Synthesis of novel 4,6-disubstituted quinazoline derivatives, their anti-inflammatory and anti-cancer activity (cytotoxic) against U937 leukemia cell lines

Article abstract of DOI:10.1016/j.ejmech.2007.06.010

In view of the link between use of NSAIDs and altered cancer incidence and a growing evidence of COX-II implication in angiogenesis, a novel series of 4,6-disubstituted quinazoline derivatives have been synthesized starting from anthranilic acid derivatives 1 through conventional methods. Initially acylation followed by cyclisation to obtain benz-oxazinones 2 which on further treatment with ammonia yielded the crucial intermediate, 2-substituted benzamide (3). The products were subsequently cyclised to obtain quinazolones 4, chlorinated 5, then hooked to various optically pure α-amino acids to have 4,6-disubstituted quinazoline derivatives 6. All the derivatives 6 are screened for anti-inflammatory and anti-cancer activity against U937 leukemia cell lines. Some of the compounds exhibited promising anti-cancer activity with reference to standard drug Etoposide.

Full text of DOI:10.1016/j.ejmech.2007.06.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 846e852
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 4,6-disubstituted quinazoline derivatives,
their anti-inflammatory and anti-cancer activity (cytotoxic)
against U937 leukemia cell lines*
b,
P. Mani Chandrika a, T. Yakaiah b, A. Raghu Ram Rao a, , B. Narsaiah
*
*
,
N. Chakra Reddy c, V. Sridhar c, J. Venkateshwara Rao c
a Medicinal Chemistry Research Division, University College of Pharmaceutical Sciences, Kakatiya University,
Warangal 506009, Andhrapradesh, India
b Fluoroorganic Division, Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500007, Andhrapradesh, India
c Biology Division, Indian Institute of Chemical Technology, Hyderabad 500007, Andhrapradesh, India
Received 3 April 2007; received in revised form 14 June 2007; accepted 15 June 2007
Available online 6 July 2007
Abstract
In view of the link between use of NSAIDs and altered cancer incidence and a growing evidence of COX-II implication in angiogenesis,
a novel series of 4,6-disubstituted quinazoline derivatives have been synthesized starting from anthranilic acid derivatives 1 through conventional
methods. Initially acylation followed by cyclisation to obtain benz-oxazinones 2 which on further treatment with ammonia yielded the crucial
intermediate, 2-substituted benzamide (3). The products were subsequently cyclised to obtain quinazolones 4, chlorinated 5, then hooked to
various optically pure a-amino acids to have 4,6-disubstituted quinazoline derivatives 6. All the derivatives 6 are screened for anti-inflammatory
and anti-cancer activity against U937 leukemia cell lines. Some of the compounds exhibited promising anti-cancer activity with reference to
standard drug Etoposide.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Anthranilic acids; Acylation; Amination; Chlorination; Quinazoline derivatives; a-Amino acids
1. Introduction
been an area of intense investigation over the past couple of
decades [4]. This led to the genesis of a substantial body of ev-
idence supporting a role of cyclooxygenase -2 (COX-2), the
enzyme implicated in the incidence of inflammatory disorders
in carcinogenesis. Several studies have reported an inverse re-
lationship for both colon [5] and breast cancers [6e8]. A num-
ber of studies have shown over expression of COX-2 in solid
malignancies [9]. The effects of specific COX-2 inhibitors
have been tested in animal models of angiogenesis, and cele-
coxib, a specific COX-2 inhibitor, has been shown to cause
inhibition of the angiogenic response in fibroblast growth
factor-induced rat corneal angiogenesis [10]. In addition,
forced over expression of COX-2 is sufficient to induce tumor-
igenesis in transgenic mice [11]. This data have provided us
a basis for designing molecules with a potential of anti-
inflammatory and anti-tumour activities.
The main target of NSAID drug action is the cyclooxyge-
nase (COX) enzyme, which catalyses the conversion of arach-
idonic acid to prostaglandin precursors important in
inflammatory processes. Two isozymes, which are the prod-
ucts of two separate genes, exist in humans: (i) COX-1, the
constitutive form; and (ii) COX-2, the inducible form [1,2].
This isoenzyme is induced in response to cytokines and
bacterial endotoxins [3].
A possible link between use of non-steroidal anti-inflam-
matory agents (NSAIDs) and altered incidence of cancer has
*
IICT communication no. 070325.
* Corresponding authors. Tel.: þ91 40 27193630; fax: þ91 40 27160387.
E-mail address: narsaiah@iict.res.in (B. Narsaiah).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.010

P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
847
In general quinazoline derivatives are known to possess re-
markable anti-inflammatory activity as NOS-II [10,12], NFKB
[13], TNF-a [14] IMPDH-II [15], MAPK [16], IL-6 [17],
PDE-3 [18] and PDE-4 [19] inhibitors. Quinazoline deriva-
tives are also found to show bronco-dilatory [20], anti-allergic
[21] properties. Primarily non-steroidal anti-inflammatory
drug (NSAID) such as acetyl salicylic acid was identified as
a well established first generation drug in the treatment of
pain and inflammation. Over a period phenyl butazone is con-
sidered as second generation NSAID and showed significant
improvement over first generation. Subsequently proquazone
and fluproquazone [22] emerged as third generation NSAIDs
and are superior in safety, efficacy and are comparable with In-
domethacin drug. In addition, quinazoline derivatives also
have a therapeutic benefit as an anti-invasive agent with poten-
tial for activity in early and advanced solid tumors, metastatic
bone disease and leukemias [23,24]. Some of the known qui-
nazoline derivatives exhibited remarkable anti-cancer activity
[25e32]. However, search is continuously on to identify
a more potent lead molecule as these molecules are developing
resistance over a period. Based on the importance of these
molecules, our attention was attracted towards synthesis of
novel quinazoline derivatives in order to find a more potent
molecule. Quinazoline derivatives are synthesized mainly
starting from anthranilic acid [15,33e37], benzonitrile [34]
and so on with an appropriate substituent to have specific func-
tionality and activity. In continuation of our efforts towards
synthesis of potential molecules like quinazolines [33,38,39],
pyrido pyrimidines [40], pyrimido[1,2-b]indazoles [41,42],
we have synthesized several new 4,6-disubstituted quinazoline
derivatives and screened for anti-inflammatory as well as anti-
cancer activity against U937 leukemia cell lines. The com-
pounds which showed promising anti-cancer activity have
been identified in comparison with Etoposide, a standard
drug and have been reported here for the first time.
