Synthesis of 1,2,3-triazole tethered bifunctional hybrids by click chemistry and their cytotoxic studies

Article abstract of DOI:10.1007/s00044-012-0312-7

In view of the drug resistance with most of the currently used anticancer drugs, molecular hybrids of pyrazolyl chalcones and p-nitro benzyl functionalities tethered by triazole ring have been synthesised and evaluated for cytotoxic studies against three human cancer cell lines (THP, COLO-205, A-549). The results of the preliminary investigation exhibited marked dependence of the cytotoxic activity on the electronic factors. Placement of naphthyl (JGPT-11) and trimethoxy phenyl ring (JGPT-6) as ring A proved to be extremely beneficial in enhancing the cytotoxic potential. Thus we herein report the synthesis and cytotoxic studies of a new class of molecular hybrids. Detailed investigation on the biological mechanistic insights of JGPT-11 and 6 is under progress.

Full text of DOI:10.1007/s00044-012-0312-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3160–3169
DOI 10.1007/s00044-012-0312-7
ORIGINAL RESEARCH
Synthesis of 1,2,3-triazole tethered bifunctional hybrids by click
chemistry and their cytotoxic studies
•
Jagjeet Singh Sahil Sharma A. K. Saxena
•
•
•
Kunal Nepali Preet Mohinder Singh Bedi
Received: 13 August 2012 / Accepted: 24 October 2012 / Published online: 11 November 2012
Ó Springer Science+Business Media New York 2012
Abstract In view of the drug resistance with most of the
currently used anticancer drugs, molecular hybrids of
pyrazolyl chalcones and p-nitro benzyl functionalities
tethered by triazole ring have been synthesised and eval-
uated for cytotoxic studies against three human cancer cell
lines (THP, COLO-205, A-549). The results of the pre-
liminary investigation exhibited marked dependence of the
cytotoxic activity on the electronic factors. Placement of
naphthyl (JGPT-11) and trimethoxy phenyl ring (JGPT-6)
as ring A proved to be extremely beneficial in enhancing
the cytotoxic potential. Thus we herein report the synthesis
and cytotoxic studies of a new class of molecular hybrids.
Detailed investigation on the biological mechanistic
insights of JGPT-11 and 6 is under progress.
afflictions in the world (Fadeyi et al., 2008; Rostom, 2006).
Despite the clear understanding of the molecular basis of
the disease and the sophistication of cancer chemotherapy,
yet there is no treatment that is 100 % effective against
disseminated cancer which is primarily due to the problem
of drug resistance.
Though cancer therapy interfering with a single biological
molecule or pathway has been successfully utilized (Saw-
yers, 2004; Petrelli and Giordano, 2008; Overington et al.,
2006; Szuromi et al., 2004), but the problem of drug resis-
tance and a general belief that agents modulating more than
one target could have superior efficacy compared to single
target drugs (Bode and Dong, 2009; Zhan and Liu, 2009) has
revived the interest among the researchers worldwide which
has led to the search for molecules modulating multiple
targets (Morphy and Rankovic, 2005; Hopkins, 2008).
The molecular hybridization (MH) as drug design
strategy involves fusion of bioactive molecules which leads
to the design of new hybrid architectures that maintain pre-
selected characteristics of the original templates (Viegas
et al., 2007).
Keywords Resistance ꢀ Molecular hybrids ꢀ
Cytotoxicity ꢀ Cancer ꢀ Cell lines
Introduction
Cancer, the uncontrolled, rapid, and pathological prolifer-
ation of abnormal cells, is one of the most formidable
Chalcones also known as 1,3-diaryl propenones are
reported to possess diverse array of biological activities
such as anti-malarial (Liu et al., 2003), anti-protozoal
(Nielson et al., 1998; Li et al., 1995; Liu et al., 2001), anti-
inflammatory (Ballesteros et al., 1995 Hsieh et al., 1998),
immunomodulatory (Barford et al., 2002), nitric oxide
inhibition (Rajas et al., 2002), tyronase inhibition (Nerya
et al., 2004), cytotoxic (Dimmock et al., 1998; Yit and Das,
1994; Satomi, 1993; Edwards et al., 1990; Yang et al.,
2000) and anticancer (Wattenberg et al., 1994; Kostova
et al., 1998) activities have been cited in the literature.
Recently the success of pyrazolyl chalcones as cytotoxic
agents has been reported by our group. These pyrazolyl
chalcone were designed rationally by our group while
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0312-7) contains supplementary
material, which is available to authorized users.
