Design and synthesis of some novel quinoline derivatives as anticancer and radiosensitizing agents targeting VEGFR tyrosine kinase

Article abstract of DOI:10.1002/jhet.749

Quinoline derivatives posses many types of biological activities and have been reported to show significant anticancer activity. There is a variety of mechanisms for the anticancer activity and the most distinguished mechanism is the inhibition of vascula

Full text of DOI:10.1002/jhet.749

November 2011
Design and Synthesis of Some Novel Quinoline Derivatives as
Anticancer and Radiosensitizing Agents Targeting
VEGFR Tyrosine Kinase
1269
Mostafa M. Ghorab,a* Fatma A. Ragab,b Helmy I. Heiba,c and Walid M. Ghorabc
aMedicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy,
King Saud University, Riyadh,Saudi Arabia
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
cDepartment of Drug Radiation Research, National Centre for Radiation Research and Technology,
Atomic Energy Authority, Nasr City, Cairo, Egypt
*E-mail: mmsghorab@yahoo.com
Received July 19, 2010
DOI 10.1002/jhet.749
Published online 27 July 2011 in Wiley Online Library (wileyonlinelibrary.com).
Quinoline derivatives posses many types of biological activities and have been reported to show sig-
nificant anticancer activity. There is a variety of mechanisms for the anticancer activity and the most
distinguished mechanism is the inhibition of vascular epithelial growth factor receptor tyrosine kinase
(VEGFRTK). Novel quinoline derivatives 6–12 and pyrimido[4,5-b]quinoline derivatives 16–20 are
reported herein. All the newly synthesized compounds were evaluated for their in vitro anticancer activ-
ity against human breast cancer cell line (MCF7) in which VEGFR is highly expressed. Compounds 6
and 7 with IC50 values of 8.5 lM and 21.9 lM were the most active compounds and exhibited cytotoxic
activities higher than that of the reference drug doxorubicin (IC50 ¼ 32.02 lM). The most active com-
pounds 6 and 7 were further evaluated for their ability to enhance the cell killing effect of c-radiation.
J. Heterocyclic Chem., 48, 1269 (2011).
INTRODUCTION
Protein tyrosine 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, sur-
vival, and apoptosis. Several protein tyrosine kinases
are known to be activated in cancer cells and to drive
tumor growth, angiogenesis, progression, and metastasis.
Therefore, blocking tyrosine kinase activity represents a
rational approach to cancer therapy [23].
Quinoline derivatives posses a wide range of biologi-
cal activities including anti-inflammatory [1], antilei-
shmanial [2], antifungal [3], antituberculosis [4], and
antimalarial activities [5,6]. Also, several novel quino-
line derivatives have been reported to show substantial
anticancer activities [7–14].
It has been known that quinoline derivatives may act as
anticancer agents through a variety of mechanisms such as
cell cycle arrest in the G2 phase [13], topoisomerase inhi-
bition [15], and inhibition of tubulin polymerization [16],
and the most common mechanism was the inhibition of ty-
rosine kinases [17–19], specially vascular epithelial growth
factor receptor tyrosine kinase (VEGFRTK) [20–22].
The cancer cell is characterized by oncogene-derived
tumor expression of pro-angiogenic proteins, such as
vascular endothelial growth factor (VEGF), placenta-like
growth factor, basic fibroblast growth factor, platelet-
derived endothelial growth factor, angiopoietin-2,
C
V
2011 HeteroCorporation

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M. M. Ghorab, F. A. Ragab, H. I. Heiba, and W. M. Ghorab
Vol 48
Scheme 1
hepatocyte growth factor, and insulin-like growth factor
[24]. The mentioned pro-angiogenic growth factors bind
to specific receptors that possess receptor tyrosine kinase
(RTK) activity.
nonitrile 4 in ethanol containing a catalytic amount of
triethylamine, as a base catalyst, yielded the correspond-
ing hexahydroquinoline derivative 6 via the formation of
the intermediate Michael type product 5 followed by
intramolecular cyclization. IR spectrum of compound 6
revealed bands at 3311, 3203 cm-1 (NH2), 2179 cmꢀ1
(CBN), 1656 cmꢀ1 (C¼¼O). The mass spectrum of 6
revealed a molecular ion peak m/z at 461 (Mþ, 20.36%),
with a base peak m/z at 372 (100) (Scheme 1).
Overexpression of VEGFR was found in a number
of cancers (e.g., breast), their expression levels often
correlate with vascularity, and is associated with poor
prognosis in patients [25]. Inhibitors of the VEGFRTK
are, therefore, expected to have great therapeutic poten-
tial in the treatment of malignant tumors.
Reaction of compound 6 with acetic anhydride under
reflux condition for 10 h, afforded diacetyl derivative 7a
rather than monoacetyl form 7b and cyclic system 7c. IR
spectrum of compound 7 showed band at 2208 cmꢀ1 for
(CBN) and bands at 1700, 1658 cmꢀ1 for (2C¼¼O),
We report here the synthesis of some new quinoline
derivatives 6–12 and pyrimido[4,5-b]quinoline deriva-
tives 16–20 to evaluate their anticancer activity against
a human breast cancer cell line (MCF7). We also aimed
to evaluate the ability of compounds 6 and 7 to enhance
the cell killing effect of c-radiation.
