Molecular modeling study and synthesis of novel pyrrolo[2,3-d]pyrimidines and pyrrolotriazolopyrimidines of expected antitumor and radioprotective activities

Article abstract of DOI:10.1016/j.bmc.2007.11.072

Novel pyrrolo[2,3-d]pyrimidine derivatives 4a-e, 10, 14, 15, pyrazolopyrrolopyrimidine 13, pyrrolotriazolopyrimidine 5-9, 17 and pyrrolopyrimidotriazine 18 are reported herein. The design of these compounds was based upon the molecular modeling simulation

Full text of DOI:10.1016/j.bmc.2007.11.072

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2391–2402
Molecular modeling study and synthesis of novel
pyrrolo[2,3-d]pyrimidines and pyrrolotriazolopyrimidines
of expected antitumor and radioprotective activities
Dalal A. Abou El Ella,a Mostafa M. Ghorab,b,* Eman Noaman,c
Helmy I. Heibab and Amira I. Khalilb
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Elkhalifa Elmaamoon St.,
11566, Abbasseya, Cairo 11371, Egypt
bDepartment of Drug Radiation, Research, National Center for Radiation, Research and Technology ‘‘NCRRT’’,
PO Box 29, Nasr City, Cairo, Egypt
cDepartment of Radiation Biology, NCRRT, PO Box 29, Nasr City, Cairo, Egypt
Received 1 June 2007; revised 25 September 2007; accepted 23 November 2007
Available online 20 December 2007
Abstract—Novel pyrrolo[2,3-d]pyrimidine derivatives 4a–e, 10, 14, 15, pyrazolopyrrolopyrimidine 13, pyrrolotriazolopyrimidine 5–
9, 17 and pyrrolopyrimidotriazine 18 are reported herein. The design of these compounds was based upon the molecular modeling
simulation of the fitting values and conformational energy values of the best-fitted conformers to VEGFRTK inhibitor hypothesis.
This hypothesis was generated from its corresponding lead compounds using CATALYST software. The structures of these com-
1
pounds were confirmed by microanalyses, IR, H NMR, and mass spectral data. Compounds 6 and 15 showed interesting in vitro
antitumor activity compared to doxorubicin as positive control. These results are nearly consistent with the molecular modeling
studies. Docking studies were made on compound 15 to predict its binding mode. Moreover, compound 10 exhibited a significant
radioprotective activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
In 1970, Folkman and Kerbel proposed that inhibition
of angiogenesis can prevent tumor growth.2,3 Subse-
quently, it was also recognized that metastasis can be
affected by angiogenesis. For these reasons, inhibitors
of angiogenesis are expected to be valuable drugs for
cancer therapy. The cancer cell is characterized by onco-
gene-derived tumor expression of pro-angiogenic pro-
teins, such as vascular endothelial growth factor
(VEGF), placenta-like growth factor, basic fibroblast
growth factor (FGF), platelet-derived endothelial
growth factor (PDGF), angiopoietin-2 (Ang-2), hepato-
cyte growth factor, and insulin-like growth factor
(ICF).4 The mentioned pro-angiogenic growth factors
bind to specific receptors that possess receptor tyrosine
kinase (RTK) activity.
In current cancer chemotherapy, several agents with
DNA-cleavage properties, antimitotics, antimetabolites,
inhibitors of topoisomerases, and most recently, signal
transduction inhibitors are used as drugs. Protein tyro-
sine kinases are enzymes that provide a central switch
mechanism in cellular signal transduction pathways.
As such they are involved in many cellular processes
such as cell proliferation, metabolism, survival and
apoptosis. Several protein tyrosine kinases are known
to be activated in cancer cells and to drive tumor
growth, angiogenesis, progression, and metastasis.
Blocking tyrosine kinase activity therefore represents a
rational approach to cancer therapy.1
Pyrrolo[2,3-d]pyrimidines have aroused recent attention
from chemical and biological view points since they have
useful properties as antimetabolites in purine biochemi-
cal reactions.5 Several mechanisms are also involved in
their cytotoxic activities as being antifolate inhibitors
of dihydrofolate reductases,6,7 tyrosine Kinase c-Src or
Keywords: Novel pyrrolo[2, 3-d]pyrimidine; Molecular modeling stud-
ies; Tyrosine kinase inhibitors; Antitumor and radioprotective
activities.
*
Corresponding author. Tel.: +20 2 2747413; fax: +20 2 2749298;
e-mail: mmsghorab@yahoo.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.072

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D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
Lck inhibitors,8,9 Cyclin dependent kinase (CDK) inhib-
itors10,11 or adenosine receptor antagonists.12,13
2.1.2. Docking studies
•
Regarding Vatalanib, four clusters of docked con-
On the other hand, sulfonamides have recently been re-
ported to show substantial antitumor activity in vitro
and/or in vivo.14–17
formations were obtained, the largest cluster (four
poses) was the lowest in mean docked energy
(À9.17 kcal/mol) (Table 2), and the best member
(in terms of docking score) was selected as the
docked solution for Vatalanib, examination of
the docking pose shows that Vatalanib forms a
hydrogen bond with the amidic NH in Cys919,
in addition to extensive non-polar interactions, espe-
cially with Val848, Leu840, and Leu1035 (Fig. 5).
Regarding radioprotective activity, some heterocyclic
compounds18–24 especially pyrrole and pyrrolopyrimi-
dines25,26 were proved to exhibit significant radioprotec-
tive activity. Also some sulfur-containing compounds
are known to have good radioprotective activity.27–29
˚
•
Using the RMSD tolerance of 1 A, compound
To explore the synergistic effect resulted from combining
both sulfonamides and pyrrolopyrimidines, the present
work was carried out to design and synthesize a novel
series of pyrrolo[2,3-d]pyrimidine derivatives bearing
sulfonamide moiety, for studying their structure activity
relationship and to be evaluated as antitumor and radio-
protective agents (Schemes 1–3).
15 gave ten distinct clusters. The best pose of
compound 15 showed good mapping to the
docked pose of Vatalanib (Fig. 6).