resulted in respective amide derivative 3. Compound 3 on re-
fluxing in aq. sodium hydroxide gave cyclised product quina-
zolin-4-one 4. It is further chlorinated using POCl3/PCl5 and
obtained 4-chloroquinazolone 5. In order to see the role of
substituents on rate of reaction, yield of products and subse-
quently on activity, anthranilic acid 1 was substituted with bro-
mine/iodine in fifth position and the sequence of reactions is
carried out to obtain the respective products 5. It is found
that the rate of reaction and yield of products are independent
of substituents used. The sequence of reactions are drawn in
Scheme 1 and yields of products are tabulated in Table 1.
The 4-chloro quinazolines 5 are reacted with various opti-
cally pure a-amino acids such as L-methyl glycine, L-phenyl
alanine, L-isoleucine, L-tryptophan and simple glycine in di-
methyl sulphoxide at temperature 100 ꢀC for 4e5 h and ob-
tained 4,6-disubstituted amino quinazoline derivatives 6 in
high yields. In order to obtain imidazo[1,2-c]quinazolines 7,
all the products were subjected to cyclisation under different
set of conditions such as (a) glacial CH3COOH/p-TSA at re-
flux, (b) (CH3CO)2O at 140 ꢀC, (c) NaOCH3/CH3OH at reflux,
(d) microwave irradiation condition on a silica gel solid sup-
port in presence of POCl3 at 600 W. In all the cases no cy-
clised product is formed except recovering starting material.
It is attributed to the stability of compounds and proton on
C-4 NH is not easily polarized on to ring nitrogen due to aro-
maticity. The sequence of reactions are drawn in Scheme 2 and
products are tabulated in Table 2.
3. Pharmacology
Quinazoline derivatives being considered as potent anti-
inflammatory and anti-cancer agents, several new quinazoline
derivatives 6ae6o were synthesized and screened for anti-in-
flammatory and anti-cancer activities. The response of all the
compounds to anti-inflammatory activity is moderate to poor,
however, some of the compounds showed promising anti-
cancer activity. The details of activity results are outlined below.
2. Chemistry
Anthranilic acid (2-amino benzoic acid) 1 was reacted with
benzoyl chloride in dry pyridine at 0e5 ꢀC for 4 h and ob-
tained benz-oxazinone 2 in high yields. Benz-oxazinone 2 is
further treated with aq. ammonia at room temperature and
3.1. Anti-inflammatory activity
Test compounds 6ae6o were screened for anti-inflamma-
tory activity in vivo using carageenan induced rat paw edema
O
X
X
COOH
O
aq. NH3
dry pyridine
0 -5 °C
C6H5COCl
+
R.T.
C6H5
N
NH2
O
2
1
Cl
N
O
X
X
N
X
POCl3/PCl5
reflux
aq. NaOH
reflux
NH
C6H5
NH2
NHCOC6H5
C6H5
N
5
4
3
X= H, Br, I
Scheme 1.

848
P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
Table 1
Preparation of 4-chloro quinazolines 5
activity and is close to standard positive control (Etoposide).
The activity is presumed to be due to the presence of iodine
on 6th position with L-phenylalanine on 4th position. If iodine
is replaced with bromine, the activity is drastically reduced in
compound 6h. Alternately on comparison of compounds 6k, 6l
and 6j with L-isoleucin on 4th position and Br, I and H on 6th
position showed descending order of activity. Similar trend is
followed in compounds 6n, 6o and 6m with L-tryptophan and
compounds 6e, 6f and 6d with L-methyl glycine. In conclusion
bromine on 6th position is considered to be optimum to show
anti-cancer activity except in compound 6i, which showed the
highest activity with iodine on 6th position. The cytotoxic ac-
tivity of test compounds with decreasing order of
6i > 6k > 6n > 6o > 6h > 6l > 6m > 6e > 6j is tabulated in
Table 4.
Entry
Compound no.
X
M.p. (ꢀC)
Yield (%)
1
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
H
Br
I
122
182
199
234
237
248
226
230
241
124
129
135
82
80
82
85
80
82
84
80
81
80
77
78
2
3
4
H
Br
I
5
6
7
H
Br
I
8
9
10
11
12
H
Br
I
model. The different test compounds were administered to the
animals in the test group at the dose of 100 mg/kg and stan-
dard group with Indomethacin at the dose of 10 mg/kg as1%
gum acacia suspension by oral route. Compound 6d having
L-methyl glycine on 4th position showed highest activity
among all the compounds with 9.30% of inhibition against
48% of standard Indomethacin. Next highest activity is ob-
served in the descending order of compounds 6f, 6j, 6i and
6m. In all these compounds chain length or bulkiness in amino
acid on 4th position is increased and the activity is inversely
proportional to the chain length. Rest of the compounds
showed no activity having either bromine/iodine or no bro-
mine/iodine on 6th position with different amino acids branch
on 4th position in a given concentration. There is no clear cut
trend in results to fix the optimum substituents at a specified
position. The results in descending order of activity are
tabulated in Table 3.
