Molecular modelling and synthesis of quinazoline-based compounds as potential antiproliferative agents

Article abstract of DOI:10.1248/cpb.c14-00016

In this study, four series of 4-anilinoquinazoline derivatives were designed and synthesized as potential anti-proliferative agents. Mechanism of anticancer activity was explained through molecular docking of the target compounds into epidermal growth factor receptor tyrosine kinase (EGFR-TK) active site which displayed comparable binding mode of certain compounds to that of lapatinib. Moreover, the newly synthesized compounds were tested for their anti-proliferative activity on breast carcinoma cell line (MCF-7). 6-(4-Ben-zylpiperazin-1-ylsulfonyl)-4-(4-bromoanilino)quinazoline (14g) exhibited the most potent inhibitory activity (IC₅₀=5.52 μM).

Full text of DOI:10.1248/cpb.c14-00016

454
Regular Article
Chem. Pharm. Bull. 62(5) 454–466 (2014)
Vol. 62, No. 5
Molecular Modelling and Synthesis of Quinazoline-Based Compounds as
Potential Antiproliferative Agents
Asmaa Said Ali Yassen,a,† Hosam Eldin Abd Elhamed Ahmed Elshihawy,a
,b
Mohamed Mokhtar Amin Said,a and Khaled Abouzid Mohamed Abouzid*
a Organic Chemistry Department, Faculty of Pharmacy, Suez Canal University; Ismailia 41522, Egypt: and
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University; Cairo 11566, Egypt.
Received January 14, 2014; accepted January 31, 2014
In this study, four series of 4-anilinoquinazoline derivatives were designed and synthesized as potential
anti-proliferative agents. Mechanism of anticancer activity was explained through molecular docking of the
target compounds into epidermal growth factor receptor tyrosine kinase (EGFR-TK) active site which dis-
played comparable binding mode of certain compounds to that of lapatinib. Moreover, the newly synthesized
compounds were tested for their anti-proliferative activity on breast carcinoma cell line (MCF-7). 6-(4-Ben-
zylpiperazin-1-ylsulfonyl)-4-(4-bromoanilino)quinazoline (14g) exhibited the most potent inhibitory activity
(IC50=5.52µM).
Key words quinazoline; tyrosine kinase; anti-proliferative agent
Although development of novel targeted anticancer drugs anticancer drugs such as gefitinib, erlotinib and lapatinib.
have shown a great progress in recent years, cancer remains These lead compounds are selective EGFR inhibitors ap-
the major leading cause of death in the world.1) Receptor proved by the Food and Drug Administration (FDA) for can-
protein tyrosine kinases (RPTKs) play a vital role in signal cer therapy6) (Fig. 1).
transduction pathways that regulate cell division and dif-
Structure–activity relationship (SAR) studies for the ability
ferentiation by binding of the growth factors, epidermal of 4-anilinoquinazoline to inhibit EGFR-TKs activity revealed
growth factor (EGF), platelet-derived growth factor (PDGF), that both of the quinazoline nitrogen atoms were absolutely
and vascular endothelial growth factor (VEGF). Among the essential for activity. The phenyl ring with small lipophilic
known receptor tyrosine kinases that have been identified as substituents such as chloro, bromo and trifluoromethyl was
being important in cancer is epidermal growth factor recep- also important as it occupies the lipophilic pocket. The nature
tor (EGFR).2) It is a transmembrane receptor belonging to of the linking group between the quinazoline ring and phenyl
the human epidermal growth factor receptor (HER) family side chain has a great effect on the inhibitory activity. Upon
of receptors. To date four members of this family have been replacement of nitrogen linker by oxygen or sulfur causes
identified including EGFR (HER1/erbB-1), HER2 (erbB-2/ the reduction in the activity of EGFR but it causes increase
neu), HER3 (erbB-3) and HER4 (erbB-4).2) The EGFR and in the activity of VEGFR especially in the compounds which
its family members are composed of an extracellular ligand- possess urea or thiourea moiety on the phenyl side chain.7)
binding domain, a transmembrane domain and an intracellular Finally, substitution on the quinazoline ring at positions 6 or
domain. When a ligand, such as EGF, binds to the ligand- 7 was confined to substituents preferably with polar groups.8)
binding domain, the EGFR forms a homodimer or hetero-
Recently, a large number of anticancer agents with diverse
dimer with other closely related receptors. Then, the intrinsic structures have been developed and evaluated. (E)-N-
kinase domain is activated, resulting in autophosphorylation (4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(2-nitrophenyl)-
and transphosphorylation of the receptors through their ty- acrylamide (a) demonstrated the most potent inhibitory activ-
rosine kinase domains leading to the activation of multiple ity, which could be optimized as a potential EGFR inhibi-
downstream pathways such as protein synthesis, cell growth, tor.9) Moreover, 4,6-substituted(diaphenylamino)quinazolines
survival, differentiation and apoptosis.3) Given this pivotal reported by Lu et al. displayed good antiproliferative and
role, it is not surprising that EGFR has been implicated in sev- EGFR-TK inhibitory activities particularly, 4-(4-(3-bromo-
eral human disorder including solid tumors.4) Overexpression phenylamino)quinazolin-6-ylamino)methyl)phenol (b).10) Fur-
and/or coexpression of EGFR has been found in numerous thermore, 4-anilino-6-substituted-quinazoline derivatives were
cancer types, including colon, breast, ovarian, head and neck, evaluated for EGFR-TK and tumor growth inhibitory activi-
and non-small cell lung cancers. Therefore, selective blockade ties. Compound 4-(4-chlorophenylamino-6-uredioquinazoline
of EGFR has been shown to be an effective therapeutic ap- (c) showed the most inhibitory activity. All of them, according
proach against cancers.5)
to the previous SAR, were directed toward the addition of a
Many compounds with different scaffolds were reported polar moiety at position-6 and small lipophilic groups on the
as epidermal growth factor receptor tyrosine kinase inhibi- aniline moiety11) (Fig. 2).
tors (EGFR-TKIs). Among them 4-anilinoquinazoline was the
With the aim of finding new structures which may serve
most attractive scaffold to be modified for yielding effective as a potential chemotherapeutic agents, in our approach, we
modified the molecular structure of recently reported com-
pounds in two main sites: one was 4-anilino moiety by sub-
stituting the small lipophilic group with other large functional
groups of different lipophilicity and/or steric hindrance on the
The authors declare no conflict of interest.
†Present address: 202 Nagayama Building, 5–64 Shimizu-machi, Nagasaki
852–8041, Japan.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: hosam_sh_27@hotmail.com;
abouzid@yahoo.com