2.2. Chemistry
Pyrrolo[2,3-d]pyrimidines are known to have a broad spec-
trum of biological activities including antitumor activity,
also pyrrole and pyrrolo[2,3-d] pyrimidines possess antiox-
idant properties and hence have radioprotective activity.30
2. Results and discussion
2.1. Molecular modeling
Additionally, sulfonamides have been explored as po-
tential anticancer agents. In the light of these facts and
guided by molecular modeling results, the present work
was carried out, hoping that the newly synthesized com-
pounds might exhibit significant antitumor and radio-
protective activities.
2.1.1. Generation of vascular epithelial growth factor
receptor tyrosine kinase (VEGFRTK) inhibitor hypothesis
using CATALYST software. The lead compounds I–VII
which were reported to have selective VEGFRTK inhib-
itory activity were used to generate common feature
hypothesis of VEGFRTK inhibitor (Fig. 1).8 The gener-
ated pharmacophore consisted of five features with con-
straint dimensions (Figs. 2 and 3).
Interaction of sulfanilamide with phenacyl bromide
furnished
fonamide 1,31 which was reacted with malononitrile in
ethanol in the presence of sodium ethoxide to give the
strategic starting material pyrrole derivative 231
(Scheme 1).
4-(2-oxo-2-phenyl-ethylamino)-benzenesul-
i. Two hydrogen bond acceptor (HBA) appeared as a
vector (green spherical mesh).
ii. Three hydrophobic functions (HY1, HY2, HY3) (blue
spherical meshes).
The 2-amino-3-cyano-4-phenyl-pyrrole derivative 2 was
subjected to reaction with triethylorthoformate under
reflux to give the Ethyl N-3-cyano-4-phenyl-1-(4-sulfa-
moylphenyl)-1H-pyrrol-2-yl formimidate 3. The structure
of compound 3 was confirmed by its micro-analytical
and spectral data, where the IR spectrum showed bands
at 2200 cmÀ1 and 1580 cmÀ1 attributed to the presence of
(C„N) and (C@N) groups, respectively. Moreover, the
1H NMR spectrum of compound 3 revealed a triplet at
1.3 ppm and a quartet at 4.3 ppm attributed to the ethyl
group, and a singlet at 10.5 assigned to the proton of
(N@CH) group (Scheme 1).
Molecular modeling simulation studies were then con-
ducted by measuring the compare/fit values, separately,
between the conformational models of lead vatalanib,
compounds 2–18 and ideal selective VEGFRTK inhibi-
tor hypothesis (Fig. 4). The results of the best fitting
values, as well as the conformational energy of the
best-fitted conformer with this hypothesis are given in
Table 1. The results of simulation studies have revealed
that compounds 6, 10, 14, 15 and 17 might be promising
active hit molecules.
Ph
Ph
CN
NH
Ph
CN
Ph
HN
N R
N
C OC2H5
N
NH2
N
O
SO2NH2
1
H
a
b
N
N
c
SO2NH2
SO2NH2
SO2NH2
2
3
4a-e
Scheme 1. Compounds: 4a, R = NH2; 4b, R = C2H5; 4c, R = CH2CH2OH; 4d, R = CH2CH2CH2OH; 4e, R = CH2 Ph. Reagents: a, malononitrile,
sodium ethoxide; b, triethylorthoformate; c, appropriate aliphatic amines.

D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
2393
Cl
N
Ph
N
N
N
N
Cl
SO2NH2
12
NH
N
N
O
O
Ph
O
Ph
N
N
H
Cl
N
N
Cl
H
N
N
N
f
SO2NH2
SO2NH2
11
10
R
NH
N
NH2
N
Ph
NC
N
Ph
N
NH2
N
Ph
g
N
a-e
N
N
N
N
N
N
SO2NH2
4a
SO2NHR'
R'= H
SO2NH2
13
5,R=H,
6,R=CH3,
R'= COCH3
R'= H
7,R=SH,
8,R=CH2CN, R'= H
9,R=C6H5, R'=H
h
NH
Ph
NHCSNHR`
N
N
N
SO2NH2
14, R' = C2H5
15, R' = C6H5
Scheme 2. Reagents: a, formic acid; b, acetic anhydride; c, carbon disulfide; d, ethyl cyanoacetate; e, benzoyl chloride; f, 4-chlorobenzoylchloride g,
malononitrile; h, appropiate isothiocyanate.
When the triethylorthoformate derivative 3 was re-
acted with various aliphatic amines a series of 3,4-
dihydropyrrolo[2,3-d]pyrimidin derivatives 4a–e were
obtained (Scheme 1). The structures of compounds
4a–e were established on the bases of elemental anal-
yses and spectral data. Their IR spectra showed the
disappearance of the (C„N) band. Moreover, 1H
NMR spectrum of compound 4b revealed a triplet
at 1.3 ppm and a quartet at 4.0 ppm attributed to
Compound 4a was reacted with different one-carbon
cyclizing agents to prepare some new pyrrolopyrimidine
derivatives containing [1,2,4]triazole moiety which was
reported to have a broad spectrum of biological activity
including antitumor effects32–34 (Scheme 2).
Compound 5 was obtained via reaction of compound 4a
with formic acid under reflux. Structure of pyrrolotriaz-
olopyrimidine derivative 5 was confirmed via its mass
spectrum, which showed a molecular ion peak m/z at
390 (M+, 100% base peak).
1
the ethyl group. Also, the H NMR spectrum of com-
pound 4c showed multiplet signal at 2.5 ppm assigned
to the four protons of the (2CH2) group. Regarding
compound 4a, its mass spectrum exhibited a molecular
ion peak m/z at 380 (M+, 1.78%) and a base peak at
55.
Also, reaction of compound 4a with acetic anhydride
gave the pyrrolotriazolopyrimidine derivative 6. Cycli-
zation at the imino and amino centers took place in

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D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
NH
NCOOEt
NHCOOEt
O
Ph
Ph
NH2
N
N
Ph
N
N
COOEt
N
a
N
N
N
N
N
N
SO2NH2
SO2NH2
4a
SO2NH2
16
17
O
NH
N
b
Ph
N
N
N
SO2NH2
18
O
N
N
NH
Ph
N
N
SO2NH2
19
Scheme 3. Reagents: a, ethyl chloroformate; b, ethyl chloroacetate.
addition to acetylation of the amino group of the sulfa-
moyl moiety (Scheme 2).
group followed by cyclization. The structures of com-
pound 9 was confirmed based on its mass spectrum,
which revealed a molecular ion peak m/z at 466 (M+,
1.38%) with a base peak at 103.