4. Conclusion
A series of novel quinazoline derivatives have been synthe-
sized through a facile strategy and screened for anti-inflamma-
tory and anti-cancer (cytotoxic) activities. Promising
compounds which showed anti-cancer activity have been
identified.
5. Experimental section
Melting points were recorded on Casia-Siamia (VMP-AM)
melting point apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer FT-IR 240-C spectrophotometer
1
using KBr optics. H NMR spectra were recorded on Gemini
Varian 200 MHz, Bruker AV 300 MHz and Unity 400 MHz
spectrometer in DMSO-d6 or CDCl3 using TMS as an internal
standard. Electron impact (EI) and chemical ionisation mass
spectra were recorded on a VG 7070 H instrument at 70 eV.
All reactions were monitored by thin layer chromatography
(TLC) on pre-coated silica gel 60 F254 (mesh); spots were vi-
sualized under UV light. Merck silica gel (100e200 mesh)
was used for chromatography. CHN analyses were recorded
on a Vario EL analyser.
3.2. Anti-cancer activity (cytotoxic activity)
Compounds 6ae6o were tested against U937 leukemia hu-
man lymphoma anti-cancer cell lines and showed significant
decrease in cell viability with reference to concentration.
Among all, compound 6i exhibited maximum cytotoxic
R
COOH
C6H5
HN
Cl
R
X
X
N
N
DMSO
reflux
+
H2N
R
COOH
N
C6H5
N
6
5
X= H, Br, I
N
R=H, CH3, CH2C6H5, CH(CH3)CH2CH3,
CH2
X
O
N
C6H5
N
N
H
7
Scheme 2.

P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
849
Table 2
Synthesis of 4,6-disubstituted amino quinazoline derivatives 6
Table 4
Cytotoxic activity of compounds 6ae6o
Entry Compound no.
X
R
m.p. (ꢀC) Yield (%)
Entry
Test compound
IC50 (mg/ml)b
1
6a
6b
6c
6d
6e
6f
H
Br
I
H
217
245
279
235
202
282
163
153
222
81.33
79.76
82.71
78.00
76.70
78.90
77.00
76.54
78.90
81.00
78.86
80.00
82.00
80.96
86.59
1
Etoposidea
10.56 ꢂ 0.70
16.11 ꢂ 1.12
32.02 ꢂ 1.73
37.90 ꢂ 1.10
46.11 ꢂ 1.21
50.38 ꢂ 0.92
52.36 ꢂ 0.61
74.78 ꢂ 1.80
104.24 ꢂ 1.85
120.71 ꢂ 6.09
ns
2
H
2
6i
6k
3
H
CH3
3
4
H
4
6n
6o
5
Br CH3
I
5
6
CH3
CH2C6H5
6
6h
7
6g
6h
6i
H
Br CH2C6H5
7
6l
6m
8
8
9
I
CH2C6H5
9
6e
6j
6ae6d, 6f and 6g
10
11
12
13
14
15
6j
H CH(CH3)CH2CH3 237
Br CH(CH3)CH2CH3 203
10
11
6k
6l
I
CH(CH3)CH2CH3 246
ns, Not significant.
a
Positive control.
6m
6n
6o
H
C9H8N
174
220
272
Br C9H8N
C9H8N
b
IC50 is defined as the concentration, which results in a 50% decrease in cell
number as compared with that of the control cultures in the absence of an in-
hibitor. The values represent the mean ꢂ SE of four individual observations.
I
5.1. 2-Phenyl-4H-benzo[d][1,3]oxazin-4-one (2)
through a column packed with silica gel and eluents used are
ethyl acetate/methanol (80:20).
See Ref. [33].
5.5.1.1. (2-Phenyl quinazolin-4-yl amino) acetic acid (6a).
Yield: 0.47 g (81.33%); m.p. 217 ꢀC; IR (KBr, cmꢁ1):
5.2. 2-Benzamido benzamide (3)
1
3575e3200 (OH, NH), 1720.69 (CO). H NMR (200 MHz,
See Ref. [43].
DMSO-d6): d 4.35(2H, d, J ¼ 14.81 Hz, CH2), 7.40 (4H, m,
Ar-H), 7.70 (1H, d, J ¼ 15 Hz, Ar-H), 7.85 (1H, d,
J ¼ 18.51 Hz, Ar-H), 8.00 (1H, br., s, NH), 8.10 (1H, d,
J ¼ 14.81 Hz, Ar-H), 8.55 (2H, m, Ar-H); EIMS, m/z: 280
(Mþ þ 1); Anal. Calcd. for C16H13N3O2: C, 68.81; H, 4.69;
N, 15.05; Found: C, 68.56; H, 4.38; N, 14.87.
5.3. 2-Phenyl quinazolin-4(3H )-one (4)
See Ref. [38].
5.4. 4-Chloro-2-phenyl quinazoline (5)
5.5.1.2. (6-Bromo-2-phenyl quinazolin-4-yl amino) acetic acid
(6b). Yield: 0.42 g (79.76%); m.p. 245 ꢀC; IR (KBr, cmꢁ1):
See Ref. [38].