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Fig. 1. Examples of EGFR Tyrosine Kinase Inhibitors
They were approved by FDA. As shown all of them possess the common structure: 4-anilinoquinazoline moiety.
Fig. 2. Recently Reported Anticancer Agents with Novel Structures
All of them were directed toward the addition of electron donating group at position-6 and small lipophilic groups on the aniline moiety.
basis of retaining the bulky group of the structure of lapatinib amino acids were neutralized at pH 7 2 by protonation of the
(Chart 1) and the other was the position-6 by incorporating terminal amino groups of basic amino acids and the terminal
sulphonyl polar group (Chart 2, Fig. 3).
carboxylic acid groups of acidic amino acids were deproton-
ated. The docking process involved the standard precision
docking (SP) in which ligand poses that scored bad energies
Experimental
Molecular Modeling Procedure Molecular modeling would be rejected. Partial charge cutoff was set to 0.25 for the
was carried out on Schrodinger computational software receptor and 0.15 for the ligand to soften the potential of non-
workstation using Maestro 9.3 graphic user interface (GUI). polar parts. Flexible docking was performed with no torsional
The atomic coordinates and the cartesian matrix of the poly- constraints was applied. Twenty percent of the final poses
peptide were obtained from the protein bank database PDB produced from the SP docking were subjected to the Extra
(Brookhaven protein database .2J5F). The basic and acidic Precision mode of Glide docking (XP) to perform strict and

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Vol. 62, No. 5
Reagents and conditions: (i) Reflux, 5–6h. (ii) POCl3, dry DMF, TEA, reflux, 7h. (iii) p-NH2PhNH2, TEA, rt 24h. (iv) PhNCO, C5H5N, THF, rt, 16h. (v) PhNCS,
C5H5N, THF, heat, 40°C, 2h. (vi) Glyoxalic acid, dry EtOH, rt, 5h. (vii) Acrylic acid, AcOH, stir at rt, 2h. (viii) Succinic anhydride, dioxane, reflux, 5h.
The starting material anthranilic acid (1) was conveniently cyclized to quinazolin-4(3H)-one (3) by heating it with formamide (2) at 120°C. Upon refluxing compound
(3) with freshly distilled phosphorous oxychloride, the corresponding 4-chloroquinazoline (4) was obtained. 4-Chloroquinazoline was allowed to react with p-pheneylene
diamine to produce 4-(4-aminoanilino)quinazoline (5). The key intermediate 4-(4-aminoanilino)quinazoline was readily converted to the title compounds (6), (7) series A,
(8)–(10) series B via the reaction with phenyl isocyanate, phenyl isothiocyanate, glyoxalic acid, acrylic acid and succinic anhydride, respectively.
Chart 1. The Reaction Sequence for the Synthesis of the Title Compounds (1–6)
more precise docking simulation. Aromatic rings were forbid- ppm on δ scale, Microanalytical Center, Cairo University,
den to flip and amide bonds of the polypeptide segments with Egypt. Mass spectra were recorded on a GCMP-QP1000 EX
non-polar amide bonds were penalized.12)
Mass spectrometer, Microanalytical Center, Cairo University,
Experimental Procedure All chemicals were obtained Egypt. Elemental analyses were carried out at the Microana-
from Aldrich, Fluka and Merck chemicals. Melting points lytical Center, Suez Canal University, Egypt. Reactions were
were determined on an electrothermal apparatus in open cap- monitored by thin layer chromatography (TLC) using different
illary tubes using Stuart melting point apparatus SMP10 and solvent mixtures as mobile phase and precoated aluminum
the values were uncorrected. IR spectra were determined on sheets silica gel Merck 60 (F254) as stationary phase; the spots
using KBr discs on a Vector 22 Infrared spectrophotometer were visualized by exposure to iodine vapor or UV light
1
(νmax in cm−1), with ratio (1 drug: 3 KBr). H-NMR spec- under 254 and 366nm illumination.
tra were carried out using Varian Mercury-300 (300MHz,
Quinazolin-4(3H)-one (3): A mixture of anthranilic acid 1
DMSO-d6). Spectrophotometer using tetramethylsilane (TMS) (0.7g, 5mmol) and formamide 2 (2mL) was stirred under re-
as internal standard. Chemical shift values are recorded in flux (120–140°C) for 5–6h. The mixture was allowed to cool

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Reagents and Conditions: (i) ClSO3H, heat, stir at 60°C, 7d. (ii) R2NH, TEA, reflux, 10h. (iii) POCl3, dry DMF, TEA, reflux, 7h. (iv) 4-Bromoaniline, TEA, stirring,
reflux, 13–24h. (v) Aniline, TEA, stirring, reflux, 3–7h.
Compound (3) reacted with freshly distilled chlorosulfonic acid under dry conditions to give 6-chlorosulfonylquinazolin-4(3H)-one (11). Compound (11) was allowed
to react with piperdine, cyclohexyl amine and substituted piperazines to produce 6-(N-substituted)sulfonylquinazolin-4(3H)-one (12a–g). Subsequently, the reaction of
compounds (12a–g) with freshly distilled phosphorous oxychloride to give 4-chloro derivatives (13a–g). Finally, compounds (13a–g) underwent a nucleophilic substitution
reaction with selected substituted anilines to give the title compounds (14b–d,g) series C and (15a–g) series D.
Chart 2. The Reaction Sequence for the Synthesis of the Title Compounds (14b, c, d,g, and 15a–g)
to 100°C. Water (20mL) was added dropwise to the mixture. The mixture was allowed to reach the room temperature and
The resulting precipitate was filtered, recrystallized from an- left stirring overnight. The formed precipitate was filtered and
hydrous ethanol to afford 3 as a white solid.13) Yield 64%, mp recrystallized from chloroform to give 4-(4-aminoanilino)-
220–222°C, spectral data as reported.14)
4-Chloroquinazoline (4):
4(3H)-one 3 (0.4g, 2.5mmol) in POCl3 (25mL) and N,N-
quinazoline 5 as yellow needle crystals.15) Yield 66%, mp
A
mixture of quinazolin- >300°C, spectral as reported.16)
1-Phenyl-3-(4-(quinazolin-4-ylamino)phenyl)urea (6): Phe-
dimethylforamide (DMF) (0.12mL) was stirred under reflux nyl isocyanate (0.15mL, 1.26mmol) was added dropwise
(100–105°C) for 5–6h. The excess POCl3 was removed by to a mixture of 4-(4-aminoanilino)quinazoline 5 (0.15g,
vacuo. The residue was poured into a mixture of chloroform 0.63mmol) and pyridine (0.16g, 1.89mmol) in THF. The mix-
(50mL), ice water (80mL) and triethylamine (5mL) then ture was stirred for 16h. The resulting precipitate was filtered
neutralized with saturated NaHCO3 solution. The chloroform and recrystallized from methanol to afford a greenish white
layer was separated, dried over Na2SO4 and filtered. The crystalline powder of 1-phenyl-3-(4-(quinazolin-4-ylamino)-
1
solvent was removed by distillation to give yellow solid of phenyl)urea 6. Yield 80%, mp >300°C. H-NMR (DMSO-d6)
4-chloroquinazoline 4. The resulting compound was stored δ: 12.57 (1H, s, exch with D2O), 12.50 (1H, s, exch with D2O),
at 0°C without a further purification.13) Yield 62.5%, mp 9.18 (1H, s, exch with D2O), 7.45–6.92 (14H, m). IR (KBr)
100–102°C, spectral data as reported.14)
cm−1: 3298, 1637, 1561, 1505. MS m/z: 355 (M+), 212, 125, 111,
4-(4-Aminoanilino)quinazoline (5): A solution of 4-chloro- 93. Anal. Calcd for C21H17N5O: C, 70.97; H, 4.82; N, 19.71.
quinazoline 4 (0.16g, 0.95mmol) in chloroform (10mL) was Found: C, 70.63; H, 4.54; N, 19.50.
cooled in an ice-bath for 30min. A solution of p-pheneylene-
1-Phenyl-3-(4-(quinazolin-4-ylamino)phenyl)thiourea
(7):
diamine (0.1g, 0.95mmol), triethylamine (1mL) in chloroform Phenyl isothiocyanate (0.17mL, 1.26mmol) was added drop-
was added dropwise to the reaction mixture in about 15min. wise to a mixture of 4-(4-aminoanilino)quinazoline 5 (0.15g,