J. Singh ꢀ S. Sharma ꢀ K. Nepali (&) ꢀ P. M. S. Bedi
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar, Punjab 143005, India
e-mail: kunal_8855@rediffmail.com
A. K. Saxena
Cancer Pharmacology Division, Indian Institute of Integrative
Medicine, Jammu, India
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Med Chem Res (2013) 22:3160–3169
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Experimental
H3C
N
O
N
The reagents were purchased from Sigma-Aldrich, Loba
and CDH, India and used without further purification. All
yields refer to isolated products after purification. Products
were characterized by comparison with authentic samples
and by spectroscopic data (IR, 1H NMR). The spectra were
measured in CDCl3 and DMSO-d6 relative to TMS
(0.00 ppm). IR (KBr pallets) spectra were recorded on a
Fourier transform infrared (FT-IR) Thermo spectropho-
tometer. Melting points were determined in open capillar-
ies and were uncorrected. HRMS were obtained on
micrOTOF-Q 11 instrument.
Stilbene
1,3-Heterocycle
Fig. 1 Stilbene core replaced by 1,3-heterocycles
modifying the stilbene core of combretastatin with a 1,3-
heterocycle (Fig. 1) (Nepali et al., 2011. Thus pyrazolyl
chalcones have been selected as one of the functionality for
the molecular hybridization process.
The other functionality chosen was benzyl derivatives,
as cytotoxic potential of benzyl derivatives particularly
the one with nitro group at para position is well explored.
The nitro group of nitro arenes and aryls is largely
responsible for cytotoxicity of these derivatives (Juneja
et al., 1995).
General procedure
Procedure for the synthesis of 1,3-diarylpropenones
Concentrated sulfuric acid (2.5 ml) was added slowly to
the stirring solution of various substituted benzaldehydes
(1 mmol) and acetophenones (1 mmol) in glacial acetic
acid in a 500-ml round bottom flask. The stirring was
continued for 48 h keeping the temperature of reaction
mixture below 5 °C. The completion of the reaction was
monitored by TLC. After the completion, the reaction
mixture was poured onto crushed ice and extracted thrice
with ethylacetate. The organic layer was pooled and con-
centrated under reduced pressure. The concentrated residue
was adsorbed on silica gel (60–120 mesh) and the desired
Numerous reports on the cytotoxic potential of triazoles
further attracted us towards their selection as a linker for
the two functionalities (Avanzo et al., 2012; Bhat et al.,
2008; Dos Anjos et al., 2009; Hu et al., 2012; Kamal et al.,
2011; Li et al., 2011; Singh et al., 2012; Song et al., 2010).
Thus with this background, triazole tethered hybrids of
these functionalities have designed, synthesised and
evaluated for cytotoxic evaluation in the present study
(Fig. 2).
O
C
Fig. 2 Hybridization process
O
N
N
CH3
N
N C CH3
N3
OCH3
O
NO2
PNB-azide
R
O
R
Propargylated-NAP
Propargylated-NAP
Hybridization
O
O
N
N C CH3
N
N C CH3
OCH3
O
R
O
R
N
N
N
N
N
N
NO2
O2N
Hybrids
NAP - N-Acetyl pyrazole
PNB - p-Nitro benzyl
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Med Chem Res (2013) 22:3160–3169
product was isolated by column chromatography employ-
ing increasing % age of ethylacetate in hexane.
300 MHz, d, TMS = 0): 8.35 (1H, s), 8.24 (2H, d,
J = 6.6 Hz), 7.70 (2H, d, J = 8.7 Hz), 7.51 (2H, d,
J = 8.4 Hz), 7.29 (2H, d, J = 7.2 Hz), 7.23 (1H, d,
J = 6.6 Hz), 7.08–7.22 (4H, m), 5.78 (2H, s), 5.50 (1H, dd,
J = 4.2 and 11.4 Hz), 5.21 (2H, s), 3.82 (1H, dd, J = 11.4
and 17.7 Hz), 3.08 (1H, dd, J = 4.2 and 17.7 Hz), 2.49 (3H,
s). 13C NMR (CDCl3, 75.4 MHz): 167.34, 158.23, 146.23,
146.71, 141. 21, 140.13, 128.67, 128. 36, 127. 93.125.06,
123.54, 114.62, 63.23, 60.51, 53.21, 30.46. Anal. Calcd for
C27H24N6O4: C, 65.31; H, 4.87; N, 16.93. Found: C, 65.64;
H, 4.39; N, 17.21. HRMS m/z: 519.0087 (M??Na)
Procedure for the synthesis of N-acetyl pyrazole (NAP)
A mixture of diarylpropenone (1 mmol) in acetic acid and
hydrazine hydrate (1 mmol) was refluxed for 4 h. The
mixture was then poured onto crushed ice to get crude
pyrazole, which was purified by crystallization with
methanol to afford the desired product.
Procedure for the synthesis of O-propargylated NAP
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-3(4-bromophenyl)-4,5-
dihydropyrazol-1-yl)ethanone (JGPT-2)
To the stirred suspension of K2CO3 (1.5 mmol) in dry
DMF, the pyrazole (1 mmol) was added. To the reaction
mixture propargylbromide (1.5 mmol) was added. The
reaction was kept on stirring at 0 °C under anhydrous
condition for 24 h. After completion of reaction, as evident
by TLC, the reaction mixture was poured onto crushed ice.
