Synthesis, characterization and anticancer evaluation of novel tri-arm star shaped 1,3,5-triazine hydrazones

Article abstract of DOI:10.1016/j.molstruc.2011.12.023

A series of novel trisubstituted triazine hydrazones [N₃C ₃(OC₆H₄-p-CHNNHC(O)C₆H ₄-p-X)₃] (X = H, Br, Cl, F, OH, OCH₃, CH ₃, NO₂, NH₂) were prepared by a three-fold condensation reaction of 2,4,6-tris(4-formylphenoxy)-1,3,5-triazine with p-substituted benzoic acid hydrazides [NH₂NHC(O)C₆H ₄-p-X] with excellent yields. The structures were confirmed by elemental analysis, FT-IR, ¹H, ¹³C, 2D-HSQC NMR and mass spectrometry (MALDI-TOF). These derivatives bearing hydrolysable hydrazone linkages were evaluated for their in vitro antiproliferative activity against the human liver carcinoma cell line (HepG2) and human cervix carcinoma cell line (HeLa).

Full text of DOI:10.1016/j.molstruc.2011.12.023

Journal of Molecular Structure 1011 (2012) 121–127
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, characterization and anticancer evaluation of novel tri-arm star
shaped 1,3,5-triazine hydrazones
Shrinath S. Machakanur a, Basavaraj R. Patil a, Dayananda S. Badiger a, Raghavendra P. Bakale a,
Kalagouda B. Gudasi a, , S.W. Annie Bligh b
⇑
a Department of Chemistry, Karnatak University, Pavate Nagar, Dharwad, Karnataka 580 003, India
b Institute for Health Research and Policy, London Metropolitan University, Holloway Road, London N7 8DB, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2011
Received in revised form 10 December 2011
Accepted 13 December 2011
Available online 22 December 2011
A series of novel trisubstituted triazine hydrazones [N3C3(AOC6H4-p-CH@NANHAC(O)AC6H4-p-X)3]
(X = H, Br, Cl, F, OH, OCH3, CH3, NO2, NH2) were prepared by a three-fold condensation reaction of 2,4,6-tris
(4-formylphenoxy)-1,3,5-triazine with p-substituted benzoic acid hydrazides [NH2ANHAC(O)AC6H4-p-X]
with excellent yields. The structures were confirmed by elemental analysis, FT-IR, 1H, 13C, 2D-HSQC NMR
and mass spectrometry (MALDI-TOF). These derivatives bearing hydrolysable hydrazone linkages were
evaluated for their in vitro antiproliferative activity against the human liver carcinoma cell line (HepG2)
and human cervix carcinoma cell line (HeLa).
Keywords:
s-Triazine
Cyanuric chloride
Ó 2011 Elsevier B.V. All rights reserved.
2,4,6-Tris(4-formylphenoxy)-1,3,5-triazine
Hydrazone
HepG2
HeLa
1. Introduction
functional groups on the substituents attached to the 1,3,5-triazine
ring system have drawn considerable interest. Recently hyper-
The synthetic utility and much of the industrial importance of
triazine heterocycle arises from the ease by which nucleophilic aro-
matic substitutions occur on halogenated 1,3,5-triazines [1,2]. In
the synthesis of numerous compounds bearing the s-triazine core,
2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) has been largely
used as a starting material due to the ease of displacement of chlo-
rine atoms by various nucleophiles. The systematic replacement of
chlorine atoms under controlled temperature enhances the utility
of this low-cost and readily available reagent for the preparation
of mono-, di- and trisubstituted s-triazines [2,3] and offers access
to a multitude of useful molecules, from medicinal chemistry to
materials [4,5]. 1,3,5-Triazine-containing compounds are powerful
chelating agents and have been used for the preparation of various
metal complexes [6] and liquid crystals [7]. s-Triazine derivatives
have proven their great potential in supramolecular chemistry
[8]. Recently triazines have found extensive use as reagents in the
conversion of functional groups [9]. Stepwise selective substitution
pattern of cyanuric chloride is successfully utilized in the prepara-
tion of triazine based dendrimers [10].
branched polymers have been synthesized using the reactivity of
peripheral functional groups on the aryl substituents appended on
s-triazine AB2 type monomer structural units [11,12]. 2,4,6-Tris(4-
formylphenoxy)-1,3,5-triazine (Tripod) possessing three reactive
peripheral aldehyde groups is used as a template for molecular
imprinting of solid surfaces [13] and the synthesis of a three-helix
bundle protein [14]. It is also used for immobilization of proteases
[15]. Recently Tripod has been utilized for the preparation of Schiff
bases [16], tripodal-benzimidazole [17] and star-shaped small
monomer containing terminal cyanovinylene 4-nitrophenyls arms
[18]. Although some examples of formation of thiosemicarbazones
by the condensation of Tripod with thiosemicarbazides is described
[19], to our knowledge formation of hydrazones by the reaction of
aromatichydrazideswithTripod has notbeen reported and deserves
further exploration.