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Vol. 62, No. 5
Fig. 3. Design for Lapatinib Congeners
Replacement of bulky side chain at position-4 of the aniline ring with urea, thiourea, glycine, β-alanine and succinamic acid. Replacement of electron donating group at
position-6 with N-substituted sulfonyl derivatives of cyclohexyl amine, piperdine and substituted piperazines.
0.63mmol) and pyridine (0.16g, 1.89mmol) in dry THF 1613, 1519. MS m/z: 308 (M+), 307, 291, 263, 235, 55. Anal.
(30mL). The mixture was heated to 40°C for 2h. The solvent Calcd for C17H16N4O2: C, 66.22; H, 5.23; N, 18.17. Found: C,
was removed under reduced pressure to give yellow residue of 66.58; H, 5.45; N, 18.55.
1-phenyl-3-(4-(quinazolin-4-ylamino)phenyl)thiourea 7 which
4-Oxo-4-(4-(quinazolin-4-ylamino)phenylamino)butanoic
was purified by column chromatogrphy using 100% hexane Acid (10): 4-(4-Aminoanilino)quinazoline 5 (9.44g, 40mmol)
as eluent to give white crystals. Yield 30%, mp 150–152°C. and succinic anhydride (4g, 40mmol) were refluxed in diox-
1H-NMR (DMSO-d6) δ: 9.63 (1H, s, exch with D2O), 8.60 ane (20mL) for 5h. The mixture was cooled to room tem-
(1H, s, exch with D2O), 8.50 (1H, s, exch with D2O), 8.49–6.61 perature and diethyl ether was added to the mixture. A white
(14H, m). IR (KBr) cm−1: 3281, 1591, 1486, 1307. MS m/z: 371 precipitate was formed, filtered and washed with cold water
(M+), 294, 279, 235, 217, 71. Anal. Calcd for C21H17N5S: C, (3×5mL) and subsequently dissolved in HCl (16mL, 16%
67.90; H, 4.61; N, 18.85. Found: C, 67.54; H, 4.23; N, 18.60.
v/v). After standing at room temperature the crystallized suc-
2-(4-(Quinazolin-4-ylamino)phenylimino)acetic Acid (8): cinic acid was removed by filtration and the pH of the mixture
Glyoxalic acid (1.84g, 20mmol) in dry ethanol (100mL) was was adjusted to 5 by addition of 10% NaOH (12mL). A gray
added to a stirred solution of 4-(4-aminoanilino)quinazoline precipitate was formed, filtered and purified by column chro-
5 (4.72g, 20mmol) in dry ethanol (100mL). The mixture matography using ethanol–ethyl acetate (15:1) to give white
1
was stirred for 5h. The solvent was removed under reduced needle crystals. Yield 50%, mp >300°C. H-NMR (DMSO-d6)
pressure to give brown solid which was purified by column δ: 11.90 (1H, s, exch with D2O), 10.90 (1H, s, exch with D2O),
chromatography using chloroform–methanol (3:1) to give 9.33 (1H, s, exch with D2O), 8.80–6.80 (9H, m), 3.9–2.54 (4H,
1
brown crystals. Yield 37%, mp >300°C. H-NMR (DMSO-d6) m). IR (KBr) cm−1: 3421, 2900, 1596, 1492. MS m/z: 336 (M+),
δ: 10.20 (1H, s, exch with D2O), 9.70 (1H, s, exch with D2O), 277, 235, 221, 189, 86. Anal. Calcd for C18H16N4O3: C, 64.28;
8.45–6.48 (9H, m), 6.5 (1H, s). IR (KBr) cm−1: 3386, 2922, H, 4.790; N, 16.66. Found: C, 64.00; H, 4.46; N, 16.26.
1619. MS m/z: 292 (M+), 291, 279, 247, 234, 97. Anal. Calcd
6-Chlorosulfonylquinazolin-4(3H)-one (11): Quinazolin-
for C16H12N4O2: C, 65.75; H, 4.14; N, 19.17. Found: C, 66.15; H, 4(3H)-one 3 (73g, 0.5mol) was added gradually to cold solu-
4.52; N, 19.50.
tion of chlorosulfonic acid (174mL, 2.49mol) at 12–15°C. The
3-(4-(Quinazolin-4-ylamino)phenylamino)propanoic
Acid temperature was raised to room temperature then the mixture
(9): Acrylic acid (0.3mL, 4.2mmol) and acetic acid (1mL) is heated at 60°C for 7d. The syrupy liquid was poured slowly
were stirred for 1h in an ice bath. 4-(4-Aminoanilino)- with stirring into ice water and was neutralized with satu-
quinazoline 5 (1g, 4.2mmol) was added gradually with rated NaHCO3 solution. The mixture was extracted with ethyl
stirring to the reaction mixture. The mixture was allowed acetate (3×10mL). The ethyl acetate layer was separated and
to reach room temperature then stirring continued for 2h. water was removed under reduced pressure to give a brown
The mixture was evaporated under vaccuo. The residue was solid. The resulting compound 6-chlorosulfonylquinazolin-
washed with cold water (1×5mL) and filtered to give a brown 4(3H)-one 11 was stored at 0°C without further purification.
solid which was purified by column chromatography using Yield 76%, mp >300°C.
ethanol–chloroform (4:1) to give brown crystals. Yield 40%,
General Procedure for the Synthesis of 6-[(N-Substituted)
1
mp 240°C. H-NMR (DMSO-d6) δ: 11.92 (1H, s, exch with sulfonyl]quinazolin-4(3H)-one (12a–g): 6-Chlorosulfonylquin-
D2O) 10.95 (1H, s, exch with D2O), 8.08–6.86 (9H, m), 5.95 azolin-4(3H)-one 11 (0.25g, 1mmol) was added to a stirred
(1H, s), 3.99–2.90 (4H, m). IR (KBr) cm−1: 3500, 2961, 3681, solution of the appropriate amine (1mmol) and triethylamine

