Antiangiogenic and antitumor agents: Design, synthesis, and evaluation of novel 2-amino-4-(3-bromoanilino)-6-benzylsubstituted pyrrolo[2,3-d]pyrimidines as inhibitors of receptor tyrosine kinases

Article abstract of DOI:10.1016/j.bmc.2003.08.034

Several different classes of growth factor receptors containing tyrosine kinases (RTK) are directly or indirectly involved in angiogenesis. Inhibition of these RTKs has provided a new paradigm in the treatment of tumors by restricting their growth and metastasis. We have designed, synthesized and evaluated eleven novel 2-amino-4-(3-bromoanilino)-6-substituted benzyl pyrrolo[2,3-d]pyrimidines as the first in a series of RTK inhibitors. These analogues were synthesized from appropriate α-bromomethylbenzyl ketones by cyclocondensation with 2,6-diamino-4-pyrimidone to afford the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-d]pyrimidines. Chlorination of the 4-position followed by displacement with 3-bromoaniline afforded the target compounds. In some instances, the 2-amino moiety of the pyrrolo[2,3-d] pyrimidines was protected prior to the chlorination and displacement followed by deprotection. The compounds were evaluated as inhibitors of vascular endothelial growth factor receptors VEGFR-2 (Flk-1, KDR) and VEGFR-1 (Flt-1); epidermal growth factor receptor (EGFR); and platelet-derived growth factor receptor-β (PDGFR-β). Selected compounds were also evaluated against the growth of A431 cells (which overexpress EGFR) in culture and as inhibitors of angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay. In each evaluation, a known standard compound was used as a comparison. Of the 11 analogues, five were more potent or equipotent as compared to standard compounds against the growth factor receptors. Two analogues showed superior inhibition of A431 cells in culture compared to the standard compounds. Three analogues were equipotent with the standard compound in the CAM assay and four of the analogues were dual inhibitors of RTKs. The structure-activity relationship for inhibition of different RTKs was quite distinct and different, and for VEGFR-2 and EGFR diametrically opposite. The inhibitory data against the RTKs in this study demonstrates that variation of the substituent(s) in the benzyl ring of these 2-amino-4-anilino 6-benzyl pyrrolo[2,3-d]pyrimidines does indeed control both the potency and specificity of inhibitory activity against RTKs.

Full text of DOI:10.1016/j.bmc.2003.08.034

Bioorganic & Medicinal Chemistry 11 (2003) 5155–5170
Antiangiogenic and Antitumor Agents:
Design, Synthesis, and Evaluation of Novel 2-Amino-4-(3-
bromoanilino)-6-benzylsubstituted Pyrrolo[2,3-d]pyrimidines as
Inhibitors of Receptor Tyrosine Kinases
Aleem Gangjee,a,* Jie Yang,a Michael A. Ihnatb and Shekhar Kamatb
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
bDepartment of Cell Biology, School of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Received 16 May 2003; accepted 15 August 2003
Abstract—Several different classes of growth factor receptors containing tyrosine kinases (RTK) are directly or indirectly involved
in angiogenesis. Inhibition of these RTKs has provided a new paradigm in the treatment of tumors by restricting their growth and
metastasis. We have designed, synthesized and evaluated eleven novel 2-amino-4-(3-bromoanilino)-6-substituted benzyl pyrrolo[2,3-
d]pyrimidines as the first in a series of RTK inhibitors. These analogues were synthesized from appropriate a-bromomethylbenzyl
ketones by cyclocondensation with 2,6-diamino-4-pyrimidone to afford the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-d]pyr-
imidines. Chlorination of the 4-position followed by displacement with 3-bromoaniline afforded the target compounds. In some
instances, the 2-amino moiety of the pyrrolo[2,3-d]pyrimidines was protected prior to the chlorination and displacement followed
by deprotection. The compounds were evaluated as inhibitors of vascular endothelial growth factor receptors VEGFR-2(Flk-1,
KDR) and VEGFR-1 (Flt-1); epidermal growth factor receptor (EGFR); and platelet-derived growth factor receptor-b (PDGFR-
b). Selected compounds were also evaluated against the growth of A431 cells (which overexpress EGFR) in culture and as inhibitors
of angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay. In each evaluation, a known standard compound
was used as a comparison. Of the 11 analogues, five were more potent or equipotent as compared to standard compounds against
the growth factor receptors. Two analogues showed superior inhibition of A431 cells in culture compared to the standard com-
pounds. Three analogues were equipotent with the standard compound in the CAM assay and four of the analogues were dual
inhibitors of RTKs. The structure–activity relationship for inhibition of different RTKs was quite distinct and different, and for
VEGFR-2and EGFR diametrically opposite. The inhibitory data against the RTKs in this study demonstrates that variation of the
substituent(s) in the benzyl ring of these 2-amino-4-anilino 6-benzyl pyrrolo[2,3-d]pyrimidines does indeed control both the potency
and specificity of inhibitory activity against RTKs.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Thus angiogenesis and metastasis contribute to the poor
prognosis seen in patients with highly angiogenic
tumors.5 Inhibition of tumor angiogenesis has thus
provided an attractive target for the development of
antiangiogenesis agents as antitumor agents. Anti-
angiogenic therapy is targeted to non-tumor cells
(endothelial cells) which are expected to have less ability
to mutate in order to produce resistance compared with
tumor cells. Thus, antiangiogenesis agents have afforded
new paradigms for the treatment of cancer.6,7
Angiogenesis is the formation of new blood vessels from
existing vasculature.1 Angiogenesis occurs during
development and in normal adults during wound heal-
ing, pregnancy and corpus luteum formation. Human
disease states associated with angiogenesis include
retinopathies, endometriosis, psoriasis, atherosclerosis,
rheumatoid arthritis and the growth and metastasis of
tumors.2 Angiogenesis plays a pivotal role in the growth
of solid tumors and their invasion and metastasis.3,4
Angiogenesis requires the transduction of signals from
the extracellular domain of endothelial cells to the
nucleus which are, either directly or indirectly, receptor
mediated and some of the receptors are receptor tyrosine
*Corresponding author. Tel.: +1-412-396-6070; fax: +1-412-396-
5593; e-mail: gangjee@duq.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.034

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
kinases (RTK). RTKs are transmembrane receptors
consisting of an extracellular ligand binding domain, a
hydrophobic transmembrane domain and a cytoplasmic
domain which contains regulatory regions and the cat-
alytic tyrosine kinase domain with binding sites for both
ATP and substrate which allows for autophos-
phorylation and is the critical step in signal transduction
pathways. RTK activation often requires the formation
of homodimers or heterodimers with other RTKs. Non-
receptor tyrosine kinases do not have an extracellular
domain and are usually dimers.7,8
has been in clinical trials and ZD6474 which is currently
in clinical trials29ꢀ33 as an antitumor agent.
PDGFR-a, PDGFR-b and PDGF have been impli-
cated, indirectly, in inducing VEGF secretion and hence
in angiogenesis.34 Inhibition of PDGFR mediated sig-
naling has direct and indirect effects on tumor growth
and metastasis.35ꢀ37 Over expression of PDGFR has
been observed in glioblastomas.23,38,39 ST1-571 (Glee-
vec) is a potent inhibitor of PDGFR and a v-Abl inhi-
bitor. It has shown excellent anti-leukemia activity in
the clinic, has been approved for the treatment of
chronic myeloid leukemia and metastatic malignant
gastrointestinal stromal tumors, and is under investiga-
tion in the treatment of glioblastomas which exploits its
PDGFR inhibitory activity.40
RTKs consist of families of growth factor receptors
such as the platelet-derived growth factor receptor
(PDGFR), fibroblast growth factor receptor (FGFR),
vascular endothelial growth factor receptor (VEGFR),
and epidermal growth factor receptor (EGFR) among
several others. Several small molecule inhibitors of
RTKs are currently in clinical trials as antitumor agents
and the majority of these are targeted at the ATP bind-
ing site of tyrosine kinases.9,10 Some examples of these
compounds, along with preclinical compounds, are pre-
sented in Figure 1.
Recent reports41 suggest that inhibition of VEGFR-1
(Flt-1) could be a therapeutic target not only for tumor
angiogenesis but also for the inflammation associated
with tumors. Thus VEGFR-1 is also a viable target
against cancer.
Single RTK inhibition by small molecules is a possible
mechanism of cancer therapy. Simultaneous targeting of
two or more RTKs represents a novel approach for
(antiangiogenic) therapy of tumors. These RTKs are
present on endothelial cells (VEGFR, PDGFR) in
tumor cells (FGFR, PDGFR) and pericytes (FGFR,
PDGFR) thus simultaneous inhibition of more than one
RTK could provide synergistic effects against
tumors.42ꢀ44
EGFR is overexpressed in several tumors.11ꢀ23 Thus,
inhibition of EGFR is a worthwhile goal for cancer
therapy and several inhibitor of EGFR such as ZD1839
(Iressa) and OS1774 (Tarceva) have completed clinical
trials as antitumor agents. ZD1839 has recently been
approved by the FDA for non-small cell lung cancer.
VEGFR-2(Flk-1/KDR) is the principal receptor that
mediates VEGF stimulation in angiogenesis. The recep-
tors for VEGF are almost exclusively expressed on
endothelial cells. Targeted inhibition or disruption of
VEGFR-2results in abrogation of angiogenesis and
tumor growth.24,25 In addition, VEGF and VEGFRs
are overexpressed in many tumor types.26ꢀ28 Several
inhibitors of VEGFR-2have provided antitumor activ-
ity. Notable among these are the pyrroloindolinones
and quinazolines exemplified by SU5416 (Fig. 1) which
The ATP-binding site of RTKs has been shown to be a
viable target for rational drug design.10 Of these, the
most successful have been those based on the quinazo-
line and indoline scaffolds which are ATP-competitive
(Fig. 1). Excellent reviews for quinazolines;45 pyr-
ido[d]pyrimidines;46,47 and general reviews of tyrosine
kinase inhibitors9,10 in cancer treatment have been
published.
Figure 1. RTK inhibitors.

