Synthesis and anticancer activity of some new s-triazine derivatives

Article abstract of DOI:10.1007/s00044-013-0584-6

New s-triazine derivatives 13a-h were synthesized for the structure-activity relationship studies as potent anticancer agents. The prepared analogues were evaluated for their in vitro inhibitory activity against the growth of PA-1 (Ovarian cancer), A549 (Lung cancer), MCF-7 (Breast cancer), and HT-29 (Colon cancer). Tri-substituted s-triazine derivatives (13e-h) with morpholino group on s-triazine scaffold exhibited potent anticancer activities compared to di-substituted s-triazine derivatives. Compounds 13e-h also showed relatively selective PA-1 and HT-29 cancer cell inhibition over other cancer cell lines. Structure-activity relationships provided useful insights in these classes of compounds and paved the way to design novel analogues with more potency.

Full text of DOI:10.1007/s00044-013-0584-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0584-6
ORIGINAL RESEARCH
Synthesis and anticancer activity of some new s-triazine
derivatives
G. Jagadeesh Kumar
E. Srihari Shweta Shrivastava
Kolupula Srinivas V. Jayathirtha Rao
•
H. V. S. Sriramkumar Bomma
•
•
•
V. G. M. Naidu
•
•
Received: 10 December 2012 / Accepted: 13 March 2013
Ó Springer Science+Business Media New York 2013
Abstract New s-triazine derivatives 13a–h were
synthesized for the structure–activity relationship studies
as potent anticancer agents. The prepared analogues were
evaluated for their in vitro inhibitory activity against the
growth of PA-1 (Ovarian cancer), A549 (Lung cancer),
MCF-7 (Breast cancer), and HT-29 (Colon cancer).
Tri-substituted s-triazine derivatives (13e–h) with mor-
pholino group on s-triazine scaffold exhibited potent
anticancer activities compared to di-substituted s-triazine
derivatives. Compounds 13e–h also showed relatively
selective PA-1 and HT-29 cancer cell inhibition over other
cancer cell lines. Structure–activity relationships provided
useful insights in these classes of compounds and paved the
way to design novel analogues with more potency.
Introduction
s-Triazine is a six-membered heterocyclic ring, with three
nitrogens situated at 1st, 3rd and 5th positions. Its analogues,
melamine, cyanuric acid and cyanuric chloride are important
starting compounds for various materials with wide range of
applications in textile, plastic, pharmaceuticals and rubber
industries. These compounds are also used as pesticides,
dyestuffs, optical bleaches, explosives and surface active
agents (Bartholomew, 1996; Comins and O’Connor, 1988;
Quirke, 1984; Smolin and Rapport, 1959). s-Triazine
compounds have been studied extensively and are the subject
of many reviews (Blotny, 2006; Giacomelli et al., 2004;
Sathiakumar et al., 2011; Therrien, 2010). This s-triazine
scaffold attracted many researchers, as its symmetrical
structure facilitates to synthesize diverse set of analogues
(ease of synthesis) such as 2, 4, 6-mono, di- or tri-substituted,
symmetrical and nonsymmetrical compounds bearing
different substituentsandinparticular, cyanuricchlorideisthe
most important one in this aspect (Blotny, 2006). The s-tri-
azine scaffold also provides the basis for the design of bio-
logically relevant molecules with widespread application as
therapeutics. For example, these compounds possess potent
antiprotozoal (Klenke et al., 2001), antimalarial (Manohar
et al., 2010; Melato et al., 2008), antiviral (Mahajan et al.,
Keywords S-triazine derivatives Á Anticancer activity Á
Structure–activity relationships Á Hit identification
G. Jagadeesh Kumar Á H. V. S. Sriramkumar Bomma Á
E. Srihari Á V. Jayathirtha Rao (&)
Crop Protection Chemicals Division, CSIR-Indian Institute
of Chemical Technology, Uppal Road Tarnaka,
Hyderabad 500607, India
2
009; Maarouf et al., 2012; Xiong et al., 2008), anticancer
Baindur et al., 2005; Leftheris et al., 2004; Manohar et al.,
010; Moon et al., 2002; Sun et al., 2010; Zheng et al., 2007),
(
e-mail: jrao@iict.res.in
2
G. Jagadeesh Kumar Á H. V. S. Sriramkumar Bomma Á
S. Shrivastava Á V. G. M. Naidu (&) Á K. Srinivas (&) Á
V. Jayathirtha Rao
National Institute of Pharmaceutical Education and Research
(NIPER), Balanagar, Hyderabad 500037, India
e-mail: vgmnaidu@niperhyd.ac.in
antimicrobial (Patel et al., 2012a, b, c; Raval et al., 2011;
Singh et al., 2011; Srinivas et al., 2005, 2006; Desai et al.,
2008), antituberculosis (Patel et al., 2012a; Sunduru et al.,
2010) etc. Recently, the s-triazine derivatives were extensively
investigated for anticancer activity with a particular target
to mTOR/PI3K pathway (Menear et al., 2009; Poulsen
et al., 2012; Tanneeru et al., 2012; Venkatesan et al., 2010;
K. Srinivas
e-mail: ksrinivas07@yahoo.com; skolupula@niperhyd.ac.in
123

Med Chem Res
Verheijen et al., 2010; Zask et al., 2010; Zhang et al., 2011).