IR spectrum of compound 6 showed a band at
1740 cmÀ1 confirming the presence of (C@O) of the
acetyl group, also its mass spectrum revealed a molecu-
lar ion peak m/z at 446 (M+, 5%) with a base peak at
404.
Also, when compound 4a was made to reaction with 4-
chlorobenzoylchloride, the dibenzoyl derivative 10
(Scheme 2) was isolated, and both the monobenzoyl 11
and the fused system 12 were eliminated from consider-
ation on the basis of elemental analyses and mass spec-
trum. IR spectrum of compound 10 revealed bands at
1680 cmÀ1 and 1700 cmÀ1, attributed to the two car-
bonyl groups. Its mass spectrum showed a molecular
ion peak m/z at 657 (M+, 4.55%) with a base peak at 139.
Carbon disulfide was used also to cyclize compound 4a
under reflux in the presence of pyridine to give com-
pound 7. Mass spectrum of compound 7 exhibited a
molecular ion peak m/z at 422 (M+, 0.34%) with a base
peak at 63.
On the other hand compound 4a reacted under reflux
with ethylcyanoacetate in the presence of ethanol con-
taining sodium ethoxide afforded the pyrrolotriazolo-
pyrimidine derivative 8 (Scheme 2).
Interaction of compound 4a with the active methylene
compound, malononitrile, afforded the fused tricyclic
compound 13 (Scheme 2). IR spectrum of compound
13 showed a band at 2200 cmÀ1 assigned to the presence
of (C„N) group. Its mass spectrum exhibited a molec-
ular ion peak m/z at 441 (M+) with a base peak at 64.
Microanalytical and spectral data were in accordance
with the expected structure, hence its IR spectrum
showed band at 2220 cmÀ1 assigned to the presence of
(C„N) group. Mass spectrum of compound 8 showed
a molecular ion peak m/z at 429 (M+, 7.71%) with a base
peak at 429.
Nucleophilic reaction of compound 4a on the highly po-
sitive carbon of the isothiocyanate RNCS yielded com-
pounds 14 and 15 (Scheme 2). Their structures were
confirmed based on their spectral data, where the IR
spectrum of compound 14 showed bands at 3460,
3330, and 3150 cmÀ1 assigned to (NH, NH2) groups,
whereas a band at 1250 cmÀ1 due to the introduction
of (C@S) group appeared in the IR spectra of both com-
Interaction of compound 4a with benzoyl chloride affor-
ded the pyrrolotriazolopyrimidine derivative 9 (Scheme
2). This assumed through benzoylation at the amino

D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
2395
Cl
F
O
S
O
O
HO
H
N
N
N
CH3
O
N
O
S
F
F
O
N
O
N
N
N
H
N
H
Br
O
O
H2N
H3C
(II)
CP-547632(III)
Gefitinib
Cl
Cl
(I)
O
C
H
N
H
N
CH3
NH
O
HO
O
N
CH3
HN
N
H
O
S
O
NH
N
N
H3C
H3C
N
F
O
O
N
N
H
N
H
N
CH3
(IV)
SU11248( VII)
SU6668(VI )
Vatalanib ( V )
Figure 1. Structures of selective VEGFRTK inhibitory lead compounds I–VII used for generation of common feature VEGFRTK inhibitor
hypothesis.
Condensation of compound 4a with ethyl chlorofor-
mate afforded 4-(2-Oxo-9-phenyl-7H-pyrrolo[3,2-e][1,2,4]
triazolo[1,5-c]pyrimidin-7-yl) benzenesulfonamide 17
(Scheme 3). The formation of compound 17 proceeded
via initial formation of the intermediate 16 followed by
intramolecular cyclization via loss of one mole ethanol.
Structure of compound 17 was established on the bases
of microanalytical and spectral data. IR spectrum
revealed bands at 3360, 3330 cmÀ1 for NH2 group,
2930, 2895 cmÀ1 for CH aliph., 1710 cmÀ1 for C@O (es-
ter) and at 1680 cmÀ1 for C@O (amide). Moreover, mass
spectrum of compound 17 showed a molecular ion peak
m/z at 480 (M+, 6.09%) and a base peak at 227.
Figure 2. Generated hypothesis for VEGFRTK inhibitors with five
features [three HY (pale blue) and two HBA (green)].
Reaction of compound 4a with ethyl chloroacetate in
refluxing sodium ethoxide solution yielded the pyrrol-
o[2,3-d]pyrimidotriazinederivative 18 rather than its iso-
meric structure 19 (Scheme 3).
Structure of 18 was suggested rather than structure 19
based on assumption that the reaction basic condition
allowed it to proceed through formation of sodium salt
on the less basic imino nitrogen atom, and elimination
of sodium chloride followed by cyclization.35 In addi-
tion, IR spectrum of the isolated product 18 showed a
carbonyl band at 1620 cmÀ1, which was at less fre-
1
quency than that expected for structure 19. Also, H
NMR spectrum of compound 18 revealed singlet at
4.3 for the (CH2CO) group. Moreover, the mass spec-
trum showed a molecular ion peak m/z at 419 (MÀ1,
Figure 3. Constraint distances and torsion angel of VEGFRTK
hypothesis.
2.81) with a base peak at 80.
1
pounds 14 and 15. Furthermore, H NMR spectrum of
compound 14 revealed a triplet at 1.2 ppm and a quartet
at 4.0 ppm attributed to the ethyl group. The mass spec-
trum of compound 15 showed a molecular ion peak m/z
at 515 (M+, 3.65%) with a base peak at 77.
2.3. Antitumor activity
Doxorubicin, the reference drug used in this study, is
one of the most effective antitumor agents used to pro-
duce regressions in acute leukemia’s, Hodgkin’s disease,

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D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
Figure 4. Mapping of VEGFRTK inhibitor hypothesis with Vatalanib and compound 15.