1
3200e2900 (OH, NH), 1673.92 (CO). H NMR (200 MHz,
DMSO-d6): d 4.35 (2H, d, J ¼ 13.33 Hz, CH2), 7.50 (3H, m,
Ar-H), 7.70e7.90 (2H, m, Ar-H), 8.20 (1H, d, J ¼ 20 Hz,
Ar-H), 8.45 (1H, br, s, NH), 8.52 (2H, m, Ar-H); EIMS, m/
z: 359(Mþ þ 1); Anal. Calcd. for C16H12BrN3O2: C, 53.65;
H, 3.38; N, 11.73; Found: C, 53.30; H, 3.30; N, 11.51.
5.5. 2-Phenyl-4,6-disubstituted quinazoline derivatives
(6ae6o)
5.5.1. General procedure
A stirred mixture of 4-chloro-2-phenyl 6-substituted quina-
zolines (4.5 mmol), optically pure a-amino acid (5 mmol) and
potassium carbonate (20 mmol) in DMSO-d6 (20 ml) was
heated at 100 ꢀC for 4e5 h. After cooling to room tempera-
ture, 1 N HCl was added till pH 4. The precipitated solid,
crude product was collected by filtration. It is purified passing
5.5.1.3. (6-Iodo-2-phenyl quinazoline-4-yl amino) ethane per-
oxoic acid (6c). Yield: 0.36 g (82.71%); m.p. 279 ꢀC; IR
(KBr, cmꢁ1): 3575e3200 (OH, NH), 1631(CO). 1H NMR
(200 MHz, DMSO-d6): d 4.40 (2H, d, J ¼ 13.79 Hz, CH2),
7.50e7.70 (3H, m, Ar-H), 7.90 (1H, d, J ¼ 13.79 Hz, Ar-H),
8.17 (2H, d, J ¼ 20 Hz, Ar-H), 8.30 (1H, d, J ¼ 20 Hz, NH),
8.37e8.42 (2H, m, Ar-H); EIMS, m/z: 406 (Mþ þ 1); Anal.
Calcd. for C16H12IN3O2: C, 47.43; H, 2.99; N, 10.37; Found:
C, 47.60; H, 3.25; N, 10.55.
Table 3
Anti-inflammatory activity of compounds 6ae6o
Entry
Test compound
% Of inhibition
1
2
3
4
5
6
7
Indomethacin
6d
48.0
9.30
6.50
4.10
3.70
1.80
0.0
5.5.1.4. (2-Phenyl quinazolin-4-yl amino) propanoic acid (6d).
Yield: 0.40 g (78%); m.p. 235 ꢀC; IR (KBr, cmꢁ1): 3550e
3000 (OH, NH), 1669.45 (CO). 1H NMR (200 MHz,
DMSO-d6): d 1.30 (3H, d, J ¼ 20 Hz, CH3), 4.20 (1H, qui,
J ¼ 5.56 Hz, CH), 7.55 (4H,m, Ar-H), 7.73 (2H, m, Ar-H),
8.25 (3H, m, Ar-H), 12.30 (1H, br., s, NH); EIMS, m/z: 294
6f
6j
6i
6m
6ae6c, 6e, 6g, 6h, 6k, 6l, 6n and 6o
Test compound, 100 mg/kg; Indomethacin, 10 mg/kg.

850
P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
(Mþ þ 1); Anal. Calcd. C17H15N3O2: C, 69.61; H, 5.15; N,
(200 MHz, DMSO-d6): d 1.02 (3H, t, J ¼ 6.66 Hz, CH3),
1.10 (3H, d, J ¼ 6.67 Hz, CH3), 1.44 (1H, m, CH2), 1.78
(1H, m, CH2), 2.20 (1H, m, CH), 4.80 (1H, t, J ¼ 6.60 Hz,
CH), 7.45 (4H, m, Ar-H), 7.70e7.85 (3H, m, Ar-H), 8.37
(1H, d, J ¼ 13.34 Hz, NH), 8.55 (2H, m, Ar-H); EIMS, m/z:
336 (Mþ þ 1); Anal. Calcd. for C20H21N3O2: C, 71.62; H,
6.31; N, 12.53; Found: C, 71.75; H, 6.40; N, 12.71.
14.33. Found: C, 69.35; H, 4.96; N, 14.01.
5.5.1.5. (6-Bromo-2-phenyl quinazolin-4-yl amino) propanoic
acid (6e). Yield: 0.39 g (76.70%); m.p. 202 ꢀC; IR (KBr,
cmꢁ1): 3150e3100 (OH, NH), 1673.63 (CO). 1H NMR
(200 MHz, DMSO-d6): d 1.60 (3H, d, J ¼ 12.90 Hz, CH3),
4.80 (1H, qui, J ¼ 8.06 Hz, CH), 7.60 (4H, m, Ar-H), 7.80
(2H, m, Ar-H), 8.35 (2H, m, Ar-H), 12.20 (1H, br., s, NH);
EIMS, m/z: 373 (Mþ þ 1); Anal. C17H14BrN3O2: C, 54.86;
H, 3.79; N, 11.29; Found: C, 54.53; H, 3.53; N, 11.01.
5.5.1.11. 2-(6-Bromo-2-phenyl quinazolin-4-yl amino)-3-
methyl pentanoic acid (6k). Yield: 0.42 g (78.86%); m.p.