1
whereas the H-NMR spectrum of compound 7 revealed
one singlet at 2.4 ppm of the acetyl groups. Interaction of
compound 6 with concentrated sulfuric acid at room tem-
perature caused partial hydrolysis of the cyano group
yielding the corresponding amide derivative 8, which
was confirmed by the disappearance of the cyano group
in the IR spectrum and presence of two carbonyl groups
at 1678, 1648 cmꢀ1. 1H-NMR spectrum showed the pres-
ence of singlet at 4.8 ppm for NH2 group, and at 6.2 ppm
for the CONH2 group. Refluxing compound 6 in triethy-
lorthoformate furnished the formimidate derivative 9,
which showed the absence of bands corresponding to the
RESULTS AND DISCUSSION
Chemistry. Several compounds were designed
with the aim of exploring their anticancer activity
(Schemes 1–3). Enaminone 3 was obtained through
condensation of 5,5-dimethyl-1,3-cyclohexandione
1
with p-bromoaniline 2. The structure of compound 3 was
supported by elemental analysis and spectral data. IR
spectrum of compound 3 revealed the presence of bands
at 3203 cmꢀ1 (NH), 3091 cmꢀ1 (CH arom.), at 2978,
2838 cmꢀ1 (CH aliph.), and 1656 cmꢀ1 (C¼¼O). Treat-
ment of enaminone 3 with 2-(4-methylbenzylidene)malo-
1
NH2 group in the IR spectrum. Also, the H-NMR spec-
trum showed the presence of significant triplet at 1.1
ppm for the CH3, quartet at 3.9 ppm of CH2 of the ethyl
group and singlet corresponding to one proton of the
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

November 2011
Design and Synthesis of Some Novel Quinoline Derivatives as Anticancer
and Radiosensitizing Agents Targeting VEGFR Tyrosine Kinase
1271
Scheme 2
N¼¼CH at 8.1 ppm. Fusion of compound 6 with succinic
anhydride resulted pyrrolidine derivative 10. IR spectrum
of compound 10 revealed the presence of bands at 1794,
1733, 1668 cmꢀ1 attributed to carbonyl groups. Mass
spectrum of compound 10 exhibited a molecular ion peak
at m/z 545 (Mþ, 8.09%) and a base peak at m/z 77. The
imidazolyl derivative 11 was obtained by refluxing com-
pound 6 with ethylenediamine in the presence of carbon
disulfide as a catalyst for 6 h. The reaction proceeded via
intramolecular cyclization through the elimination of 1
mol of ammonia. Structure of compound 11 was estab-
lished on the basis of elemental analysis and spectral
data. IR spectrum of compound 11 revealed the absence
of (CBN) band. Additionally, its mass spectrum exhib-
ited a molecular ion peak at m/z 504 (Mþ, 6.34%) with a
base peak at m/z 86. The 2-(3-oxobutanamido)quinoline
derivative 12 was obtained via refluxing of compound 6
with ethyl acetoacetate. The reaction proceeded via the
elimination of 1 mol of ethanol. IR spectrum of com-
pound 12 revealed the presence of three carbonyl groups
1
at (1840, 1728, and 1662 cmꢀ1). H-NMR spectrum of
compound 12 showed a singlet at 2.4 ppm for COCH3,
singlet at 4.8 ppm for COCH2 and singlet at 10.1 ppm
corresponding to NH (Scheme 2).
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M. M. Ghorab, F. A. Ragab, H. I. Heiba, and W. M. Ghorab
Vol 48
Scheme 3
Reaction of compound 6 with aromatic aldehydes in
trum of compound 19 showed a doublet at 4.9 ppm for
NH pyrimidine, at 8.0 ppm for NHCO, and singlet at
6.1 ppm for CH pyrimidine (Scheme 3).
acetic acid was expected to yield the corresponding
Schiff’s bases 13. Although, what actually happened
was an intramolecular cyclization via initial formation
of intermediates 14, 15 followed by a rearrangement to
give the pyrimido[4,5-b]quinoline derivatives 16–20.
The structure of these compounds was proved on the
bases of elemental analyses and IR spectrum which
showed the absence of (CBN) group and the presence
of two bands corresponding to two carbonyl groups
ranging from (1710–1658 cmꢀ1) of compounds 16–20.