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459
(1mL) at room temperature for 1h. The reaction mixture exch with D2O), 7.95–7.92 (1H, d, J=9.3Hz), 7.9–7.7 (8H, m),
was heated under reflux for the appropriate time. The mix- 4.20 (2H, s), 3.06–2.37 (8H, m). IR (KBr) cm−1: 3439, 2929,
ture was allowed to reach room temperature, quenched in ice 1590, 1490, 1355. MS m/z: 384 (M+), 358, 318, 279, 217.
with efficient stirring. The aqueous phase was extracted with
dichloromethane (3×20mL). The dichloromethane layer was substituted sulfonyl)quinazoline (13a–g): POCl3 (25mL)
dried over Na2SO4, filtered and the solvent was removed by was added to mixture of 6-[(N-substituted)sulfonyl]-
distillation to give yellow solid, which was washed with cold quinazolin-4(3H)-one 12a–g (2.5mmol) in DMF (0.12mL).
dichloromethane to give the desired product (12a–g). The mixture was stirred under reflux (100–105°C) for the
General Procedure for the Preparation of 4-Chloro-6-(N-
a
N-Cyclohexyl-4-oxo-3,4-dihydroquinazoline-6-sulfonamide appropriate time. Excess POCl3 was removed by vacuo. The
(12a): 6-Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, residue was poured into a mixture of chloroform (50mL), ice
1mmol) was added to a stirred solution of cyclohexylamine water (80mL) and triethylamine (5mL) and the mixture was
(0.1mL, 1mmol) and triethylamine (1mL). The reaction mix- neutralized with saturated NaHCO3 solution. The chloroform
ture was heated under reflux for 15h. Yield 75% as a white layer was separated and the aqueous layer was extracted with
1
crystals, mp 95–97°C. H-NMR (DMSO-d6) δ: 9.78 (1H, s, chloroform (1×20mL). The chloroform layer was dried over
exch with D2O), 7.95–7.92 (1H, d, J=9.3Hz), 7.70–7.14 (3H, Na2SO4, filtered and the solvent was removed by distillation to
m), 4.60 (1H, s, exch with D2O), 2.6–1 (11H, m). IR (KBr) give the desired compounds 13a–g which were stored at 0°C
cm−1: 3390, 3295, 2931, 1623, 1552, 1327. MS m/z: 307 (M+), and used without any further purification for the next step.
99, 70, 56, 55.
4-Chloro-N-cyclohexylquinazoline-6-sulfonamide
(13a):
6-(Piperidin-1-ylsulfonyl)quinazolin-4(3H)-one (12b): 6- POCl3 (25mL) was added to a mixture of N-cyclohex-
Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, 1mmol) was yl-4-oxo-3,4-dihydroquinazoline-6-sulfonamide 12a (0.77g,
added to a stirred solution of piperidine (0.09mL, 1mmol) and 2.5mmol) in DMF (0.12mL). The mixture was stirred under
triethylamine (1mL). The reaction mixture was heated under reflux for 5h. Yield 80% as a white solid, mp 100–102°C.
reflux for 16h. Yield 86% as a white crystals, mp 257–259°C.
IR (KBr) cm−1: 3389, 2952, 1671, 1592, 1324. MS m/z: 293 (25mL) was added to a mixture of 6-(piperidin-1-ylsulfonyl)-
(M+), 248, 221, 148, 136.
quinazolin-4(3H)-one 12b (0.73g, 2.5mmol) in DMF
4-Chloro-6-(piperidin-1-ylsulfonyl)quinazoline (13b): POCl3
6-(Piperazin-1-ylsulfonyl)quinazolin-4(3H)-one (12c): 6- (0.12mL). The mixture was stirred under reflux for 7h. Yield
Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, 1mmol) was 80% as a white solid, mp 120–122°C.
added to a stirred solution of piperazine in toluene (0.09g,
4-Chloro-6-(piperazin-1-ylsulfonyl)quinazoline (13c): POCl3
1mmol) and triethylamine (1mL). The reaction mixture was (25mL) was added to a mixture of 6-(piperazin-1-ylsulfonyl)-
heated under reflux for 8h. Yield 60% as a yellow crystals, quinazolin-4(3H)-one 12c (0.73g, 2.5mmol) in DMF
mp 115–117°C. IR (KBr) cm−1: 3418, 3208, 2956, 1624, 1556, (0.12mL). The mixture was stirred under reflux for 6h. Yield
1363. MS m/z: 294 (M+), 224, 206, 149, 97.
55% as a yellow solid, mp 150–152°C.
6-(4-Methylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one
4-Chloro-6-(4-methylpiperazin-1-ylsulfonyl)quinazoline
(12d): 6-Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, (13d): POCl3 (25mL) was added to
a
mixture of
1mmol) was added to a stirred solution of methylpiperazine 6-(4-methylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one 12d
(0.1mL, 1mmol) and triethylamine (1mL). The reaction mix- (0.77g, 2.5mmol) in DMF (0.12mL). The mixture was
ture was heated under reflux for 16h. Yield 56% as a white stirred under reflux for 7h. Yield 50% as a yellow solid, mp
crystals, mp 160–162°C. IR (KBr) cm−1: 3408, 2923, 1657, 130–132°C.
1373. MS m/z: 308 (M+), 293, 273, 245, 219, 191.
4-Chloro-6-(4-ethylpiperazin-1-ylsulfonyl)quinazoline
6-(4-Ethylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one (13e): POCl3 (25mL) was added to mixture of
(12e): 6-Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, 6-(4-ethylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one 12e
a
1mmol) was added to a stirred solution of ethyl piperazine (0.8g, 2.5mmol) in DMF (0.12mL). The mixture was
(0.11mL, 1mmol) and triethylamine (1mL). The reaction mix- stirred under reflux for 6h. Yield 50% as a yellow solid, mp
ture was heated under reflux for 16h. Yield 55% as a white 110–112°C.
crystals, mp 130–132°C. IR (KBr) cm−1: 3428, 2855, 1647,
4-Chloro-6-(4-phenylpiperazin-1-ylsulfonyl)quinazoline
(13f): POCl3 (25mL) was added to
mixture of
12f
1375. MS m/z: 323 (M+), 308, 298, 244, 188.
a
6-(4-Phenylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one 6-(4-phenylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one
(12f): 6-Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g, (0.9g, 2.5mmol) in DMF (0.12mL). The mixture was
1mmol) was added to a stirred solution of phenyl piperazine stirred under reflux for 6h. Yield 60% as a white solid, mp
(0.2mL, 1mmol) and triethylamine (1mL). The reaction mix- 120–122°C.
ture was heated under reflux for 11h. Yield 60% as a yellow
6-(4-Benzylpiperazin-1-ylsulfonyl)-4-chloro-quinazo-
1
crystals, mp 200–202°C. H-NMR (DMSO-d6) δ: 9.36 (1H, line (13g): POCl3 (25mL) was added to a mixture of
s, exch with D2O), 7.20–6.79 (9H, m), 3.38–3.16 (8H, m). IR 6-(4-benzylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one 12g
(KBr) cm−1: 3439, 2919, 1663, 1590, 1326. MS m/z: 370 (M+), (0.96g, 2.5mmol) in DMF (0.12mL). The mixture was
344, 314, 284, 242, 76. stirred under reflux for 7h. Yield 70% as a yellow solid, mp
6-(4-Benzylpiperazin-1-ylsulfonyl)quinazolin-4(3H)-one 140–142°C.
(12g): 6-Chlorosulfonylquinazolin-4(3H)-one 11 (0.25g,
General Procedure for the Preparation of 4-(4-
1mmol) was added to a stirred solution of benzyl piperazine Bromoanilino)-6-(N-substituted sulfonyl)]quinazoline (14b–
(0.18mL, 1mmol) and triethylamine (1mL). The reaction mix- d,g): 4-Chloro-6-(N-substitutedsulfonyl)quinazoline 13b–d,g
ture was heated under reflux for 15h. Yield 80% as a white (0.95mmol) in dichloromethane (10mL) was added gradu-
1
crystals, mp 173–175°C. H-NMR (DMSO-d6) δ: 9.7 (1H, s, ally with stirring to a solution of 4-bromoaniline (0.34g,