Hydrazones containing an azomethine (AC(O)NHN@CHA)
group are important synthons for several transformations and have
gained importance due to their broad spectrum of biological activ-
ities [20,21]. Aryl hydrazones and their metal complexes have at-
tracted attention due to their therapeutic activity and application
in materials research [22,23]. Hydrazone linkage provides a suitable
system for pH-dependent release of drugs from drug-conjugates
[24]. Several hydrazide–hydrazone derivatives have been shown
to exhibit antiproliferative activities and act as cytotoxic agents
In addition to the selective reactivity of three chlorine atoms of
cyanuric chloride towards a variety of nucleophiles, the reaction of
⇑
Corresponding author. Tel.: +91 836 2215286x28; fax: +91 836 2771275.
E-mail address: kbgudasi@gmail.com (K.B. Gudasi).
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.12.023

122
S.S. Machakanur et al. / Journal of Molecular Structure 1011 (2012) 121–127
with the ability to prevent cell progression in cancerous cells
through different mechanisms [25] and also exhibited antimicro-
bial [26,27] and antitumoral [28] activities.
2.2.3. General procedure for the preparation of triazine hydrazone
compounds 3a–i
Tripod (1) (0.106 g, 0.24 mmol) was added to a solution of ben-
zoic hydrazide (2a) (0.109 g, 0.8 mmol) in tetrahydrofuran (75 mL)
and the reaction mixture was stirred under reflux for 15–18 h. The
solvent was removed by evaporation under reduced pressure. The
residue was filtered under suction and washed several times with
hot tetrahydrofuran. The resulting solid (3a) was dried in vacuo at
40 °C for 4 h. The same general procedure was followed for the
compounds 3b–i.
The triazine scaffold has provided basis for the design of biolog-
ically relevant molecules with broad biomedical value as therapeu-
tics. It was reported that some of the triazine derivatives possess
potent biological activity [29–31]. In continuation of our work on
star shaped hydrazones [32], in this article we present the synthe-
sis, characterization and in vitro anticancer efficacy of a series of
s-1,3,5-triazine hydrazones prepared by the three-fold condensa-
tion of Tripod with p-substituted benzoic hydrazides against hu-
man liver carcinoma cell line (HepG2) and human cervix
carcinoma cell line (HeLa).
2.2.4. Experimental details of 3a-i
2.2.4.1. 2,4,6-tris[4-{(E)-[20(phenylcarbonyl)hydrazinylidene]methyl}
phenoxy]-1,3,5-triazine (3a). Yield: 96.8%. m.p. 255–258 °C. IR (KBr)
cmꢀ1: 3239 (NAH), 1655 (C@O), 1607 (C@N), 1573 (C@NTriazine),
1367 (CAOAAr). 1H NMR (400 MHz, DMSO-d6, d ppm): 7.34 (d, 6H,
J = 7.2 Hz, C2H), 7.50 (d, 6H, J = 7.2 Hz, C3H), 7.59 (d, 3H, J = 7.6 Hz,
C10H), 7.77 (t, 6H, J = 7.6 Hz, J = 8.0 Hz, C9H), 7.88 (d, 6H, J = 8.0 Hz,
C8H), 8.48 (s, 3H, C5H), 11.84 (s, 3H, NH). 13C NMR (DMSO-d6, d
ppm): 121.95 (C2), 124.85 (C8), 127.46 (C9), 128.40 (C3), 131.70
(C4), 132.25 (C10), 133.33 (C7), 146.82 (C5), 152.44 (C1), 163.09 (C6),
172.92 (CT). MS (MALDI-TOF) m/z: 818.50 (M + Na)+. Anal. Calcd.
For C45H33N9O6: C, 67.92; H, 4.18; N, 15.84%. Found: C, 67.83; H,
4.12; N, 15.76%.