Table 1. Fit and energy values and in vitro cytotoxic activity of some newly synthesized compounds
Compound
Fit value
Energy K/calÀ1
Non viable cells (%)
Concentration (lmol/mL)
a
IC50 (nM/ml)
200
100
75
50
25
2
2.13
1.5
7.39
0.1
100
90
100
60
80
80
80
0
100
15
20
20
15
0
80
0
40
0
88.75
100
5
6
2.97
1.78
1.78
1.73
0.9
10.00
0.46
0
100
80
5
0
75.78
90.25
90.25
>200
>200
26.5
—
7
0
0
8
90
0
0
0
9
13
0.44
7.78
5.47
0.13
—
0
0
0
0
0
0
0
15
Vatalanib
Doxorubicin
2.95
5.00
—
100
—
95
—
75
80
—
64
60
—
55
50
—
20
100
81.5
a IC50 value which corresponds to the compound concentration causing 50% mortality in net cells.
Table 2. Docked energies and estimated Ki values for Vatalanib and
compound 15
vation of scavenger enzymes activities induced by
reactive oxygen species (ROS). Oxidative stress occurs
in living organisms when the production of ROS exceeds
the ability of organisms to prevent their accumula-
tion.37,38 Such elevation of LPx is accompanied by decline
in GSH content and in the activity of related antioxidant
enzyme SOD. Additionally LPx can be initiated by hydro-
gen abstraction from lipid molecules by lipid radiolytic
products. This leads to permeability changes, secondary
alteration in membrane proteins, and other sequences.39,40
Compound
Docked energy
Ki
Vatalanib
15
À9.41
À9.54
+1.37eÀ06
+2.25eÀ06
and other lymphomas. The relationship between sur-
vival ratio and drug concentration was plotted to obtain
the survival curve of Ehrlich Ascites Carcinoma (EAC)
cell line. The response parameter calculated was IC50 va-
lue (Table 1), which corresponds to the compound con-
centration causing 50% mortality in net cells.
2.5. Effect of compound 10 and/or c-irradiation on lipid
peroxidation and antioxidant status
2.5.1. Glutathione level in blood (GSH). As summarized
in Table 3, a significant depletion in GSH level was
observed in blood of irradiated mice compared to their
corresponding controls. While mice exposed to c-irradi-
ation and treated with compound 10 showed significant
increase of GSH content in blood.
Compounds 6 and 15 showed significant in vitro antitu-
mor activity compared to the reference drug Doxorubi-
cin. One must mention that compound 15 (IC50, 26.5)
possesses thioureido moiety which is known to have
antitumor activity.36
Also, fusion of pyrrolopyrimidine in a tricyclic structure
namely pyrrolotriazolopyrimidine 6 (IC50, 67.5) enhances
the antitumor activity which agrees with the literature.
2.5.2. Lipid peroxidation content (MDA) in plasma. As
shown in Table 3, mice exposed to c-irradiation showed
significant elevation in Malonaldialdehyde (MDA) level
in plasma compared to the control values. Treatment with
compound 10 prior to irradiation exhibited a higher
reduction in MDA levels in blood as compared to irradi-
ated group.
2.4. Radioprotective activity
Radiation exposure significantly increases lipid peroxida-
tion (LPx), such increase seems to be the result of inacti-

D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
2397
Figure 5. The proposed binding mode of Vatalanib inside the active site of VEGFT kinase resulting from docking. The most important amino acids
are shown together with their respective numbers. Vatalanib forms only hydrogen bond with Cys919 through its nitrogen (HB acceptor) in pyridine
moiety. The other interactions are hydrophobic bonding with Val848, Leu840, and Leu1035 amino acids.
Figure 6. The proposed binding mode of compound 15 inside the active site of VEGFT kinase resulting from docking. The most important amino
acids are shown together with their respective numbers. Compound 15 forms three hydrogen bonds with Cys919 and Glu917 through its
isothiocyanate moiety and other hydrogen bond with Glu885 through its sulfonamide moiety. The other interactions are hydrophobic bonding with
Val848 and Leu840 amino acids.
Table 3. Effect of compound 10 administration on blood glutathione (GSH) content and plasma lipid peroxide concentrations (LPx) of normal and
irradiated mice
Groups
GSH mg/dl
LPx lmol/ml
Control
CMC
Mean SE% of change
Mean SE#
78.59 5.45 (100%)
75.45 0.28 (96.15%)
38.98 1.03*** (49.59%)
138.07 13.75** (175.68%)
303.51 35.99*** (386.19%)
78.05 0.79 (100%)
80 1.18 (103.9%)
101.29 0.11*** (129.78%)
80.96 2.23 (103.7%)
80.32 1.00 (100.9%)
Rad
Compound 10
Compound 10 + Rad
Mean SE#
Mean SE#
Mean SE#
Each value is the mean of eight mice SE.
#
Percentage of change from control group.
High significance at P < 0.01 and very high significance
CMC: Carboxy methyl cellulose.
**
***
at P < 0.001.
3. Conclusion
zenesulfonamide moieties exhibited promising in vitro
cytotoxic activity against (EAC) cell line. Compounds
6 and 15 showed the highest in vitro cytotoxic activity
when compared to other tested compounds and Doxo-
The present data showed that some compounds combin-
ing both pyrrole or pyrrolo[2,3-d]pyrimidine and ben-

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D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
rubicin as a reference drug. Additionally, compound 10
showed significant radioprotective activity. Docking
studies revealed that compound 15 showed binding
mode similar to vatalanib which was reported to have
VEGFRTK inhibitory effects.
the file and hydrogens were added, then Kollman
charges were calculated. No further geometry
optimization was performed.
•
•
Non-polar hydrogens were merged with the par-
ent atoms for both the ligands and the receptor.
The grid maps were generated for a box centered
on the ATP binding site (extension relative to the
original pdb file coordinates: x: 27.2–42.2, y:
26.0–41.0, z: 9.0–24.0), and the grid points spac-
4. Experimental protocols
˚
4.1. Catalyst molecular modeling experiments
All molecular modeling work was performed on Silicon
ing was kept to the default value of 0.375 A.