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Vol. 62, No. 5
1.95mmol) and triethylamine (1mL) in dichloromethane substituted sulfonyl)quinazoline 13a–g (0.95mmol) in di-
(10mL) at 0°C. The mixture was allowed to reach room tem- chloromethane (10mL) was added gradually with stirring to
perature then heated to reflux for appropriate time. The sol- a solution of aniline (0.18mL, 1.95mmol) and triethylamine
vent was removed under reduced pressure and the residue was (1mL) in dichloromethane (10mL) at 0 ᵒC. The mixture was
washed with dichloromethane (2×10mL) and purified with allowed to reach room temperature then heated under reflux
column chromatography using ethyl acetate–petroleum ether for the appropriate time. The solvent was removed under
(3:1) to give the listed compounds 14b–d,g.
reduced pressure and the residue was washed with dichloro-
4-(4-Bromoanilino)-6-(piperidin-1-ylsulfonyl)quinazoline methane (2×10mL) and purified with column chromatography
(14b): 4-Chloro-6-(piperidin-1-ylsulfonyl)quinazoline 13b using the appropriate eluent to give the desired compounds
(0.29g, 0.95mmol) in dichloromethane (10mL) was added 15a–g.
gradually with stirring to a solution of 4-bromoaniline (0.34g,
N-Cyclohexyl-4-(phenylamino)quinazoline-6-sulfonamide
1.95mmol) and triethylamine (1mL) in dichloromethane (15a): 4-Chloro-N-cyclohexylquinazoline-6-sulfonamide 13a
(10mL) at 0°C. The mixture was allowed to reach room tem- (0.3g, 0.95mmol) in dichloromethane (10mL) was added grad-
perature then heated to reflux for 24h. Yield 60% as a white ually with stirring to a solution of aniline (0.18mL, 1.95mmol)
1
needle crystals, mp 140–142°C. H-NMR (DMSO-d6) δ: 9.10 and triethylamine (1mL) in dichloromethane (10mL) at 0°C.
(1H, s, exch with D2O), 7.28–6.62 (8H, m), 3.26–1.54 (10H, The mixture was allowed to reach room temperature then
m). IR (KBr) cm−1: 3472, 2983, 1620, 1356. MS m/z: 447 (M+), heated under reflux for 5h. The residue was purified with
414, 374, 286, 228, 86. Anal. Calcd for C19H19BrN4O2S: C, column chromatography using ethyl acetate–petrolum ether
51.01; H, 4.28; N, 12.52. Found: C, 50.62; H, 3.97; N, 12.29.
(3:1) as eluent. Yield 49% as a white crystals, mp 110–112°C.
4-(4-Bromoanilino)-6-(piperazin-1-ylsulfonyl)quinazoline 1H-NMR (DMSO-d6) δ: 9.36 (1H, s, exch with D2O), 7.95–7.92
(14c): 4-Chloro-6-(piperazin-1-ylsulfonyl)quinazoline 13c (1H, d, J=9.3Hz), 7.80–6.80 (8H, m), 4.50 (1H, s, exch with
(0.29g, 0.95mmol) in dichloromethane (10mL) was added D2O), 2.99–1.11 (11H, m). IR (KBr) cm−1: 3428, 2922, 1642,
gradually with stirring to a solution of 4-bromoaniline (0.34g, 1548, 1375. MS m/z: 382 (M+), 354, 312, 270, 231, 71. Anal.
1.95mmol) and triethylamine (1mL) in dichloromethane Calcd for C20H22N4O2S: C, 62.80; H, 5.80; N, 14.65. Found: C,
(10mL) at 0°C. The mixture was allowed to reach room tem- 63.10; H, 6.05; N, 14.87.
perature then heated to reflux for 13h. Yield 50% as a yellow
4-Anilino-6-(piperidin-1-ylsulfonyl)quinazoline (15b): 4-
1
crystals, mp 180–182°C. H-NMR (DMSO-d6) δ: 9.36 (1H, s, Chloro-6-(piperidin-1-ylsulfonyl)quinazoline
13b (0.29g,
exch with D2O), 7.95–7.92 (1H, d, J=9.3Hz), 7.70–6.96 (7H, 0.95mmol) in dichloromethane (10mL) was added gradually
m), 4.13–1.31 (8H, m), 2.20 (1H, s, exch with D2O). IR (KBr) with stirring to a solution of aniline (0.18mL, 1.95mmol) and
cm−1: 3467, 2923, 1624, 1463, 1178. MS m/z: 447 (M+), 414, triethylamine (1mL) in dichloromethane (10mL) at 0°C. The
379, 336, 289, 57. Anal. Calcd for C18H18BrN5O2S: C, 48.22; H, mixture was allowed to reach room temperature then heated
4.05; N, 15.62. Found: C, 48.34; H, 4.30; N, 15.98.
under reflux for 7h. The residue was purified with column
4-(4-Bromoanilino)-6-(4-methylpiperazin-1-ylsulfonyl)- chromatography using ethyl acetate–petrolum ether (3:1) as
quinazoline (14d): 4-Chloro-6-(4-methylpiperazin-1-ylsulfon- eluent. Yield 55% as a white needle crystals, mp 140–142°C.
yl)quinazoline 13d (0.3g, 0.95mmol) in dichloromethane 1H-NMR (DMSO-d6) δ: 9.36 (1H, s, exch with D2O),
(10mL) was added gradually with stirring to a solution of 7.28–6.82 (9H, m), 3.26–1.54 (10H, m). IR (KBr) cm−1: 3471,
4-bromoaniline (0.34g, 1.95mmol) and triethylamine (1mL) 2984, 1620, 1356. MS m/z: 368 (M+), 315, 280, 235, 138, 121,
in dichloromethane (10mL) at 0°C. The mixture was al- 86. Anal. Calcd for C19H20N4O2S: C, 61.94; H, 5.47; N, 15.21.
lowed to reach room temperature then heated to reflux for Found: C, 61.58; H, 5.21; N, 15.06.
1
19h. Yield 49% as a white crystals, mp 180–182°C. H-NMR
4-Anilino-6-(piperazin-1-ylsulfonyl)quinazoline (15c): 4-
(0.29g,
(DMSO-d6) δ: 9.18 (1H, s, exch with D2O), 7.95–7.92 (1H, d, Chloro-6-(piperazin-1-ylsulfonyl)quinazoline 13c
J=9.3Hz), 7.8–7.4 (1H, dd, J=9.3, 2.4Hz), 7.4–6.6 (6H, m), 0.95mmol) in dichloromethane (10mL) was added gradually
3.3–2.03 (11H, m). IR (KBr) cm−1: 3395, 2923, 1623, 1461, with stirring to a solution of aniline (0.18mL, 1.95mmol) and
1370. MS m/z: 460 (M+), 432, 390, 295, 86. Anal. Calcd for triethylamine (1mL) in dichloromethane (10mL) at 0°C. The
C19H20BrN5O2S: C, 49.36; H, 4.36; N, 15.15. Found: C, 49.68; mixture was allowed to reach room temperature then heated
H, 4.72; N, 15.15.
under reflux for 4h. The residue was purified with column
6-(4-Benzylpiperazin-1-ylsulfonyl)-4-(4-bromoanilino)- chromatography using ethyl acetate–petrolum ether (3:1)
quinazoline (14g): 6-(4-Benzylpiperazin-1-ylsulfonyl)-4-chlo- as eluent. Yield 50% as a yellow crystals, mp 110–112°C.
ro-quinazoline 13g (0.95mmol) in dichloromethane (10mL) 1H-NMR (DMSO-d6) δ: 9.18 (1H, s, exch with D2O), 7.95–7.92
was added gradually with stirring to a solution of 4-bromoani- (1H, d, J=9.3Hz), 8.51–6.81 (8H, m), 4.70–1.31 (8H, m), 2.0
line (0.34g, 1.95mmol) and triethylamine (1mL) in dichloro- (1H, s, exch with D2O). IR (KBr) cm−1: 3425, 2922, 1597,
methane (10mL) at 0°C. The mixture was allowed to reach 1493, 1375. MS m/z: 369 (M+), 343, 309, 281, 266, 57. Anal.
room temperature then heated to reflux for 20h. Yield 65% Calcd for C18H19N5O2S: C, 58.52; H, 5.18; N, 18.96. Found: C,
1
as a white needle crystals, mp 150–152°C. H-NMR (DMSO- 58.25; H, 4.91; N, 18.80.
d6) δ: 9.3 (1H, s, exch with D2O), 7.95–7.92 (1H, d, J=9.3Hz),
4-Anilino-6-(4-methylpiperazin-1-ylsulfonyl)quinazoline
7.89–7.13 (12H, m), 4.15 (2H, s), 3.5–3.2 (8H, m). IR: (KBr) (15d): 4-Chloro-6-(4-methylpiperazin-1-ylsulfonyl)quinazoline
cm−1: 3396, 2923, 1623, 1370. MS m/z: 537 (M+), 513, 495, 13d (0.3g, 0.95mmol) in dichloromethane (10mL) was added
458, 365, 58. Anal. Calcd for C25H24BrN5O2S: C, 55.76; H, gradually with stirring to a solution of aniline (0.18mL,
4.49; N, 13.01. Found: C, 55.49; H, 4.12; N, 12.68.
1.95mmol) and triethylamine (1mL) in dichloromethane
General Procedure for the Preparation of 4-Anilino-6-(N- (10mL) at 0°C. The mixture was allowed to reach room tem-
substituted sulfonyl)quinazoline (15a–g): 4-Chloro-6-(N- perature then heated under reflux for 3h. The residue was pu-