Some of the compounds possessing s-triazine scaffold reported
in the literature are presented in Fig. 1. Our continuous interest in
the field of bioactive heterocyclic compounds (Kumar et al.,
coupling reaction between mono-substituted s-triazine
derivatives (8–10 & 12) and one equivalent of compound 3
in the presence of K CO (Scheme 3). Compounds 13e–
2
3
h were synthesized by the replacement of third chlorine
atom of di-substituted s-triazine derivatives (13a–d) in the
presence of K CO (Scheme 4). All the intermediates and
2010, 2011; Ravinder et al., 2012; Rao et al., 2011; Srinivas
et al., 2009), we have got interest in new s-triazine derivatives as
cancer therapeutics. In the context of identification of new
chemical entities (NCEs) for cancer therapy, we have selected
s-triazine as core scaffold as many reports indicate its signifi-
cance (Fig. 1) and structural modifications were made at three
positions (1st, 3rd and 5th positions) with various pharmaco-
phores. In this article, synthesis of new s-triazine derivatives is
presented. All the synthesized compounds were screened against
four cancer cell lines and discussed to develop the structure–
activity relationships (SAR) of this series. Among all, com-
pounds 13e–h exhibited potent inhibitory activity against
ovarian (PA-1) and colon (HT-29) cancer cell lines
2
3
final compounds were purified by column chromatography
and characterized by spectroscopic techniques.
Biology
The anticancer activities of all the synthesized compounds
were evaluated by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide] method using PA-1
(Ovarian cancer), A549 (Lung cancer), MCF-7 (Breast
cancer) and HT-29 (Colon cancer) cell lines. The effect of
compounds on the cell viability for each cell line after
exposure to different concentrations were depicted in the
Fig. 2 and their respective IC50 values are presented in
Table 1. Synthesized compounds can be classified into two
categories based on the presence of the chlorine atom and
morpholino group i.e. di-substituted s-triazine derivatives
(13a–d) and tri-substituted s-triazine derivatives (13e–h),
respectively. Among the two categories, tri-substituted
s-triazine derivatives were more active against in all tested
human cancer cell lines than di-substituted s-triazine
derivatives which suggest the key role of morpholino group
for activities. In case of PA-1 cell lines, among 13e–h,
compounds 13e–g are more potent and are equally potent
as Doxorubucin. This indicates the importance of substi-
tuted aniline groups on s-triazine compared to benzylamine
group. The activity difference in compounds 13e and 13h is
more than one magnitude (13e: 0.61 ± 0.16 lM and 13h:
10.5 ± 0.51 lM) and this attributes to bridge between
Chemistry
4-(Benzo[d]oxazol-2-yl) aniline (3) was prepared from the
simple condensation of o-amino phenol (1) with p-amino
benzoic acid in the presence of polyphosphoric acid at
2
00 °C (Scheme 1) (Chua et al., 1999). Nucleophilic sub-
stitution of one of the chlorines of the cyanuric acid (4)
with substituted anilines (5–7 & 11) in the presence of
diisopropylehtylamine (DIPEA) yielded mono-substituted
triazine derivatives 8–10 & 12, respectively, in good yields
(
mono substitution w.r.t. cyanuric chloride) and the syn-
thetic scheme is presented in Scheme 2 (Zheng et al.,
007). Compounds 8–10 and 12 were reported in the lit-
2
erature (Hunter et al., 1994; McKay et al., 2006; Maga
et al., 2011). Synthesis of compounds 13a–d involves a
Fig. 1 List of potent anticancer
