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

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,^aMichael A. Ihnat^band Shekhar Kamat^b

^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.

Introduction

Thus angiogenesis and metastasis contribute to the poor

prognosis seen in patients with highly angiogenic

tumors.⁵Inhibition 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.¹Angiogenesis 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.²Angiogenesis 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

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 trials^29ꢀ33as an antitumor agent.

PDGFR-a, PDGFR-b and PDGF have been impli-

cated, indirectly, in inducing VEGF secretion and hence

in angiogenesis.³⁴Inhibition of PDGFR mediated sig-

naling has direct and indirect eﬀects on tumor growth

and metastasis.^35ꢀ37Over expression of PDGFR has

been observed in glioblastomas.^23,38,39ST1-571 (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.^9,10Some examples of these

compounds, along with preclinical compounds, are pre-

sented in Figure 1.

Recent reports⁴¹suggest 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.^11ꢀ23Thus,

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.^24,25In addition, VEGF and VEGFRs

are overexpressed in many tumor types.^26ꢀ28Several

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.¹⁰Of 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;⁴⁵pyr-

ido[d]pyrimidines;^46,47and general reviews of tyrosine

kinase inhibitors^9,10in 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 proposed⁴⁹and

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-NH₂moiety, 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-NH₂group to exploit this

H-bonding site. A 2-NH₂pyrazolopyrimidine was

reported by Traxler et al.,⁵³however the authors do not

ascribe a binding for the 2-NH₂moiety. We have uti-

lized this 2-NH₂group 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) domain⁵⁰with 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-NH₂of 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),⁵²followed 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-NH₂group 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-NH₂and 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.⁴⁸For 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,¹⁰has 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 structure⁵¹and

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-NH₂with 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, we⁵⁷proposed 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 50.⁵⁸The 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-NH₂moiety.

To our knowledge this 2-NH₂substitution has not been

explored in the literature to any signiﬁcant extent. The

rationale for including the 2-NH₂group 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,^61,62there 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

POCl₃and a base PhNMe₂. The 2-amino-4-oxo-6-sub-

stituted benzyl pyrrolo[2,3-d]pyrimidines 24–27, 29, 31

and 33 in POCl₃with PhNMe₂at 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 PhNMe₂and

distillation of POCl₃prior to use as suggested in the lit-

erature.⁶⁰However, protection of the 2-amino groups of

28, 30, 32 and 34 with the pivaloyl group followed by

reﬂux with POCl₃aﬀorded 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]pyrimidines⁶³and 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.⁶⁴In 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.⁶⁸Finally, 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.⁶⁹In 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 IC₅₀values 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.^65,66Compounds 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-CH₃and 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. IC₅₀values (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

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

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

reports⁴¹suggest its involvement in tumor angiogenesis

and inﬂammation associated with tumors.^70ꢀ72Thus we

also evaluated compounds 1–11 as inhibitors of

5162

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 P₂O₅at 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.^42ꢀ44On the basis of the data dis-

cussed above, compound 5 shows potent inhibition of

VEGFR-2(IC 0.6 mM) as well as PDGFRb (IC₅₀8.9

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)⁷³at 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

NaHCO₃aqueous 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 R_f0.74 (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 R_f0.71 (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 R_f0.53 (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 R_f

270 ^ꢁC. TLC R_f0.50 (CHCl₃/CH₃OH, 5:1). H NMR

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 R_f0.58 (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 R_f0.42(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 R_f0.60 (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 R_f0.56 (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 R_f0.76

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 R_f0.51 (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

.

(C₁₃H₁₀N₄OCl₂0.6H₂O): C, H, N, Cl.

product 26 (430 mg, 49%) as a yellow solid. Mp: 290 ^ꢁC.

1

TLC R_f0.52(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,

5164

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 R_f0.50 (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 R_f

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 R_f0.42(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 R_f0.52(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,

(C₁₃H₁₀N₄OCl₂0.1CH₃OH): 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 R_f0.53

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 R_f0.45 (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 R_f

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.

.

(C₁₇H₁₄N₄0.4H₂O): 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

R_f0.65 (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 R_f

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 R_f

.

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 R_f0.70 (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).

.