The docking itself was performed using the
Lamarckian Genetic Algorithm, with a transla-
•
˚
tional increment of 0.5 A, and both quaternion
Graphic (SGI), Fuel workstation (500 MHz,
R
14000 ATM processor, 512 MB memory) using the cata-
lyst package of Molecular Simulation (version 4.8), un-
der an IRIX 6.8 operating system, at the Faculty of
Pharmacy, Ain Shams University. A generalized visual-
izer, confirm, info, HipHop, compare/fit, force field was
used throughout.
and torsional angles were incremented by 15 A
˚
each. The generation size was set to 150 individ-
uals per generation, and the rest of the docking
parameters were kept to the default values. Ten
runs of the Genetic algorithm were performed,
thus for each compound, we got 10 docked
poses, which were subsequently clustered relative
to the original MMFF structure using root mean
Training sets, lead compounds I–VII, were selected.
Molecules were built within the catalyst and conforma-
tional models for each compound were generated auto-
matically using the poling algorithm. This emphasizes
representative coverage over a 20 kcal molÀ1 energy
range above the estimated global energy minimum and
the best searching procedure was chosen. The training
molecules with their associated conformational models
were submitted to catalyst by using default common fea-
tures hypothesis generation by using HipHop com-
mands. All needed features for VEGFRTK inhibitors
were selected from the Dictionary List: HB Acceptor,
Hydrophobic (HY) and HB donor. By this step, we
specified the expected features required for the activity
of VEGFRTK inhibitors. Then, a Generate Hypothesis
order was given to the computer. Finally, the Process
data were collected, and the Process log files were exam-
ined which showed that 10 hypotheses were generated.
˚
square deviation (RMSD) tolerance of 1.0 A.
The poses were evaluated using the default
empirical scoring function in Autodock.
4.3. Chemistry
Melting points are uncorrected and were determined on
a Stuart melting point apparatus (Stuart Scientific, Red-
hill, UK). Elemental analyses (C, H, N) were performed
on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Nor-
walk, CT, USA) at the Microanalytical Unit of Cairo
University. All compounds were within 0.4% of the
theoretical values. IR spectra (KBr) were measured on
(Pye Unicam SP1000 IR spectrophotometer Thermo-
1
electron, Egelbach, Germany). H NMR spectra were
obtained on a Bruker 300 MHz NMR spectrophotome-
ter (Bruker, Munich, Germany) in DMSO-d6 as a sol-
vent, using tetramethyl-silane (TMS) as internal
standard. Mass spectra were run on Varian MAT 311-
A70 eV (Varian, Fort Collins, USA).
All the 10 generated hypotheses were analyzed. The
assessment of the ideal hypothesis among the generated
ones indicated that hypothesis ranked number 2 was the
ideal one.
4.3.1. 4-(2-Oxo-2-phenyl-ethylamino)-benzenesulfon-
amide (1). A mixture of sulfanilamide (1.72 g, 0.01 mol)
with phenacyl bromide (2 g, 0.01 mol) was refluxed in
dimethylformamide (30 ml); in the presence of triethyl-
amine (three drops); for 3 h. The solid obtained was
filtered and recrystallized from ethanol31 (88% yield).
M.p.: 218–220 ꢁC as reported,31 IR (KBr, cmÀ1): 3474,
3259, 3130 (NH2,NH), 1676 (C@O), 1306, 1195 (SO2).
1H NMR, (DMSO-d6): d 4.80 (d, 2H, CH2), 6.60–7.70
(m, 11H, Ar–H + NH2), 8.10 (s, 1H, NH), C14H14N2O3S
(290).
4.2. Docking studies
•
•
•
The docking was performed using Autodock
3.05.41
The docking was performed on two compounds;
Vatalanib and 15.
The structures were minimized by Merck Molec-
ular force field (MMFF) to a gradient of
4.5 · 10À5 using Spartan’06. The optimized
geometries were exported as Sybyl mol2 file
which were exported to Autodock tools. Gastei-
ger charges were calculated for each molecule
and the torsional degrees of freedom were set
to the maximum number of rotatable bonds.
The receptor structure was downloaded from the
protein databank (pdb) [www.rcsb.org], pdb
entry 1VR2. Water molecules were deleted from
4.3.2. 4-(2-Amino-3-cyano-4-phenyl-pyrrol-1-yl)benzene-
sulfonamide (2). A mixture of compound 1 (2.9 g,
0.01 mol) with malononitrile (0.7 g, 0.01 mol) was re-
fluxed in ethanol (30 ml) containing sodium ethoxide
(0.5 g) for 3 h. The reaction mixture was acidified with
diluted HCl. The solid obtained was recrystallized from
ethanol31 (85% yield), m.p. 200–202 ꢁC as reported,31 IR
(KBr, cmÀ1): 3400, 3340, 3230 (NH2), 2200 (C„N),
•
1
1350, 1170 (SO2), H NMR, (DMSO-d6): 6.20 (br. s,

D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
2399
2H, NH2), 6.90 (s, 1H, pyrrole-H), 7.30–7.90 (m, 11H,
Ar–H + SO2NH2), MS (m/z): 338 (M+, 33.9%), 258
(9.7%), 155 (100%), 139 (2.9%), C17H14N4O2S (338).
for 4 h and the reaction mixture was then concentrated.
The separated crystals were recrystallized from dioxane
(63% yield), m.p. 260–262 ꢁC, IR (KBr, cmÀ1): 3300,
3190 (NH2), 1580 (C@N), 1350, 1160 (SO2), MS (m/z):
390 (M+, 100% base peak), Anal. Found: C, 58.80; H,
3.80; N, 21.70%; C19H14N6O2S Calcd C, 58.46; H,
3.59; N, 21.54%.