Mass spectrum of compound 16 exhibited a molecular
ion peak at m/z 645 (Mþ, 2.08%) with a base peak at m/
z 183, mass spectrum of compound 17 exhibited a mo-
lecular ion peak at m/z 645 (Mþ, 4.46%) with a base
peak at m/z 44, mass spectrum of compound 18 exhib-
ited a molecular ion peak at m/z 603 (Mþ, 10.68%) with
a base peak at m/z 43, mass spectrum of compound 19
exhibited a molecular ion peak at m/z 600 (Mþ, 1.4%)
with a base peak at m/z 55 and mass spectrum of com-
pound 20 exhibited a molecular ion peak at m/z 589
In vitro anticancer screening. All the newly synthe-
sized compounds were evaluated for their in vitro cyto-
toxic activity against human breast cancer cell line,
MCF7. Doxorubicin, the reference drug used in this
study, is one of the most effective antitumor agents
used to produce regressions in acute leukemia’s, Hodg-
kin’s disease, and other lymphomas. The relationship
between surviving fraction and drug concentration was
plotted to obtain the survival curve of human breast
cancer cell line (MCF7). The response parameter cal-
culated was the IC50 value, which corresponds to the
concentration required for 50% inhibition of cell viabil-
ity. Table 1 shows the in vitro cytotoxic activity of the
synthesized compounds, where some compounds exhib-
ited significant activity compared with the reference
drug. From Table 1, we can observe that the quinoline
derivative 6 (IC50 ¼ 8.5 lM) and the diacetyl form of
quinoline derivative 7 (IC50 ¼ 21.9 lM) exhibited a
remarkable cytotoxic activity when compared with the
1
(Mþ, 8.4%) with a base peak at m/z 43. H-NMR spec-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

November 2011
Design and Synthesis of Some Novel Quinoline Derivatives as Anticancer
and Radiosensitizing Agents Targeting VEGFR Tyrosine Kinase
1273
Table 1
In vitro anticancer screening of the newly synthesized compounds (6–20) against human breast cancer cell line (MCF7) .
Compound concentration (lM)
10
25
50
100
Compound
Surviving fraction (Mean 6 SE)a
IC50 (lM)
Doxorubicin
0.451 6 0.02
0.276 6 0.01
0.561 6 0.01
0.525 6 0.03
0.904 6 0.03
0.819 6 0.01
0.816 6 0.01
0.921 6 0.01
0.643 6 0.01
0.872 6 0.01
0.952 6 0.01
0.812 6 0.02
0.808 6 0.3
0.352 6 0.02
0.130 6 0.03
0.167 6 0.01
0.436 6 0.07
0.685 6 0.01
0.425 6 0.02
0.738 6 0.04
0.171 6 0.01
0.523 6 0.01
0.579 6 0.01
0.657 6 0.01
0.473 6 0.01
0.541 6 0.04
0.290 6 0.01
0.280 6 0.03
0.143 6 0.01
0.205 6 0.01
0.250 6 0.01
0.209 6 0.01
0.208 6 0.01
0.264 6 0.01
0.246 6 0.01
0.174 6 0.01
0.173 6 0.01
0.197 6 0.01
0.245 6 0.01
0.354 6 0.01
32.02
8.5
6
7
8
0.176 6 0.01
0.102 6 0.01
0.163 6 0.01
0.355 6 0.01
0.119 6 0.01
0.253 6 0.01
0.158 6 0.01
0.264 6 0.02
0.394 6 0.04
0.359 6 0.01
0.232 6 0.01
0.238 6 0.01
21.9
33.1
53
9
10
11
12
16
17
18
19
20
38.6
52.3
36.73
40.1
38.3
52.8
44.4
50.8
a Each value is the mean of three values 6 standard error.
reference drug doxorubicin (IC50
¼
32.02 lM).
6 and 7, to improve the cell killing effect of c-irradia-
tion, was studied. From the results obtained in Table 1,
compound 6 showed an in vitro cytotoxic activity with
IC50 value of 8.5 lM, when the cells were subjected to
different concentrations of the compound alone.
Although when the cells were subjected to the same
concentrations of compound 6, and irradiated with a sin-
gle dose of c-radiation at a dose level of 8 Gy, as shown
in Table 2, the IC50 value was synergistically decreased
to 1.57 lM (Fig. 1). Similarly, compound 7 showed
IC50 value of 21.9 lM when used alone, as shown
in Table 1. The IC50 values were decreased to 3.14 lM
after irradiation (Fig. 2).
Although compound 8 (IC50 ¼ 33.1 lM) is nearly as
active as doxorubicin.
Radiosensitizing evaluation. The rationale for com-
bining chemotherapy and radiotherapy is based mainly
on two ideas, one being spatial cooperation, which is
effective if chemotherapy is sufficiently active to era-
dicate subclinical metastases and if the primary local tu-
mor is effectively treated by radiotherapy. In this regard,
no interaction between radiotherapy and chemotherapy
is required. The other idea is the enhancement of ra-
diation effects by direct enhancement of the initial
radiation damage by incorporating drugs into DNA, in-
hibiting cellular repair, accumulating cells in a radiosen-
sitive phase or eliminating radioresistant phase cells,
eliminating hypoxic cells, or inhibiting the accelerated
repopulation of tumor cells. Virtually, all chemothera-
peutic agents have the ability to sensitize cancer cells to
the lethal effects of ionizing radiation [26]. Conse-
quently, the tendency of the most two active compounds
Molecular modeling. Generation of VEGFRTK inhi-
bitor hypothesis using CATALYST software. The lead com-
pounds I–IV, which were reported to have selective
VEGFRTK inhibitory activity, were used to generate
common feature hypothesis of VEGFRTK inhibitor
(Fig. 3) [27]. The generated pharmacophore consisted of
five features with constraint dimensions (Fig. 4).