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5157
Molecular modelling
appropriately orient the other parts of the molecule in
the ATP binding site. This modeling alignment, with
ATP from IRK modeled into VEGF-2is shown in Fig-
ure 2 and is based on the proposed general pharmaco-
phore for RTKs.10,49
Several crystal structures of RTKs have been published.
However, crystal structures of VEGFR2or PDGFR
with ATP or with quinazoline or pyrrolo[2,3-d]pyr-
imidine inhibitors are unknown. After this study was
completed a report of the crystal structure of EFGR
with quinazoline inhibitors was recently published.48
Using available X-ray crystal structures of c-AMP
dependent protein kinase and FGFR, a general phar-
macophore model of the ATP and ATP-competitive
inhibitor binding site of RTKs has been proposed49 and
refined.10
An important additional binding site in the Hinge
region which involves a hydrogen-bonding backbone
carbonyl that would be bonded to a 2-amino group on
the pyrimidine ring of an inhibitor of a quinazoline,
pyrido[2,3-d]pyrimidines or pyrrolo[2,3-d]pyrimidine
scaffold is shown as the carbonyl of Cys919 in the model
in Figure 2. ATP, which lacks this 2-NH2 moiety, does
not use this site. For EGFR this residue corresponds to
Met 769 and for VEGFR2, Cys 919. None of the RTK
inhibitors in the literature, which contain a pyrrolopyr-
imidine ring, has a 2-NH2 group to exploit this
H-bonding site. A 2-NH2 pyrazolopyrimidine was
reported by Traxler et al.,53 however the authors do not
ascribe a binding for the 2-NH2 moiety. We have uti-
lized this 2-NH2 group as an important integral part of
the design of pyrrolo[2,3-d]pyrimidine analogues as
RTK inhibitors to provide additional binding.
The model proposed consists of an Adenine region
which is a hydrophobic binding site for the adenine ring
of ATP as well as for the heterocyclic scaffold of inhi-
bitors such as quinazolines and pyridopyrimidines. The
N1- and N6-amino nitrogens of the adenine ring of
ATP are hydrogen bonded with a donor acceptor sys-
tem with two amino acid residues of the Hinge region.
For EGFR these residues are Gln 767 and Met 769.10
For the structure based design of RTK inhibitors, we
carried out a sequence homology alignment using
SYBYL 6.7 and its Align Structure Using Homology
option of the known crystal structures of the insulin
receptor kinase (IRK) domain50 with bound ATP and
The ATP binding site also contains a Hydrophobic
region I (Fig. 2). This pocket extends in the direction of
the lone pair of the N7 of ATP and is not used by ATP
for binding. This pocket accommodates the phenyl or
heterocyclic ring of inhibitor molecules attached to the
4-NH2 of the pyrimidine ring of quinazolines, pyr-
idopyrimidines or pyrrolopyrimidines. Thus, in Figure
1, the 2-Cl, 4-F-phenyl ring of ZD1839 is accom-
modated in this region. The size of this pocket varies in
different RTKs and is an area that could be exploited
for selectivity. Thus several substituted phenyl rings and
the unoccupied or apo X-ray crystal structure of
51
VEGFR-2(Flk1, KDR)
(and the inhibitor bound
FGFR X-ray crystal structure),52 followed by the Fit
option to further align the Hinge region. For VEGFR-2
the corresponding Hinge region residues are Glu 917
and Cys 919 (Fig. 2). These are important binding sites
for ATP and ATP-competitive inhibitors and serve to
anchor the heterocyclic portion of the molecule and
Figure 2. ATP from IRK modeled into VEGFR-2using SYBYL 6.7.

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
heterocyclic rings have been utilized on a variety of
pyrimidine containing scaffolds to afford potent, and in
some cases specific and multiple inhibitors of various
RTKs.9,10,45ꢀ47,54ꢀ56
was anticipated to increase binding and consequently
potency. The second moiety included in our design was
the 4-anilinophenyl ring with a 3-bromo substitution.
This moiety has provided potent inhibition of RTKs
and is accommodated in the Hydrophobic region I or
the Sugar pocket depending on the mode of attachment
(i.e., Mode 1 or 2, Fig. 3) for our proposed pyrrolo[2,3-
d]pyrimidines. As the first analogues in a series of RTK
inhibitors, we elected to keep these two substitutions
(i.e., the 2-NH2 group and the 4-amino-(3-Br-phenyl)
constant. The purpose was to determine if variation in
the phenyl ring of the 6-benzyl moiety of the pyr-
rolo[2,3-d]pyrimidine scaffold would allow for specifi-
city and/or for multiple inhibition of various RTKs,
with the 2-NH2 and 4-amino-(3-Br)phenyl groups being
constant. Thus we designed compounds 1–11 which
contain an amino moiety at the 2-position, a meta-
bromo aniline at the 4-position and a benzyl substitu-
tion at the 6-position.
The Sugar pocket (Fig. 2) in the ATP binding site
accommodates the sugar moiety of ATP and is hydro-
philic but not entirely so. The mode of binding of the
4-anilinophenyl quinazoline based RTK inhibitors,
shown in Figure 1, are proposed similar to the binding
of ATP. Alternate modes of binding of quinazolines
such as Tarceva have recently been reported.48 For the
6-5 pyrrolo[2,3-d]pyrimidines such as CGP59326 (Fig.
1) two different modes of binding are also possible. The
first is where the pyrrolo[2,3-d]pyrimidine binds like
ATP and the quinazolines, where the m-Cl-phenyl ring
binds in Hydrophobic region I is termed Mode 1 (Fig.
3). An alternate mode of binding proposed by the
Novartis group,10 has the m-Cl-phenyl moiety binding
in the Sugar pocket. In this mode, the 7-position pyr-
rolo NH of CGP59326 forms one of the bidentate
H-bonds with the Hinge region, substituting for the
4-NH of ATP or quinazolines; the N1 nitrogen of
CGP59326 forms the second bidentate H-bond and
mimics the N3-nitrogen of quinazoline, this is termed
Mode 2. These are illustrated, without the RTK in
Figure 3.
Compounds 1–11 were modeled in the VEGFR-2kinase
binding site using the apo X-ray crystal structure51 and
the alignment of the ATP from Figure 2. Figure 4 shows
a stereo view of the binding of a low energy conformer
(SYBYL 6.7 Minimize and Search Options) of com-
pound 5 superimposed on ATP (not shown) modeled in
VEGFR-2. In the Hinge region, the anilino NH makes a
H-bond with the backbone carbonyl of Glu 917, the N3
with the NH of Cys 919 and the 2-NH2 with the car-
bonyl of Cys 919. The 3-Br-phenyl ring lies in the
Hydrophobic region I which consists of Leu 1035 and
Cys 1045. The 6-benzyl ring lies in the Sugar Pocket and
the ortho-methyl group of the benzyl ring makes
hydrophobic contact with Val 848. Since these are
models, constructed using homology alignment with
known X-ray crystal structures, the exact mode of
binding of 1–11 cannot be predicted, however this
model does provide a general template for the design of
RTK (VEGFR-2) inhibitors.
In 1994, we57 proposed similar dual binding modes for
6-5 fused ring furo[2,3-d]pyrimidines and pyrrolo[2,3-
d]pyrimidines for inhibitors of folate related enzymes.
Thus Mode 2is obtained from Mode 1 by rotating 180 ꢁ
around the C2–H bond. In the absence of X-ray crystal
structures of any pyrrolo[2,3-d]pyrimidines with any
RTK in the literature, the exact mode(s) of binding of
these analogues are not known and it is possible that
different RTKs may bind the pyrrolo[2,3-d]pyrimidine
molecules in different or both Modes 1 and 2shown
below.
The Phosphate binding region (Fig. 2) of the RTK
model is exposed to solvent and can be exploited to
impart water solubility to the inhibitors.
Results and Discussion
The target 2-amino-4-(3-bromoanilino)-6-substituted-
pyrrolo[2,3-d]pyrimidines 1–11 were synthesized via
the displacement of the 4-chloro analogues with
3-bromo-aniline 50.58 The 4-chloro precursors were in
turn synthesized from the corresponding 2-amino-4-
oxo-pyrrolo[2,3-d]pyrimidines.59,60
On the basis of the general pharmacophore model,10,49
adapted to VEGFR-2, presented in Figure 2, we
embarked on the design of RTK inhibitors using the
pyrrolo[2,3-d]pyrimidine scaffold with a 2-NH2 moiety.
To our knowledge this 2-NH2 substitution has not been
explored in the literature to any significant extent. The
rationale for including the 2-NH2 group in our com-
pounds was that it provides a third H-bonding moiety
in the Hinge region of RTKs, as described above, and
Though there are several reported methods for the
synthesis of the 2-amino-4-oxo-pyrrolo[2,3-d]pyr-
imidines,61,62 there is no report for the preparation of a
Figure 3.

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5159
Figure 4. Stereoview: model of compound 5 superimposed on the ATP structure (not shown) in VEGFR-2.
single-carbon bridged side chain at the 6-position. We
initially attempted the Fischer indole cyclization
(Scheme 1) to form the pyrrolo[2,3-d]pyrimidine nucleus
using the appropriate methyl benzyl ketone and the
hydrazine from 2-amino-4-oxo-6-chloropyrimidine
which afforded the hydrazone. Fischer indole cyclization
under a variety of neutral, acidic and various tempera-
ture conditions did not afford the desired compounds.
Cylocondensation of the a-bromomethyl benzyl ketones
12–22 with 2,6-diamino-4-oxo-pyrimidine 23 was suc-
cessful under different solvent (DMF, DMSO, water)
and temperature (room temperature to 80 ꢁC) condi-
tions both with and without base (Et3N, K2CO3). The
optimized conditions were room temperature in DMF
for 3 days which afforded the 2-amino-4-oxo-6-sub-
stituted benzyl pyrrolo[2,3-d]pyrimidines 24–34 in
45–60% yield. With 24–34 in hand, the conversion of
the 4-oxo to the corresponding 4-chloro was attempted
with POCl3, SOCl2, or PCl5/POCl3. None of these
reagents afforded the desired product, instead decom-
position of the starting material was observed. Clearly
the 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-
d]pyrimidines 24–34 were sensitive to the strong acidic
conditions. Thus the chlorination was attempted with
POCl3 and a base PhNMe2. The 2-amino-4-oxo-6-sub-
stituted benzyl pyrrolo[2,3-d]pyrimidines 24–27, 29, 31
and 33 in POCl3 with PhNMe2 at reflux afforded
2-amino-4-chloro-pyrrolo[2,3-d]pyrimidines 43–49 in
25–45% yield. The low yield of this step was probably,
in part, due to the absence of drying the PhNMe2 and
distillation of POCl3 prior to use as suggested in the lit-
erature.60 However, protection of the 2-amino groups of
28, 30, 32 and 34 with the pivaloyl group followed by
reflux with POCl3 afforded a smooth conversion to the
2-pivaloylamino-4-chloro-6-substituted benzyl pyr-
rolo[2,3-d]pyrimidines 39–42 in 75–81% yield. 59
The failure of the Fischer indole reaction prompted the
search for alternate synthetic methodology. We have
reported the cyclocondensation of a-haloketones with
substituted pyrimidines to afford a variety of pyr-
rolo[2,3-d]pyrimidines63 and furo[2,3-d]pyrimidines,57
and envisioned that the a-bromomethyl benzyl ketones
with 2,6-diamino-4-oxo-pyrimidine would afford the
desired 2-amino-4-oxo-6-substituted benzyl pyrrolo[2,3-
d]pyrimidine 24–34. The precursor a-bromomethyl ben-
zyl ketones 12–22 were obtained from the correspond-
ing substituted phenyl acetic acids or acid chlorides via
a three-step sequence (Scheme 2). First the a-diazoke-
tones were formed from the acid chlorides with diazo-
methane. Treatment of the a-diazoketones with HBr gas
was reported to afford the a-bromoketones.64 In our
case, 47.5% aqueous HBr worked just as well to afford
the a-bromomethyl benzyl ketones 12–22 and no
O-demethylated intermediates 16 and 22, as would be
expected from HBr gas, were obtained.
Scheme 1.