2. Experimental
2.1. Materials and measurements
IR spectra were recorded in KBr matrix using an Impact-410
Nicolet (USA) FT-IR spectrometer in 4000–400 cmꢀ1 range. The
1H and 13C NMR spectra were recorded in DMSO-d6 solvent on
BRUKER AV-400 MHz High Resolution Multinuclear FT-NMR Spec-
trometer at room temperature. The 1H and 13C NMR chemical shifts
were measured using SiMe4 as an internal standard at d = 0 ppm. In
1H NMR spectrum, DMSO-d6 solvent residual peak was observed at
2.49 ppm and water peak at 3.30 ppm. In 13C NMR spectrum
DMSO-d6 solvent residual peak was observed at 39.50 ppm.
Elemental analyses (C, H, N) were carried using the Leco Model
Truespec CHNS Analyser. The GCMS spectrum was measured with
SHIMADZU GCMS-QP2010S spectrometer. The MALDI-TOF mass
spectrum was measured with a Voyager-DE STR spectrometer with
2.2.4.2. 2,4,6-tris[4-{(E)-[2’(4-bromophenylcarbonyl)hydrazinylidene]
methyl}phenoxy]-1,3,5-triazine (3b). Yield: 98.0%. m.p. > 300 °C. IR
(KBr) cmꢀ1: 3232(NAH), 1656 (C@O), 1614(C@N), 1567 (C@NTriazine),
1364 (CAOAAr). 1H NMR (400 MHz, DMSO-d6, d ppm): 7.33 (d, 6H,
J = 7.1 Hz, C2H), 7.70 (d, 6H, J = 7.1 Hz, C3H), 7.76 (d, 6H, J = 7.6 Hz,
C9H), 7.83 (d, 6H, J = 7.6 Hz, C8H), 8.46 (s, 3H, C5H), 11.89 (s, 3H,
NH). 13C NMR (DMSO-d6, d ppm): 121.98 (C2), 125.50 (C10), 128.31
(C8), 129.66 (C3), 131.43 (C4), 132.14 (C9), 132.35 (C7), 147.17 (C5),
152.52 (C1), 162.11 (C6), 172.93 (CT). MS (MALDI-TOF) m/z: 1052.23
(M + Na)+. Anal. Calcd. For C45H30Br3N9O6: C, 52.35; H, 2.93; N,
12.21%. Found: C, 52.31; H, 2.89; N, 12.26%.
a-cyano-4-hydroxycinnamic acid as the matrix. Melting points
were determined in an open capillary on a Gallenkamp melting
point apparatus and are uncorrected.
Solvents were purified by standard methods [33]. Cyanuric
chloride was procured from Sigma Aldrich and used as received.
The purity of compounds were checked by thin-layer chromatogra-
phy (TLC) on aluminum-backed silica gel plates.
2.2.4.3. 2,4,6-tris[4-{(E)-[2’(4-chlorophenylcarbonyl)hydrazinylidene]
methyl} phenoxy]-1,3,5-triazine (3c). Yield: 98.0%. m.p. 289–293 °C.
IR (KBr) cmꢀ1: 3237 (NAH), 1655 (C@O), 1601 (C@N), 1569
(C@NTriazine), 1363 (CAOAAr). 1H NMR (400 MHz, DMSO-d6, d
ppm): 7.36 (d, 6H, J = 7.1 Hz, C2H), 7.58 (d, 6H, J = 7.6 Hz, C9H),
7.81 (d, 6H, J = 7.1 Hz, C3H), 7.92 (d, 6H, J = 7.6 Hz, C8H), 8.47 (s,
3H, C5H), 11.95 (s, 3H, NH). 13C NMR (DMSO-d6, d ppm): 121.94
(C2), 126.27 (C9), 126.45 (C8), 129.44 (C3), 131.09 (C4), 132.07
(C7), 136.49 (C10), 147.04 (C5), 152.43 (C1), 161.92 (C6), 172.96
(CT). MS (MALDI-TOF) m/z: 920.38 (M + Na)+. Anal. Calcd. For
2.2. Synthesis
2.2.1. 2,4,6-tris(4-formylphenoxy)-1,3,5-triazine (1) [N3C3(AOC6H4-p-
CHO)3]
Tripod (1) was synthesized according to reported method [34].
Yield: 61%, m.p. 174–176 °C. IR (KBr, cmꢀ1): 1702 (C@O), 1569
(C@NTriazine). 1H NMR (400 MHz, CDCl3, d ppm): 7.31 (d, 6H, C2H),
7.93 (d, 6H, C3H), 10.00 (s, 3H, CHO). 13C NMR (CDCl3, d ppm):
122.59 (C2), 131.69 (C3), 134.85 (C4), 156.07 (C1), 173.62 (CT),
190.99 (CHO). MS m/z: 441 (M+). Anal. Calc. For C24Hl5N3O6: C,
65.31; H, 3.43; N, 9.52%. Found: C, 65.23, H, 3.38; N, 9.46%.