(C₂₂H₂₂N₄O₂0.3H₂O): 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 R_f0.43

(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 R_f0.8 (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 R_f0.47 (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

R_f0.80 (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 R_f0.44

(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 R_f0.75 (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 POCl₃5 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

POCl₃and 0.1 mL of PhNMe₂were 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 NH₃H₂O, 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 NH₃H₂O. 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

R_f0.83 (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 R_f0.50 (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. (C₁₃H₁₀N₄Cl₂0.2H₂O):

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 R_f0.51 (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 R_f0.53 (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

R_f0.43 (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

C₂₁H₂₀N₅BrO₂453.0800, found 453.0778.

yellow solid. Mp: >250 ^ꢁC. TLC R_f0.52(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 R_f0.52(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 R_f

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 R_f0.53 (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 C₁₉H₁₄N₅BrCl₂460.9810, 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

R_f0.49 (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 R_f0.53 (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 R_f0.42(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 R_f0.45 (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 R_f

.

with NH₃H₂O, 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 C₁₉H₁₄N₅BrCl₂460.9810, 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 R_f0.51

(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 R_f0.46 (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 R_f0.50 (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 R_f0.50

(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 37^ꢁC in a humidiﬁed envir-

onment containing 5% CO₂using 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 R_f0.47

(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.⁶⁸Brieﬂy, 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 IC₅₀value of

8.2ꢂ0.65 mM. Data are graphed as a percent of cells

receiving growth factor alone and IC₅₀values 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 IC₅₀values listed in the text.

Chorioallantoic membrane (CAM)assay of angiogenesis

The CAM assay is a standard assay for testing anti-

angiogenic agents.⁶⁹The CAM assay used in these

studies was modiﬁed from a procedure by Sheu⁷⁴and

Brooks⁷⁵and as published previously.⁷⁶Brieﬂy, 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 IC₅₀values 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

C₁₃H₁₀N₄OCl₂0.6H₂O

.

C₁₃H₁₀N₄OCl₂0.1CH₃OH

C₁₇H₁₄N₄O 0.4H₂O

C₁₇H₁₄N₄O 0.8H₂O

.

C₁₉H₁₆N₄O 0.1H₂O

C₁₆H₁₈N₄O₄

.

C₂₂H₂₂N₄O₂0.3H₂O

C₂₂H₂₁N₄OCl

.

C₁₃H₁₀N₄Cl₂0.2H₂O

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

443.0746

393.0572

427.0218

453.0778

460.9790

460.9805

443.0758

C₁₉H₁₅N₅BrCl

C₂₁H₂₀N₅BrO₂

C₁₉H₁₄N₅BrCl₂

C₂₃H₁₈N₅Br

References and Notes

J. A.; Hayes, R. L.; Wong, A. J. Cancer Res. 1995, 55,

5536.

1. Folkman, J. New Eng. J. Med. 1971, 285, 1182.

2. Folkman, J. Breast Cancer Res. Treat. 1995, 36, 109.

3. Bohle, A. S.; Kalthoﬀ, H. Langebeck’s Arch. Surg. 1999,

384, 133.

4. Hanahan, D.; Folkman, J. Cell 1996, 1996, 353.

5. Fox, S. B.; Harris, A. L. In The New Angiotherapy; Fan, T.

P., Kohn, E. C.; Eds; Humana: Totowa, 2002; p 151.

6. Cherrington, J. M.; Strawn, L. M.; Shawver, L. K. Adv.

Can. Res. 2000, 2000, 1.

7. Sun, L.; McMahon, G. Drug Discov. Today 2000, 5, 344.

8. Shauver, L. K.; Lipson, K. E.; Fong, T. A. T.; McMahon,

G.; Strawn, L. M.. Receptor Tyrosine Kinases in Angiogenesis

in the New Angiotherapy; Humana: Totowa, 2002; p 409.

9. Traxler, P.; Bold, G.; Buchdunger, E.; Caravatti, G.; Furet,

P.; Manley, P.; O’Reilly, T.; Wood, J.; Zimmermann, J. Med.

Res. Rev. 2001, 21, 499.

15. Cohen, D. W.; Simak, R.; Rair, W. R.; Melamed, J.;

Scher, H. L.; Cordon-Cardo, C. J. Urol. 1994, 152, 2120.

16. Grandis, J. R.; Melhem, M. F.; Gooding, W. E.; Day, R.;

Holst, W. A.; Wagener, M. M.; Drenning, S. D.; Tweardy,

D. J. J. Natl. Cancer Inst. 1998, 90, 824.

17. Morishigie, K. I.; Kurachi, H.; Ameniya, K.; Fujita, Y.;

Yamamoto, T.; Mikaye, A.; Tanizawa, O. Cancer Res. 1991,

51, 5322.