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
Scheme 2. (a) SOCl2, benzene, reflux, 1 h; (b) CH2N2/Et2O, 30 min; (c) 47.5% HBr, 70–80 ꢁC, 1 h; (d) DMF, rt, 3 days; (e) Piv2O, DMF, DMAP,
Et3N, 60–70 ꢁC, 2days; (f) POCl 3; (g) POCl3, PhNMe2 (h) i-PrOH, 2–3 drops of concd HCl, reflux, 30 min, (i) 15% KOH, 1,4-dioxane, reflux, 10 h.
The displacement of the 4-chloro moiety of 39–42 or
43–49 with 3-bromo aniline 50 to give the target com-
pounds 1–11 occurred as reported in the literature.58
The reaction condition requires 2–3 drops of concd HCl
without which no reaction occurred, even at reflux for
24 h. Compared with the unprotected compounds 43–49
where the yields of 1, 2, 3, 4, 6, 8 and 10 were 70–80%,
the reaction of the 2-amino protected compounds 39–42
occurred faster and afforded a quantitative yield of the
pivaloyl protected target compounds. However, depro-
tection of the 2-pivaloyl group did not occur under mild
basic conditions, such as 1 N NaOH in THF or metha-
nol at room temperature or high temperature (100 ꢁC).
Stronger conditions of reflux in 1,4-dioxane with 15%
KOH for 10 h was required for deprotection to afford 5,
7, 9, and 11.
linear approximation of cell number.68 Finally, the
effect of selected compounds on blood vessel formation
was assessed using the chicken embryo chorioallantoic
membrane (CAM) assay, a standard test for angiogen-
esis.69 In this assay, purified angiogenic growth factors
are placed locally on a vascularized membrane of a
developing chicken embryo together with possible inhi-
bitors. Digitized images of the vasculature are taken at
48 h after growth factor administration and the number
of vessels per unit area evaluated as a measure of vas-
cular density.
Since the IC50 values of compounds vary under different
assay conditions, we used a standard (control) com-
pound in each of the evaluations. For VEGFR-2the
standard was SU5416; for VEGFR-1 the standard was
(4-chloro-2-fluoro-phenyl)-(6,7-dimethoxy-quinazolin-4-
yl)-amine; for EGFR the standard was PD153035; for
PDGFR the standard was AG1295; for the cytotoxicity
study against the growth of A431 cells in culture the
standard was PD153035. Cisplatin and SU5416 were
also used along with the other standard analogue in the
A431 assay. SU5416 was also used as the standard for
the antiangiogenic CAM assay.
Kinase activity of the compounds were evaluated using
human tumor cells known to express high levels of
EGFR, Flk1, Flt1, PDFGRb, and FGFR1 using a
phosphotyrosine ELISA.65,66 Compounds known to
inhibit the particular RTKs were used as positive con-
trols for these assays. The effect of compounds on cell
proliferation was measured using A431 cancer cells,
shown to overexpress EGFR, because EGFR has been
shown to play a role in the overall survival of the cells.67
Cell proliferation was assessed using CYQUANT1, a
DNA intercalating dye that has been shown to give a
In the VEGFR-2assay ( Table 1), the 2-CH3 and the
2,5-diOMe substituted compounds 2 and 5 were the
most potent and were 4- and 8-fold more potent,

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5161
Table 1. IC50 values (mM) of kinase inhibition, A431 cytotoxicity and inhibition of the CAM assay
Compd
VEGFR-2
inhibition
EGFR
inhibition
A431
cytotoxicity
PDGFR-b
inhibition
VEGFR-1
inhibition
CAM angiogenesis
inhibition
1
2
3
4
5
6
7
8
>50
1.67ꢂ0.3 (2)
9.19ꢂ1.8 (2)
4.31ꢂ1.75 (2)
17.42ꢂ3.9 (2)
12.62ꢂ3.3 (2)
0.23ꢂ0.06 (2)
19.77ꢂ5.6 (2)
>50
31.8ꢂ6.3 (2)
1.21ꢂ0.42(2)
>50
>50
>50
>50
>50
>50
>50
26.8ꢂ4.1(2)
42.7ꢂ6.1 (2)
31.1ꢂ5.8 (2)
>50
0.25ꢂ0.04 (2)a
5.58ꢂ0.69 (2)
8.28ꢂ0.53 (2)
0.62ꢂ0.21 (3)
28.11ꢂ9.9 (2)
>50
1.21ꢂ0.23 (2)
1.70ꢂ0.34 (3)
<0.1 (toxic)
1.32ꢂ0.42(2)
10.8ꢂ3.2(2)
28.6ꢂ5.1 (2)
>50
8.92ꢂ1.6 (2)
17.0ꢂ5.6 (2)
>50
>50
>50
>50
14.7ꢂ3.4 (2)
2.8ꢂ1.1 (3)
33.5ꢂ6.2(2)
>50
33.2ꢂ5.9 (2)
23.5ꢂ5.2(2)
42.1ꢂ18.5 (2)
19.2 ꢂ4.2(5)
12.6 ꢂ2.9 (6)
12.6ꢂ4.8 (2)
>50
5.08ꢂ0.83 (2)
19.2ꢂ4.3 (2)
15.2ꢂ2.9 (2)
>50
0.053ꢂ0.007 (2)
9
10
11
>50
1.24ꢂ0.21 (2)
6.16ꢂ1.2 (2)
>50
5.97ꢂ0.78 (2)
9.42ꢂ1.9 (2)
2.43ꢂ0.32(15)
0.052ꢂ0.008 (2)
0.43ꢂ0.03 (2)
>50
SU5416
PD153035
PD168393
AG1295
VEGFRkinase
Inhibitor
0.032 ꢂ0.005 (13)
0.24 ꢂ0.042(16)
6.2ꢂ1.6 (7)
17.7ꢂ2.3 (8)
aNumber in parentheses is n.
respectively, than the standard, SU5416. The 1-naph-
thyl analogue 8, the 2-Cl analogue 3 and the 4-biphenyl
analogue 10 were equipotent and were about one half as
potent as SU5416. The other analogues (11, 7 and 4)
were 3- to 12-fold less potent than SU5416 or inactive
(1, 7 and 9). These results indicate that a 2-substitution
of a Me or OMe on the phenyl ring afford the most
potent analogues. The 1-naphthyl, also a 2-substituted
phenyl, is reasonably potent as is the 2-Cl analogue.
Thus ortho or 2-substitutions are conducive to potent
VEGFR-2inhibitory activity and electron donating
groups are better than electron withdrawing groups (2
vs 3). Both the electronic nature and the location of the
substituents on the phenyl ring were important for
VEGFR-2inhibitory activity. A comparison of the
1-naphthyl and 2-naphthyl analogues 8 and 9 reveals a
more than 10-fold difference in activity indicating that a
2,3-disubstituted phenyl is much better than a 3,4-di-
substituted phenyl. In addition, the decrease in inhibi-
tory activity of the 2-naphthyl vs the 1-naphthyl may
also indicates a subtle distinction of bulk tolerance at
the VEGFR-2kinase domain.
analogues were mono ortho substituted or 2,5-di-
substituted analogues with electron donating groups.
Cytotoxicity studies against the growth of A431 (human
cancer cells that overexpress EGFR) in culture provided
interesting results. Two of the standard compounds
SU5416, and PD153035 along with cisplatin were com-
pared with analogues 1–11. The most potent analogue
was the 2-Me compound 2. This was 16-, 10.5- and
7-fold more potent than SU5416, PD153035 and cis-
platin, respectively. Though this analogue was not the
most potent against EGFR. The most potent analogue
against EGFR, compound 6, was the second most
active analogue against A431 cells, and was 7-, 4.5- and
3-fold more potent than the standard compounds
SU5416, PD153035 and cisplatin, respectively. All of
the other analogues were less potent than the standard
compounds. The data for most of the analogues do
correlate EGFR inhibitory activity (or inactivity) with
A431 inhibitory activity (or inactivity). However, there
is no relative correlation for 1, 3 and 9. This could
reflect transport and/or other factors which influence
the cytotoxic activity against cells in culture. Thus two
analogues, 2 and 6, were remarkably potent against the
growth of A431 cells in culture and were from 3- to
16-fold more potent than the standard compounds.
Against EGFR the most potent compound was the 2,4-
diCl analogue 6 and it was equipotent with the standard
EGFR inhibitor, PD153035. The other analogues had
comparatively lower inhibitory effects and ranged from
6- to 100-fold less potent than PD153035. Removal of
either of the Cl moieties of 6 to afford 3 or 4 leads to a
considerable loss in inhibitory activity as does moving
the 2-Cl to the 3-position (compound 7). Remarkably,
Against PDGFRb compounds 5, 6, and 11 were similar
in activity to the standard analogue AG1295 with
activities of 1.5-, 3- and 2-fold less potent, respectively,
than AG1295. All of the monosubstituted analogues,
the unsubstituted analogue and the 1- and 2-naphthyl
analogues were essentially inactive. This suggests that
for PDGFRb inhibition a di- or trisubstituted phenyl
ring is necessary and that electron donating groups were
slightly better than electron withdrawing groups.
the 2-naphthyl analogue (also
a 3,4-disubstituted
phenyl) was significantly more potent (16.5-fold) than 7.
In addition, comparison of the 1- and 2-naphthyl ana-
logues 8 and 9 provides a greater than 42-fold difference
in activity indicating that, in contrast to VEGFR-2, for
EGFR a 2-naphthyl group is much better than a
1-naphthyl and again may reflect subtle differences in
bulk tolerance at the EGFR kinase domain. Thus, for
EGFR a 2,4-disubstituted phenyl with electron with-
drawing groups provided the most potent analogues
and was in contrast to VEGFR-2where the most potent
The involvement of Flt1 (VEGFR-1) in angiogenesis
has not been clearly delineated; however, recent
reports41 suggest its involvement in tumor angiogenesis
and inflammation associated with tumors.70ꢀ72 Thus we
also evaluated compounds 1–11 as inhibitors of