C45H30Cl3N9O6: C, 60.11; H, 3.36; N, 15.84%. Found: C, 60.04; H,
3.29; N, 15.90%.
2.2.4.4. 2,4,6-tris[4-{(E)-[2’(4-florophenylcarbonyl)hydrazinylidene]
methyl} phenoxy]-1,3,5-triazine (3d). Yield: 89.7%. m.p. 212–
216 °C. IR (KBr) cmꢀ1: 3236 (NAH), 1655 (C@O), 1605 (C@N),
1569 (C@NTriazine), 1366 (CAOAAr). 1H NMR (400 MHz, DMSO-d6,
d ppm): 7.34 (d, 6H, J = 8.0 Hz, C2H), 7.79 (d, 6H, J = 8.0 Hz, C9H),
7.97 (d, 6H, J = 8.0 Hz, C3H), 7.99 (d, 6H, J = 8.0 Hz, C8H), 8.46 (s,
3H, C5H), 11.91 (s, 3H, NH). 13C NMR (DMSO-d6, d ppm): 115.26
(C9), 121.93 (C2), 128.14 (C8), 129.65 (C3), 130.20 (C7), 132.12 (C4),
146.80 (C5), 152.39 (C1), 161.93 (C6), 165.03 (C10), 172.86 (CT). MS
(MALDI-TOF) m/z: 872.47 (M + Na)+. Anal. Calcd. For C45H30F3N9O6:
C, 63.60; H, 3.56; N, 14.83%. Found: C, 63.66; H, 3.62; N, 14.76%.
2.2.2. Synthesis of p-substituted benzoic hydrazides 2a-i
Methyl benzoates were synthesized from their respective
p-substituted benzoic acids, using excess of dry methanol in pres-
ence of H2SO4. p-Substituted benzoic acid hydrazides (2a–i) were
prepared by reaction of the corresponding methyl benzoates
(10 mmol) with hydrazine hydrate 99% (50 mmol) in methanol un-
der reflux for 4–6 h. The excess solvent was removed under vacuum
and the residue was filtered under suction, washed with water and
dried. The spectral and analytical data of benzoic hydrazide (2a)
[35], 4-bromobenzoic hydrazide (2b) [36], 4-chlorobenzoic hydra-
zide (2c) [37], 4-fluorobenzoic hydrazide (2d) [35], 4-hydroxyben-
zoic hydrazide (2e) [38], 4-methoxybenzoic hydrazide (2f) [39],
4-methylbenzoic hydrazide (2g) [37], 4-nitrobenzoic hydrazide
(2h) [37] and 4-aminobenzoic hydrazide (2i) [38] are in good agree-
ment with literature values.
2.2.4.5. 2,4,6-tris[4-{(E)-[2’(4-hydroxyphenylcarbonyl)hydrazinylid-
ene]methyl} phenoxy]-1,3,5-triazine (3e). Yield: 98.6%. m.p. 276–
281 °C. IR (KBr) cmꢀ1: 3410 (OAH), 3251 (NAH), 1641 (C@O),
1608 (C@N), 1571 (C@NTriazine), 1374 (CAOAAr). 1H NMR

S.S. Machakanur et al. / Journal of Molecular Structure 1011 (2012) 121–127
123
J = 8.4 Hz, C8H), 8.46 (s, 3H, C5H), 11.71 (s, 3H, NH). 13C NMR
(DMSO-d6, d ppm): 55.36 (OCH3), 113.64 (C9), 121.95 (C2), 125.34
(C7), 128.13 (C8), 129.49 (C3), 132.38 (C4), 146.21 (C5), 152.33
(C1), 161.96 (C6), 162.56 (C10), 172.93 (CT). MS (MALDI-TOF) m/z:
908.53 (M + Na)+. Anal. Calcd. For C48H39N9O9: C, 65.08; H, 4.44;
N, 14.23%. Found: C, 65.16; H, 4.54; N, 14.32%.
Table 1
Effect of test compounds on growth of HepG2 and HeLa cell lines incubated for 24 h in
vitro.
Test compounds
% Inhibition
HepG2
HeLa
3a
3b
3c
3d
3e
3f
3g
3h
3i
26.31
8.42
6.41
13.23
19.46
28.36
18.19
16.45
9.87
21.39
8.36
23.35
2.2.4.7. 2,4,6-tris[4-{(E)-[2’(4-methylphenylcarbonyl)hydrazinylidene]
methyl}phenoxy]-1,3,5-triazine (3g). Yield: 97.6%. m.p. 266–270 °C.