May 2014
461
rified with column chromatography using ethyl acetate–hexane intensity was measured in an enzyme-linked immunosorbent
(1:1) as eluent. Yield 57% as a white crystals, mp 140–142°C. assay (ELISA). Percentage of the surviving and inhibition
1H-NMR (DMSO-d6) δ: 9.9 (1H, s, exch with D2O), 7.95–7.92 were tabulated.17)
(1H, d, J=9.3Hz), 7.80–7.42 (1H, dd, J=9.3, 2.4Hz), 7.40–6.60
Cytotoxicity Test: Cell Culture: MCF-7 human breast can-
(7H, m), 3.32–2.03 (11H, m). IR (KBr) cm−1: 3175, 2972, 1599, cer cells was grown in RPMI-1640 medium, supplemented
1494, 1379. MS m/z: 382 (M+), 348, 318, 249, 164, 79. Anal. with 10% heat inactivated fetal bovine serum (FBS), 50 units/
Calcd for C19H21N5O2S: C, 59.51; H, 5.52; N, 18.26. Found: C, mL of penicillin and 50g/mL of streptomycin and maintained
59.32; H, 5.19; N, 17.86.
at 37°C in a humidified atmosphere containing 5% CO2. The
4-Anilino-6-(4-ethylpiperazin-1-ylsulfonyl)quinazoline cells were maintained as “monolayer culture” by serial sub-
(15e): 4-Chloro-6-(4-ethylpiperazin-1-ylsulfonyl)quinazoline culturing.
13e (0.3g, 0.95mmol) in dichloromethane (10mL) was added
Sulforhodamine B Colorimetric Assay (SRB) Cytotoxicity
gradually with stirring to a solution of aniline (0.18mL, Assay: Cytotoxicity was determined using SRB method as
1.95mmol) and triethylamine (1mL) in dichloromethane previously described by Skehan et al.17) Exponentially growing
(10mL) at 0°C. The mixture was allowed to reach room tem- cells were collected using 0.25% trypsin–EDTA and seeded in
perature then heated under reflux for 3h. The residue was 96-well plates at 1000–2000 cells/well in RPMI-1640 supple-
purified with column chromatography using ethyl acetate– mented medium. After 24h, cells were incubated for 72h with
hexane (1:1) as eluent. Yield 53% as a white crystals, mp various concentrations of the tested compounds. Following
1
135–137°C. H-NMR (DMSO-d6) δ: 9.18 (1H, s, exch with 72h treatment, the cells will be fixed with 10% trichloroace-
D2O), 7.36–7.24 (9H, m), 3.5–2.4 (13H, m). IR (KBr) cm−1: tic acid for 1h at 4°C. Wells were stained for 10min at room
3175, 2921, 1598, 1494, 1376. MS m/z: 397 (M+), 374, 316, 287, temperature with 0.4% SRB dissolved in 1% acetic acid. The
217, 56. Anal. Calcd for C20H23N5O2S: C, 60.43; H, 5.83; N, plates were air dried for 24h and the dye was solubilized with
17.62. Found: C, 60.67; H, 6.17; N, 17.84.
Tris–HCl for 5min on a shaker at 1600rpm. The optical den-
4-Anilino-6-(4-phenylpiperazin-1-ylsulfonyl)quinazoline sity (OD) of each well was measured spectrophotometrically
(15f): 4-Chloro-6-(4-phenylpiperazin-1-ylsulfonyl)quinazoline at 564nm with an ELISA microplate reader (ChroMate-4300,
13f (0.37g, 0.95mmol) in dichloromethane (10mL) was added FL, U.S.A.). The IC50 values were calculated according to
gradually with stirring to a solution of aniline (0.18mL, the equation for Boltzman sigmoidal concentration–response
1.95mmol) and triethylamine (1mL) in dichloromethane curve using the nonlinear regression fitting models (Graph
(10mL) at 0°C. The mixture was allowed to reach room tem- Pad, Prism Version 5).
perature then heated under reflux for 4h. The residue was
purified with column chromatography using ethyl acetate–
Results and Discussion
petrolum ether (3:1) as eluent. Yield 54% as a yellow crystals,
Chemistry The synthetic pathway of the title com-
1
mp 110–112°C. H-NMR (DMSO-d6) δ: 9.78 (1H, s, exch with pounds is outlined in Charts 1 and 2. The starting ma-
D2O), 7.95–7.92 (1H, d, J=9.3Hz), 7.7–7.2 (14H, m), 3.4–2.1 terial anthranilic acid (1) was conveniently cyclized to
(8H, m). IR (KBr) cm−1: 3742, 2913, 1598, 1494, 1411. MS m/z: quinazolin-4(3H)-one (3) by heating with formamide (2)
445 (M+), 417, 435, 266, 168, 93. Anal. Calcd for C24H23N5O2S: at 120°C.14) Upon refluxing compound (3) with phospho-
C, 64.70; H, 5.20; N, 15.72. Found: C, 65.00; H, 5.55; N, 15.87. rous oxychloride gave compound 4-chloroquinazoline (4).13)
4-Anilino-6-(4-benzylpiperazin-1-ylsulfonyl)quinazoline 4-Chloroquinazoline was allowed to react with p-pheneylene
(15g): 6-(4-Benzylpiperazin-1-ylsulfonyl)-4-chloro-quinazo- diamine to produce 4-(4-aminoanilino)quinazoline (5).15)
line 13g (0.38g, 0.95mmol) in dichloromethane (10mL) was Compounds 1-phenyl-3-(4-(quinazolin-4-ylamino) phenyl)-
added gradually with stirring to a solution of aniline (0.18mL, urea (6), 1-phenyl-3-(4-(quinazolin-4-ylamino) phenyl)thiourea
1.95mmol) and triethylamine (1mL) in dichloromethane (7), 2-(4-(quinazolin-4-ylamino)phenylimino)acetic acid (8),
(10mL) at 0°C. The mixture was allowed to reach room tem- 3-(4-(quinazolin-4-ylamino)phenylamino)propanoic acid (9)
perature then heated under reflux for 8h. The residue was and 4-oxo-4-(4-(quinazolin-4-ylamino)phenylamino)butanoic
purified with column chromatography using ethyl acetate– acid (10) were synthesized from intermediate compound
petrolum ether (3:1) as eluent. Yield 60% as a white needle 4-(4-aminoanilino)quinazoline using different reagents as re-
1
crystals, mp 170–172°C. H-NMR (DMSO-d6) δ: 9.186 (1H, s, ported in Chart 1.
exch with D2O), 7.95–7.92 (1H, d, J=9.3Hz), 7.91–7.10 (13H,
Moreover, compound 6-chlorosulfonylquinazolin-4(3H)-
m), 4.15 (2H, s), 3.30–2.37 (8H, m). IR (KBr) cm−1: 3396, one (11) was synthesized from compound (3) by reacting
2913, 1625, 1365. MS m/z: 459 (M+), 308, 290, 279, 203, 57. with chlorosulfonic acid under dry conditions. Subsequently
Anal. Calcd for C25H25N5O2S: C, 65.34; H, 5.48; N, 15.24. compound (11) reacted with piperidine, cyclohexyl amine
Found: C, 65.00; H, 5.09; N, 14.91.
and substituted piperazines to produce 6-(N-substituted)-
Biological Evaluations Sensitivity Test: Cells were plated sulfonylquinazolin-4(3H)-one (12a–g). This series then
in 96-multiwellplate (104 cells/well) for 24h before treat- reacted with phosphorous oxychloride to produce 4-chloro
ment with the compounds to allow attachment of cell to the derivatives (13a–g).
wall of the plate. A single concentration of the compounds
Finally, compounds (13a–g) underwent a nucleophilic sub-
under test was added to the cell monolayer. Monolayer cells stitution reaction in the presence of substituted anilines to
were incubated with the compounds for 48h at 37°C and in give the title compounds (14b–d,g) and (15a–g) in Chart 2.
atmosphere of 5% CO2. After 48h, cells were fixed, washed
The structure of the synthesized final products was con-
1
and stained with Sulforhodamine-B stain. Excess stain was firmed by elemental analyses, IR, H-NMR and mass spec-
washed with acetic acid and attached stain was recovered with tral data. Chart 1, IR spectra, generally, showed a peak of
Tris ethylenediaminetetraacetic acid (EDTA) buffer. Color secondary amine at 3300–3500cm−1 due to the coupling of