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
VEGFR-1. The results are indicated in Table 1. The
bulky 1- and 2-naphthyl analogues 8 and 9 were equi-
potent with the standard VEGFR-1 inhibitor 4-(4-
chloro, 2-fluoro phenyl)amino-6,7-dimethoxyquinazo-
line and indicates a preference for bulk on the phenyl
ring. However, both the biphenyl and triOMe analogues
10 and 11 were inactive against VEGFR-1. Compounds
3, 4 and 5 were about one half as potent as 8 and 9. The
remaining analogues were inactive.
are also being evaluated in appropriate animal models
for solid tumors.
Experimental
Melting points were determined on a Mel-Temp II
melting point apparatus with FLUKE 51 K/J electronic
thermometer and were uncorrected. Nuclear magnetic
resonance spectra for proton (1H) were recorded on a
Bruker WH-300 (300 MHz) spectrometer. The chemical
shift values were expressed in ppm (parts per million)
relative to tetramethylsilane as internal standard;
s=singlet, d=double, t=triplet, q=quartet, m=multi-
plet, br=broad singlet. The relative integrals of peak
areas agreed with those expected for the assigned struc-
tures. High-resolution mass spectra (HRMS), using
Electron Impact (EI), were recorded on a VG Autospec
(Fisons Instruments) micromass (EBE Geometry) dou-
ble focusing mass spectrometer. Thin-layer chromato-
graphy (TLC) was performed on Polygram Sil
G/UV254 silica gel plates with fluorescent indicator,
and the spots were visualized under 254 and 366 nm
illumination. Proportions of solvents used for TLC were
by volume. Column chromatography was performed on
230–400 mesh silica gel purchased from Aldrich Chem-
ical Co., Milwaukee, WI, USA. All evaporations were
carried out in vacuo with a rotary evaporator. Analy-
tical samples were dried in vacuo (0.2mmHg) in an
Abderhalden drying apparatus over P2O5 at 75–110 ꢁC.
Elemental analysis was performed by Altlantic Micro-
labs, Norcross, GA, USA. Element compositions are
within ꢂ0.4% of calculated values. Fractional moles of
water or organic solvents frequently found in some
analytical samples could not be prevented despite 24–48
h of drying in vacuo and were confirmed where possible
On the basis of their activity against VEGFR-2selected
compounds were evaluated in the CAM angiogenesis
assay. Two compounds, 8 and 10 were equipotent with
SU5416 in inhibiting bFGF/VEGF induced blood ves-
sel formation. Compound 11 was the next most potent
analogue. The other analogues were much less potent
than SU5416. The two most potent VEGFR-2inhibi-
tors, 2 and 5, had good activity in the CAM assay but
were not the most potent. Compounds 8 and 10 were
reasonably potent against VEGFR-2and were also
highly active in the CAM assay.
Multiple inhibitors of RTKs are also of interest in the
treatment of cancer.42ꢀ44 On the basis of the data dis-
cussed above, compound 5 shows potent inhibition of
VEGFR-2(IC 0.6 mM) as well as PDGFRb (IC50 8.9
50
mM) kinase inhibition and hence is a potent dual inhi-
bitor, with activities which supercede both standard
compounds. Compounds 3 and 8 can also be classified
as dual inhibitors of VEGFR-1 (Flt) and VEGFR-2
kinases. Compound 9 has both EGFR and VEGFR-1
kinase inhibition inhibitory activity and can also be
considered a dual acting agent.
Conclusion
1
by their presence in the H NMR spectra. All solvents
and chemicals were purchased from Aldrich Chemical
Co. and Fisher Scientific and were used as received.
Eleven novel 2-amino-4-anilino 6-methylphenyl sub-
stituted pyrrolo[2,3-d]pyrimidines 1–11 were designed
and synthesized as RTK inhibitors. The biological
evaluation showed several analogues had remarkable
inhibitory activity against VEGFR-2, EGFR, A431
proliferation, PDGFRb, VEGFR-1 and in the CAM
angiogenesis assay. Of the analogues evaluated, five
(compounds 1, 2, 5, 6, and 8) were much more potent or
equipotent as compared to the standard compounds.
Two analogues, 2 and 6, showed potent cytotoxic effects
against A431 cells in culture and two analogues, 8 and
10, demonstrated high antiangiogenic activity in the
CAM assay. In addition, four analogues (compounds
3, 5, 8, and 9) showed dual inhibitory activity. The
structure–activity relationship for inhibition of different
RTKs was quite distinct and different, and for
VEGFR-2and EGFR diametrically opposite. The
inhibitory data against the RTKs in this study demon-
strates that variation of the substituent(s) in the benzyl
ring of these 2-amino-4-anilino 6-benzyl pyrrolo[2,3-
d]pyrimidines does indeed control both the potency
and specificity of inhibitory activity against RTKs.
Using the analogues described in this study as leads, an
extensive SAR and structure-based design and synth-
esis of further RTK inhibitors are currently in pro-
gress. In addition, selected compounds from this study
General procedure for the synthesis of compounds 24–34
A solution of substituted phenyl acetic acid (5 mmol) in
5 mL of dry benzene and 5 mL of thionyl chloride was
refluxed for 1 h and the colorless solution was evapo-
rated in vacuo. The resulting acid chloride was dissolved
in 8 mL of ether and added dropwise with stirring to
30–40 mL of ethereal diazomethane (about 13 mmol,
made from 20 mmol of nitromethyl urea)73 at 0–5 ꢁC.
The yellow solution was allowed to stand at room tem-
perature for 1 h, then 5 mL of 47.5% aqueous HBr was
added dropwise to the solution and the yellow mixture
was heated at 70–80 ꢁC (oil bath) for 1 h. The reaction
was cooled to room temperature and the ether layer was
separated, washed with water (10 mL), saturated
NaHCO3 aqueous solution (10 mL) and water (10 mL)
and dried (anhydrous Na2SO4). The solution was eva-
porated to afford the a-bromomethyl benzyl ketones
12–22. The compounds 12–22 were not purified further
and were used directly in the next step.
The a-bromomethyl benzyl ketone 12–22 was placed
in a 50-mL flask and an equivalent amount of

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5163
2,6-diaminopyrimidin-4-one 22 and 5 mL dry DMF
were added. The mixture was stirred at room tempera-
ture for 3 days. After half or 1 day, the reaction became
a brown or dark solution, at the third day of the reac-
tion, a solid could be precipitated out in some cases. To
the reaction mixture was added 1 g of silica gel and the
solvent was evaporated in vacuo to afford a dry plug.
This plug was placed on the top of 45ꢃ150 mm silica gel
column and eluted with CHCl3/MeOH (a gradient elu-
tion, 2% MeOH in CHCl3, and 5% MeOH in CHCl3).
Fractions containing the product (TLC) were pooled
and evaporated to afford the pure 2-amino-4-oxo-6-
substituted benzyl pyrrolo[2,3-d]pyrimidine 24–34.
2H, NH2), 7.25–7.45 (m, 4H, Ar–H), 10.16 (s, 1H, NH),
.
10.97 (s, 1H, NH). Anal. (C13H11N4OCl 0.8H2O): C, H,
N, Cl.
2-Amino-4-oxo-6-(4-chlorobenzyl)-pyrrolo[2,3-d]pyrimi-
dine (27). From 4-chlorophenylacetic acid (0.86 g, 5
mmol), 1-bromo-3-(4-chlorophenyl)-acetone 15 was
obtained using the general procedure described above as
a yellow oil (0.63 g, 51%). TLC Rf 0.74 (hexane/EtOAc,
1
3:1). H NMR (CDCl3): d 3.88 (s, 2H, CH2), 3.91 (s,
2H, CH2), 7.31–7.12(dd, 4H, Ar–H).
Using the general procedure described above, com-
pound 15 (500 mg, 2mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
compound 27 (670 mg, 54%) as a yellow solid. Mp:
2-Amino-4-oxo-6-benzyl-pyrrolo[2,3-d]pyrimidine (24).
From phenyl acetyl chloride (0.77 g, 5 mmol), 1-bromo-
3-phenyl-acetone 12 was obtained using the general
procedure described above as a light-yellow oil (0.63 g,
59%). TLC Rf 0.71 (hexane/EtOAc, 3:1). 1H NMR
(CDCl3): d 3.90 (s, 2H, CH2), 3.93 (s, 2H, CH2), 7.30
(m, 5H, Ar–H).
255 ꢁC. TLC Rf 0.53 (CHCl3/CH3OH, 5:1). H NMR
1
(DMSO-d6): d 3.81 (s, 2H, CH2), 5.86 (s, 1H, C5-CH),
6.02(s, 2H, NH 2), 7.20–7.35 (m, 4H, Ar–H), 10.17 (s,
1H, NH), 10.94 (s, 1H, NH). Anal. (C13H11N4OCl): C,
H, N, Cl.
Using the general procedure described above, com-
pound 12 (600 mg, 3.75 mmol) reacted with an equiva-
lent amount of 2,6-diaminopyrimidin-4-one 23 to afford
the compound 24 (400 mg, 45%) as a yellow solid. Mp:
2-Amino-4-oxo-6-(2,5-dimethoxybenzyl)-pyrrolo[2,3-
d]pyrimidine (28). From 2,5-dimethoxyphenylacetic acid
(0.98 g, 5 mmol), 1-bromo-3-(2,5-dimethoxyphenyl)-
acetone 16 was obtained using the general procedure
described above as a yellow oil (0.52g, 45%). TLC Rf
270 ꢁC. TLC Rf 0.50 (CHCl3/CH3OH, 5:1). H NMR
1
1
(DMSO-d6): d 3.81 (s, 2H, CH2), 5.85 (s, 1H, C5-CH),
5.98 (s, 2H, NH2), 7.10–7.30 (m, 5H, Ar–H), 10.15 (s, 1H,
NH), 10.95 (s, 1H, NH). Anal. (C13H12N4O): C, H, N.
0.46 (hexane/EtOAc, 3:1). H NMR (CDCl3): d 3.75 (s,
6H, 2ꢃOCH3), 3.80 (s, 2H, CH2), 4.18 (s, 2H, CH2),
6.84–6.70 (m, 3H, Ar–H).
2-Amino-4-oxo-6-(2-methylbenzyl)-pyrrolo[2,3-d]pyrimi-
dine (25). From 2-methylphenylacetic acid (0.75 g, 5
mmol), 1-bromo-3-(2-methylphenyl)-acetone 13 was
obtained using the general procedure described above as
a yellow oil (0.71 g, 62%). TLC Rf 0.58 (hexane/EtOAc,
Using the general procedure described above, com-
pound 28 (510 mg, 2.2 mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
compound 28 (230 mg, 40%) as a yellow solid. Mp:
ꢁ
1
275 C. TLC Rf 0.42(CHCl /CH3OH, 5:1). H NMR
3
1
3:1). H NMR (CDCl3): d 2.25 (s, 3H, CH3), 3.85 (s,
2H, CH2), 3.95 (s, 2H, CH2), 7.10–7.22 (m, 4H, Ar–H).
(DMSO-d6): d 3.64 (s, 3H, OCH3), 3.68 (s, 3H, OCH3),
3.73 (s, 2H, CH2), 5.76 (s, 1H, C5-CH), 5.97 (s, 2H,
NH2), 6.66–6.76 (m, 2H, Ar–H), 6.86–6.89 (m, 1H,
Ar–H), 10.13 (s, 1H, NH), 10.85 (s, 1H, NH). Anal.
(C15H16N4O3): C, H, N.
Using the general procedure described above, com-
pound 13 (700 mg, 3 mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
compound 25 (400 mg, 52%) as a yellow solid. Mp:
2-Amino-4-oxo-6-(2,4-dichlorobenzyl)-pyrrolo[2,3-d]pyri-
midine (29). From 2,4-dichlorophenylacetic acid (1.02 g,
5 mmol), 1-bromo-3-(2,4-dichlorophenyl)-acetone 17
was obtained using the general procedure described
above as a white solid (0.72g, 51%). Mp: 70–73 ꢁC.
TLC Rf 0.60 (hexane/EtOAc, 3:1). 1H NMR (CDCl3): d
3.95 (s, 2H, CH2), 4.05 (s, 2H, CH2), 7.20–7.45 (m, 3H,
Ar–H).
290 ꢁC. TLC Rf 0.56 (CHCl3/CH3OH, 5:1). H NMR
1
(DMSO-d6): d 2.32 (s, 3H, CH3), 3.90 (s, 2H, CH2), 5.85
(s, 1H, C5-CH), 6.03 (s, 2H, NH2), 7.15–7.30 (m, 4H,
Ar–H), 10.10 (s, 1H, NH), 10.94 (s, 1H, NH). Anal.
(C14H14N4O): C, H, N.
2-Amino-4-oxo-6-(2-chlorobenzyl)-pyrrolo[2,3-d]pyrimi-
dine (26). From 2-chlorophenylacetic acid (0.86 g, 5
mmol), 1-bromo-3-(2-chlorophenyl)-acetone 14 was
obtained using the general procedure described above as
a white solid (0.81 g, 65%). Mp: 38–41 ꢁC. TLC Rf 0.76
Using the general procedure described above, com-
pound 17 (710 mg, 2.5 mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
compound 29 (440 mg, 56%) as a yellow solid. Mp:
1
(hexane/EtOAc, 3:1). H NMR (CDCl3): d 4.05 (s, 2H,
CH2), 4.10 (s, 2H, CH2), 7.20–7.50 (m, 4H, Ar–H).
265 ꢁC. TLC Rf 0.51 (CHCl3/CH3OH, 5:1). H NMR
1
(DMSO-d6): d 3.94 (s, 2H, CH2), 5.85 (s, 1H, C5-CH),
7.25–7.60 (m, 3H, Ar–H), 8.24 (s, br., 2H, NH2), 10.60
(s, 1H, NH), 11.20 (s, 1H, NH). Anal.
Using the general procedure described above, com-
pound 14 (800 mg, 3.2mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
.
(C13H10N4OCl2 0.6H2O): C, H, N, Cl.
product 26 (430 mg, 49%) as a yellow solid. Mp: 290 ꢁC.
1
TLC Rf 0.52(CHCl /CH3OH, 5:1). H NMR (DMSO-
3
2-Amino-4-oxo-6-(3,4-dichlorobenzyl)-pyrrolo[2,3-d]pyri-
midine (30). From 3,4-dichlorophenylacetic acid (1.02g,
d6): d 3.92(s, 2H, CH 2), 5.77 (s, 1H, C5-CH), 6.01 (s,