4.3.3. Ethyl N-3-cyano-4-phenyl-1-(4-sulfamoylphenyl)-
1H-pyrrol-2-yl-formimidate (3). A mixture of compound
2 (3.4 g, 0.01 mol) and triethylorthoformate (20 ml) was
refluxed for 5 h. The reaction mixture was cooled and
then poured onto ice cold water. The formed residue
was recrystallized from dioxane (75% yield), m.p. 120–
122 ꢁC, IR (KBr, cmÀ1): 3350, 3230 (NH2), 2200
(C„N), 1580 (C@N), 1340, 1155(SO2), 1H NMR
(DMSO-d6) d: 1.3 (t, 3H, CH3), 4.3 (q, 2H, CH2), 5.0
(s, 1H, CH pyrrole), 7.3–8.2 (m, 11H, Ar–H + NH2),
10.5 (s, 1H, N@CH). Anal. Found: C, 60.60; H, 4.20;
N, 14.40%; C20H18N4O3S Calcd C, 60.91; H, 4.56; N,
14.21%.
4.3.6. 4-(2-Methyl-9-phenyl-7H-pyrrolo[3,2-e][1,2,4]triaz-
olo[1,5-c]pyrimidin-7-yl)benzenesulfonamide (6). A solu-
tion of 4a (3.8 g, 0.01 mol) in acetic anhydride (20 ml)
was refluxed for 4 h and the reaction mixture was then
concentrated. The separated crystals were recrystallized
from dioxane (65% yield), m.p. 268–270 ꢁC, IR (KBr,
cmÀ1): 3230 (NH), 1740 (C@O), 1350, 1160 (SO2), MS
(m/z): 446 (M+, 5%), 404 (100% base peak), Anal.
Found: C, 59.30; H, 4.20; N, 18.50%; C22H18N6O3S
Calcd C, 59.19; H, 4.04; N, 18.83%.
4.3.4. 4-(3-Amino-4-imino-5-phenyl-3,4-dihydropyrrolo[2,
3-d]pyrimidin-7-yl)benzenesulfonamide(4a),4-(3-ethyl-4-
imino-5-phenyl-3,4-dihydropyrrolo[2,3-d]pyrimidin-7-yl)ben-
zenesulfonamide(4b), 4-(3-2 hydroxyethyl-4-imino-5-phe-
nyl-3,4-dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesul-
fonamide(4c), 4-(3-3 hydroxypropyl-4-imino-5-phenyl-3,
4-dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesulfonamide
(4d), and 4-(3-benzyl-4-imino-5-phenyl-3,4-dihydropyrrol-
4.3.7. 4-(2-Mercapto-9-phenyl-7H-pyrrolo[3,2-e][1,2,4]
triazolo[1,5-c]pyrimidin-7-yl)benzenesulfonamide (7). A
solution of 4a (3.8 g, 0.01 mol) in carbon disulfide
(2 ml) was refluxed for 8 h in the presence of pyridine
(20 ml), the reaction mixture was then concentrated
and poured into ice cold water. The separated solid
was filtered off and recrystallized from ethanol (82%
yield), m.p. 259–260 ꢁC, IR (KBr, cmÀ1): 3300, 3230,
(NH2), 1580 (C@N), 1350, 1150 (SO2).
o[2,3-d]pyrimidin-7-yl)benzenesulfonamide(4e). A mix-
ture of 3 (3.9 g, 0.01 mol) and the appropriate
aliphatic amines (0.01 mol) was stirred in ethanol at
room temperature for 30 min, the solid formed was fil-
tered, and recrystallized from dioxane, 4a: (87% yield),
m.p. 206–207 ꢁC, IR (KBr, cmÀ1) 4a: 3450, 3315, and
3150 (NH, NH2), 1580 (C@N), 1360, 1170 (SO2), MS
(m/z) 4a: 381 (M + 1, 1.78%), 55 (100%), 194 (3.56%),
116(6.87%), Anal. Found: C, 56.50; H, 4.60; N,
21.80%; C18H16N6O2S Calcd C, 56.84; H, 4.21; N,
22.11%. 4b: (70% yield), m.p. 237–239 ꢁC. IR (KBr,
cmÀ1): 3460, 3330, 3200 (NH, NH2), 1600 (C@N),
MS (m/z): 422 (M+, 0.34%), 63 (100%). Anal. Found: C,
54.30; H, 3.10; N, 19.60%; C19H14N6O2S2Calcd C,
54.03; H, 3.32; N, 19.91%.
4.3.8. 4-(2-Cyanomethyl-9-phenyl-7H-pyrrolo[3,2-e][1,2,
4]triazolo[1,5-c]pyrimidin-7-yl)benzenesulfonamide (8). A
mixture of 4a (3.4 g, 0.01 mol) and ethyl cyanoacetate
(1.3 g, 0.01 mol) was refluxed for 8 h in ethanol (30 ml)
containing sodium ethoxide (0.23 g, 0.01 mol ). The
reaction mixture was then acidified with diluted HCl.
The separated solid was filtered, and recrystallized from
dioxane (76% yield), m.p. 264–266 ꢁC, IR (KBr, cmÀ1):
3300, 3230 (NH2), 2220 (C„N), 1590 (C@N), MS (m/z):
429 (M+, 7.71%), 429(100%). Anal. Found: C, 58.50; H,
3.20; N, 22.60%; C21H15N7O2S2 Calcd C, 58.74; H, 3.49;
N, 22.84%.
1
1360, 1170 (SO2), H NMR (DMSO-d6) d: 1.3 (t, 3H,
CH3), 4.0 (q, 2H, CH2), 5.0 (s, 2H, NH2), 7.3–7.7 (m,
11H, Ar–H + NH + CH pyrrole), 8.1 (s, 1H, CH pyrim-
idine), Anal. Found: C, 61.30; H, 4.50; N, 17.50%;
C20H19N5O2S Calcd C, 61.07; H, 4.83; N, 17.81%. 4c:
(78% yield), m.p. 260–262 ꢁC, IR (KBr, cmÀ1): 3400,
3310, 3190 (NH, NH2), 1590 (C@N), 1360, 1170
(SO2), 1H NMR, (DMSO-d6) d: 2.5 (m, 4H, 2CH2),
6.4 (s, 1H, CH pyrrole), 7.2–8.3 (m, 12H, Ar–
H + NH + SO2NH2), 9.2 (s, 1H, CH pyrimidine), Anal.
Found: C, 58.30; H, 4.50; N, 17.40%; C20H19N5O3S
Calcd C, 58.68; H, 4.65; N, 17.11%. 4d: (80% yield),
m.p. 220–222 ꢁC, IR (KBr, cmÀ1): 3450, 3315, and
3150 (NH, NH2), 1580 (C@N), 1360, 1170 (SO2). Anal.