18. Rusch, V.; Klimstra, D.; Venkatraman, E.; Pisters,

P. W. T.; Langenfeld, J.; Dmitrovsky, E. Clin. Cancer Res.

1997, 3, 515.

19. Thogersen, V. B.; Jorgensen, P. E.; Sorensen, B. S.; Bross,

P.; Orntoft, T.; Wolf, H.; Nexo, E. Scand. J. Clin. Lab Invest.

1999, 59, 267.

20. Shin, D. M.; Ro, J. Y.; Hong, W. K.; Hittelamn, W. N.

Cancer Res. 1994, 54, 3153.

10. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.

11. Salomon, D. S.; Brandt, R.; Ciadiello, F.; Normanno, N.

Crit. Rev. Oncol. Haematol. 1995, 19, 183.

21. Tateishi, M.; Ishida, T.; Mitsudomi, T.; Kaneko, S.;

Sugimachi, K. Cancer Res. 1990, 50, 7077.

22. Gorgoulis, V.; Aninos, D.; Mikou, P.; Kanavaros, P.;

Karameris, A.; Joardanoglu, J.; Rasidakis, A.; Veslemes, M.;

Ozanne, B.; Spandidos, D. A. Anticancer Res. 1992, 12,

1183.

12. Gullick, W. J. Br. Med. Bull. 1991, 47, 87.

13. Woodburn, J. R. Pharmacol. Ther. 1999, 82, 241.

14. Moscatello, D. K.; Holgado-Mudruga, M.; Godwin,

A. K.; Ramirez, G.; Gunn, G.; Zoltick, P. W.; Biegel,

23. Fleming, T. P.; Saxena, A.; Clark, W. C.; Robertson, J. T.;

5170

A. Gangjee et al. / Bioorg. Med. Chem. 11 (2003) 5155–5170

Oldﬁeld, E. H.; Aaronson, S. A.; Ali, I. U. Cancer Res. 1992,

52, 4550.

24. Millauer, B.; Longhi, M. P.; Plate, K. H.; Shawver, L. K.;

Risau, W.; Ullrich, A.; Strawn, L. M. Cancer Res. 1996, 56,

1615.

25. Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.;

Phillips, H. S.; Ferrara, N. Nature 1993, 362, 841.

26. Anan, K.; Morosaki, T.; Katano, M. Surgery 1996, 119,

333.

27. Brown, L. F.; Jackman, R. W.; Tognazzi, K.; Manseau,

E. J.; Senger, D. R.; Dvorak, H. F. Cancer Res. 1993, 53, 4727.

28. Takahashi, A.; Sasski, H.; Kim, S. J.; Tobisu, K.-I.;

Kakizoe, T.; Tsukamoto, T.; Kumamoto, Y.; Sugimura, T.;

Terada, M. Cancer Res. 1994, 54, 4233.

Bridges, A. J.; Cody, D. R.; Zhou, H.; Fry, D. W.; McMi-

chael, A.; Kraker, A. J.; Denny, W. A. J. Med. Chem. 1996,

39, 1823.

47. Thompson, A. M.; Bridges, A. J.; Fry, D. W.; Kraker,

A. J.; Denny, W. A. J. Med. Chem. 1995, 38, 3780.

48. Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. J. Biol.

Chem. 2002, 277, 46265.

49. Furet, P.; Caravatti, G.; Lydon, N.; Priestle, J.; Sowadski, J.;

Trinks, U.; Traxler, P. J. Comput. Aided Mol. Des. 1995, 9, 465.

50. Hubbard, S. R. EMBO J. 1997, 16, 5573.

51. McTigue, M. A.; Wichersham, J. A.; Pinko, C.; Sho-

walter, R. E.; Parast, C. V.; Tempczyk-Tussell, A.; Gehring,

M. R.; Mroczkowski, B.; Kan, C. C.; Villafrance, J. E.;

Applelt, K. Structure (London) 1999, 7, 319.

29. Shaheen, R. M.; Davis, D. W.; Liu, W.; Zebrowski, B. K.;

Wilson, M. R.; Bucana, C. D.; McConkey, D. J.; McMahon,

G.; Ellis, L. M. Cancer Res. 1999, 59, 5412.

52. Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.;

Hirth, P.; Yeh, B. K.; Hubbard, S. R.; Schlessinger, J. Science

1997, 276, 955.

30. Mendel, D. B.; Laird, A. D.; Smolich, B. D.; Blake, R. A.;

Liang, C.; Hannah, A. L.; Shaheen, R. M.; Ellis, L. M.;

Weitman, S.; Shawver, L. K.; Cherrington, J. M. Anti-Cancer

Drug Des. 2000, 15, 2 9.

31. Hennequin, L. F.; Thomas, A. P.; Johnston, C.; Stokes, E.

S. E.; Ple, P.; Wedge, S. R.; Ogilvie, D. J.; Kendrew, J.; Cur-

wen, J. O.; Dukes, M. AACR 92nd Annual Meeting, New

Orleans, LA, March 24–28, 2001; abstract no. 3152.

32. Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.;

Hennequin, L. F.; Stokes, E. S. E.; Curry, B. AACR 91st

Annual Meeting, San Francisco, CA, April 1–5, 2000; abstract

no. 3610.

33. Wedge, S. R.; Ogilvie, D. J.; Dukes, M.; Kendrew, J.;

Hennequin, L. F.; Stokes, E. S. E.; Curry, B.; Wadsworth, P.;

Checkley, D.; Tessier, J.; Waterton, J. 11th NCI-EORTC-

AACR Symposium, Amsterdam, The Netherlands, 2000.

34. Brogi, E.; Wu, T.; Namiki, A.; Isner, J. M. Circulation

1994, 90, 649.

53. Traxler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett,

H.; Buchdunger, E.; Meyer, T.; Mueller, M.; Furet, P. J. Med.

Chem. 1997, 40, 3601.

54. Fry, D. W.; Kraker, A. J.; McMichael, A.; Ambroso,

L. A.; Nelson, J. M.; Leopold, W. R.; Connors, R. W.;

Bridges, A. J. Science 1994, 265, 1093.

55. Ward, W. H. J.; Cook, P. N.; Slater, A. M.; Davies, D. H.;

Holdgate, G. A.; Green, L. R. Pharmacology 1994, 48, 659.

56. Rewcastle, G. W.; Bridges, A. J.; Fry, D. W.; Rubin, J. A.;

Denny, W. A. J. Med. Chem. 1997, 40, 1820.

57. Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L.;

Queener, S. F.; Barrows, L. R. J. Med. Chem. 1994, 37, 1169.

58. Rewcastle, G. W.; Palmer, B. D.; Bridges, A. J.; Sho-

walter, H. D. H.; Sun, L.; Nelson, J.; McMichael, A.; Kraker,

A. J.; Fry, D. W.; Denny, W. A. J. Med. Chem. 1996, 39, 918.

59. Shih, C.; Gosset, L. S. Heterocycles 1993, 35, 825.

60. Seela, F.; Steker, H.; Driller, H.; Bindig, U. Liebigs Ann.

Chem. 1987, 15.

35. Fry, D. W. Pharmacol. Ther. 1999, 82, 207.

36. Taylor, C. C. Endocrinology 2000, 141, 1545.

37. Shawver, L. K.; Schwartz, D. P.; Mann, E.; Chen, H.;

Tsai, J.; Chu, L.; Taylorson, L.; Longhi, M.; Meredith, S.;

Germain, L.; Jacobs, J. S.; Tang, C.; Ulrich, A.; Bereens,

M. E.; Hersh, E.; McMahon, G.; Hirth, K. P.; Powell, T. J.

Clin. Cancer Res. 1997, 3, 1167.

61. Gangjee, A. US Patent 5,939,420, CAN 131:157766, 1999.

62. Kondo, Y.; Watanabe, R.; Sakamoto, T.; Yamanaka, H.

Chem. Pharm. Bull. 1989, 37, 2933.

63. Gangjee, A.; Mavandadi, F.; Kisliuk, R. L.; McGuire,

J. J.; Queener, S. F. J. Med. Chem. 1996, 39, 4563.

64. McPhee, W. D.; Klingsberg, E. Org. Synth. 1955, Coll.

Vol. III, 119.

38. Maxwell, M.; Naber, S. P.; Wolfe, H. J.; Galanopoulos,

T.; Hedley-Whyte, E. T.; Black, P. M.; Antoniades, H. N. J.

Clin. Invest. 1990, 86, 131.