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5 mmol), 1-bromo-3-(3,4-dichlorophenyl)-acetone 18
was obtained using the general procedure described
above as a white solid (0.64 g, 45%). Mp: 70–72 ꢁC.
TLC Rf 0.50 (hexane/EtOAc, 3:1). 1H NMR (CDCl3): d
3.89 (s, 2H, CH2), 3.91 (s, 2H, CH2), 7.05–7.45 (m, 3H,
Ar–H).
Using the general procedure described above, com-
pound 21 (800 mg, 2.7 mmol) reacted with equivalent
2,6-diaminopyrimidin-4-one 23 to afford the compound
33 (410 mg, 48%) as a yellow solid. Mp: 275 ꢁC. TLC Rf
0.51 (CHCl3/CH3OH, 5:1). 1H NMR (DMSO-d6): d
3.86 (s, 2H, CH2), 5.90 (s, 1H, C5-CH), 6.00 (s, 2H,
NH2), 7.31–7.46 (m, 4H, Ar–H), 7.56–7.64 (m, 5H, Ar–
H), 10.16 (s, 1H, NH), 10.96 (s, 1H, NH). Anal.
Using the general procedure described above, com-
pound 18 (630 mg, 2.2 mmol) reacted with an equivalent
amount of 2,6-diaminopyrimidin-4-one 23 to afford the
compound 30 (450 mg, 65%) as a yellow solid. Mp:
.
(C19H16N4O 0.1H2O): C, H, N.
2-Amino-4-oxo-6-(3,4,5-trimethoxybenzyl)-pyrrolo[2,3-
d]pyrimidine (34). From 3,4,5-trimethoxy-phenylacetic
acid (1.13 g, 5 mmol), 1-bromo-3-(3,4,5-trimethoxy-
phenyl)-acetone 22 was obtained using the general pro-
cedure described above as a white solid (0.66 g, 51%).
Mp: 50–53 ꢁC. TLC Rf 0.42(hexane/EtOAc, 3:1). 1H
NMR (CDCl3): d 3.68 (s, 3H, OCH3), 3.86 (s, 6H,
2ꢃOCH3), 3.95 (s, 2H, CH2), 4.05 (s, 2H, CH2),
7.20–7.45 (s, 2H, Ar–H).
290 ꢁC. TLC Rf 0.52(CHCl /CH3OH, 5:1). H NMR
1
3
(DMSO-d6): d 3.83 (s, 2H, CH2), 5.92(s, 1H, C5-CH),
6.01 (s, 2H, NH2), 7.21-7.55 (m, 3H, Ar–H), 10.17 (s,
1H,
NH),
10.94
(s,
1H,
(C13H10N4OCl2 0.1CH3OH): C, H, N, Cl.
NH).
Anal.
.
2-Amino-4-oxo-6-(1-naphthylmethyl)-pyrrolo[2,3-d]pyri-
midine (31). From 1-naphthylacetic acid (0.93 g, 5
mmol),
obtained using the general procedure described above as
1-bromo-3-(1-naphthyl)-acetone
19
was
Using the general procedure described above, com-
pound 22 (950 mg, 3.67 mmol) reacted with an equiva-
lent amount of 2,6-diaminopyrimidin-4-one 23 to afford
the compound 34 (360 mg, 44%) as a yellow solid. Mp:
a white solid (0.65g, 55%). Mp: 53–56 ꢁC. TLC Rf 0.53
1
(hexane/EtOAc, 3:1). H NMR (CDCl3): d 3.86 (s, 2H,
CH2), 4.38 (s, 2H, CH2), 7.55 (m, 4H, Ar–H), 7.90 (m,
3H, Ar–H).
1
235ꢃC. TLC Rf 0.45 (CHCl3/CH3OH, 5:1). H NMR
(DMSO-d6): d 3.61 (s, 3H, OCH3), 3.74 (s, 2H, CH2),
3.77 (s, 6H, 2ꢃOCH3), 5.90 (s, 1H, C5-CH), 5.98 (s, 2H,
NH2), 6.58 (s, 2H, Ar–H), 10.15 (s, 1H, NH), 10.91 (s,
1H, NH). Anal. (C16H18N4O4): C, H, N.
Using the general procedure described above, com-
pound 19 (470 mg, 2.23 mmol) reacted with equivalent
2,6-diaminopyrimidin-4-one 23 to afford the compound
31 (470 mg, 72%) as a yellow solid. Mp: 255 ꢁC. TLC Rf
0.47 (CHCl3/CH3OH, 5:1). 1H NMR (DMSO-d6): d
4.30 (s, 2H, CH2), 5.75 (s, 1H, C5-CH), 6.05 (s, 2H,
NH2), 7.35–7.55 (m, 4H, Ar–H), 8.80–7.15 (m, 3H, Ar–
H), 10.15 (s, 1H, NH), 11.05 (s, 1H, NH). Anal.
General procedure for the synthesis of compounds 35–38
2-Amino-4-oxo-6-substituted-pyrrolo[2,3- d]pyrimidine
28, 30, 32, 34 (1–2mmol), Piv 2O (3 equivalence),
DMAP (0.05 equivalence), Et3N (5 equivalence), and 5
mL of dry DMF were placed in a 50-mL round-bottom
flask. The mixture was stirred at 60–70 ꢁC for 2days,
the solid was dissolved completely and formed a dark
solution. Then about 500 mg silica gel was added
and the solvent was evaporated in vacuo to afford a
dry plug. This plug was placed on the top of 15ꢃ150
mm silica gel column and eluted with 1% methanol
in chloroform. Fractions containing the product were
pooled and evaporated to afford pure 2-pivaloyla-
mino-4-oxo-6-substitued-pyrrolo[2,3-d]pyrimidine 35–
38.
.
(C17H14N4 0.4H2O): C, H, N.
2-Amino-4-oxo-6-(2-naphthylmethyl)-pyrrolo[2,3-d]pyri-
midine (32). From 2-naphthylacetic acid (0.93 g, 5
mmol),
1-bromo-3-(2-naphthyl)-acetone
20
was
obtained using the general procedure described above
as a white solid (0.78 g, 60%). Mp: 80–82.5 ꢁC. TLC
Rf 0.65 (hexane/EtOAc, 3:1). 1H NMR (CDCl3): d
3.90 (s, 2H, CH2), 4.10 (s, 2H, CH2), 7.20–8.80 (m,
7H, Ar–H).
Using the general procedure described above, com-
pound 20 (780 mg, 2.96 mmol) reacted with equivalent
2,6-diaminopyrimidin-4-one 23 to afford the compound
32 (450 mg, 54%) as a yellow solid. Mp: 270 ꢁC. TLC Rf
0.50 (CHCl3/CH3OH, 5:1). 1H NMR (DMSO-d6): d
4.00 (s, 2H, CH2), 5.90 (s, 1H, C5-CH), 6.05 (s, 2H,
NH2), 7.95–7.50 (m, 7H, Ar–H), 10.20 (s, 1H, NH),
2-Pivaloylamino-4-oxo-6-(2-naphthylmethyl)-pyrrolo[2,3-
d]pyrimidine (35). Compound 35 was synthesized from
2-amino-4-oxo-6-(2-naphthylmethyl)-pyrrolo[2,3-d]pyr-
imidine 32 (580 mg, 2.0 mmol) using the general
procedure described above to afford 35 450 mg
(60%) as an off yellow solid. Mp: 230 ꢁC, TLC Rf
.
11.00 (s, 1H, NH). Anal. (C17H14N4O 0.8H2O): C, H,
N.
1
0.38 (CHCl3/CH3OH, 10:1). H NMR (DMSO-d6): d
1.17 (s, 9H, C(CH3)3), 4.12(s, 2H, CH 2), 6.12(s, 1H,
C5-CH), 7.41–7.88 (m, 7H, Ar–H), 10.76 (s, 1H,
NH), 11.56 (s, 1H, NH), 11.82(s, 1H, NH). Anal.
2-Amino-4-oxo-6-(4-phenylbenzyl)-pyrrolo[2,3-d]pyrimi-
dine (33). From 4-biphenylacetic acid (1.06 g, 5 mmol),
1-bromo-3-(4-biphenyl)-acetone 21 was obtained using
the general procedure described above as white solid
(0.85 g, 59%). Mp: 71–73 ꢁC. TLC Rf 0.70 (hexane/
EtOAc, 3:1). H NMR (CDCl3): d 3.90 (s, 2H, CH2),
4.00 (s, 2H, CH2), 7.28–7.50 (m, 5H, Ar–H), 7.52–7.65
(dd, 4H, Ar–H).
.
(C22H22N4O2 0.3H2O): C, H, N.
2-Pivaloylamino-4-oxo-6-(2,5-dimethoxybenzyl)-pyr-
rolo[2,3-d]pyrimidine (36). Compound 36 was synthe-
sized from 2-amino-4-oxo-6-(2,5-dimethoxybenzyl)-
pyrrolo[2,3-d]pyrimidine 28 (300 mg,1.0 mmol) using
1