462
Vol. 62, No. 5
Fig. 4. Docking of Lapatinib in the ATP Binding Site of EGFR-TK
Lapatinib docking pose showed a maximum fitting to the receptor cavity where the quinazoline ring be inserted nicely inside the pocket. The anilino portion is oriented
deep in the hydrophobic region I. The modeling also suggested that there were also two hydrogen bonds observed. One between N-1 of quinazoline ring and Met-793 and
the other between N-3 of quinazoline ring and a water molecule that bridged the gap between N-3 and Asp-855.
Fig. 5. Docking of Compound (14g) in the ATP Binding Site of EGFR-TK (PDB Code: 2J5F)
Compound (14g) showed a docking pose similar to the docking pose of the lapatinib in the receptor. Two hydrogen bonds were observed, one between N-1 of quinazo-
line and Met-793 while, the other between N-3 of quinazoline and a water molecule of crystallization.
4-chloroquinazoline (4) with p-phenylene diamine. Mass spec- the presence of a downfield exchangeable singlet with D2O
tra showed the major fragments according to their expected corresponding to OH of the carboxylic group (COOH). One
1
molecular formula. H-NMR spectra of compounds (6) and (7) singlet shielded proton was also observed upfield correspond-
1
displayed the presence of two downfield exchangeable singlets ing to N=CH of the imine group. H-NMR Spectra of com-
with D2O corresponding to two NH of urea and thiourea moi- pounds (9) and (10) showed the presence of three downfield
1
ety respectively. H-NMR spectrum of compound (8) showed exchangeable singlets with D2O corresponding to OH of the

May 2014
463
Fig. 6. Docking of Compound (6) in the ATP Binding Site of EGFR-TK (PDB Code: 2J5F)
Compound (6) showed a docking pose similar to the docking pose of the lapatinib in the receptor. Two hydrogen bonds were observed, one between N-1 of quinazoline
and Met-793 while, the other between N-3 of quinazoline and a water molecule of crystallization.
Fig. 7. Docking of Compound (10) in the ATP Binding Site of EGFR-TK (PDB Code: 2J5F)
Compound (10) showed a docking pose similar to the docking pose of the lapatinib in the receptor. Two hydrogen bonds were observed, one between N-1 of quinazoline
and Met-793 while, the other between N-3 of quinazoline and a water molecule of crystallization.