IR (KBr) cmꢀ1: 3237 (NAH), 1653 (C@O), 1610 (C@N), 1569
(C@NTriazine), 1365 (CAOAAr). 1H NMR (400 MHz, DMSO-d6, d
ppm): 2.36 (s, 9H, CH3), 7.28 (d, 6H, J = 7.1 Hz, C9H), 7.33 (d, 6H,
J = 7.4 Hz, C2H), 7.76 (d, 6H, J = 7.4 Hz, C8H), 7.80 (d, 6H,
J = 7.4 Hz, C3H), 8.47 (s, 3H, C5H), 11.77 (s, 3H, NH). 13C NMR
(DMSO-d6, d ppm): 20.98 (CH3), 121.95 (C2), 127.59 (C8), 128.20
(C9), 128.92 (C7), 130.43 (C3), 132.32 (C4), 141.74 (C10), 146.55
(C5), 152.39 (C1), 162.92 (C6), 172.93 (CT). MS (MALDI-TOF) m/z:
860.54 (M + Na)+. Anal.Calcd. For C48H39N9O6: C, 68.81; H, 4.69;
N, 15.05%. Found: C, 68.73; H, 4.72; N, 15.13%.
4.32
36.24
34.69
52.34
58.91
30.77
(400 MHz, DMSO-d6, d ppm): 6.83 (d, 6H, J = 7.1 Hz, C2H), 7.32 (d,
6H, J = 7.6 Hz, C9H), 7.74 (d, 6H, J = 7.6 Hz, C8H), 7.78 (d, 6H,
J = 7.1 Hz, C3H), 8.45 (s, 3H, C5H), 10.07 (s, 3H, OH), 11.63 (s, 3H,
NH). 13C NMR (DMSO-d6, d ppm): 114.94 (C9), 121.92 (C2),
128.09 (C7), 128.62 (C8), 129.64 (C4), 132.46 (C3), 145.80 (C5),
152.27 (C1), 159.18 (C10), 161.96 (C6), 172.93 (CT). MS (MALDI-
TOF) m/z: 866.48 (M + Na)+. Calcd. For C45H33N9O9: C, 64.05; H,
3.94; N, 14.94%. Found: C, 64.13; H, 3.85; N, 14.84%.
2.2.4.8. 2,4,6-tris[4-{(E)-[20(4-nitrophenylcarbonyl)hydrazinylidene]
methyl} phenoxy]-1,3,5-triazine (3h). Yield: 98.6%. m.p. 225–
229 °C. IR (KBr) cmꢀ1: 3214 (NAH), 1662 (C@O), 1604 (C@N),
1568 (C@NTriazine), 1366 (CAOAAr). 1H NMR (400 MHz, DMSO-d6,
d ppm): 7.34 (d, 6H, J = 7.1 Hz, C2H), 7.79 (d, 6H, J = 7.1 Hz, C3H),
8.10 (d, 6H, J = 7.4 Hz, C8H), 8.30 (d, 6H, J = 7.4 Hz, C9H), 8.49 (s,
3H, C5H), 12.10 (s, 3H, NH). 13C NMR (DMSO-d6, d ppm): 122.54
(C2), 124.03 (C9), 128.96 (C8), 129.61 (C3), 132.48 (C4), 139.44
(C7), 148.47 (C5), 149.69 (C10), 153.19 (C1), 161.91 (C6), 173.45
(CT). MS (MALDI-TOF) m/z: 953.45 (M + Na)+. Anal. Calcd. For
2.2.4.6. 2,4,6-tris[4-{(E)-[20(4-methoxyphenylcarbonyl)hydrazinylid-
ene]methyl}phenoxy]-1,3,5-triazine (3f). Yield: 97.8%. m.p. 258–
262 °C. IR (KBr) cmꢀ1: 3238 (NAH), 1649 (C@O), 1607 (C@N),
1570 (C@NTriazine), 1365 (CAOAAr). 1H NMR (400 MHz, DMSO-d6,
d ppm): 3.82 (s, 9H, OCH3), 7.01 (d, 6H, J = 8.0 Hz, C2H), 7.32 (d,
6H, J = 8.4 Hz, C9H), 7.75 (d, 6H, J = 8.0 Hz, C3H), 7.88 (d, 6H,
Scheme 1. Synthetic pathway for tri-arm 1,3,5-triazine hydrazones (3a–i).