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5165
the general procedure described above to afford 36 330
mg (85%) as a yellow solid: Mp: 250 ꢁC. TLC Rf 0.43
(CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d 1.16 (s,
9H, C(CH3)3), 3.64 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),
3.85 (s, 2H, CH2), 5.96 (s, 1H, C5-CH), 6.64–6.92(m,
3H, Ar–H), 10.76 (s, 1H, NH), 11.45 (s, 1H, NH), 11.80
(s, 1H, NH).
dimethoxybenzyl)-pyrrolo[2,3-d]pyrimidine 36 (300 mg,
0.86 mmol) using the general procedure described above
to afford 40 270 mg (80%) as an off white solid. Mp:
129–131.5 ꢁC. TLC Rf 0.8 (CHCl3/CH3OH, 10:1). H
1
NMR (DMSO-d6): d 1.20 (s, 9H, C(CH3)3), 3.65 (s, 3H,
OCH3), 3.72(s, 3H, OCH ), 3.97 (s, 2H, CH2), 6.01 (s,
1H, C5-CH), 6.78–7.00 (m, 3H, Ar–H), 9.99 (s, 1H,
NH), 12.35 (s, 1H, NH).
3
2-Pivaloyamino-4-oxo-6-(3,4-dichlorobenzyl)-pyrrolo[2,3-
d]pyrimidine (37). Compound 37 was synthesized from
2- amino - 4 - oxo - 6 - (3,4 - dichlorobenzyl) - pyrrolo[2,3 -
d]pyrimidine 30 (570 mg,1.8 mmol) using the general
procedure described above to afford 37 300 mg (65%) as
a yellow solid. Mp: 250 ꢁC. TLC Rf 0.47 (CHCl3/
2-Pivaloylamino-4-chloro-6-(3,4-dichlorobenzyl)-pyr-
rolo[2,3-d]pyrimidine (41). Compound 41 was synthe-
sized from 2-pivaloylamino-4-oxo-6-(3,4-dichlorobenzyl)-
pyrrolo[2,3-d]pyrimidine 37 (280 mg, 0.7 mmol) using
the general procedure described above to afford 41 220
mg (75%) as an off white solid. Mp: 120–123 ꢁC. TLC
Rf 0.80 (CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d
1.29 (s, 9H, C(CH3)3), 3.95 (s, 2H, CH2), 6.19 (s, 1H,
C5-CH), 6.76–7.23 (m, 3H, Ar–H), 8.06 (s, 1H, NH),
10.84 (s, 1H, NH).
1
CH3OH, 10:1). H NMR (DMSO-d6): d 1.16 (s, 9H,
C(CH3)3), 3.97 (s, 2H, CH2), 6.14 (s, 1H, C5-CH), 7.23–
7.58 (m, 3H, Ar–H), 10.75 (s, 1H, NH), 11.51 (s, 1H,
NH), 11.83 (s, 1H, NH).
2-Pivaloylamino-4-oxo-6-(3,4,5-trimethoxybenzyl)-pyr-
rolo[2,3-d]pyrimidine (38). Compound 38 was synthe-
sized from 2-amino-4-oxo-6-(3,4,5-trimethoxybenzyl)-
pyrrolo[2,3-d]pyrimidine 34 (400 mg, 1.2mmol) using
the general procedure described above to afford 38 460
mg (92%) as a yellow solid. Mp: 240 ꢁC. TLC Rf 0.44
(CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d 1.23 (s,
9H, C(CH3)3), 3.48 (s, 3H, OCH3), 3.66 (s, 6H,
2ꢃOCH3), 3.73 (s, 2H, CH2), 6.60 (s, 1H, C5-CH), 6.78
(s, 2H, Ar–H), 10.73 (s, 1H, NH), 11.50 (s, 1H, NH),
11.80 (s, 1H, NH).
2-Pivaloylamino-4-chloro-6-(3,4,5-trimethoxybenzyl)-pyr-
rolo[2,3-d]pyrimidine (42). Compound 42 was synthe-
sized from 2- pivaloylamino - 4 - oxo - 6 - (3,4,5 -
trimethoxybenzyl)-pyrrolo[2,3-d]pyrimidine 38 (400 mg,
0.96 mmol) using the general procedure described above
to afford 42340 mg (81%) as an off white solid. Mp:
98–101 ꢁC. TLC Rf 0.75 (CHCl3/CH3OH, 10:1). 1H
NMR (DMSO-d6): d 1.21 (s, 9H, C(CH3)3), 3.61 (s, 3H,
OCH3), 3.75 (s, 6H, 2ꢃOCH3), 3.98 (s, 2H, CH2), 6.24
(s, 1H, C5-CH), 6.67 (s, 2H, Ar–H), 9.98 (s, 1H, NH),
12.34 (s, 1H, NH).
General procedure for the synthesis of compounds 39–42
General procedure for the synthesis of compounds 43–49
50-mL round-bottom flask was placed 2-pivaloylamino-4-
oxo-6-substitued-pyrrolo[2,3-d]pyrimidine 35–38 (about 1
mmol) and POCl3 5 mL. The mixture was then heated
and kept refluxing for 2h. After evaporation of the
excess of POCl3, ice-cold water was added. The reac-
2-Amino-4-oxo-6-substitued-pyrrolo[2,3-d]pyrimidine 24,
25, 26, 27, 29, 31, and 33 (about 1 mmol), 5 mL of
POCl3 and 0.1 mL of PhNMe2 were placed in a 50-mL
round-bottom flask. The mixture was heated to reflux
and kept stirring for 4 h. After evaporation of the excess
of POCl3, ice-cold water was added and the reaction
.
tion mixture was neutralized with NH3 H2O, and
extracted with CHCl3 (3ꢃ50 mL). The organic phase
was combined and dried with Na2SO4. Concentration
of the chloroform afforded a brown solid that was dis-
solved in chloroform (2–3 mL) again and was placed to
the top of a 15ꢃ150 mm column and eluted with 0.1%
methanol in chloroform. Fractions containing the pro-
duct were pooled and evaporated to afford pure 2-piva-
loylamino-4-chloro-6-substitued-pyrrolo[2,3-d]pyrimidine
39–42.
.
mixture was neutralized with NH3 H2O. The pre-
cipitated solid was collected by filtered, and the filtrate
was extracted with CHCl3 (3ꢃ50 mL). The organic phase
was combined and dried (Na2SO4). Concentration of the
chloroform afforded another solid. Combined these
solids and dissolved in methanol, then 500 mg silica gel
was added and removed the solvent in vacuo to afford a
dry plug. This plug was placed on the top of a 15ꢃ150
mm column and eluted with 2% methanol in chloroform.
Fractions containing the product were pooled and eva-
porated to afford pure 2-amino-4-chloro-6-substituted-
pyrrolo[2,3-d]pyrimidine 43–49.
2 - Pivaloylamino - 4 - chloro - 6 - (2 - naphthylmethyl)- pyr-
rolo[2,3-d]pyrimidine (39). Compound 39 was synthe-
sized from 2-pivaloylamino-4-oxo-6-(2-naphthylmethyl)-
pyrrolo[2,3-d]pyrimidine 35 (400 mg, 1.06 mmol) using
the general procedure described above to afford 39 350
mg (80%) as an off white solid. Mp: 151–153 ꢁC. TLC
Rf 0.83 (CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d
1.28 (s, 9H, C(CH3)3), 4.19 (s, 2H, CH2), 6.20 (s, 1H,
C5-CH), 7.20-7.80 (m, 7H, Ar–H), 8.03 (s, 1H, NH),
10.04 (s, 1H, NH). Anal. (C22H21N4OCl): C, H, N, Cl.
2-Amino-4-chloro-6-(2-chlorobenzyl)-pyrrolo[2,3-d]pyri-
midine (43). Compound 43 was synthesized from 2-
amino-4-oxo-6-(2-chlorobenzyl)-pyrrolo[2,3-d]pyrimidine
26 (300 mg, 1.1 mmol) using the general procedure
described above to afford 43 140 mg (45%) as a light
yellow solid. Mp: 240 ꢁC. TLC Rf 0.50 (CHCl3/CH3OH,
10:1). 1H NMR (DMSO-d6): d 4.05 (s, 2H, CH2), 5.77 (s,
1H, C5-CH), 6.45 (s, 2H, NH2), 7.28–7.46 (m, 4H,
2-Pivaloylamino-4-chloro-6-(2,5-dimethoxybenzyl)-pyr-
rolo[2,3-d]pyrimidine (40). Compound 40 was syn-
thesized from 2- pivaloylamino - 4 - oxo - 6 - (2,5-
.
Ar–H), 11.51 (s, 1H, NH). Anal. (C13H10N4Cl2 0.2H2O):
C, H, N, Cl.

