (31 mg, 1.31 mmol), using the general proce-
dure described above, gave 19a: yield 14%, mp 113.6 °C; 1H NMR
(DMSO-d6): d 3.61 (s, 3H, OCH3), 3.69 (s, 6H, 2 ꢁ OCH3), 5.10 (s,
2H, CH2), 5.99 (br s, 2H, NH2, exch), 6.60 (s, 1H, C5–CH), 6.60 (s,
2H, Ar–H), 6.90 (s, 1H, C6–CH), 7.12–8.12 (m, 4H, Ar–H), 9.09 (s,
1H, NH, exch). TLC Rf 0.85 (CHCl3/CH3OH, 10:1 with two drops con-
centrated NH4OH); Anal. (C22H22N5BrO3) C, H, N, Br.
5.9. N4-(3-Bromophenyl)-7-(2,4-dichlorobenzyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (14a)
Reaction of 29 (100 mg, 0.32 mmol), 30f (125 mg, 0.64 mmol)
and sodium hydride (7 mg, 0.32 mmol), using the general proce-
dure described above, gave 14a: yield 24%, mp 191.4 °C; TLC Rf
0.86 (CHCl3/CH3OH, 5:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6) d 5.26 (s, 2H, CH2), 6.05 (br s, 2H, NH2, exch),
6.58 (d, J = 6 Hz, 1H, Ar–H), 6.69 (s, 1H, C5–CH), 6.89 (s, 1H, C6–
CH), 7.13–7.35 (m, 3H, Ar–H), 7.67 (s, 1H, Ar–H), 8.07–8.11 (m,
2H, Ar–H), 9.18 (s, 1H, NH, exch). Anal. (C19H14N5BrCl2) C, H, N,
Br, Cl.
5.15. 4-Chloro-7-(2-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-
2-amine (31)
To a round bottomed flask was added 27 (60 mg, 0.35 mmol)
and dissolved in 5 ml of DMF. The solution was cooled to 0 °C
and sodium hydride (9 mg, 0.33 mmol) was added. After stirring
at 0 °C for 1 h 30c (81 mg, 0.39 mmol) was added. The reaction
was continued at rt for 2 h, at the end of which the reaction was
quenched with water. The water phase was extracted with ethyl-
acetate. The organic phase was dried over sodium sulfate and evap-
orated under reduced pressure. The residue was dissolved in
methylene chloride, 250 mg silica gel was added to the solution
which was then evaporated to dryness to form a plug. The silica
gel plug obtained was loaded onto a silica gel column and eluted
with 4:1 hexane/ethylacetate. Fractions corresponding to the prod-
uct (TLC) were pooled and evaporated to dryness under reduced
pressure to afford the product 31 as a white solid: yield 48%, mp
148 °C; TLC Rf 0.23 (hexane/EtOAc, 4:1); 1H NMR (DMSO-d6): d
5.32 (s, 2H, CH2), 6.39 (d, J = 4 Hz, 1H, C5–CH), 6.65–6.73 (bs, 2H,
NH2, exch), 6.71 (d, J = 16 Hz, 1H, Ar-H), 7.17–7.18 (d, 1H, C6–
CH), 7.22–7.50 (m, 4H, Ar–H). Anal. (C13H10Cl2N4) C, H, N, Cl.
5.10. N4-(3-Bromophenyl)-7-(3,4-dichlorobenzyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (15a)
Reaction of 29 (200 mg, 0.65 mmol), 30g (256 mg, 1.31 mmol)
and sodium hydride (15 mg, 0.62 mmol), using the general proce-
dure described above, gave 15a: yield 16%, mp 136.3 °C; TLC Rf
0.78 (CHCl3/CH3OH, 10:1 with two drops concentrated NH4OH);
1H NMR (DMSO-d6) d 5.20 (s, 2H, CH2), 6.02 (br s, 2H, NH2, exch),
6.64 (d, J = 3 Hz, 1H, C5–CH), 6.92 (d, J = 3 Hz, 1H, C6–CH), 7.09–
8.10 (m, 7H, Ar–H), 9.13 (s, 1H, NH, exch). Anal.
(C19H14BrCl2N5ꢀ0.25CHCl3) C, H, N, Br, Cl.
5.11. N4-(3-Bromophenyl)-7-(1-naphthylmethyl)-7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine (16a)
Reaction of 29 (500 mg, 1.64 mmol), 30h (579 mg, 3.28 mmol)
and sodium hydride (39 mg, 1.64 mmol), using the general proce-

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
2453
6. Molecular modeling and computational studies
Molecular Probes (Eugene, OR). All other chemicals were from Sig-
ma Chemical unless otherwise noted.