compounds possessing
s-triazine scaffold
N
F2HC
H
N
N
N
OH
N
N
N
S
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
N
N
N
N
O
A
B
C
D
Altrtamine (Hexalene) Triethylene Melamine (TEM)
Foster BJ, et. al., 1986 Luippold HE, et. al., 1978 Yaguch S, et. al., 2006
ZSTK474 (Phase 1)
Baindur N, et. al., 2005
O
N
O
H3CO
O
N
NH
HN
O
N
OH
H3CO
N
N
N
O
N
N
N
O
N
N
N
N
H
H
N
N
N
H
H3CO
O
E
F
G
Keith A, et. al., 2009
Moon HS, et. al., 2002
Leftheris K, et. al., 2004
1
23

Med Chem Res
s-triazine and substituted group i.e. –NH– and –NH–CH2–
groups, respectively. The di-substituted s-triazine deriva-
tives (13a–d) were inactive against A549 (lung cancer) cell
lines, whereas tri-substituted derivatives have exhibited
relatively better inhibitory activity. Among the compounds
(Fig. 1) and these are more potent than reference com-
pound, doxorubicin. Among the 13e–h, compound 13e–
g are more potent with IC50 of 0.27 ± 0.02, 0.14 ± 0.04
and 0.31 ± 0.15 lM, respectively and compound 13h is
relatively less potent with IC50 at 0.92 ± 0.51 lM
(Table 1). The disparity in magnitude of activity within
13e–h is due to the structural dissimilarity i.e. the presence
of anilino and substituted anilino groups in case of 13e–
g and the presence of benzylamino group in case of 13h. In
summary, compounds 13e–h exhibited selective cytotox-
icity in HT-29 & PA-1 when compared to other cell line
(A549 and MCF-7) and compound 13f is found to be most
potent in respective cell lines. Structure–activity relation-
ships (SAR) reveals that the presence of morpholino group,
substituted aniline groups along with benzoxazole moiety
on s-triazine ring are essential to exhibit potent anticancer
activity. Structural modifications on aniline group and
benzoxazole group can provide more insights and give
better hits/leads and focus of our future work is on struc-
tural modifications on these classes of compounds to
1
3e–h, compound 13f is relatively potent with IC50 at
2.9 ± 2.43 lM and other compounds (13e, 13g–
1
h) exhibited twofolds less inhibitory activity. In case of
MCF-7 cell lines, compounds 13f and 13h are more potent
than reference compound, doxorubicin. Among the all
compounds in the series, 13h is most potent. Compounds
13e and 13g exhibited moderate anticancer activity against
MCF-7 cell lines. Compounds 13e–h exhibited potent
anticancer activity against HT-29 (Colon cancer) cell lines
NH2
NH2
N
PPA
H2N
2
00°C
OH
O
COOH
1
2
3
O
Scheme 1 Synthesis of 4-(benzo[d]oxazol-2-yl) aniline
O
N
N
N
N
N
1
3a, 13b, 13c
H2N
X
Cl
N
K CO / dry DMF
N
N
X
2
3
H
H
1
00°C / 24 h
N
N
DIPEA
CH2Cl2 / -78°C
13e: X = H
3f: X= OCH3
3g: X = F
H
N
1
1
Cl
N
X
H
5
6
7
: X = H
: X= OCH3
: X = F
8
9
1
: X = H
: X= OCH3
0: X = F
Cl
O
O
N
N
N
14
Cl
N
Cl
O
1
3d
Cl
4
N
N
N
H2N
K CO / dry DMF
2
3
N
N
1
1
1
00°C / 24 h
N
N
N
H
H
DIPEA
CH2Cl2 / -78°C
Cl
N
N
H
12
13 h
Scheme 2 Synthesis of compounds 8–10 & 12
Scheme 4 Synthesis of compounds 13e–h
Scheme 3 Synthesis of
compounds 13a–d
O
Cl
N
8
, 9, 10
K2CO3 / THF
0-80°C / 24 h
N
N
N
N
N
X
7
H
H
1
1
1
3a: X = H
3b: X= OCH3
3c: X = F
O
N
NH2
3
O
Cl
N
12
N
N
K2CO3 / THF
0-80°C / 24 h
N
N
N
7
H
H
13d
123

Med Chem Res
Table 1 Chemical structures of compounds 13a–h and their inhibitory effects on the growth of tumour cell lines
R3
N
N
R1
N
R2
Comp-
ound
R
1
R
2
R
3
IC50 (µM)
a
b
c
d
PA-1
A549
MCF-7
552±±ꢁ2±1
HT-29
ꢁ±26±±265
1
1
1
1
1
3a
3b
3c
3d
3e
N
Cl
Cl
Cl
Cl
169±±2.7 ±±5±526ꢀ
1.±±62±1 ±.7±ꢁ297
176±ꢁ2±1 ±5ꢀ±62±1
195±52ꢀꢁ ±96±±2ꢀ5
0261±0216 ±±20±1211
NH
HN
HN
HN
HN
HN
O
N
±692.±6297 1ꢁ9±52ꢀ.