Found: C, 59.30; H, 4.60; N, 16.30%; C21H21N5O3S
Calcd C, 59.57; H, 4.96; N, 16.55%. 4e: (65% yield),
m.p. 160–162 ꢁC, IR (KBr, cmÀ1): 3440, 3325, 3200
(NH, NH2), 1585 (C@N), 1360, 1170 (SO2). Anal.
Found: C, 65.70; H, 4.30; N, 15.60%; C25H21N5O3S
Calcd C, 65.93; H, 4.62; N, 15.38%.
4.3.9. 4-(2,9-Diphenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,
5-c]pyrimidin-7-yl)benzenesulfonamide (9). A mixture of
4a (3.4 g, 0.01 mol) and benzoyl chloride (10 ml) was re-
fluxed for 2 h. The reaction mixture was then concen-
trated and the formed precipitate was filtered off and
recrystallized from dioxane, (70% yield), m.p. 268–
270 ꢁC, IR (KBr, cmÀ1): 3340, 3195 (NH2), 1585 (C@N),
1350, 1160 (SO2). MS (m/z): 466 (M+, 1.38%), 103
(100%), 77 (15.4%). Anal. Found: C, 64.70; H, 3.60; N,
18.40%; C25H18N6O2S Calcd C, 64.38; H, 3.86; N, 18.03%.
4.3.10. 3-(4-Chlorobenzoylamino)-4-(4-chlorobenzoylami-
no)5-phenyl-7-(4-sulfamoylphenyl)-3,4-dihydropyrrolo[2,3-d]
pyrimidin (10). A mixture of compound 4a (3.8 g,
0.01 mol) and 4-chlorobenzoylchloride (10 ml) was re-
fluxed for 3 h, the reaction mixture was then concentrated
and the obtained solid was recrystallized from dioxane
4.3.5. 4-(9-phenyl-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]
pyrimidin-7-yl)benzenesulfonamide (5). A solution of 4a
(3.8 g, 0.01 mol) in formic acid (20 ml) was refluxed

2400
D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
(80% yield), m.p. 223–224 ꢁC, IR (KBr, cmÀ1): 3340,
3240, 3230, (NH, NH2), 1700, 1680, (2 C@O), 1310,
1150 (SO2). MS (m/z): 657 (M+, 4.55%), base peak at
139 (100%). Anal. Found: C, 58.50; H, 3.10; N, 12.40%;
C32H22N6O4SCl2 Calcd C, 58.36; H, 3.34; N, 12.77%.
ane (80% yield), m.p. >300 ꢁC, IR (KBr, cmÀ1): 3450,
3315 (NH2), 1620 (C@O), 1580 (C@N), 1350, 1160
(SO2), 1H NMR (DMSO-d6): d 4.3 [s, 2H, CH2CO],
6.7 [s, 1H, CH pyrrole], 7.1–7.9 [m, 11H, Ar–
H + SO2NH2], 8.3 [s, 1H, CH pyrimidine], 9.2 [s, 1H,
NH], MS (m/z): 419 (M-1, 2.81%), 80 (100%). Anal.
Found: C, 57.30; H, 3.60; N, 19.70%; C20H16N6O3S
Calcd C, 57.14; H, 3.81; N, 20.00%.
4.3.11. 4-(8-Amino-9-cyano-1-phenyl-3H-pyrazolo[1,5-
c]pyrrolo[3,2-e]pyrimidin-3-yl)benzenesulfonamide (13).
A mixture of 4a (3.8 g, 0.01 mol) and malononitrile
(0.7 g, 0.01 mol) was refluxed in ethanol (30 ml) contain-
ing sodium ethoxide (0.23 g, 0.01 mol ) the reaction mix-
ture was then acidified with diluted HCl. The obtained
residue was filtered and recrystallized was from ethanol,
(61% yield), m.p. 152–153 ꢁC, IR (KBr, cmÀ1): 3460,
3330 (NH2), 2200 (C„N), 1340, 1160 (SO2). MS
(m/z): 441 (M+, 4.87%), base peak at 64 (100%). Anal.
Found: C, 58.60; H, 3.20; N, 22.50%; C21H15N7O2S
Calcd C, 58.74; H, 3.49; N, 22.84%.
4.4. Biological testing
4.4.1. Antitumor activity. Ehrlich Ascites Carcinoma
cells (EAC) were obtained by needle aspiration of ascitic
fluid from the preinoculated mice under aseptic
conditions. Tumor cells suspension (2.5 · 106 per ml)
was prepared in saline. Tested compounds solutions
were prepared by dissolving 10 lmol of the tested com-
pounds in a mixture of 7 ml DMSO and 3-ml saline.
Some compounds failed to be dissolved in either DMSO
or DMF so could not be screened as cytotoxic agents
using this method.
4.3.12. 4-(3-(3-Ethyl thioureido)-4-imino-5-phenyl-3,4-
dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesulfonamide
(14), 4-(3-(3-phenyl thioureido)-4-imino-5-phenyl-3,4-
dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesulfonamide
(15). A mixture of 4a (3.8 g, 0.01 mol) and the appropri-
ate isothiocyanate derivatives (RNCS) was refluxed in
ethanol for 3 h, the reaction mixture was concentrated,
and the separated crystals were recrystallized from etha-
nol, 14: (70% yield), m.p. 258–260 ꢁC, IR (KBr, cmÀ1):
3460, 3330, and 3150 (NH, NH2), 3100 (CH arom.),
In a set of sterile test tubes 200, 100, 75, 50 and 25 lL of
each tested compound were mixed with 100 lL of tumor
cell suspension, then completed to 1 mL with saline to
obtain a solution of 200, 100, 75, 50 and 25 lM, respec-
tively, for each tested compound. The test tubes were
incubated at 37 ꢁC for 2 h. Trypan blue exclusion test
was carried out to calculate the percentage of non-viable
cells after 2 h of incubation.42 The results of in vitro
cytotoxic activity experiments are presented in Table 1.