124
S.S. Machakanur et al. / Journal of Molecular Structure 1011 (2012) 121–127
C45H30N12O12: C, 58.07; H, 3.25; N, 18.06%. Found: C, 58.02; H,
3.32; N, 18.13%.
and incubation was continued for another 4 h. The medium was
discarded and the cells were washed with sterile PBS and then
the resultant formazan blue crystals were dissolved in 500 lL
2.2.4.9. 2,4,6-tris[4-{(E)-[20(4-aminophenylcarbonyl)hydrazinylidene]
methyl} phenoxy]-1,3,5-triazine (3i). Yield: 97.3%. m.p. 267–270 °C.
IR (KBr) cmꢀ1: 3219 (NAH), 1656 (C@O), 1605 (C@N), 1558
(C@NTriazine), 1360 (CAOAAr). 1H NMR (400 MHz, DMSO-d6, d
ppm): 5.46 (s, 6H, NH2), 7.13 (d, 6H, J = 8.4 Hz, C9H), 7.30 (d, 6H,
J = 7.6 Hz, C2H), 7.76 (d, 6H, J = 7.6 Hz, C3H), 7.81 (d, 6H,
J = 8.4 Hz, C8H), 8.48 (s, 3H, C5H), 11.86 (s, 3H, NH). 13C NMR
(DMSO-d6, d ppm): 115.04 (C9), 122.48 (C2), 123.62 (C7), 128.59
(C8), 130.03 (C3), 131.54 (C4), 147.36 (C5), 152.45 (C10), 152.92
(C1), 162.71 (C6), 172.91 (CT). MS (MALDI-TOF) m/z: 863.53
(M + Na)+. Anal. Calcd. For C45H36N12O6: C, 64.28; H, 4.32; N,
19.99%. Found: C, 64.27; H, 4.35; N, 19.90%.
DMSO and the optical density (OD) was measured at 570 nm (ref-
erence filter 690 nm) using an automatic microplate reader. Tripli-
cate readings were obtained for each sample and optical density
values of the three wells were averaged for one sample. The results
were recorded and expressed as % inhibition in cell growth of Hu-
man liver carcinoma cell line (HepG2) and human cervix carci-
noma cell line (HeLa) compared with the blank control where
cells incubated with DMSO (Table 1).
3. Results and discussion
3.1. Synthesis of tri-arm 1,3,5-triazine hydrazones
Tri-arm star shaped hydrazones with different substitutions at
para positions emanating from 1,3,5-triazine core were synthe-
sized as shown in Scheme 1. The initial reaction consists of grafting
of three hydroxybenzaldehyde groups onto the 1,3,5-triazine core.
The reaction of 2,4,6-trichloro-1,3,5-triazine with the sodium salt
of 4-hydroxybenzaldehyde yielded 2,4,6-tris(4-formylphenoxy)-
1,3,5-triazine. The second step consists of the condensation of 3
equivalents of benzoic hydrazides 2a–i with the aldehydic func-
tional groups of Tripod in THF for 15–18 h at refluxing temperature
and affords the corresponding trifunctionalized hydrazones 3a–i.
Due to the limited solubility of 4-hydroxybenzoic hydrazide and
4-nitrobenzoic hydrazide in THF the duration of the reaction for
the synthesis of 3e and 3h is comparatively more.
2.3. Protocols for anticancer activity
Under a sterile condition, human liver carcinoma cell line
(HepG2) and human cervix carcinoma cell line (HeLa) were grown
in RPMI 1640 supplemented with 10% fetal bovine serum (FBS).
Cytotoxicity of compounds 3a–i at 100 lM concentration was
tested against HepG2 and HeLa cell lines using MTT assay [40,41].
Each compound was initially solubilized in dimethyl sulfoxide
(DMSO), however, each final dilution contained less than 1% DMSO.
The cells at approximately 80% confluence were selected for
trypsinization. The cells were harvested by removing the medium
and then 1 mL of trypsin-EDTA (200 mg/L for EDTA, 500 mg/L for
trypsin in a ratio (1:250) is added and incubated at 37 °C for about
5 min. The cells were detached from the plate and collected in a
centrifuge tube and centrifuged at 1000 rpm for 5 minutes. Super-
natant solution was removed and the cells were resuspended in
10 mL RPMI-1640 culture medium. Cell number was determined
using hemocytometer. The cell suspension was diluted to required
concentration of 5 ꢁ 104 cells/mL. 24-well microplates were
All products were generally obtained in high yields and were
characterized by FT-IR, 1H, 13C and 2D-HSQC NMR spectroscopy
and MALDI-TOF mass spectrometry.