H3CO
F
NH
O
N
672ꢀ±ꢁ2ꢀ1
6621±±2ꢀ7
ꢀ.±±25ꢀ
ꢀꢀ±ꢁ259
NH
O
N
NH
O
N
O
10256±1256 02±.±020±
NH
O
N
O
N
O
02±5±0219 1±29±±2ꢀꢁ
02ꢀ5±02±1 ±62.±026.
1025±0251 ±ꢁ2ꢀ±ꢂ12.0
026ꢀ±021ꢁ 1277±0256
d
62ꢀ7±02±1
021ꢀ±020ꢀ
1
1
1
3f
H3CO
F
NH
HN
HN
HN
N
O
N
O
1±2ꢁ9±12ꢁ7 02ꢁ1±0215
3g
3h
NH
N
O
NH
N
O
±2ꢀ6±0215
1029±12.6
029±±0251
12.6±02±ꢁ
N
Doxorubicinꢂ((Referenceꢂcompound)
a
b
c
Ovarian cancer cells; Lung cancer cells; Breast cancer cells, Colon cancer cells
improve the anticancer activity and on understanding of
this activity through mechanistic studies.
Experimental
Chemistry
Conclusions
Synthesis of 4-(benzo[d]oxazol-2-yl) aniline (3)
Chua et al., 1999; Shi et al., 1996)
(
In conclusion, two series of s-triazine derivatives i.e. di-
substituted (13a–d) and tri-substituted derivatives (13e–
h) were synthesized as anticancer agents. All the synthe-
sized compounds were screened against four cancer cell
lines viz. PA-1 (Ovarian cancer), A549 (Lung cancer),
MCF-7 (Breast cancer) and HT-29 (Colon cancer). This
preliminary study demonstrated that tri-substituted deriva-
tives 13e–h exhibited potent anticancer activity over the di-
substituted derivatives. Among these derivatives, com-
pound 13f is the most potent in respective cell lines.
Structure–activity relationships in these classes of com-
pounds laid foundations to develop more potent com-
pounds as leads for cancer chemotherapy.
To a stirring solution of polyphosphoric acid (PPA; 85 g),
2-Aminothiophenol 1 (5.78 g, 0.053 mol), para amino
benzoic acid 2 (7.26 g, 0.053 mol) were added, heated at
220 °C for 4 h. After confirming the reaction completion
by TLC (Thin Layer Chromatography), it was cooled, and
poured into ice-cold 10 % aqueous sodium carbonate. The
solid product was collected, washed with water and
recrystallized with methanol water. Yield: 65 %; mp
1
180–183 °C; H NMR (CDCl , 300 MHz): d 8.07–8.03 (d,
3
2H, J 8.3 Hz), 7.73–7.67 (m, 1H), 7.55–7.49 (m, 1H),
7.31–7.27 (m, 2H), 6.78–6.74 (d, 2H, J 9.0 Hz) and 4.05
(brs, 2H); ESI–MS: m/z 211 (M ? H).