Table 2
In vitro anticancer screening of compounds 6 and 7 against human breast cancer cell line (MCF7) in combination with c-radiation.
Compound concentration (lM) þ irradiation (8 Gy)
10
25
50
100
Compd. no.
Control
Irradiated (8 Gy)
Surviving fraction (Means 6 SE)a
IC50 (lM)
6
7
1.000
1.000
0.927 6 0.02*
0.927 6 0.02*
0.147 6 0.01*
0.193 6 0.04*
0.082 6 0.04*
0.134 6 0.01*
0.036 6 0.01*
0.084 6 0.01*
0.021 6 0.01*
0.065 6 0.01*
1.57
3.14
a Each value is the mean of three values 6 standard error.
* P < 0.001.
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M. M. Ghorab, F. A. Ragab, H. I. Heiba, and W. M. Ghorab
Vol 48
Figure 2. Survival curve for MCF7 cell line for compound 7 alone or
in combination with c-irradiation (8 Gy). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
Figure 1. Survival curve for MCF7 cell line for compound 6 alone or
in combination with c-irradiation (8 Gy). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
pounds 6, 8, 11, and 17–20 were filtered during mapping
process, so they might act with another mechanism other
than VEGFRTK inhibition. On the other hand, compound
7, 10, 12, and 16 showed a remarkable fit values with a
considerable IC50, so these compounds might be promis-
ing anticancer molecules targeting VEGFRTK.
i.. Two hydrogen bond acceptor (HBA) appeared as a
vector (green spherical mesh).
ii.. Three hydrophobic functions (HY1, HY2, and HY3)
(blue spherical meshes).
Docking studies. Regarding Vatalanib, four clusters
of docked conformations were obtained, the largest
cluster (four poses) was the lowest in mean docked
energy, and the best member (in terms of docking
score) was selected as the docked solution for Vatala-
nib, examination of the docking pose shows that Vata-
lanib forms two hydrogen bonds, one with Lys868
through its chlorine atom in benzene ring and the other
Molecular modeling simulation studies were then con-
ducted by measuring the compare/fit values, separately,
between the conformational models of lead Vatalanib,
compounds 6–20, and ideal selective VEGFRTK inhibi-
tor hypothesis (Fig. 5). The results of the best fitting val-
ues as well as the conformational energy of the best-fitted
conformer with this hypothesis are given in Table 3. The
results of simulation studies have revealed that com-
Figure 3. Structures of selective VEGFRTK inhibitory lead compounds I–IV used for generation of common feature VEGFRTK inhibitor hypothesis.
Journal of Heterocyclic Chemistry
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November 2011
Design and Synthesis of Some Novel Quinoline Derivatives as Anticancer
and Radiosensitizing Agents Targeting VEGFR Tyrosine Kinase
1275
are quinoline derivatives 6 and the diacetylated form 7.
Additionally, compound 8 is nearly as active as doxoru-
bicin. Because it was reported that quinoline derivatives
may exhibit potent VEGFRTK inhibition activity, which
is considered to be an interesting target for the design of
anticancer agents, the results obtained from the anti-
cancer screening may give a suggestion that some of the
synthesized compounds may act as VEGFRTK inhibi-
tors and this may contribute in part to their anticancer
activity. Docking studies revealed that compound 7
showed binding mode similar to Vatalanib, which was
reported to have VEGFRTK inhibitory effects. More-
over, the most two active compounds 6 and 7 showed
the ability to sensitize cancer cells to the lethal effects
of ionizing radiation.
Figure 4. Generated hypothesis for VEGFRTK inhibitors with five
features [three HY (pale blue) and two HBA (green)] with constraint
distances and torsion angel of VEGFRTK hypothesis. [Color figure
can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
EXPERIMENTAL
Melting points are (ꢁC, uncorrected) and were determined
on Buchi melting point apparatus (B-540). Elemental analyses
(C, H, and N) were performed on Perkin-Elmer 2400 analy-
ser (Perkin-Elmer, Norwalk, CT) at Microanalytical Laborato-
ries of the Faculty of Science, Cairo University. All
compounds were within 60.4% of the theoretical values. The
IR spectra were measured on Nicolet 380 FTIR spectrometer.
1H-NMR spectra were obtained on a Bruker proton NMR-
Avance 300 (300, MHZ), in dimethyl sulfoxide (DMSO)-d6as
a solvent, using tetramethylsilane (TMS) as internal standard.
Splitting patterns were designated as follows: s: singlet; d:
doublet; t: triplet; m: multiplet. Mass spectra were run on HP
Model MS-5988 (Hewlett Packard).