464
Vol. 62, No. 5
carboxylic group (COOH) and NH of NH–CH2–CH2 group in the gap between N-3 and Asp-855. The binding models of the
compound (9) and NH of the amide group in compound (10). most active inhibitor in each series and EGFR were shown in
A set of multiplets was also observed upfield corresponding Figs. 5–7. Compound (14g) showed low free energy of bind-
to aliphatic side chain. Chart 2, IR spectra, generally, showed ing −6kJ/mol and a high inhibition in the cancer cell growth
a peak of sulfonamide moiety at 1363cm−1 and a peak of (IC50=6.735 µM). The root mean square deviation (RMSD) for
secondary amine at 3300–3500cm−1 due to the coupling of the compound was 1.6Å. Compound (14g) showed a dock-
4-chloro-derivatives (13a–g) with primary aromatic amines. ing pose similar to the docking pose of the lapatinib in the
Mass spectra showed the major fragment according to their receptor. Two hydrogen bonds were observed, one between
1
expected molecular formula. H-NMR Spectra were charac- N-1 of quinazoline and Met-793 while, the other between N-3
terized by two important regions: a highly shielded region of quinazoline and a water molecule of crystallization. Com-
consisting of aliphatic multiplet signals corresponding to cy- pound (6) showed a moderate free energy of binding −4.75 k J/
clohexane or piperdine ring or substituted piperazine ring at δ mol and a moderate inhibition in the cancer cell growth
1–4ppm and deshielded region consisting of aromatic and/or (IC50=21.71 µM). The RMSD value was 1.9Å. Compound (6)
heteroaromatic multiplets corresponding to quinazoline ring, also showed a docking pose similar to the docking pose of
4-anilino moiety and benzene ring at δ 6–9ppm.
the lapatinib in the receptor. Two hydrogen bonds were also
Molecular Docking Molecular docking was performed observed, one between N-1 of quinazoline and Met-793 while,
on the binding model based on the EGFR-lapatinib complex the other between N-3 of quinazoline and a water molecule of
structure (2J5F.pdb). There is a direct proportionality between crystallization. Compound (10) also showed a moderate free
the free energy of binding of the inhibitors (anticancer agents) energy of binding −3kJ/mol and a weak to moderate inhibi-
and their activity. The smaller the free energy of binding, the tion in the cancer cell growth (IC50=34.91 µM). The RMSD
more tight is the binding of the inhibitors to their binding value was 1.9Å. Compound (10) also showed a docking pose
domain with the inhibition of cancer cell growth.12) Lapatinib similar to the docking pose of the lapatinib in the receptor.
was docked inside the receptor as illustrated in Fig. 4. Lapa- Two hydrogen bonds were observed between N-1 of quinazo-
tinib docking pose showed a maximum and plausible fitting line and Met-793 while, the other between N-3 of quinazoline
to the receptor cavity where the quinazoline ring was inserted and a water molecule of crystallization.
nicely inside the pocket. The anilino portion was oriented
Biological Activity The anticancer activity was evaluated
deep inside the hydrophobic region I. The model also suggest- via a two-stage process, beginning with measurement of the
ed that there were also two hydrogen bonds observed between sensitivity of series A, B, C and D via selection of one com-
N-1 of quinazoline ring and Met-793 and the other between pound representing each series from our newly synthesized
N-3 of quinazoline ring and a water molecule that bridged compounds against a panel of eight human cancer cell lines
Fig. 8. Sensitivity Test of Series A, B, C and D against 8 Human Carcinoma Cell Lines
Most of the newly synthesized derivatives displayed a significant inhibitory activity against breast carcinoma cell line (MCF-7).

May 2014
465
Table 1. In Vitro Cytotoxic Activity of the Synthesized Compounds
against MCF-7
hand, in series B, It was noticed that elongation and branch-
ing of the investigated substituents increased their ability to
inhibit EGFR. Elongation allowed deep penetration in the
hydrophobic region I while branching suggested several possi-
bilities for hydrogen bonds. Therefore, compound (10) showed
a higher activity than its congeners (8) and (9). Chart 2, the
quinazoline core was modified at position-6 of the quinazoline
ring with N-substituted sulfonyl derivatives, with the aim of
improving physical properties (solublization) and conferring a
more favorable pharmacokinetic profile.18) It was noticed that
series C showed a higher cytotoxic activity than series D. This
was because that the phenyl ring of the aniline was tilted out
of the plane from the quinazoline allowing the bromine atom
to fit more precisely into the hydrophobic region I. We may
conclude that compound containing halogen at aniline ring
showed a better cytotoxic activity than those without halogen.
In series C, it was also noticed that the presence of one or
more rotamer allowed a free rotation of this side chain so as
to be close to the amino acid residue to increase the chance of
the interaction. Therefore, compound (14g) showed the highest
cytotoxic activity in series C.
Compound
Series
IC50 (µM)
6
7
A
21.71
24.25
64.09
45.87
34.91
9.48
A
8
B
9
B
10
B
14b
14c
14d
14g
15a
15b
15c
15d
15e
15f
15g
Lapatinib
C
C
6.735
18.26
5.52
C
C
D
82.48
>100
>100
>100
>100
>100
>100
3
D
D
D
D
D
D
Reference
Series C was the most active series followed by series A, then series B. Series D
was the least cytotoxic as it showed high IC50.
Conclusion
The present work led to the development of four series of
representing different tumor types, namely liver carcinoma novel anticancer molecules containing 4-anilinoquinazoline
cell line (Hep G2), cervical carcinoma cell line (Hela), colon pharmacophore. Eight cell lines were used to measure cy-
carcinoma cell line (HCT-116), breast carcinoma cell line totoxic sensitivity of the proposed quinazoline derivatives.
(MCF-7), larynx carcinoma cell line (Hep2), intestinal carci- Most of tested compounds showed good activity against breast
noma cell line (Caco-2), breast carcinoma cell line (T-47D) cancer (MCF-7) with IC50 range of 5.52–34.91µg/mL. The
and prostate carcinoma cell line (PC-3) at a single dose of best cytotoxic result was obtained with series C especially;
100 µg/mL, followed by the evaluation of potential cytotox- compound (14g) that showed the most potent inhibitory activ-
icity of the new compounds at five different concentrations ity. Molecular docking studies supported the strong inhibitory
(0.1, 1, 10, 100, 1000µg/mL) against the most promising cell activity of compound (14g) and help to design novel potent
lines, corresponding to the maximum percentage of inhibition inhibitors.
achieved at the single dose experiment. The initial screening
effect i.e., sensitivity test indicated that, most of the newly
Acknowledgment Our deep appreciation to Dr. Ahmad
synthesized derivatives displayed a significant inhibitory ac- Esmat, Pharmacology Department, Faculty of Pharmacy, Ain
tivity against breast carcinoma cell line (MCF-7) (Fig. 8), so Shams University for his help in carrying out cytotoxic activ-
the cytotoxic activity of target compounds against (MCF-7) ity.
cells was determined using Sulforhodamine-B assay using
labatinib as a reference. Compounds were tested over a range
of concentrations from 0.1 to 1000µg/mL, and the calculated
IC50 values, that is, the concentration (µg/mL) of a compound
that was able to cause 50% cell death with respect to the con-
trol, were reported. The results were summarized in Table 1.
As shown in Table 1. Among the compounds tested for their
cytotoxicity against the breast carcinoma cell line (MCF-7),
compound (14g) showed the best inhibitory activity and its
inhibitory rate was 85% at 100µg/mL and 64% at 10µg/mL.
Compounds (14c) and (14b) also displayed a remarkable activ-
ity at micromole order of magnitude, with IC50 values of 6.735
and 9.48, respectively, followed by compounds (14d), (6) and
(7). The remaining compounds were the least cytotoxic as
they showed high IC50. The cytotoxic activity of the tested
compounds could be correlated to structural modification.
Chart 1, the quinazoline core was modified at the position-4 of
the aniline ring with different bulky substituents such as urea,
thiourea, amide or glycine, with the aim of strengthening the
ligand receptor interaction and therefore increasing affinity. In
series A, there was no obvious difference between urea and
thiourea moiety both showed good activities. On the other
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