1
23

Med Chem Res
Fig. 2 Dose response of
compounds (13a–13h) against
a, b PA-1, c, d A549, e, f MCF-
(
a) 100
(b) 100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
7
, g, h HT-29 cancer cell lines
0
0
0
50
50
50
100
150
200
250
250
250
300
300
300
0
0
0
0
10
20
30
40
40
80
50
Concentrationꢂ(µM)
Concentrationꢂ(µM)
1
1
ꢁa
ꢁb
1ꢁc
1ꢁd
1ꢁe
1ꢁh
1ꢁf
DOX
1ꢁg
(
c) 100
(d) 100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
100
150
200
10
20
30
50
Concentrationꢂ(µM)
Concentrationꢂ(µM)
1
1
ꢁa
ꢁb
1ꢁc
1ꢁd
1ꢁe
1ꢁf
1ꢁh
DOX
1
ꢁg
(
e) 100
(f) 100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
100
150
200
20
40
60
100
Concentrationꢂ(µM)
Concentrationꢂ(µM)
1
ꢁa
1ꢁc
1ꢁe
1ꢁf
1ꢁh
1
ꢁb
1ꢁd
DOX
1
ꢁg
(
g) 100
(h) 100
8
6
4
2
0
0
0
0
0
80
60
40
20
0
0
50
100
150
200
10
20
30
40
50
Concentrationꢂ(µM)
Concentrationꢂ(µM)
1
1
ꢁa
ꢁb
1ꢁc
1ꢁd
1
1
1
ꢁe
1ꢁh
ꢁf
DOX
ꢁg
123

Med Chem Res
General procedure for synthesis of mono-substituted
cyanuric chloride derivatives (Zheng et al., 2007)
1H), 10.00 (brs, 1H), 8.13–8.07 (m, 3H,), 7.90 (s, 1H),
7.70–7.55 (m, 4H), 7.5 (s, 1H), 7.35 (m, 2H), 6.90 (m, 2H)
1
and 3.82 (s, 3H); C NMR (75 MHz, CDCl ? DMSO-
3
3
Compounds 8, 9, 10 and 12 were synthesized using cyan-
uric chloride 4 (10 g, 0.054 mol) in dichloromethane
d6): d 165.1, 164.3, 164.3, 155.0, 151.9, 142.3, 142.2,
133.3, 133.2, 127.1, 123.4, 122.2, 121.9, 119.8, 119.7,
?
113.8, 79.6, 79.3, 79.1, 78.7; ESI–MS: m/z 445 [M ? H] ;
(DCM), N,N- Di- isopropyl ethylamine (DIPEA). DIPEA
(9.26 ml, 0.054 mol) was added slowly drop wise to the
?
HRMS (ESI) m/z Calcd. for C H ClN O [M ? H]
18
2
3
6 2
reaction mixture at -78 °C (maintained by using acetone
and dry ice) for 10 min then it was allowed to stir for
445.1102, found 445.1112.
2
N -(4-(benzo[d]oxazol-2-yl)phenyl)-6-chloro-N -(4-fluoro-
4
1
0 min. Aniline 5 (4.94 ml, 0.054 mol) or 4-methoxyani-
line 6 (6.66 g, 0.054 mol) or 4-fluoroaniline 7 (5.19 ml,
.054 mol) or benzyl amine 11 (5.9 ml, 0.054 mol) were
phenyl)-1,3,5-triazine-2,4-diamine (13c):
mp: 268–269 °C; IR (KBr): 3,384, 3,111, 1,508, 1,242 and
Yield: 77 %;
0
-
1 1
added respectively and allowed to stir for 15 min. The
reaction was monitored by TLC. After completing the
reaction, the reaction mixture was filtered, washed with
water, extracted with chloroform, the organic layer was
dried over Na SO and then concentrated to get the desired
995 cm ; H NMR (300 MHz, DMSO-d ): d 9.42 (brs,
6
1H), 9.16 (brs, 1H), 8.18–8.12 (m, 4H), 7.69–7.67 (m, 2H),
7.59–7.57 (m, 1H), 7.32–7.29 (m, 3H), 7.05–7.01 (t, 2H,
1
3
J 8.5, 17.0 Hz); C NMR (75 MHz, CDCl ? DMSO-d ):
3
6
2
4
d 162.1, 160.5, 148.5, 140.1, 132.8, 126.0, 122.9, 122.6,
121.5, 119.2, 118.5, 117.6, 113.3, 108.6, 93.9; ESI–MS:
mono-substituted s-triazines in excellent yield. Compounds
?
m/z 433 [M ? H] ; HRMS (ESI) m/z Calcd. for
8
–10 and 12 were reported in the literature (Hunter et al.,
994; McKay et al., 2006; Maga et al., 2011).