203 ꢀC; IR (KBr, cmꢁ1): 3400e3000 (OH, NH), 1675.09
1
(CO). H NMR (200 MHz, DMSO-d6): d 0.95e1.05 (3H, t,
5.5.1.6. (6-Iodo-2-phenyl quinazolin-4-yl amino) propanoic
acid (6f). Yield: 0.49 g (78.90%); m.p. 282 ꢀC; IR (KBr,
cmꢁ1): 3150e3100 (OH, NH), 1673 (CO). 1H NMR
(200 MHz, DMSO-d6): d 1.65 (3H, d, J ¼ 10 Hz, CH3), 4.85
(1H, qui., J ¼ 5 Hz, CH), 7.50 (4H, m, Ar-H), 7.70 (2H, m,
Ar-H), 8.25 (2H, m, Ar-H), 12.50 (1H, br., s, NH); EIMS,
m/z: 420 (Mþ þ 1); Anal. Calcd. for C17H14IN3O2: C, 48.71;
H, 3.37; N, 10.02; Found: C, 48.90; H, 3.61; N, 10.26.
J ¼ 8.61 Hz, CH3), 1.10 (3H, d, J ¼ 13.79 Hz, CH3), 1.45
(1H, m, CH2), 1.80 (1H, m, CH2), 2.15 (1H, m, CH), 4.75
(1H, t, J ¼ 10.34 Hz, CH), 7.48 (4H, m, Ar-H), 8.12 (1H, d,
J ¼ 13.79 Hz, Ar-H), 8.30 (1H, m, Ar-H), 8.60 (2H, m, Ar-
H), 8.81 (1H, br., s, NH); EIMS, m/z: 415 (Mþ þ 1); Anal.
Calcd. for C20H20BrN3O2: C, 57.98; H, 4.87; N, 10.14; Found:
C, 57.81; H, 4.70; N, 9.98.
5.5.1.12. 2-(6-Iodo-2-phenylquinazolin-4-yl amino)-3-methyl
5.5.1.7. 3-Phenyl-2-(2-phenyl quinazolin-4-yl amino) propa-
noic acid (6g). Yield: 0.40 g (77%); m.p. 163 ꢀC; IR (KBr,
cmꢁ1): 3350e3100 (OH, NH), 1635.13 (CO). 1H NMR
(200 MHz, DMSO-d6): d 3.40 (2H, d, J ¼ 6.67 Hz, CH2),
5.00 (1H, quartet, J ¼ 4.45 Hz, CH), 7.12 (1H, d,
J ¼ 13.34 Hz, Ar-H), 7.20 (2H, t, J ¼ 6.67 Hz, Ar-H), 7.37
(2H, d, J ¼ 13.34 Hz, Ar-H), 7.45 (4H, m, Ar-H), 7.70 (1H,
t, J ¼ 6.67 Hz, Ar-H), 7.78 (1H, d, J ¼ 13.33 Hz, Ar-H), 8.27
(1H, d, J ¼ 13.33 Hz, NH), 8.45 (3H, m, Ar-H); EIMS, m/z:
370 (Mþ þ 1); Anal. Calcd. for C23H19N3O2: C, 74.78; H,
5.18; N, 11.37; Found: C, 74.93; H, 5.37; N, 11.61.
pentanoic acid (6l). Yield: 0.45 g (80.0%); m.p. 246 ꢀC; IR
1
(KBr, cmꢁ1): 3100e2965 (OH, NH), 1675 (CO). H NMR
(200 MHz, DMSO-d6): d 0.95e1.05 (3H, t, J ¼ 8.60 Hz,
CH3), 1.10 (3H, d, J ¼ 13.78 Hz, CH3), 1.45 (1H, m, CH2),
1.80 (1H, m, CH2), 2.15 (1H, m, CH), 4.75 (1H, t,
J ¼ 10 Hz, CH), 7.48 (4H, m, Ar-H), 8.12 (1H, d,
J ¼ 13.79 Hz, Ar-H), 8.30 (1H, m, Ar-H), 8.60 (2H, m, Ar-
H), 8.81 (1H, br., s, NH); EIMS, m/z: 415 (Mþ þ 1); Anal.
Calcd. for C20H20IN3O2: C, 52.07;H, 4.37; N, 9.11; Found:
C, 52.31; H, 4.60; N, 9.29.
5.5.1.13. 2-Phenyl quinazolin-4-(2-amino-3-(1H-indol-3-yl)
5.5.1.8. 2-(6-Bromo-2-phenyl quinazolin-4-yl amino)-3-phenyl
propanoic acid) (6m). Yield: 0.48 g (82.0%); m.p. 174 ꢀC;
1
propanoic acid (6h). Yield: 0.50 g (76.54%); m.p. 153 ꢀC;
IR (KBr, cmꢁ1): 3300e3000 (OH, NH), 1632.64 (CO). H
1
IR (KBr, cmꢁ1): 3300e3000 (OH, NH), 1727.61 (CO). H
NMR (200 MHz, DMSO-d6): d 3.50 (2H, d, J ¼ 16.67 Hz,
CH2), 5.0(1H, q, J ¼ 8.34 Hz, CH), 6.95 (3H, m, Ar-H), 7.20
(2H, m, Ar-H), 7.40 (4H, m, Ar-H), 7.70 (3H, m, Ar-H),
8.45 (2H, m, Ar-H), 9.60 (1H, d, J ¼ 16.67 Hz, NH), 10.65
(1H, s, NH); EIMS, m/z: 409 (Mþ þ 1); Anal. Calcd. for
C25H20N4O2: C, 73.51; H, 4.94; N, 13.72; Found: C, 73.70;
H, 5.18; N, 13.91.