3-(4-Bromophenylamino)-5,5-dimethylcyclohex-2-enone 3. A
mixture of 5,5-dimethylcyclohexane-1,3-dione 1 (1.4 g, 0.01
mol) and 4-bromoaniline 2 (1.7 g, 0.01 mol) in ethanol (20
mL) was refluxed for 5 h. The reaction mixture was cooled
then poured onto cold water. The obtained solid was separated
and crystallized from dioxane to give 3. Yield, 79%; m.p.
128–130ꢁC; IR (KBr, cmꢀ1): 3203 (NH), 3099 (CH arom.),
2978, 2838 (CH aliph.), 1656 (C¼¼O). Anal. Calcd. for
with Arg1032 through its nitrogen atom in phthalazine
˚
ring (Fig. 6). Using the RMSD tolerance of 1 A, com-
pound 7 gave 10 distinct clusters. The best pose of
compound 7 showed good mapping to the docked pose
of Vatalanib, its docking pose shows that formation of
a hydrogen bond with Lys868 through its cyclohexa-
none oxygen atom (Fig. 7).
CONCLUSIONS
As breast cells are known to overexpress VEGFR,
which leads to continuous activation of the VEGFR
pathway involved in cell proliferation; therefore, we
measured antitumor activity of the compounds in vitro
on human breast carcinoma cell line (MCF-7). Two of
the tested compounds exhibited potent inhibitory activity
against MCF-7 cell line compared with other tested
compounds and doxorubicin as a reference drug which
Figure 5. Mapping of VEGFRTK inhibitor hypothesis with Vatalanib and compound 7. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
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M. M. Ghorab, F. A. Ragab, H. I. Heiba, and W. M. Ghorab
Vol 48
Table 3
Fit and energy values of some newly synthesized compounds
in a comparison with Vatalanib.
Compound
Absolute energy (k/calꢀ1
)
Fit value
7
9
10
12
16
124.189
121.354
112.509
101.164
80.02
3.073
3.31
3.421
3.50
3.555
3.925
Vatalanib
74.243
C14H16BrNO: C, 57.16; H, 5.48; N, 4.76. Found: C, 57.50; H,
5.10; N, 4.40.
2-Amino-1-(4-bromophenyl)-7,7-dimethyl-5-oxo-4-p-tolyl-
1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile 6. A mixture
of compound 3 (2.94 g, 0.01 mol) and 2-(4-methylbenzy-
lidene)malononitrile 4 (1.68 g, 0.01 mol) in ethanol (20 mL)
containing three drops of triethylamine was refluxed for 6 h.
The reaction mixture was filtered, whereas hot and the solid
product was recrystallized from dioxane to give 6. Yield, 75%;
m.p. 262–264ꢁC; IR (KBr, cmꢀ1): 3311, 3203 (NH2), 3099
(CH arom.), 2978, 2838 (CH aliph.), 2179 (CBN), 1656
Figure 7. The proposed binding mode of compound 7 inside the active
site of VEGFRT kinase resulting from docking. The most important
amino acids is shown together with their respective numbers. Com-
pound 7 forms one hydrogen bond with Lys868 through its oxygen
atom in cyclohexanone ring. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
1
(C¼¼O). H-NMR (DMSO-d6) d: 0.6, 0.9 [2s, 6H, 2CH3], 1.9–
2.2 [m, 4H, 2CH2], 2.34 [s, 3H, CH3 tolyl], 4.6 [s, 1H, CH],
5.4 [s, 2H, NH2, D2O exchangeable], 7.1–8.0 [m, 8H, ArAH].
MS, m/z (%): 461 [Mþ] (20.36), 372 (100). Anal. Calcd. for
C25H24BrN3O: C, 64.94; H, 5.23; N, 9.09. Found: C, 64.71; H,
5.10; N, 9.20.
N-acetyl-N-(1-(4-bromophenyl)-3-cyano-7,7-dimethyl-5-
oxo-4-p-tolyl-1,4,5,6,7,8-hexahydroquinolin-2-yl)acetamide 7. A
solution of compound 6 (4.61 g, 0.01 mol) in acetic anhydride
(30 mL) was refluxed for 10 h. The reaction mixture was then
concentrated and the solid separated was recrystallized from
ethanol to give 7. Yield, 36 %; m.p. 120–122ꢁC; IR (KBr,
cmꢀ1): 3090 (CH arom.), 2928, 2880 (CH aliph.), 2208
(CBN), 1700, 1658 (2C¼¼O). MS, m/z (%): 545 [Mþ] (2.04),
78 (100). 1H-NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3],
1.8–2.2 [m, 4H, 2CH2], 2.3 [s, 3H, CH3 tolyl], 2.4 [s, 6H,
2COCH3], 4.6 [s, 1H, CH], 7.0–7.7 [m, 8H, ArAH]. Anal.
Calcd. for C29H28BrN3O3: C, 63.74; H, 5.23; N, 7.69. Found:
C, 63.40; H, 5.10; N, 7.49.