?
C H ClFN O [M ? H] 433.8376, found 433.8362.
1
2
2
15
6
2
4
N -(4-(benzo[d]oxazol-2-yl)phenyl)-N -benzyl-6-chloro-1,3,5-
General procedure for synthesis of di-substituted
s-triazines
triazine-2,4-diamine (13d):
Yield: 76 %; mp: 278-
2
79 °C; IR (KBr): 3,268, 3,111, 2,993, 1,240 and
-
1 1
Di-substituted cyanuric chloride derivatives (13a–d), were
synthesized using mono-substituted cyanuric chloride 8
987 cm ; H NMR (75 MHz, CDCl ): d 8.30–8.26 (m,
3
1H), 8.22–8.17 (m, 2H), 7.78–7.74 (m, 3H), 7.58–7.56 (m,
(
(
1 eq), 9 (1 eq), 10 (1 eq) and 12 (1 eq) in dry THF
30 ml), K CO (2 eq). The reaction mixture was allowed
1H), 7.40–7.39 (m, 2H), 7.37–7.31 (m, 4H), 6.05 (brs, 1H),
1
3
2
3
5.77 (brs, 1H) and 4.72–4.66 (t, 2H, J 5.2, 10.5 Hz);
NMR (75 MHz, CDCl ? DMSO-d ): d 148.9, 140.9,
C
to stir for 5 min then the benzoxazolephenylamine 3 (1 eq)
was added and refluxed for 24 h at 70–80 °C. After con-
firming the reaction completion by TLC, K CO was
3
6
140.6, 126.9, 126.8, 126.4, 125.6, 125.5, 118.4, 118.3,
2
3
118.0, 117.9, 117.8, 109.1, 109.0; ESI–MS: m/z 429
?
[M ? H] ; HRMS (ESI) m/z Calcd. for C H ClN O
decanted and the THF was removed, water was added and
extracted with EtOAc. The organic layer was dried over
Na SO , concentrated and purified to give the desired di-
2
3
18
6
?
[M ? H] 429.8737, found 429.8725.
2
4
substituted s-triazines.
General procedure for synthesis of tri-substituted
s-triazines
2
N -(4-(benzo[d]oxazol-2-yl)phenyl)-6-chloro-N -phenyl-1,3,5-
4
triazine-2,4-diamine (13a): Yield: 80 %; mp: 263–265 °C;
Tri-substituted s-triazine derivatives (13e–h) were syn-
thesized from di-substituted cyanuric chloride derivatives
13a–d (1 eq) in dry DMF (20 ml), K CO (2 eq). The
-
1
IR (KBr): 3,273, 3,109, 1,610, 1,585, 1,243 and 989 cm
1
;
H NMR (300 MHz, DMSO-d ): d 10.32 (brs, 1H), 10.03
6
2
3
(
brs, 1H) 8.11 (m, 2H), 7.93 (m, 2H), 7.74–7.68 (m, 2H),
reaction mixture was allowed to stir for 5 min then mor-
pholine 14 (1 eq) was added and stirred for 24 h at 100 °C.
The reaction was monitored by TLC. After completion of
the reaction, the mixture was poured into ice-cold water,
extracted with ehtylacetate and dried over Na SO . Solvent
1
3
.57 (m, 1H) and 7.32 (m, 4H); C NMR (75 MHz,
7
DMSO-d ): d 162.1, 150.1, 150.0, 142.0, 141.5, 128.8,
1
6
28.5, 127.8, 125.0, 124.7, 120.6, 120.4, 119.4, 110.8,
?
10.7; ESI–MS: m/z 415 [M ? H] ; HRMS (ESI) m/z
1
2
4
?
Calcd. for C H ClN O [M ? H] 415.8471, found
2
2
16
6
was evaporated and solid was purified to get desired
product in good yield.
4
15.8342.