1
1250 (C@S), 1350, 1170 (SO2), H NMR (DMSO-d6):
d 1.20 (t, 3H, CH3), 4.0 (q, 2H, CH2), 6.2 (s, 1H, CH
pyrrole), 7.3–8.0 (m, 11H, Ar–H + SO2NH2), 8.3 (s,
1H, CH pyrimidine), 9.3(s, 3H, +3NH). Anal. Found:
C, 53.60; H, 4.20; N, 21.20%; C21H21N7O2S2 Calcd C,
53.96; H, 4.49; N, 20.98%.
4.4.2. Radioprotective activity. This study was conducted
to evaluate the potency of one of the synthesized com-
pounds 10 as protective agent against c-irradiation-in-
duced toxicity. Female Swiss albino mice were injected
intraperitoneally with suspension of the tested com-
pound in carboxy methylcellulose once every other day
for a total of three injections during 7 days. Each injec-
tion was given 30 min prior to exposure to a single dose
of whole body c-irradiation at a dose level of 6 Gy. Li-
pid peroxide level (LPx) and Glutathione content (GSH)
were estimated in blood of animals at the end of the
experiment.
Compound 15: (81% yield), m.p. 270–271 ꢁC, IR (KBr,
cmÀ1) 15: 3450, 3320, 3130 (NH, NH2), 3080 (CH
arom.), 1245 (C@S), 1350, 1170 (SO2), MS (m/z): 515
(M+, 3.65%), 481(61.13%), 77 (100%), 65(53.16%). Anal.
Found: C, 58.50; H, 4.40; N, 19.30%; C25H21N7O2S2
Calcd C, 58.25; H, 4.08; N, 19.03%.
4.3.13. 4-(2-Oxo-9-phenyl-7H-pyrrolo[3,2-e][1,2,4]triazol-
o[1,5-c]pyrimidin-7-yl)benzenesulfonamide (17). A mix-
ture of 4a (3.8 g, 0.01 mol) and ethyl chloroformate
(1.4 g, 0.015 mol) in benzene (20 ml) was refluxed for
5 h. The reaction mixture was then filtered off and recrys-
tallized from ethanol (80% yield), m.p. >300 ꢁC, IR (KBr,
cmÀ1): 3360, 3330 cmÀ1 (NH2), 2983, 2895 cmÀ1 (CH
aliph.), 1710 cmÀ1 (C@O ester), 1680 cmÀ1 (C@O amide),
1350, 1150 (SO2). MS (m/z): 478 (M+, 6.09%), 227
(100%), 174(56.09%), 134(61.07%), 73 (53.14%). Anal.
Found: C, 55.50; H, 4.50; N, 17.30%; C22H18N6O5S Calcd
C, 55.23; H, 3.76; N, 17.57%.
4.4.2.1. Chemicals and facilities. All chemicals and re-
agents were of the highest grade commercially available.
Facilities including animal house and biochemical
equipments have been made available by the National
Center for Radiation Research and Technology
(NCRRT), Cairo, Egypt.
4.4.2.2. Animals. Female Swiss albino mice weighing
25–30 g were used in this study. Mice were housed at a
constant temperature (24 2 ꢁC) with alternating 12-h
light and dark cycles and fed standard laboratory food
and water.
4.3.14. 4-(2-Oxo-10-phenyl-3,4-dihydro-pyrrolo[2,3-d]pyrim-
ido[3,4-e][1,2,4]triazine-8-yl)benzenesulfonamide (18). A
mixture of 4a (3.8 g, 0.01 mol) and ethyl chloroacetate
(1.2 g, 0.01 mol) was refluxed in ethanol (30 ml) contain-
ing sodium ethoxide (0.23 g, 0.01 mol) for 10 h, the reac-
tion mixture was then cooled and poured into ice cold
water, the solid separated was recrystallized from diox-
4.4.2.3. Irradiation. Whole body c-irradiation was per-
formed at the NCRRT, Cairo, Egypt, using Gamma cell-
40 (Cesium-137 source). Animals were irradiated at three
doses, every other day 30 min after drug injection at dose
level of 6 Gy delivered at a dose rate of 0.86 Gy/min.

D. A. Abou El Ella et al. / Bioorg. Med. Chem. 16 (2008) 2391–2402
2401
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suspended in carboxy methylcellulose (CMC) and given
to mice by intraperitoneal injection (ip) of the maxi-
mum tolerated dose (150 mg/kg body weight) once
every other day for a total of three injections during
7 days.
´
´
12. Esteve, C.; Nueda, A.; Dıaz, J. L.; Beleta, J.; Cardenas, A.;
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4.4.2.5. Experimental design. From the beginning of
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Each group consists of 10 animals. All experimental ani-
mals were categorized as follows:
14. Owa, T.; Yoshino, H.; Okauchi, T.; Yoshimatsu, K.;
Ozawa, Y.; Sugi, N. H.; Nagasu, T.; Koyanagi, N.; Kitoh,
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1. Control: Animals served as untreated control group.
2. CMC: Animals were treated by ip injection of
Carboxymethylcellulose.
3. Rad: Animals were subjected to three doses; every
other day of whole body c-irradiation at a dose level
of 6 Gy starting from day 10.
4. Compound 10: Animals were treated by ip injection
compound
of
Carboxymethylcellulose.
suspension
of
10
in
5. Compound 10 + Rad: Mice injected ip with com-
pound 10 and subjected to whole body c-irradiation.
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4.4.2.6. Sample collection. Animals were fasted for
16 h prior to each sampling. Samples were collected
after 1 day post last irradiation dose. Whole blood was
collected by heart puncture after light anesthesia using
heparinized syringes. One part was used for glutathione
(GSH) estimation. The separated plasma from heparin-
ized blood was used for the determination of lipid per-
oxide as malondialdehyde (MDA).
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4.4.2.7. Analytical procedures. Lipid peroxide (Lpx)
level in plasma was ascertained by the formation of
MDA and measured as described by Yoshioka et al.43
GSH content was determined according to Beutler
et al.44
ˇ
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4.4.2.8. Statistical analysis. Student’s t test45 was used
for the evaluation of the biochemical parameters.
29. Ghorab, M. M.; Osman, A. N.; Noaman, E.; Heiba, H. I.;
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References and notes
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