NMR (200 MHz, DMSO-d6): d 3.30 (2H, d, J ¼ 14.81 Hz,
CH2), 4.95 (1H, quartet, J ¼ 4.93 Hz, CH), 7.15e7.30 (5H,
m, Ar-H), 7.45e7.60 (3H, m, Ar-H), 7.90 (2H, m, Ar-H),
8.05 (1H, br., s, NH), 8.15 (2H, m, Ar-H), 9.32 (1H, d,
J ¼ 14.81 Hz, Ar-H); EIMS, m/z: 449.3 (Mþ þ 1); Anal.
Calcd. C23H18BrN3O2: C, 61.62; H, 4.05; N, 9.37; Found: C,
61.84; H, 4.29; N, 9.61.
5.5.1.14. 6-Bromo-2-phenyl quinazolin-4-(2-amino-3-(1H-in-
dol-3-yl) propanoic acid) (6n). Yield: 0.45 g (80.96%); m.p.
220 ꢀC; IR (KBr, cmꢁ1): 3300e2950 (OH, NH), 1632 (CO).
1H NMR (200 MHz, DMSO-d6): d 3.50 (2H, d, J ¼ 12 Hz,
CH2), 5.18 (1H, q, J ¼ 8.0 Hz, CH), 6.95 (3H, m, Ar-H),
7.30 (2H, m, Ar-H), 7.55 (4H, m, Ar-H), 8.0 (3H, m, Ar-H),
8.20 (1H, m, Ar-H), 9.50 (1H, d, J ¼ 16 Hz, NH), 10.55
(1H, s, NH); EIMS, m/z: 409 (Mþ þ 1); Anal. Calcd.
C25H19BrN4O2: C, 61.61; H, 3.93; N, 11.50; Found: C,
61.45; H, 3.70; N, 11.32.
5.5.1.9. 2-(6-Iodo-2-phenyl quinazolin-4-yl amino)-3-phenyl
propanoic acid (6i). Yield: 0.45 g (78.90%); m.p. 222 ꢀC;
1
IR (KBr, cmꢁ1): 3450e3100 (OH, NH), 1673.90 (CO). H
NMR (200 MHz, DMSO-d6): d 3.40 (2H, d, J ¼ 10 Hz,
CH2), 5.10 (1H, quartet, J ¼ 6.67 Hz, CH), 7.20e7.35 (2H,
m, Ar-H), 7.40e7.48 (2H, m, Ar-H), 7.50e7.65 (7H, m, Ar-
H), 8.25 (1H,br, s, NH), 8.48 (2H, m, ArH); EIMS, m/z: 496
(Mþ þ 1); Anal. Calcd. for C23H18IN3O2: C, 55.77; H, 3.66;
N, 8.48; Found: C, 55.50; H, 3.47; N, 8.29.
5.5.1.10. 3-Methyl-2-(2-phenyl quinazolin-4-yl amino)penta-
noic acid (6j). Yield: 0.40 g (81.0%); m.p. 237 ꢀC; IR (KBr,
cmꢁ1): 3400e3000 (OH, NH), 1635.46 (CO). 1H NMR
5.5.1.15. 6-Iodo-2-phenyl quinazolin-4-(2-amino-3-(1H-indol-
3-yl) propanoic acid (6o). Yield: 0.5 g (86.59%); m.p.
272 ꢀC; IR (KBr, cmꢁ1): 3300e2900 (OH, NH), 1632 (CO).

P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
851
1H NMR (200 MHz, DMSO-d6): d 3.55 (2H, d, J ¼ 12.10 Hz,
Acknowledgements
CH2), 5.15 (1H, q, J ¼ 8.12 Hz, CH), 7.0 (3H, m, Ar-H), 7.22
(2H, m, Ar-H), 7.35 (4H, m, Ar-H), 7.60 (1H, m, Ar-H), 8.40
(3H, m, Ar-H), 9.40 (1H, d, J ¼ 16.10 Hz, NH), 10.50 (1H, s,
NH); EIMS, m/z: 409 (Mþ þ 1); Anal. Calcd. for
C25H19IN4O2: C, 56.19; H, 3.58; N, 10.49; Found: C, 56.36;
H, 3.82; N, 10.63.
Authors are thankful to the Principal, Dr. G. Achaiah, Uni-
versity College of Pharmaceutical Sciences, Kakatiya Univer-
sity, Dr. J.S. Yadav, Director, IICT, Hyderabad, Dr. S. Narayan
Reddy, Head, Fluoroorganic Division, IICT for their constant
encouragement and facilities. One of the authors, (P.M.C.) is
grateful to AICTE, New Delhi for the grant of research fellow-
ship under Quality Improvement Programme (Q.I.P.).
5.6. Pharmacology
5.6.1. Assay method of anti-inflammatory activity in vivo
The anti-inflammatory activity of the test compounds was
evaluated in Wistar rats by carrageenan induced rat paw
edema model employing the method of Winter et al. (1963)
[44] and Diwan et al. (1989) [45]. Male Wistar rats were
used for the study. Animals were fasted overnight and were di-
vided into control, standard and different test groups each con-
sisting of three animals. Rats in the control group received the
vehicle solution without drugs. One hour after test drugs ad-
ministration, rats in all the groups were challenged with
0.1 ml of 1% carageenan in the sub plantar region of left
hind paw. A zero hour paw volume was measured for the
rats using digital plethysmometer (Ugo Basile, Italy) before
the administration of carrageenan for all the groups. Paw vol-
umes were again measured 3 h after the challenge of cara-
geenan. The percent inhibition of paw volume for each rat
in treated groups was calculated by comparing with mean
paw volume of control group and expressed as mean (ꢂSE)
percent inhibition of paw volume for each test group.