2
N -(4-(benzo[d]oxazol-2-yl)phenyl)-6-chloro-N -(4-methoxy-
4
2
4
N -(4-(benzo[d]oxazol-2-yl)phenyl)-6-morpholino-N -phe-
nyl-1,3,5-triazine-2,4-diamine (13e): Yield: 82 % mp:
259–260 °C; IR (KBr): 3,402, 3,299, 2,925–2,855 and
phenyl)-1,3,5-triazine-2,4-diamine (13b):
mp: 275–276 °C; IR (KBr): 3,278, 3,185, 2,835, 1,232 and
Yield: 82 %;
-
1
1
-1 1
9
92 cm ; H NMR (300 MHz, DMSO-d ): d 10.36 (brs,
1,242 cm ; H NMR (300 MHz, DMSO-d ): d 9.08 (brs,
6
6
1
23

Med Chem Res
1
H), 8.67 (brs, 1H), 8.14–8.11 (d, 2H, J 8.6 Hz), 7.98–7.96
d, 2H, J 8.4 Hz), 7.72–7.70 (t, 3H, J 7.9, 5.3 Hz),
Biological evaluation
(
7
.61–7.58 (t, 1H, J 7.1, 14.3 Hz), 7.35–7.27 (m, 4H),
.03–6.98 (m, 1H), 3.84 (d, 4H, J 3.2 Hz), 3.75 (d, 4H,
Materials
7
1
3
J 3.3 Hz);); C NMR (75 MHz, CDCl ? DMSO-d ): d
PA-1 (Ovarian cancer), A549 (Lung cancer) cell lines
MCF-7 (Breast cancer) and HT-29 (Colon cancer) cell line
were obtained from the National center for Cell science
(NCCS), Pune, India. MEM, DMEM, RPMI, MTT [3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide],
Trypsin–EDTA were purchased from Sigma Chemicals Co
3
6
1
1
1
64.5, 163.9, 163.9, 162.4, 150.0, 143.7, 141.6, 139.8,
28.3, 127.8, 124.8, 124.6, 122.1, 122.0, 119.5, 119.3,
?
18.9, 10.6, 65.9, 43.4; ESI–MS: m/z 466 [M ? H] ;
?
HRMS (ESI) m/z Calcd. for C H N O [M ? H]
2
6 24 7 2
4
66.5065, found 466.5023.
(
st. Louis, MO), Fetal bovine serum were purchased from
2
4
N -(4-(benzo[d]oxazol-2-yl)phenyl)-N -(4-methoxyphenyl)-6-
morpholino-1,3,5-triazine-2,4-diamine (13f): Yield: 80 %;
mp: 252–253 °C; IR (KBr): 3,400, 3,307, 2,963, 2,922,
Gibco, USA, 96-well flat bottom tissue culture plates were
purchased from Tarson.
-
1 1
2
9
7
,855 and 1,227 cm ; H NMR (300 MHz, DMSO-d ): d
.52 (brs, 1H), 9.08 (brs, 1H), 8.04–7.99 (d, 2H, J 8.3 Hz),
.97–7.92 (m, 2H), 7.72–7.66 (m, 2H), 7.53–7.50 (d, 2H,
Maintenance of cell lines
6
PA-1 (Ovarian cancer) A549 (Lung cancer) cell line were
grown as adherent in MEM medium, MCF-7 (Breast can-
cer) cell line was grown as adherent in DMEM medium
and HT-29 (Colon cancer) cell line was grown as adherent
in RPMI medium supplemented with 10 % fetal bovine
serum, 100 lg/ml penicillin, 200 lg/ml streptomycin,
2 mM L-glutamine, and culture was maintained in a
J 8.3 Hz), 7.35–7.26 (m, 2H), 3.8 (m, 7H) and 3.7 (d, 4H,
1
J 4.5 Hz); C NMR (75 MHz, CDCl ? DMSO-d ): d
3
3
6
1
1
5
64.6, 163.8, 154.4, 151.4, 141.6, 132.6, 126.6, 122.8,
21.6, 121.3, 119.2, 113.3, 79.0, 78.8, 78.6, 78.1, 65.9,
?
4.9, 43.3; ESI–MS: m/z 496 [M ? H] ; HRMS (ESI) m/z
?
Calcd. for C H N O [M ? H] 496.2325, found
27 26 7 3
4
66.2091.
humidified atmosphere with 5 % CO .