The X-ray crystal structure of VEGFR2 at 1.71 Å resolution was
obtained from the protein database (PDB ID 1YWN).38 This crystal
structure contains VEGFR2 in complex with a novel 4-amino-
furo[2,3-d]pyrimidine. Docking studies were performed using the
docking suite of Molecular Operating Environment software (MOE
2008.10)37 and Flexx 3.1.2.45 After addition of hydrogen atoms,
the protein was then ‘prepared’ using the LigX function in MOE.
LigX is a graphical interface and collection of procedures for con-
ducting interactive ligand modification and energy minimization
in the active site of a flexible receptor. In LigX calculations, the
receptor atoms far from the ligand are constrained and not allowed
to move while receptor atoms in the active site of the protein are
allowed to move but are subject to tether restraints that discour-
age gross movement.37 This procedure was performed with the de-
fault settings. Ligands were built using the molecule builder
function in MOE and were energy minimized to its local minima
using the MMF94X forcefield to a constant of 0.05 kcal/mol. Li-
gands were docked into the active site of the prepared protein
using the docking suite as implemented in MOE. The docking
was restricted to the active site pocket residues using Alpha trian-
gle placement method. Refinement of the docked poses was carried
out using the Forcefield refinement scheme and scored using Affin-
ity dG scoring system. Around 30 poses were returned for each
compound at the end of each docking run. The docked poses were
manually examined in the binding pocket to ensure quality of
docking and to confirm absence of steric clashes with the amino
acid residues of the binding pocket.
To validate the utility of MOE 2008.10 for docking ligands into
the active site, the native ligand in the crystal structure (PDB:
1YWN) was built using the molecule builder, energy minimized
using MMFF94x forcefield to a gradient of 0.05 kcal/mol and
docked into the active site using the above parameters. The best
docked pose of the native ligand displayed an RMSD of 0.6031 Å
compared to the crystal structure pose. MOE 2008.10 was thus val-
idated for our docking process.
Docking with Flexx 3.1.2 was carried out in by defining the ac-
tive site as a sphere of ꢂ6.5 Å from the ligand. The protonation
states utilized for the proteins and the ligands were calculated
using the default settings. Water molecules in the active site were
permitted to rotate freely. Ligands for docking were prepared using
MOE and energy minimized using the MMF94X forcefield to a con-
stant of 0.05 kcal/mol. Triangle matching was used as the place-
ment method and the docked poses were scored using default
settings. The docked poses were exported and visualized in MOE.
The best docked pose had an RMSD of 0.7686 Å. Flexx 3.1.2 was
thus validated for our docking process.
6.3. 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).
6.4. Phosphotyrosine ELISA
Cells used were tumor cell lines naturally expressing high levels
of EGFR (A431), Flk-1 (U251), Flt-1 (A498), PDGFR-b (SF-539), and
FGFR-1 (NIH OVCAR-8). Expression levels at the RNA level were de-
rived from the NCI Developmental Therapeutics Program (NCI-
DTP) web site public molecular target information (http://
www.dtp.nci.nih.gov/mtargets/mt_index.html). Briefly, cells at
60–75% confluence were placed in serum-free medium for 18 h
to reduce the background of phosphorylation. Cells were always
>98% viable by Trypan blue exclusion. Cells were then pretreated
for 60 min with 10, 3.33, 1.11, 0.37, and 0.12 lM compounds fol-
lowed 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) containing 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 for 30 min at 60 °C and
further incubation in 70% ethanol for an additional 30 min. Cells
were further exposed to block (TBS with 1% BSA) for 1 h, washed,
and then a horseradish peroxidase (HRP)-conjugated phosphotyro-
sine (PY) antibody added overnight. The antibody was removed,
cells were washed again in TBS, exposed to an enhanced luminal
ELISA substrate (Pierce Chemical, Rockford, IL), and light emission
measured using a UV product (Upland, CA) BioChemi digital dark-
room. The known RTK-specific kinase inhibitor, PD153035, was
used as a positive control compound for EGFR kinase inhibition;
SU5416 for Flk-1 kinase inhibition; AG1295 for PDGFR-b kinase
inhibition; and CB676475 (4-[(40-chloro-20-fluoro)phenylamino]-
6,7-dimethoxyquinazoline) was used as a positive control for both
Flt-1 and Flk-1 kinase inhibition. Data were graphed as a percent of
cells receiving growth factor alone and IC50 values were estimated
from two to three separate experiments (n = 8–24) using hand-
drawn probit plots. In each case, the activity of a positive control
inhibitor did not deviate more than 10% from the IC50 values listed
in the text.