References
[1] W.L. Xie, J.G. Chipman, D.L. Robertson, R.L. Erikson, D.L. Somons,
Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 2692e2696.
[2] D.A. Kujubu, B.S. Fletcher, B.C. Vernum, R.W. Lim, H.R. Herschman,
J. Biol. Chem. 266 (1991) 12866e12872.
[3] H.R. Herschman, Biochim. Biophys. Acta 1299 (1996) 125e140.
[4] R.G. Mehta, R.C. Moon, Anticancer Res. 11 (1991) 593e596.
[5] M.J. Thun, M.M. Namboodiri, C.W. Heath Jr., N. Engl. J. Med. 325
(1991) 1593e1596.
[6] R.E. Harris, M.M. Namboodiri, W.B. Farrar, Epidemiology 7 (1996)
203e205.
[7] A.J. Minn, G.P. Gupta, P.M. Siegel, P.D. Bos, W. Shu, D.D. Giri,
A. Viale, A.B. Olshen, W.L. Gerald, J. Massague, Nature 436 (2005)
518e524.
[8] Y. Kang, W. He, S. Tulley, G.P. Gupta, I. Serganova, C.R. Chen,
K.M. Tadorova, R. Blasberg, W.L. Gerald, J. Massague, Proc. Natl.
Acad. Sci. U.S.A. 102 (2005) 13909e13914.
[9] J.L. Masferrer, K.M. Leahy, A.T. Koki, B.S. Zwiefel, S.L. Settle,
B.M. Woerner, D.A. Edwards, A.G. Flickinger, R.J. Moore, K. Seibert,
Cancer Res. 60 (2000) 1306e1311.
[10] R.E. Harris, G.A. Alshafie, H. Abou-Issa, K. Seibert, Cancer Res. 60
(2000) 2101e2103.
5.6.2. Assay method of cytotoxic activity in vitro
[11] C.H. Liu, S.H. Chang, K. Narko, O.C. Trifan, M.T. Wu, E. Smith,
C. Handenschild, T.F. Lane, T. Hla, J. Biol. Chem. 276 (2001) 18563e
18569.
U937 human histocytic lymphoma cell line was obtained
from cell line bank of National Center for Cellular Sciences
(NCCS), Pune, India. These cells were cultured in RPMI-
1640 media containing 10% fetal bovine serum at 37 ꢀC,
CO2 incubator in the presence or absence of test compounds.
Cytotoxicty was measured using the MTT [3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl tetrasolium bromide] assay, according
to the method of Mosmann (1983) [46]. Briefly: the cells
(2 ꢃ 104) were seeded in each well containing 0.1 ml of
RPMI medium in 96 well plates. After 24 h different test con-
centrations (10e100 mg/ml) were added and cell viability was
assessed after two days, 10 ml per well of MTT (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide; 5 mg/ml;
stock solution, Sigma) was added to the wells. The plates
were incubated at 37 ꢀC for additional 4 h. The medium was
discarded and the formazan blue, which formed in the cells
was dissolved with 100 ml of DMSO. The rate of color produc-
tion was measured at 570 nm in a spectrophotometer (Spectra
MAX Plus; Molecular Devices; supported by SOFTmax PRO-
3.0). All experiments were conducted under the standard lab-
oratory illumination. The percent inhibition of cell viability
was determined with reference to the control values (without
test compound). The data were subjected to linear regression
analysis and the regression lines were plotted for the best fit.
The IC50 (inhibition of cell viability) concentrations were cal-
culated using the respective regression equation.
[12] A.C. Tinker, H.G. Beaton, N.B. Smith, T.R. Cook, S.L. Cooper, L.F. Rae,
K. Hallam, P. Hamley, T. McInally, D.J. Nicholls, A.D. Pimm,
A.V. Wallace, J. Med. Chem. 46 (2003) 913e916.
[13] Moorthy S.S. Palanki, J. Mark Suto, Patent WO-9901441, Appl WO 98
e US 13483, CAN 130:125085.
[14] M. Tobe, Y. Isobe, H. Tomizawa, M. Matsumoto, F. Obara, T. Nagasaki,
H. Hayashi, Bioorg. Med. Chem. Lett. 11 (2001) 545e548.
[15] G.M. Buckley, N. Davies, H.J. Dyke, P.J. Gilbert, D.R. Hannah,
A.F. Yaughan, C.A. Hunt, W.R. Pitt, R.H. Profit, N.C. Ray,
M.D. Richard, A. Sharpe, A.J. Taylor, J.M. Whitworth, S.C. Williams,
Bioorg. Med. Chem. Lett. 15 (2005) 751e754.
[16] A. Schlapbach, R. Heng, F.D. Padov, Bioorg. Med. Chem. Lett. 14 (2004)
357e360.
[17] C.D. Timothy, H. Xiao, M. Louise Smith, R. Michael Degraffenreid, Jay
Powers, S. Craig Tomooka, D.L. Clark, Xiaolin Hao, J.C. Juan, Marc La-
belle, P.C. Nigel Walker, L. Adrian Gill, X. Francisco Talamas, S. Shar-
ada Labadie, Patent WO 2003048152, Appl. WO 2002 e US 39134,
CAN 139:36536.
[18] V.D. Piaz, M.P. Giovannoni, Eur. J. Med. Chem. 35 (2000) 463e480.
[19] H.B. Cottam, H. Shih, L.R. Tehrani, B. Wasson, D.A. Carson, J. Med.
Chem. 39 (1996) 2e9.