2
2
4
N -(4-(benzo[d]oxazol-2-yl)phenyl)-N -(4-fluorophenyl)-6-
morpholino-1,3,5-triazine-2,4-diamine (13g): Yield: 84 %;
mp: 246–247 °C; IR (KBr): 3,424, 3,297, 2,924–2,856,
Preparation of samples for cytotoxicity
Stock solution for compounds 13a–13d of 30 mM and
compounds 13e–13f of 10 mM stock solution in DMSO
were prepared, from the above stock various dilutions were
made with sterile PBS to get required concentration.
-
1 1
1
,508 and 1,243 cm ; H NMR (75 MHz, DMSO-d ): d
6
8
.22–8.19 (d, 2H, J 8.3 Hz), 7.75–7.72 (m, 3H), 7.58–55
m, 1H), 7.53–7.48 (m, 2H), 7.37–7.31 (m, 2H), 7.08–7.02
m, 3H), 6.99 (brs, 1H) 6.79 (brs, 1H) 3.84 (d, 4H,
(
(
1
3
J 4.5 Hz) and 3.76 (d, 4H, J 4.5 Hz);); C NMR
(
Cytotoxicity screening using MTT assay
300 MHz, CDCl ? DMSO-d ): d 164.6, 164.0, 163.9,
3
6
1
51.4, 143.8, 141.7, 139.8, 134.8, 128.3, 126.7, 123.2,
MTT [3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetra-
zolium bromide] assay is a standard colourimetric assay for
measuring cellular proliferation. MTT is a tetrazolium salt,
which is yellow in colour and is photosensitive. MTT [3-(4,
1
22.0, 121.9, 121.4, 120.1, 119.5. 118.3, 110.9, 6539, 43.3;
?
ESI–MS: m/z 484 [M ? H] ; HRMS (ESI) m/z Calcd. for
?
C H FN O [M ? H] 484.1970, found 484.1891.
2
6
23
7 2
5
- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bro-
2
N -(4-(benzo[d]oxazol-2-yl)phenyl)-N -benzyl-6-morpho-
4
mide] is taken by the living cells and reduced by a mito-
chondrial dehydrogenase enzyme to a purple formazan
product that is impermeable to the cell membrane. Solu-
bilisation with solvents like DMSO leads to liberation of
product and amount of purple formazan product is directly
lino-1,3,5-triazine-2,4-diamine (13h): Yield: 78 %; mp:
2
1
40–242 °C; IR (KBr): 3,403, 3,296, 2,924, 2,853 and
-1 1
,241 cm ; H NMR (300 MHz, DMSO-d ): d 9.33–9.29
6
(
brs, 2H), 8.19–8.16 (d, 2H, J 8.3 Hz), 7.71–7.68 (d, 2H,
J 8.3 Hz), 7.52–7.49 (d, 2H), 7.29–7.27 (m, 2H), 7.1 (m,
H), 7.09–7.06 (m, 1H), 6.86 (s, 1H), 6.66–6.62 (d, 2H,
J 7.9 Hz), 3.78–3.76 (d, 4H, J 4.5 Hz), 3.72–3.70 (d, 4H,
4
related to the cell viability. 1 9 10 Cells (counted by
3
Trypan blue exclusion dye method)) in 96-well plates were
incubated with compounds and standard Cisplatin with
series of concentrations for 24 h at 37 °C in MEM with
10 % FBS medium. Then, the above media was replaced
with 90 ll of fresh serum free media and 10 ll of MTT
reagent (5 mg/ml) and plates were incubated at 37 °C for
4 h, there after the above media was replaced with 200 ll
of DMSO and incubated at 37 °C for 10 min. The
1
3
J4.9 Hz) and 3.55 (s, 2H););
CDCl ? DMSO-d ): d 136.3, 163.1, 150.0, 138.6, 126.3,
C NMR (75 MHz,
3
6
1
4
25.6, 125.2, 125.0, 121.1, 119.9, 117.6, 117.5, 64.4, 42.0,
?
1.6; ESI–MS: m/z 480 [M ? H] ; HRMS (ESI) m/z
?
Calcd. for C H N O [M ? H] 480.5331, found
27 26 7 2
4
80.2103.
123

Med Chem Res
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