2-Amino-1-(4-bromophenyl)-7,7-dimethyl-5-oxo-4-p-tolyl-
1,4,5,6,7,8-hexahydroquinoline-3-carboxamide 8. A solution
of compound 6 (4.61 g, 0.01 mol) in concentrated H2SO4 (10
mL) was stirred for 6 h at room temperature. The reaction
mixture was then poured onto ice cold water. The obtained
solid was recrystallized from ethanol to give 8. Yield, 66 %;
m.p. 172–174ꢁC; IR (KBr, cmꢀ1): 3385, 3182 (NH2), 3052
1
(CH arom.), 2937, 2850 (CH aliph.), 1678, 1648 (2C¼¼O). H-
NMR (DMSO-d6) d: 0.6, 0.9 [2s, 6H, 2CH3], 1.8–2.2 [m, 4H,
2CH2], 2.3 [s, 3H, CH3 tolyl], 4.6 [s, 1H, CH], 4.8 [s, 2H,
NH2, D2O exchangeable], 6.2 [s, 2H, CONH2], 7.0–8.0 [m,
8H, ArAH]. Anal. Calcd. for C25H26BrN3O2: C, 62.50; H,
5.46; N, 8.75. Found: C, 62.15; H, 5.89; N, 8.54.
(E)-ethyl N-1-(4-bromophenyl)-3-cyano-7,7-dimethyl-5-oxo-
4-p-tolyl-1,4,5,6,7,8-hexahydroquinolin-2-ylformimidate 9. A
solution of compound 6 (4.61 g, 0.01 mol) in triethylorthofor-
mate (10 mL) was refluxed for 8 h. The reaction mixture was
cooled and then poured onto ice water. The solid precipitate
Figure 6. The proposed binding mode of Vatalanib inside the active
site of VEGFRT kinase resulting from docking. The most important
amino acids are shown together with their respective numbers. Vatala-
nib forms two hydrogen bonds, one with Arg1032 through its nitrogen
(HB acceptor) in phthalazine moiety and the other with Lys868
through its chlorine atom in benzene ring. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

November 2011
Design and Synthesis of Some Novel Quinoline Derivatives as Anticancer
and Radiosensitizing Agents Targeting VEGFR Tyrosine Kinase
1277
was filtered and recrystallized from ethanol to give 9. Yield,
(NH), 3080 (CH arom.), 2956, 2876 (CH aliph.), 1707, 1657
(2C¼¼O). MS, m/z (%): 645 [Mþ] (2.08), 183 (100). Anal.
Calcd. for C32H29Br2N3O2: C, 59.37; H, 4.52; N, 6.49. Found:
C, 59.66; H, 4.99; N, 6.89.
2-(3-Bromophenyl)-10-(4-bromophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 17. Yield, 74%; m.p. 88–90ꢁC; IR (KBr, cmꢀ1): 3390
(NH), 3080 (CH arom.), 2956, 2876 (CH aliph.), 1707, 1657
(2C¼¼O). MS, m/z (%): 645 [Mþ] (4.46),) 44 (100). Anal.
Calcd. for C32H29Br2N3O2: C, 59.37; H, 4.52; N, 6.49. Found:
C, 59.61; H, 4.92; N, 6.93.
10-(4-Bromophenyl)-2-(2-chlorophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 18. Yield, 74%; m.p. 72–74ꢁC; IR (KBr, cmꢀ1): 3390
(NH), 3080 (CH arom.), 2956, 2876 (CH aliph.), 1707, 1657
(2C¼¼O). MS, m/z (%): 603 [Mþ] (10.68), 43 (100). Anal.
Calcd. for C32H29BrClN3O2: C, 63.74; H, 4.85; N, 6.97.
Found: C, 63.22; H, 4.35; N, 6.50.
10-(4-Bromophenyl)-2-(4-chlorophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 19. Yield, 41%; m.p. 58–60ꢁC; IR (KBr, cmꢀ1): 3390
(NH), 3080 (CH arom.), 2956, 2876 (CH aliph.), 1707, 1657
(2C¼¼O). 1H-NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3],
1.9–2.0 [m, 4H, 2CH2], 2.3 [s, 3H, CH3 tolyl], 4.5 [s, 1H,
CH], 4.9 [d, 1H, NH pyrimidine], 6.1 [s, 1H, CH pyrimidine],
7.1–7.9 [m, 8H, ArAH], 8.0 [d, 1H, NHCO]. MS, m/z (%):
600 [Mþ] (1.4), 55 (100). Anal. Calcd. for C32H29BrClN3O2:
C, 63.74; H, 4.85; N, 6.97. Found: C, 63.29; H, 4.40; N, 6.53.
10-(4-Bromophenyl)-2-(4-fluorophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 20. Yield, 64%; m.p. 100–104ꢁC; IR (KBr, cmꢀ1): 3390
(NH), 3080 (CH arom.), 2956, 2876 (CH aliph.), 1707, 1657
(2C¼¼O). MS, m/z (%): 589 [Mþ] (8.4), 43 (100). Anal. Calcd.
for C32H29BrFN3O2: C, 65.53; H, 4.98; N, 7.16. Found: C,
65.53; H, 4.43; N, 7.56.