[20] D.P. Jindal, R.S. Bhatti, S. Ahlawat, R. Gupta, Eur. J. Med. Chem. 37
(2002) 419e425.
[21] J.C. Sircar, T. Capiris, S.J. Kesten, D.J. Herzig, J. Med. Chem. 24 (1981)
735e742.
[22] J.W. Perrine, W.J. Houlihan, E.I. Takesue, Arzneim. Forsch./Drug Res.
34 (1984) 879e885.
[23] A.J. Ashcroft, F.E. Davies, G.J. Morgan, Lancet Oncol. 4 (2003) 284e
292.

852
P.M. Chandrika et al. / European Journal of Medicinal Chemistry 43 (2008) 846e852
[24] C. Marcelli, D. Chappard, J.F. Rossi, J. Jaubert, C. Alexandre,
P. Dessauw, P. Baldet, R. Bataille, Cancer 62 (1988) 1163e1170.
[25] A.E. Wakeling, S.P. Guy, J.R. Woodburn, S.E. Ashton, B.J. Curry,
A.J. Barker, K.H. Gibson, Cancer Res. 62 (2002) 5749e5754.
[26] S.T.A. Rashood, I.A. Aboldahab, M.N. Nagi, L.A. Abouzeid,
A.A.M.A. Aziz, S.G.A. Hamide, K.M. Youssef, A.M.A. Obaid,
H.I.E. Subbagh, Bioorg. Med. Chem. 14 (2006) 8608e8621.
[27] L.F. Hennequin, J. Allen, J. Breed, J. Curwen, M. Fennell, T.P. Green,
C.L. Brempt, R. Morgentin, R.A. Norman, A. Oliver, L. Otterbein,
P.A. Ple, N. Warin, G. Costello, J. Med. Chem. 49 (2006) 6465e6488.
[28] F.H. Jung, G. Pasquet, C.L. Brempt, J.M. Lohmann, N. Warin, F. Renaud,
H. Germain, C.D. Savi, N. Roberts, T. Johnson, C. Dousson, G.B. Hill,
A.A. Mortlock, N. Heron, R.W. Wilkinson, S.R. Wedge, S.P. Heaton,
S. Brightwell, J. Med. Chem. 49 (2006) 955e970.
[33] A.R.R. Rao, R.H. Bahekar, Indian J. Chem. 38 (1999) 434e439.
[34] K. Ozaki, Y. Yamada, T. Oine, T. Ishizuka, Y. Iwasawa, J. Med. Chem. 28
(1985) 568e576.
[35] Y.S. Sadanandam, K. Ram Mohan Reddy, A. Bhaskar Rao, Eur. J. Med.
Chem. 22 (1987) 169e173.
[36] A. Kumar, S. Sharma, Archana, Kiran Bajaj, S. Sharma, H. Panwar,
T. Singh, V.K. Srivastava, Bioorg. Med. Chem. 11 (2003) 5293e5299.
[37] P. Rani, Archana, V.K. Srivastava, A. Kumar, Indian J. Chem. 41B (2002)
2642e2646.
[38] A.R.R. Rao, R.H. Bahekar, Arzneim. Forsch./Drug Res. 51 (2001) 284e
292.
[39] D. Maitraie, G.V. Reddy, V.V.V.N.S.R. Rao, S.R. Kanth, S. Rao,
B. Narsaiah, J. Fluorine Chem. 73 (2002) 118.
[40] S.R. Kanth, G.V. Reddy, K.H. Kishore, P.S. Rao, B. Narsaiah,
U.S.N. Murthy, Eur. J. Med. Chem. 41 (2006) 1011e1016.
[41] T. Yakaiah, B.P.V. Lingaiah, B. Narsaiah, B. Shireesha, B. Ashok Kumar,
S. Gururaj, T. Parthasarathy, B. Sridhar, Bioorg. Med. Chem. Lett. 17
(2007) 3445e3453.
[29] S. Jantova, M. Urbancikova, T. Maliar, M. Mikulasova, P. Ranko,
L. Cipak, J. Kubikova, S. Stankovsky, K. Spirkova, Neoplasma 48
(2001) 52e60.
[30] A.J. Bridger, H. Zhou, D.R. Cody, G.W. Rewcastle, A.M. Michael,
H.D. Showalter, D.W. Fry, A.J. Kraker, W.A. Denny, J. Med. Chem.
39 (1996) 267e276.
[42] T. Yakaiah, B.P.V. Lingaiah, B. Narsaiah, K. Pranay Kumar,
U.S.N. Murthy, Eur. J. Med. Chem. 43 (2008) 341e347.
[43] S.A. Rocco, J.E. Barbarini, R. Rittner, Synthesis 3 (2004) 429e435.
[44] C.A. Winter, E.A. Risley, G.W. Nuss, J. Pharmacol. Exp. Ther. 141
(1963) 369e376.
[45] P.V. Diwan, I. Karwande, I. Margaret, P.B. Sattur, Indian J. Pharmacol.
21 (1989) 1.
[46] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
[31] I.M. Sielecki, T.L. Johnson, J. Liu, J.K. Muckelbauer, R.H. Grafstrom,
S. Cox, J. Boylan, C.R. Burton, H. Chen, A. Smallwood, C. Chang,
M. Boisclair, P.A. Benfield, G.L. Trainor, S.P. Seitz, Bioorg. Med.
Chem. Lett. 11 (2001) 1157e1160.
[32] S. Jantova, L. Ruzekova, K. Spirkova, S. Stankovsky, Biol. Brat. 52
(1997) 445e450.