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A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
2-Amino-4-chloro-6-benzyl-pyrrolo[2,3-d]pyrimidine (44).
Compound 44 was synthesized from 2-amino-4-oxo-6-
benzyl-pyrrolo[2,3-d]pyrimidine 24 (300 mg, 1.25 mmol)
using the general procedure described above to afford
44 81 mg (25%) as a light yellow solid. Mp: 230 ꢁC.
TLC Rf 0.51 (CHCl3/CH3OH, 10:1). 1H NMR (DMSO-
d6): d 3.93 (s, 2H, CH2), 5.89 (s, 1H, C5-CH), 6.44 (s,
2H, NH2), 7.31–7.44 (m, 5H, Ar–H), 11.46 (s, 1H, NH).
d]pyrimidines 39–42 (about 0.2mmol), 3-bromo-aniline
50 (1.5 equivalence), i-PrOH 10 mL, and 2–3 drops of
concd HCl were placed in a 50-mL flask. The mixture
was heated to reflux 45 min. After evaporation of the
solvent, the solid was dissolved in 1,4-dioxane 10 mL,
and 2mL of 15% KOH aqueous solution was added.
The reaction mixture was heated to reflux for 10 h.
After remove of the solvent to form a syrup, water (20
mL) was added and extracted with chloroform (3ꢃ50
mL) Combined the organic phase and dried with anhy-
drous Na2SO4. Concentration of the chloroform affor-
ded a solid that was dissolved in methanol, and then 250
mg silica gel was added and removed the solvent in
vacuo to afford a dry plug. This plug was placed on the
top of a 15ꢃ150 mm column and eluted with 2%
methanol in chloroform. Fractions containing the pro-
duct were pooled and evaporated to afford pure 2-
amino-4-(3-bromoanilino)-6-subsituted-pyrrolo[2,3-
d]pyrimidine 5, 7, 9, and 11.
2-Amino-4-chloro-6-(2-methylbenzyl)-pyrrolo[2,3-d]pyri-
midine (45). Compound 45 was synthesized from 2-
amino-4-oxo-6-(2-methylbenzyl)-pyrrolo[2,3-d]pyrimidine
25 (200 mg, 0.78 mmol) using the general procedure
described above to afford 45 60 mg (28%) as a light
yellow solid. Mp: 250 ꢁC. TLC Rf 0.53 (CHCl3/CH3OH,
1
10:1). H NMR (DMSO-d6): d 3.91 (s, 2H, CH2), 5.69
(s, 1H, C5-CH), 6.42(s, 2H, NH 2), 7.13–7.16 (m, 4H,
Ar–H), 11.46 (s, 1H, NH).
2-Amino-4-chloro-6-(4-chlorobenzyl)-pyrrolo[2,3-d]pyri-
midine (46). Compound 46 was synthesized from 2-
amino-4-oxo-6-(4-chlorobenzyl)-pyrrolo[2,3-d]pyrimidine
27 (137 mg, 0.5 mmol) using the general procedure
described above to afford 46 51 mg (35%) as a light
2-Amino-4-(3-bromoanilino)-6-(2,5-dimethoxybenzyl)-
pyrrolo[2,3-d]pyrimidine (5). Compound 5 was synthe-
sized from 2-pivaloylamino-4-chloro-6-(2,5-dimethoxy-
benzyl)-pyrrolo[2,3-d]pyrimidine 40 (80 mg, 0.20 mmol)
using the general procedure described above to afford 5
50 mg (55%) as an off white solid. Mp: 190–192 ꢁC. TLC
Rf 0.43 (CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d
3.67 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.83 (s, 2H,
CH2), 5.73 (s, 2H, NH2), 6.06 (s, 1H, C5-CH), 6.75–7.18
(m, 5H, Ar–H), 8.00–8.10 (m, 2H, Ar–H), 8.89 (s, 1H,
NH), 10.86 (s, 1H, NH). HRMS (EI): calcd for
C21H20N5BrO2 453.0800, found 453.0778.
yellow solid. Mp: >250 ꢁC. TLC Rf 0.52(CHCl
/
3
1
CH3OH, 10:1). H NMR (DMSO-d6): d 3.93 (s, 2H,
CH2), 5.91 (s, 1H, C5-CH), 6.40 (s, 2H, NH2), 7.28–7.38
(dd, 4H, Ar–H), 11.45 (s, 1H, NH).
2-Amino-4-chloro-6-(2,4-dichlorobenzyl)-pyrrolo[2,3-
d]pyrimidine (47). Compound 47 was synthesized from
2 - amino - 4 - oxo - 6 - (2,4 - dichlorobenzyl) - pyrrolo[2,3 -
d]pyrimidine 29 (300 mg, 0.97 mmol) using the general
procedure described above to afford 47 95 mg (30%) as
a light yellow solid. Mp: >255 ꢁC. TLC Rf 0.52(CHCl 3/
2-Amino-4-(3-bromoanilino)-6-(3,4-dichlorobenzyl)-pyr-
rolo[2,3-d]pyrimidine (7). Compound 7 was synthesized
from 2-pivaloylamino-4-chloro-6-(3,4-dichlorobenzyl)-
pyrrolo[2,3-d]pyrimidine 41 (100 mg, 0.24 mmol) using
the general procedure described above to afford 7 68 mg
(61%) as an off white solid. Mp: 215–217.5 ꢁC. TLC Rf
1
CH3OH, 10:1). H NMR (DMSO-d6): d 4.17 (s, 2H,
CH2), 5.82(s, 1H, C5-CH), 6.48 (s, 2H, NH 2), 7.33–7.66
(m, 3H, Ar–H), 11.53 (s, 1H, NH).
1
2- Amino - 4- chloro -6 -(1 -naphthylmethyl)- pyrrolo[2,3 -
d]pyrimidine (48). Compound 48 was synthesized from
2-amino-4-oxo-6-(1-naphthylmethyl)-pyrrolo[2,3-d]pyr-
imidine 31 (400 mg, 1.37 mmol) using the general pro-
cedure described above to afford 48 125 mg (30%) as an
off white solid. Mp: >250 ꢁC. TLC Rf 0.53 (CHCl3/
0.46 (CHCl3/CH3OH, 10:1). H NMR (DMSO-d6): d
3.93 (s, 2H, CH2), 5.77 (s, 2H, NH2), 6.13 (s, 1H,
C5-CH), 7.05–7.60 (m, 5H, Ar–H), 7.99–8.10 (m, 2H,
Ar–H), 8.92(s, 1H, NH), 10.94 (s, 1H, NH). HRMS (EI):
calcd for C19H14N5BrCl2 460.9810, found 460.9805.
1
CH3OH, 10:1). H NMR (DMSO-d6): d 4.11 (s, 2H,
CH2), 5.95 (s, 1H, C5-CH), 6.40 (s, 2H, NH2), 7.44–7.89
(m, 7H, Ar–H), 11.50 (s, 1H, NH).
2-Amino-4-(3-bromoanilino)-6-(2-naphthylmethyl)-pyr-
rolo[2,3-d]pyrimidine (9). Compound 9 was synthesized
from 2-pivaloylamino-4-chloro-6-(2-naphthylmethyl)-
pyrrolo[2,3-d]pyrimidine 39 (160 mg, 0.4 mmol) using
the general procedure described above to afford 9 130
mg (73%) as an off white solid. Mp: 207–210 ꢁC. TLC
Rf 0.49 (CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d
4.09 (s, 2H, CH2), 5.77 (s, 2H, NH2), 6.13 (s, 1H,
C5-CH), 7.04–7.51 (m, 9H, Ar–H), 7.99–8.09 (m, 2H,
Ar–H), 8.89 (s, 1H, NH), 11.00 (s, 1H, NH). Anal.
(C23H18N5Br): C, H, N, Br.
4-Amino-4-chloro-6-(4-phenylbenzyl)-pyrrolo[2,3-d]pyri-
midine (49). Compound 49 was synthesized from 2-
amino-4-oxo-6-(4-phenylbenzyl)-pyrrolo[2,3-d]pyrimidine
33 (300 mg, 0.95 mmol) using the general procedure
described above to afford 49 98 mg (31%) as a light yel-
low solid. Mp: >260 ꢁC. TLC Rf 0.53 (CHCl3/CH3OH,
10:1). 1H NMR (DMSO-d6): d 3.77 (s, 2H, CH2), 5.75 (s,
1H, C5-CH), 6.22 (s, 2H, NH2), 7.11–7.26 (m, 5H,
Ar–H), 7.39–7.44 (dd, 4H, Ar–H), 11.30 (s, 1H, NH).
2-Amino-4-(3-bromoanilino)-6-(3,4,5-trimethoxybenzyl)-
pyrrolo[2,3-d]pyrimidine (11). Compound 11 was syn-
thesized from 2-pivaloylamino-4-chloro-6-(3,4,5-tri-
methoxybenzyl)-pyrrolo[2,3-d]pyrimidine 42 (100 mg,
0.23 mmol) using the general procedure described above
to afford 11 67 mg (60%) as an off white solid. Mp:
General procedure for the synthesis of compounds 5, 7, 9,
11
2-Pivaloylamino-4-chloro-6-substituted-pyrrolo[2,3-

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5167
213–215 ꢁC. TLC Rf 0.42(CHCl 3/CH3OH, 10:1). 1H
from 2-amino-4-chloro-6-(4-chlorobenzyl)-pyrrolo[2,3-
d]pyrimidine 46 (30 mg, 0.1 mmol) using the general pro-
cedure described above to afford 4 36 mg (82%) as an off
white solid. Mp: 195–197.5 ꢁC. TLC Rf 0.45 (CHCl3/
CH3OH, 10:1). 1H NMR (DMSO-d6): d 4.03 (s, 2H, CH2),
5.78 (s, 2H, NH2), 6.07 (s, 1H, C5-CH), 7.05–7.49 (m, 6H,
Ar–H), 8.00–8.10 (m, 2H, Ar–H), 8.92 (s, 1H, NH), 10.97
NMR (DMSO-d6): d 3.62(s, 3H, OCH ), 3.69 (s, 6H,
3
2ꢃOCH3), 3.84 (s, 2H, CH2), 5.76 (s, 2H, NH2), 6.16 (s,
1H, C5-CH), 6.63 (s, 2H, Ar–H), 7.02–7.22 (m, 2H,
Ar–H), 8.00–8.11 (m, 2H, Ar–H), 8.93 (s, 1H, NH),
10.93 (s, 1H, NH). Anal. (C22H22N5O3Br): C, H, N, Br.
.
(s, 1H, NH). Anal. (C19H15N5BrCl 0.2(CH3)2CHOH): C,
H, N, Br, Cl.
General procedure for the synthesis of compounds 1, 2,
3, 4, 6, 8, and 10
2-Amino-4-chloro-6-sustituted-pyrrolo[2,3-d]pyrimidines
43–49 (about 0.2mmol), 3-bromo-aniline 50 (1.5 equiv),
i-PrOH 10 mL, and 2–3 drops of concd HCl were placed
in a 50-mL flask. The mixture was heated to reflux 1.5 h.
After being cooled, the reaction mixture was neutralized
2-Amino-4-(3-bromoanilino)-6-(2,4-dichlorobenzyl)-pyr-
rolo[2,3-d]pyrimidine (6). Compound 6 was synthesized
from 2- amino - 4 - chloro - 6 - (2,4 - dichlorobenzyl) - pyr-
rolo[2,3-d]pyrimidine 47 (80 mg, 0.24 mmol) using the
general procedure described above to afford 6 77 mg
(70%) as a light yellow solid. Mp: 196–198 ꢁC. TLC Rf
.
with NH3 H2O, and then the solvent was evaporated in
1
vacuo. The residue solid was dissolved in methanol,
then 250 mg silica gel was added and removed the sol-
vent in vacuo to afford a dry plug. This plug was placed
on the top of a 15ꢃ150 mm column and eluted with 2%
methanol in chloroform. Fractions containing the pro-
duct were pooled and evaporated to afford pure 2-
amino-4-(3-bromoanilino)-6-subsituted-pyrrolo[2,3 -
d]pyrimidine 1, 2, 3, 4, 6, 8, 10.
0.44 (CHCl3/CH3OH, 10:1). H NMR (DMSO-d6): d
4.01 (s, 2H, CH2), 5.81 (s, 2H, NH2), 6.04 (s, 1H,
C5-CH), 7.05–7.64 (m, 5H, Ar–H), 8.00–8.09 (m, 2H,
Ar–H), 8.91 (s, 1H, NH), 10.99 (s, 1H, NH). HRMS (EI):
calcd for C19H14N5BrCl2 460.9810, found 460.9790.
2-Amino-4-(3-bromoanilino)-6-(1-naphthylmethyl)-pyr-
rolo[2,3-d]pyrimidine (8). Compound 8 was synthesized
from 2-amino-4-chloro-6-(1-naphthylmethyl)-pyrrolo[2,3-
d]pyrimidine 48 (50 mg, 0.16 mmol) using the general
procedure described above to afford 8 50 mg (71%) as
an off white solid. Mp: 235–238 ꢁC. TLC Rf 0.51
(CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d 4.38 (s,
2H, CH2), 5.73 (s, 2H, NH), 5.96 (s, 1H, C5-CH), 7.02-
7.52(m, 9H, Ar–H), 7.83–8.04 (m, 2H, Ar–H), 8.81 (s,
1H, NH), 11.02(s, 1H, NH). HRMS (EI): calcd for
C23H18N5Br 443.0746, found 443.0758.
2-Amino-4-(3-bromoanilino)-6-benzyl-pyrrolo[2,3-d]pyri-
midine (1). Compound 1 was synthesized from 6-phenyl
methyl 2-amino-4-chloro-6-benzyl-pyrrolo[2,3- d]pyr-
imidine 44 (80 mg, 0.30 mmol) using the general proce-
dure described above to afford 1 90 mg (77%) as an off
white solid. Mp: 195–197.5 ꢁC. TLC Rf 0.46 (CHCl3/
1
CH3OH, 10:1). H NMR (DMSO-d6): d 4.05 (s, 2H,
CH2), 5.77 (s, 2H, NH2), 6.13 (s, 1H, C5-CH), 7.05–7.34
(m, 7H, Ar–H), 7.98–8.11 (m, 2H, Ar–H), 8.93 (s, 1H,
NH), 10.93 (s, 1H, NH). HRMS (EI): calcd for
C19H16N5Br 393.0589, found 393.0572.
2-Amino-4-(3-bromoanilino)-6-(4-phenylbenzyl)-pyr-
rolo[2,3-d]pyrimidine (10). Compound 10 was synthesized
from 2-amino-4-chloro-6-(4-phenylbenzyl)-pyrrolo[2,3-
d]pyrimidine 49 (50 mg, 0.15 mmol) using the general pro-
cedure described above to afford 10 58 mg (83%) as white
solid. Mp: 200–202 ꢁC. TLC Rf 0.50 (CHCl3/CH3OH,
10:1). 1H NMR (DMSO-d6): d 4.04 (s, 2H, CH2), 5.85 (s,
2H, NH2), 6.18 (s, 1H, C5-CH), 7.06–7.71 (m, 11H, Ar–
H), 7.95–8.10 (m, 2H, Ar–H), 8.99 (s, 1H, NH), 11.01 (s,
1H, NH). Anal. (C25H20N5Br): C, H, N, Br.
2-Amino-4-(3-bromoanilino)-6-(2-methylbenzyl)-pyr-
rolo[2,3-d]pyrimidine (2). Compound 2 was synthesized
from 2-amino-4-chloro-6-(2-methylbenzyl)-pyrrolo[2,3-
d]pyrimidine 45 (50 mg, 0.18 mmol) using the general
procedure described above to afford 2 56 mg (75%) as
an off white solid. Mp: 225–228 ꢁC. TLC Rf 0.50
(CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d 2.27 (s,
3H, CH3), 3.89 (s, 2H, CH2), 5.73 (s, 2H, NH2), 5.98 (s,
1H, C5-CH), 7.04–7.20 (m, 6H, Ar–H), 7.99–8.09 (m,
2H, Ar–H), 8.87 (s, 1H, NH), 10.91 (s, 1H, NH). Anal.
(C20H18N5Br): C, H, N, Br.
Cells
All cells were maintained at 37ꢁ C in a humidified envir-
onment containing 5% CO2 using media from Media-
tech (Hemden, NJ, USA). A-431 cells were from the
American Type Tissue Collection (Manassas, VA, USA).
2-Aimno-4-(3-bromoanilino)-6-(2-chlorobenzyl)-pyr-
rolo[2,3-d]pyrimidine (3). Compound 3 was synthesized
from 2-amino-4-chloro-6-(2-chlorobenzyl)-pyrrolo[2,3-
d]pyrimidine 43 (35 mg, 0.12mmol) using the general
procedure described above to afford 3 45 mg, 88%) as
an off white solid. Mp: 208–211 ꢁC. TLC Rf 0.47
(CHCl3/CH3OH, 10:1). 1H NMR (DMSO-d6): d 3.91 (s,
2H, CH2), 5.77 (s, 2H, NH2), 6.10 (s, 1H, C5-CH),
7.05–7.45 (m, 6H, Ar–H), 8.00–8.10 (m, 2H, Ar–H),
8.91 (s, 1H, NH), 10.94 (s, 1H, NH). HRMS (EI): calcd
for C19H15N5BrCl 427.0199, found 427.0218.
Chemicals
All growth factors (bFGF, VEGF, EGF, PDGF-BB)
were purchased from Peprotech (Rocky Hill, NJ, USA).
PD153035, SU5416, AG1295 and VEGF kinase inhi-
bitor (4-[40 -chloro-20 -fluoro)phenylamino]-6,7-dime-
thoxyquinazoline) were purchased from Calbiochem
(San Diego, CA, USA). The CYQUANT cells pro-
liferation assay was from Molecular Probes (Eugene,
OR, USA). All other chemicals were from Sigma
Chemical unless otherwise noted.
2-Amino-4-(3-bromoanilino)-6-(4-chlorobenzyl)-pyr-
rolo[2,3-d]pyrimidine (4). Compound 4 was synthesized