3.2. IR spectral studies
seeded with 500
5% CO2 for 24 h.
lL of cell suspension and incubated at 37 °C and
The diagnostic IR bands of hydrazones 3a–i are presented in the
experimental section. The aldehyde carbonyl groups of the core (1)
which were observed at 1705 cmꢀ1 disappeared following hydra-
zone formation, accompanied by the appearance of imine C@N
stretching frequencies in the 1601–1614 cmꢀ1 region. A broad
band at 3214–3251 cmꢀ1 is ascribed to NAH stretching frequency
After 24 h incubation, the cells were treated with the newly
synthesized compounds and then incubated for further 24 h.
100
lL of PBS solution of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide, 5 mg/mL] was added to each well,
Fig. 1. 1H NMR spectrum of 3g in DMSO-d6 (expansion of the aromatic region is shown in the box inset).

S.S. Machakanur et al. / Journal of Molecular Structure 1011 (2012) 121–127
125
Fig. 2. 13C NMR spectrum of 3g in DMSO-d6.
of the amide (ANHAC@O) moiety. A strong band at 1641–
1662 cmꢀ1 is due to the amide carbonyl (C@O) stretching frequen-
cies and the C–O–Ar absorption is observed as a distinct band at
1363–1374 cmꢀ1 this is attributed to the involvement of carbon
atom of triazine ring in ether linkage [16,17]. Furthermore, triazine
derivatives show another important band in the region 1560 and
1573 cmꢀ1 ascribed to C@N stretching vibrations of s-triazine ring
[16,17]. Thus, the IR spectral data provide strong evidences for the
formation of the 1,3,5-triazine hydrazones.
the carbons (C5) of the azomethine (HC@NAN) functions. The sig-
nal in the region 160.66–163.09 ppm is assigned to amide carbonyl
(C6). A distinct resonance in the range 172.86 to 173.45 ppm is due
to the symmetrically substituted carbon atoms of the s-triazine
ring. 13C NMR analysis agreed with the 1H NMR analysis in demon-
strating the architecture of the star shaped hydrazones. The 13C
NMR spectrum of 3g is given at Fig. 2.
From 2D HSQC NMR spectrum, it is clear that the 13C NMR reso-
nances of 3a–i in the range 152.27–153.19 and 129.64-132.48 ppm
are not having directly attached hydrogen atoms and are assigned to
C1 and C4 respectively. The resonances of C1 carbons are shifted up
field by 2.88–3.80 ppm compared to the corresponding signal at
156.07 ppm in (1). The signals of C2 were observed in the range
121.92–122.54 and C3 chemical shift were in the range 128.40–
132.46 ppm. The p-substituents at the periphery of the hydrazone
arm do not have much impact on the resonances of these inner aro-
matic ring carbons hence the variation in their chemical shift values
is much less. 2D HSQC NMR spectrum confirms that the C7 and C10
are not having directly bonded hydrogens and the resonances of
these carbons are influenced by the substituent at C10 carbon.
Depending upon the inductive and mesomeric effect of the
3.3. NMR investigations
According to the NMR spectral data, all the s-triazine hydrazone
molecules 3a–i appears to have symmetric structures in solution.
Detailed assignment of 1H and 13C NMR resonances presented in
experimental section was made based on the 2D-Heteronuclear
Single Quantum Coherence (HSQC) NMR analysis. For the descrip-
tion of NMR spectra, we used the arbitrary numbering of atoms as
in Figs. 1 and 2.
3.3.1. 1H NMR studies
1H NMR spectrum of 1 showed a singlet at 10 ppm for aldehyde
protons in addition to the two doublets at 7.31 and 7.93 ppm for
the aromatic protons. The absence of aldehydic proton resonance
and appearance of singlet peak for azomethine (AHC@NANA) pro-
tons in the range 8.45-8.49 ppm confirms the formation of hydra-
zones 3a–i. This is further supported by the resonance for
hydrazide (ACOANHAN@C) protons as a broad singlet in the
downfield region 11.63–12.10 ppm. The resonances due to aro-
matic protons appear in the range 6.83 to 8.30 ppm for the triazine
derivatives. In case of 3e, the p-hydroxy (AOH) protons resonate as
a singlet in the downfield region 10.07 ppm and 3i, the p-amine
(ANH2) protons resonate as a singlet in the region 5.46 ppm. A sin-
glet at 3.82 ppm and 2.36 ppm for 3f and 3g accounts for protons of
p-methoxy (AOCH3) and p-methyl (ACH3) groups respectively. A
representative 1H NMR spectrum of 3g is given at Fig. 1.