39. Hermanson, M.; Funa, K.; Hartman, H.; Claesson-Welsh,

L.; Heldin, C.-H. Cancer Res. 1992, 52, 3213.

65. Fong, T. A.; Shawver, L. K.; Sun, L.; Tang, C.; Tapp, H.;

Powell, T. J.; Kim, Y. H.; Schreck, R.; Wang, X.; Risau, W.;

Ullrich, A.; Hirth, K. P.; McMahon, G. Cancer Res. 1999, 59, 99.

66. Stockwell, B. R.; Haggarty, S. J.; Schreiber, S. L. Chem.

Biol. 1999, 6, 71.

40. Kilic, T.; Alberta, J. A.; Zdunek, P. R.; Acar, M.; Ian-

narelli, P.; O’Reilly, T.; Buchdunger, E.; Black, P. M.; Stiles,

C. D. Cancer Res. 2000, 60, 5143.

67. Schilder, R. J. L. H.; Monks, A.; Handel, L. M.; Fornace,

A. J.; Ozols, R. F.; Fojo, A. T.; Hamilton, T. C. Int. J. Cancer

1990, 45, 416.

41. Luttun, A.; Tjwa, M.; Moons, L.; Wu, Y.; Angelillo-

Scherrer, A.; Liao, F.; Nagy, J. A.; Hooper, A.; Priller, J.; De

Klerck, B.; Compernolle, V.; Daci, E.; Bohlen, P.; Dewerchin,

M.; Herbert, J.-M.; Fava, R.; Matthys, P.; Carmeliet, G.;

Collen, D.; Dvorak, H. F.; Hicklin, D. J.; Carmeliet, P. Nat.

Med. 2002, 8, 831.

42. Shaheen, R. M.; Tseng, W. W.; Davis, D. W.; Liu, W.;

Reinmuth, N.; Vellagas, R.; Wieczorek, A. A.; Ogura, Y.;

McConkey, D. J.; Drazan, K. E.; Bucana, C. D.; McMahon,

G.; Ellis, L. M. Cancer Res. 2001, 61, 1464.

43. Levitt, M. L.; Koty, P. P. Invest. New Drugs 1999, 17, 213.

44. Rusnak, D. W.; Lackey, K.; Aﬄeck, K.; Wood, E. R.;

Alligood, K. J.; Rhodes, N.; Keith, B. R.; Murray, D. M.;

Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer Ther.

2001, 1, 86.

68. Wilson, S. M.; Barsoum, M. J.; Wilson, B. W.; Pappone,

P. A. Cell Prolif. 1999, 32, 131.

69. Vu, M. T.; Smith, C. F.; Burger, P. C.; Klintworth, G. K.

Lab. Invest. 1985, 53, 499.

70. Koolwijk, P.; Peters, E.; Van Der Vecht, B.; Hornig, C.;

Weich, H. A.; Alitalo, K.; Hicklin, D. J.; Wu, Y.; Witte, L.;

Van Hinsbergh, V. W. Angiogenesis 2001, 4, 53.

71. Bussolati, B.; Dunk, C.; Grohman, M.; Kontos, C. D.;

Mason, J.; Ahmed, A. Am. J. Pathol. 2001, 159, 993.

72. Gille, H.; Kowalski, J.; Li, B.; LeCouter, J.; Moﬀat, B.;

Zioncheck, T. F.; Pelletier, N.; Ferrara, N. J. Biol. Chem.

2001, 276, 3222.

73. Arndt, F. Org. Synth. 1943, Coll. Vol. II, 165.

74. Sheu, J. R.; Fu, C. C.; Tsai, M. L.; Chung, W. J. Anti-

cancer Res. 1998, 18, 4435.

45. Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.;

Cody, D. R.; McMichael, A.; Fry, D. W. J. Med. Chem. 1995,

38, 3482.

75. Brooks, P. C.; Montgomery, A. M.; Cheresh, D. A.

Methods Mol. Biol. 1999, 129, 257.

76. Marks, M. G.; Shi, J.; Fry, M. S.; Xiao, Z.; Trzyna, M.;

Pokala, V.; Ihnat, M. A.; Li, P.-K. Biol. Pharm. Bull. 2002, 25, 597.

46. Rewcastle, G. W.; Palmer, B. D.; Thompson, A. M.;

Article Doi

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

DOI: 10.1016/j.bmc.2003.08.034

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