6.5. CYQUANT cell proliferation assay
Docking studies were performed for 11 and 11a in MOE and
Flexx using a similar procedure. Poses from the docking experi-
ment performed in Flexx were exported and visualized in MOE.
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 a UV product
BioChemi digital darkroom. A positive control used for cytotoxicity
in each experiment was cisplatin, with an apparent average IC50
6.1. 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).
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.
6.2. Chemicals
All growth factors (bFGF, VEGF, EGF, and PDGF-b) were pur-
chased from Peprotech (Rocky Hill, NJ). PD153035, SU5416,
AG1295, and CB676475 (4-[(40-chloro-20-fluoro)phenylamino]-
6,7-dimethoxyquinazoline) were purchased from Calbiochem
(San Diego, CA). The CYQUANT cell proliferation assay was from
6.6. Statistics
All analysis was done using Prism 4.0. (GraphPad Software, San
Diego, CA).

2454
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 2444–2454
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groups of four male NCr nu/nu mice at 8 weeks of age without tu-
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10, 15 and 20 mg/kg 8 three times weekly on Monday AM,
Wednesday noon and Friday PM. Weights were taken and animals
observed for acute distress during the first 96 h after injection. No
apparent toxicity and no significant weight loss (P >0.05; one-way
ANOVA with Repeated Measures Post-Test) was observed through-
out a 6 week period after drug administration.
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6.8. COLO-205. human metastatic colon mouse xenograft
One million COLO-205 (liver colonizing; American Type Culture
Collection) human metastatic colon cancer cells were injected SQ
into the lateral flank of athymic NIH-II male mice, 8 weeks in
age. Animals are monitored every other day for the presence of tu-
mors. At the time in which most tumors are measurable by calipers
(days 8–11 after implantation), animals with tumors are randomly
sorted into treatment groups. DMSO stocks (30 mM) of drugs are
further dissolved into sterile water for injection and the optimal
dose (from MTD studies) injected intraperitoneally (IP). The length
(long side), width (short side) and depth of the tumors were mea-
sured using digital Vernier Calipers each Monday, Wednesday, and
Friday. Tumor volume is calculated using the formula length ꢁ
width ꢁ depth. Tumor growth rate is calculated using a linear
regression analysis algorithm with the software GraphPad Prism
4.0.c (GraphPad software, San Diego, CA). At the experiment’s
end, animals were humanly euthanized using carbon dioxide, tu-
mors and liver excised, fixed in 20% neutral buffered formalin for
8–10 h, embedded into paraffin, and hematoxylin-eosin (H&E)
stain of 3–4 separate tissue sections completed to span the tu-
mor/liver. Together with the OUHSC Department of Pathology core,
metastases per liver lobe were counted using the H&E stained sec-
tions. The metastases could be seen as purple clusters of disorga-
nized cells on the highly organized largely pink liver. Together
with the OUHSC Department of Pathology core, blood vessels per
unit area on primary tumors are also counted in 5 fields at 100x
magnification and averaged.
36. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
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Acknowledgments
39. Gangjee, A.; Zaware, N.; Raghavan, S.; Ihnat, M.; Shenoy, S.; Kisliuk, R. L. J. Med.
Chem. 2010, 53, 1563.
40. Traxler, P. M.; Furet, P.; Mett, H.; Buchdunger, E.; Meyer, T.; Lydon, N. J. Med.
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Chem. 2005, 48, 710.
This work was supported, in part, by the National Institutes of
Health and National Cancer Institute Grant CA98850 (A.G.) and
the Duquesne University Adrian Van Kaam Chair in Scholarly
Excellence (AG).
42. Choowongkomon, K.; Sawatdichaikul, O.; Songtawee, N.; Limtrakul, J. Molecules
2010, 15, 4041.
Supplementary data
43. Schroeder, M. C.; Hamby, J. M.; Connolly, C. J. C.; Grohar, P. J.; Winters, R. T.;
Barvian, M. R.; Moore, C. W.; Boushelle, S. L.; Crean, S. M.; Kraker, A. J.; Driscoll,
D. L.; Vincent, P. W.; Elliott, W. L.; Lu, G. H.; Batley, B. L.; Dahring, T. K.; Major, T.
C.; Panek, R. L.; Doherty, A. M.; Showalter, H. D. H. J. Med. Chem. 1915, 2001, 44.
44. Shaheen, R. M.; Davis, D. W.; Liu, W.; Zebrowski, B. K.; Wilson, M. R.; Bucana, C.
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45. Flexx, 3.1.2 BioSolveIT GmbH.
Supplementary data (elemental analysis and high-resolution
mass spectra (HRMS) (ESI)) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2012.01.029.
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