In vitro anticancer screening. Human tumor breast cell
line (MCF7) was used in this study. The cytotoxic activity was
measured in vitro for the newly synthesized compounds using
the Sulfo-Rhodamine-B stain (SRB) assay using the method of
Skehan et al. [28]. The in vitro anticancer screening was done
by the pharmacology unit at the National Cancer Institute,
Cairo University.
Cells were plated in 96-multiwell microtiter plate (104cells/
well) for 24 h before treatment with the compound(s) to allow
attachment of cell to the wall of the plate. Test compounds
were dissolved in DMSO and diluted with saline to the appro-
priate volume. Different concentrations of the compound under
test (0, 10, 25, 50, and 100 lM/mL) were added to the cell
monolayer. Triplicate wells were prepared for each individual
dose. Monolayer cells were incubated with the compound(s)
for 48 h at 37ꢁC and in humidified incubator with 5% CO2.
After 48 h, cells were fixed, washed, and stained for 30 min
with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. Excess
unbounded dye was removed by four washes with 1% acetic
acid, and attached stain was recovered with Tris ethylene dia-
mine tetra-acetic acid (EDTA) buffer. Color intensity was
measured in an enzyme-linked immunosorbent assay (ELISA)
reader. The relation between surviving fraction and drug con-
centration is plotted to get the survival curve for human breast
tumor cell line after the specified time. The molar concentra-
tion required for 50% inhibition of cell viability (IC50) was
81%; m.p. 190–192ꢁC; IR (KBr, cmꢀ1): 3052 (CH arom.),
1
2959, 2890 (CH aliph.), 2208 (CBN), 1632 (C¼¼O). H-NMR
(DMSO-d6) d: 0.6, 0.9 [2s, 6H, 2CH3], 1.1 [t, 3H, CH3 ethyl],
1.9–2.2 [m, 4H, 2CH2], 2.3 [s, 3H, CH3 tolyl], 3.9 [q, 2H,
CH2 ethyl], 4.6 [s, 1H, CH], 7.1–7.9 [m, 8H, ArAH], 8.1 [s,
1H, N¼¼CH]. Anal. Calcd. for C28H28BrN3O2: C, 64.87; H,
5.44; N, 8.11. Found: C, 64.54; H, 5.13; N, 8.50.
1-(4-Bromophenyl)-2-(2,5-dioxopyrrolidin-1-yl)-7,7-dimethyl-
5-oxo-4-p-tolyl-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile 10. A
mixture of compound 6 (4.61 g, 0.01 mol) and succinic anhy-
dride (1 g, 0.01 mol) was fused together in an oil bath at
250ꢁC for 15 min, athe fused mass was dissolved in dimethyl-
formamide and poured onto ice cold water, and the solid
obtained was crystallized from ethanol to give 10. Yield, 47%;
m.p. 144–146ꢁC; IR (KBr, cmꢀ1): 3085 (CH arom.), 2960,
2880 (CH aliph.), 2213 (CBN), 1794, 1733, 1668 (3C¼¼O).
1H-NMR (DMSO-d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.9–2.2 [m,
4H, 2CH2], 2.3 [s, 3H, CH3 tolyl], 4.8 [s, 1H, CH], 5.2 [t, 4H,
2CH2 pyrrolidine, J ¼ 6.2 Hz], 7.0–8.0 [m, 8H, ArAH]. MS,
m/z (%): 545 [Mþ] (8.09), 77 (100). Anal. Calcd. for
C27H26BrN3O3: C, 63.98; H, 4.81; N, 7.72. Found: C, 63.78;
H, 4.61; N, 7.52.
2-Amino-1-(4-bromophenyl)-3-(4,5-dihydro-1H-imidazol-
2-yl)-7,7-dimethyl-4-p-tolyl-4,6,7,8-tetrahydroquinolin-5(1H)-
one 11. A solution of compound 6 (4.61 g, 0.01 mol) and eth-
ylenediamine (7 mL) was refluxed in carbon disulfide (10 mL)
for 6 h. The reaction mixture was cooled and then poured onto
ice cold water. The solid obtained was crystallized from diox-
ane to give 11. Yield, 55%; m.p. 108–110ꢁC; IR (KBr, cmꢀ1),
3271, 3225 (NH, NH2), 3033 (CH arom.), 2919, 2850 (CH
1
aliph.), 1675 (C¼¼O). H-NMR (DMSO-d6) d: 0.9, 1.0 [2s, 6H,
2CH3], 1.9–2.2 [m, 4H, 2CH2], 2.3 [s, 3H, CH3 tolyl], 4.5 [s,
1H, CH], 5.6 [s, 2H, NH2, D2O exchangeable], 6.4–6.6 [m,
4H, 2CH2 imidazole], 7.1–8.0 [m, 8H, ArAH]. MS, m/z (%):
504 [Mþ] (6.34), 86 (100). Anal. Calcd. for C27H29BrN4O: C,
64.94; H, 5.16; N, 11.08. Found: C, 64.71; H, 5.46; N, 11.30.