5168
A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
Antibodies
described.68 Briefly, cells are first treated with com-
pounds for 12h and 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 Products BioChemi digital darkroom. A Positive
control used for cytotoxicity in each experiment was
cisplatin, with an apparent average IC50 value of
8.2ꢂ0.65 mM. Data are graphed as a percent of cells
receiving growth factor alone and IC50 values estimated
from 2–3 separate experiments (n=6–15) using Probit
plots.
The PY-HRP antibody was from BD Transduction
Laboratories (Franklin Lakes, NJ, USA). Antibodies
against EGFR, PDGFRb, FGFR-1, Flk-1, and Flt-1
were purchased from Upstate Biotech (Framingham,
MA, USA).
Phosphotyrosine (PY)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) and FGFR-1 (NIH
OVCAR-8). 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/mtargets/mt_
index.html). Briefly, cells at 60–75% confluence are
placed in serum-free medium for 18 h to reduce the
background of phosphorylation. Cells were always
>98% viable by Trypan blue exclusion. Cells are then
pre-treated for 60 min with 10, 3.33, 1.11, 0.37 and 0.12
mM compound followed by 100 ng/mL EGF, VEGF,
PDGF-BB, or bFGF for 10 min. The reaction is 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 is then removed and cells fixed to the plate
by 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 phosphotyr-
osine antibody added overnight. The antibody is removed,
cells are washed again in TBS, exposed to an enhanced
luminol ELISA substrate (Pierce Chemical, Rockford, IL,
USA) and light emission measured using an UV Products
(Upland, CA, USA) BioChemi digital darkroom. The
known RTK-specific kinase inhibitor PD153035 was used
as a positive control compound for EGFR kinase inhibi-
tion, SU5416 for Flk1 kinase inhibition. AG1295 for
PDGFRb kinase inhibition, and VEGF kinase inhibitor
(4 - [40 - chloro - 20 - fluoro)phenylamino] - 6,7 - dimethox-
yquinazoline) was used as a positive control for both
Flt1 and Flk1 kinase inhibition. Data were graphed as a
percent of cells receiving growth factor alone and IC50
values estimated from 2–3 separate experiments
(n=8–24) using hand-drawn Probit plots. In every case,
the activity of a positive control inhibitor did not deviate
more than 10% from the IC50 values listed in the text.
Chorioallantoic membrane (CAM)assay of angiogenesis
The CAM assay is a standard assay for testing anti-
angiogenic agents.69 The CAM assay used in these
studies was modified from a procedure by Sheu74 and
Brooks75 and as published previously.76 Briefly, fertile
leghorn chicken eggs (CBT Farms, Chestertown, MD,
USA) are allowed to grow until 10 days of incubation.
The proangiogenic factors human VEGF-165 and
bFGF (100 ng each) are then added to saturation to a 6-
mm microbial testing disc (BBL, Cockeysville, MD,
USA) and placed onto the CAM by breaking a small
hole in the superior surface of the egg. Antiangiogenic
compounds are added 8 h after the VEGF/bFGF at
saturation to the same microbial testing disk and
embryos allowed to incubate for an additional 40 h.
After 48 h, the CAMs are perfused with 2% paraf-
ormaldehyde/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 onto Petri dishes,
and a digitized image taken using a dissecting micro-
scope (Wild M400; Bannockburn, IL, USA) at 7.5ꢃ
and SPOT Enhanced digital imaging system (Diag-
nostic Instruments, Sterling Heights, MI, USA). 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. AGM-1470 (a kind gift of the
NIH Developmental Therapeutics Program) and
SU5416 are used as a positive control for anti-
angiogenic activity. Data are graphed as a percent of
CAMs receiving bFGF/VEGF and IC50 values esti-
mated from 2–3 separate experiments (n=5–11) using
Probit plots.
Acknowledgements
This work was supported, in part, by Duquesne
University and the National Institutes of Health
grants CA098850 (A.G.), AI44661 (A.G.), AI47759
(A.G.).
CYQUANT cell proliferation assay
As a measure of cell proliferation, the CYQUANT cell
counting/proliferation assay was used as previously

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170
5169
Appendix
Elemental Analyses
Compd Formula
Calcd (%)
Found (%)
Cl
C
H
N
Cl
Br
C
H
N
Br
24
25
26
27
28
29
30
31
32
33
34
35
39
43
2
C13H12N4O
C14H14N4O
C13H11N4OCl 0.8H2O
C13H11N4OCl
C15H16N4O3
C13H10N4OCl2 0.6H2O
.
C13H10N4OCl2 0.1CH3OH
C17H14N4O 0.4H2O
C17H14N4O 0.8H2O
.
C19H16N4O 0.1H2O
C16H18N4O4
.
C22H22N4O2 0.3H2O
C22H21N4OCl
.
C13H10N4Cl2 0.2H2O
C20H18N5Br
C19H15N5BrCl 0.2(CH3)2CHOH 53.41 3.80 15.89
C23H18N5Br
C25H20N5Br
C22H22N5O3Br
64.99 5.03 23.32
66.13 5.55 22.03
54.01 4.39 19.38 12.26
56.84 4.04 20.39 12.91
59.99 5.37 18.66
48.80 3.53 17.51 22.16
50.37 3.36 17.94 22.70
68.63 5.01 18.83
67.00 5.16 18.39
71.73 5.13 17.61
58.17 5.49 16.96
69.38 6.25 14.71
67.26 5.39 14.26
52.62 3.53 18.88 23.89
58.83 4.44 17.15
64.97 5.10 23.08
66.16 5.61 21.89
53.93 4.50 19.16 12.44
56.93 4.12 20.15 12.82
59.69 5.36 18.37
48.58 3.53 17.47 22.01
50.64 3.49 17.62 22.45
68.65 4.83 18.71
66.96 5.00 18.03
71.60 5.14 17.40
.
.
.
.
58.15 5.58 16.86
69.16 6.00 14.82
66.98 5.42 14.12
52.88 3.46 18.54 24.02
9.02
9.27
19.57 58.57 4.5217.15
19.86
8.00 18.03
18.29
.
4
9
10
11
8.04 18.13 53.70 3.73 16.15
17.98 62.40 4.10 15.46
16.99 63.72 4.36 14.63
16.50 54.28 4.59 14.30
62.17 4.08 15.76
63.84 4.29 14.89
54.56 4.58 14.46
17.22
16.70
High-Resolution Mass Spectra (HRMS) (EI)
Compd
Formula
Calcd mass
Found mass
1
3
5
6
7
8
C19H16N5Br
393.0589
427.0199
453.0800
460.9810
460.9810
443.0746
393.0572
427.0218
453.0778
460.9790
460.9805
443.0758
C19H15N5BrCl
C21H20N5BrO2
C19H14N5BrCl2
C19H14N5BrCl2
C23H18N5Br
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