3.3.2. 13C NMR spectral studies
In the 13C-NMR spectrum of compound 1, the aldehyde carbon
atom is observed at 190.99 ppm. None of the 13C NMR spectra of
compounds 3a–i exhibited any signal that could be attributed to
unreacted aldehyde functional group. 13C NMR spectra of hydra-
zones showed resonance in the range 145.80–148.47 ppm due to
Fig. 3. 2D-HSQC-NMR spectrum of 3e in DMSO-d6 (expanded aromatic region is
shown in the box inset).

126
S.S. Machakanur et al. / Journal of Molecular Structure 1011 (2012) 121–127
Fig. 4. MALDI-TOF mass spectrum of 3c. Expansion is shown in the box inset.
substituent attached the C10 carbons show large variation in their
chemical shift values. The signals due to the C10 attached to bromine
(3b), chlorine (3c) and fluorine (3d) show downfield shift with
increasing electronegativity of halogen substitutions and were ob-
served at 125.50, 136.49 and 165.03 ppm respectively. In case of
3a, carbons (C10) bonded to hydrogen show resonances at 132.25
while in 3g they are attached to methyl group and resonates at
141.74 ppm. The resonances arising from C10 attached to the hydro-
xy (3e), methoxy (3f) nitro (3h) and amino (3i) groups are observed
at 159.18, 162.56, 149.69 and 152.45 ppm respectively. A represen-
tative 2D-HSQC NMR spectrum of compound 3e is shown in Fig. 3.
2,4,6-Tris(4-formylphenoxy)-1,3,5-triazine (1) possessing three
reactive terminal aldehydic functions on the substituents could
be readily elaborated to the trifunctionalized hydrazones by con-
densation with the benzoic hydrazides. IR as well as 1H, 13C and
2D-HSQC NMR spectral characteristics of triazine hydrazones are
consistent with their proposed structures. The 13C NMR spectra
of 3a–i bearing three hydrazone arms exhibited a single peak in
the range 172.86–173.45 ppm corresponding to the symmetrically
substituted carbon atoms of the s-triazine ring. Microanalysis and
MALDI-TOF mass spectrometry also proved the proposed chemical
structures. The compounds synthesized were tested for their in vi-
tro antiproliferative activity against human liver carcinoma
(HepG2) and human cervix carcinoma (HeLa) cell lines. The present
compounds exhibited lower activity against HeLa cell lines but rea-
sonably moderate in vitro cytotoxicity against HepG2 cell lines. The
compounds 3a–i have been identified as trisubstituted symmetric
triazines and are important as synthetic and structural models for
the reactions and molecular structure of the analogous triazine
dendrimers. Efforts on the synthesis and characterization of higher
generation triazine hydrazones are in progress and will be reported
in future communications.
3.4. Mass spectrometry
The single molecular nature of the hydrazone triazines 3a–i
formed by the complete reaction of all peripheral aldehyde groups
of 1 with the benzoic hydrazides was also checked by MALDI-TOF
mass spectrometry, which confirmed the expected chemical struc-
tures with m/z values corresponding to (M + Na)+ ion. MALDI-TOF
mass spectrograph of 3c is given as a representative in Fig. 4.
3.5. In vitro antiproliferative activity
Acknowledgements
The synthesized compounds were tested for their antiprolifera-
tive activity in vitro against human liver carcinoma (HepG2) and hu-
man cervix carcinoma (HeLa) cell lines. The resulting data in Table 1
indicates that the compounds behaved differently in relation to the
different cell lines. The compounds 3g and 3h bearing p-methyl and
p-nitro substituents respectively exhibit highest activity against
HepG2cells. The compounds 3c and 3i bearing p-chloro and p-amino
substituents respectively exhibited good activity against HeLa cells.
It is interesting to note that the compound 3h with p-nitro substitu-
ent exhibited least activity against HeLa cells but highest activity
against the HepG2 cells. 3a–i were not sufficiently effective against
the HeLa cell line but exhibited moderate antiproliferative activity
against HepG2 cell line.
The authors thank the NMR Research Center, Indian Institute of
Science, Bangalore, for NMR facility. Fellowship for S. S. Machakanur
and B. R. Patil by the University Grants Commission under Research
Fellowship in Sciences for Meritorious Students (UGC-RFSMS) is
gratefully acknowledged. K. B. Gudasi is thankful to the Association
of Commonwealth Universities, for Commonwealth fellowship.
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