N-(1-(4-bromophenyl)-3-cyano-7,7-dimethyl-5-oxo-4-p-tolyl-
1,4,5,6,7,8-hexahydroquinolin-2-yl)-3-oxobutanamide 12. A
solution of compound 6 (4.61 g, 0.01 mol) in ethyl acetoace-
tate (10 mL) was refluxed for 5 h. The reaction mixture was
then concentrated, the solid separated was crystallized from
ethanol to give 12. Yield, 91%; m.p. 47–49ꢁC; IR (KBr,
cmꢀ1): 3204 (NH), 3080 (CH arom.), 2959, 2833 (CH aliph.),
2210 (CBN), 1840, 1728, 1662 (3C¼¼O). 1H-NMR (DMSO-
d6) d: 0.7, 0.9 [2s, 6H, 2CH3], 1.9–2.1 [m, 4H, 2CH2], 2.3 [s,
3H, CH3 tolyl], 2.4 [s, 3H, COCH3], 4.5 [s, 1H, CH], 4.8 [s,
2H, COCH2], 7.1–7.9 [m, 8H, ArAH], 10.1 [s, 1H, NH]. Anal.
Calcd. for C29H28BrN3O3: C, 63.74; H, 5.16; N, 7.69. Found:
C, 63.90; H, 5.45; N, 7.39.
2-(Substituted)-10-(4-bromophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 16–20. A mixture of 6 (4.61 g, 0.01 mol) and aromatic
benzaldehydes (0.01 mol) in acetic acid (20 mL) was refluxed
for 5 h. The reaction mixture was cooled then poured onto ice
cold water where a precipitate was formed. The precipitate
was filtered and crystallized from dioxane to give 16–20,
respectively.
2-(2-Bromophenyl)-10-(4-bromophenyl)-8,8-dimethyl-5-p-tolyl-
2,3,7,8,9,10-hexahydropyrimido[4,5-b]quinoline-4,6(1H,5H)-
dione 16. Yield, 74%; m.p. 100–102ꢁC; IR (KBr, cmꢀ1): 3390
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1278
M. M. Ghorab, F. A. Ragab, H. I. Heiba, and W. M. Ghorab
Vol 48
calculated and compared to the reference drug doxorubicin
(CAS, 25316-40-9). The surviving fractions were expressed as
as Sybyl mol2 file, which were exported to Discovery
Studio tools. Gasteiger charges were calculated for each
molecule and the torsional degrees of freedom were set
to the maximum number of rotatable bonds. The recep-
tor structure was downloaded from the protein databank
(pdb) [www.rcsb.org], pdb entry 1VR2. Water mole-
cules were deleted from the file and hydrogens were
added, then Kollman charges were calculated. No fur-
ther geometry optimization was performed. Nonpolar
hydrogens were merged with the parent atoms for both
the ligands and the receptor. The docking itself was per-
formed using the Lamarckian Genetic Algorithm, with a
means 6 standard error and the results are given in Table 1.
Radiosensitizing evaluation. The most potent compounds
resulted from the in vitro anticancer screening; the quinoline
derivative 6 and the diacetyl form of quinoline derivative 7
were selected to be evaluated again for their in vitro anticancer
activity alone and in combination with c-radiation. This study
was conducted to evaluate the ability of these compounds to
enhance the cell killing effect of c-radiation.
Cells were subjected to a single dose of c-radiation at a
dose level of 8 Gy with a dose rate of 2 Gy/min. Irradiation
was performed in the National Cancer Institute, Cairo Univer-
sity, using Gamma cell-40 (60Co) source.
˚
translational increment of 0.5 A, and both quaternion
The surviving fractions were expressed as means 6 standard
error. The results were analyzed using 1-way ANOVA test and
given in Table 2.
˚
and torsional angles were incremented by 15 A each.
The generation size was set to 150 individuals per gen-
eration, and the rest of the docking parameters were
kept to the default values. Ten runs of the genetic algo-
rithm were performed, thus for each compound, we got
10 docked poses, which were subsequently clustered rel-
ative to the original MMFF structure using root mean
COMPUTATIONAL DETAILS
Catalyst molecular modeling experiments. All mo-
lecular modeling work was performed on Silicon Graphic
(SGI), Fuel workstation (500 MHz, R 14000 ATM proc-
essor, 512 MB memory) using the catalyst package of
Molecular Simulation (version 4.8), under an IRIX 6.8
operating system, at the Faculty of Pharmacy, Ain Shams
University. A generalized visualizer, confirm, info,
HipHop, compare/fit, force field was used throughout.
Training sets, lead compounds I–IV, 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 molꢀ1 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 commands.
All needed features for VEGFRTK inhibitors were
selected from the Dictionary List: HB acceptor, hydro-
phobic (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 examined which
showed that 10 hypotheses were generated. 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.
˚
square deviation (RMSD) tolerance of 1.0 A. The poses
were evaluated using the default empirical scoring func-
tion in Discovery Studio.
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