Precipitates were then filtered out. Propargylated pyrazole
was obtained in good yield.
% Yield: 72 %; mp: 92–94 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2, sym-
metric stretch), 1270 (C–O, stretch), 1130 (C–N, stretch); 1H
NMR (CDCl3, 300 MHz, d, TMS = 0): 8.27 (2H, d,
J = 8.1 Hz), 8.21 (2H, d, J = 7.8 Hz), 7.93 (1H, s), 7.63
(2H, d, J = 8.7 Hz), 7.52 (2H, d, J = 8.7 Hz), 7.01 (1H, d,
J = 8.1 Hz), 6.80 (1H, s), 6.61 (1H, d, J = 8.4 Hz), 5.76
(2H, s), 5.48 (1H, dd, J = 4.2 and 11.4 Hz), 5.07 (2H, s),
4.02 (1H, dd, J = 11.4 and 17.7 Hz), 3.70 (3H, m), 3.10 (1H,
dd, J = 4.2 and 17.7 Hz), 2.41 (3H, s). 13C NMR (CDCl3,
75.4 MHz): 168.34, 159.23, 147.13, 146.71, 140.19, 140.13,
128.67, 128.36, 127.93. 125.06, 123.54, 113.62, 65.31,
59.51, 55.32, 52.21, 29.46 Anal. Calcd for C28H25BrN6O5:
C, 55.55; H, 4.16; Br, 13.20; N, 13.88. Found: C, 55.31; H,
4.47; Br, 12.98; N, 14.22. HRMS m/z: 627.0091 (M??Na).
Procedure for the synthesis of p-nitrobenzyl azide (PNB
azide)
A mixture of p-nitrobenzyl bromide (1 mmol) in DMF and
sodium azide (1 mmol) was stirred for 2 h at room tem-
perature. After completion of reaction, as monitored by
TLC, reaction mixture was extracted with ethyl acetate.
Ethyl acetate layer was separated out and evaporated to get
the desired product in good yield.
Procedure for the synthesis of bifunctional (NAP–PNB)
hybrids
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-3(4 chlorophenyl)-4,5-
dihydropyrazol-1-yl)ethanone (JGPT-3)
To the stirred solution O-propargylated NAP (1 mmol) and
PNB azide (1.5 mmol) in DMF, copper sulfate
(0.055 mmol) and sodium ascorbate (0.143 mmol) was
added in succession, at room temperature. On completion
of reaction, as monitored by TLC, the reaction mixture was
poured onto crushed ice. Precipitates were then filtered out.
Desired product was obtained in good yield.
% Yield: 64 %; mp: 89–91 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), s1270 (C–O, stretch), 1130 (C–N,
1
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.21
(2H, d, J = 8.1 Hz), 8.17 (2H, d, J = 7.8 Hz), 7.81 (1H,
s), 7.32–7.50 (4H, m), 6.90 (1H, d, J = 8.4 Hz), 6.68 (1H,
s), 6.60 (1H, d, J = 8.1 Hz), 5.60 (2H, s), 5.43 (1H, dd,
J = 4.2 and 11.4 Hz), 5.03 (2H, s), 3.70 (1H, dd, J = 11.4
and 17.7 Hz), 3.75 (3H, s), 3.07 (1H, dd, J = 4.2 and
17.7 Hz), 2.46 (3H, s). 13C NMR (CDCl3, 75.4 MHz):
167.34, 159.23, 147.13, 146.71, 140.19, 140.13, 128.67,
128.36, 127.93. 125.06, 123.54, 113.62, 65.31, 59.51,
56.12, 51.19, 30.16. Anal. Calcd for C28H25ClN6O5: C,
59.95; H, 4.49; Cl, 6.32; N, 14.98. Found: C, 60.16; H,
4.24; Cl, 6.54; N, 14.71. HRMS m/z: 583.0091 (M??Na)
Characterization of the synthesized compounds
1-(3-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-4,5-dihydro-5-phenylpyrazol-1-
yl)ethanone (JGPT-1)
% Yield: 69 %; mp: 80–82 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2, sym-
metric stretch), 1130 (C–N, stretch); 1H NMR (CDCl3,
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3163
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-(4-
methoxyphenyl)pyrazol-1-yl)ethanone (JGPT-4)
60.38; H, 5.23; N, 13.63. Found: C, 60.03; H, 5.64; N,
13.37. HRMS m/z: 639.1101 (M??Na)
1-(3-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-4,5-dihydro-5-(4-
methoxyphenyl)pyrazol-1-yl)ethanone (JGPT-7)
% Yield: 73 %; mp: 91–93 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1270 (C–O, stretch), 1130 (C–N,
% Yield: 71 %, semisolid; I.R. (KBr, cm-1): 1660 (C=O,
stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1130 (C–N, stretch); 1H NMR (CDCl3,
400 MHz, d, TMS = 0): 8.24 (2H, d, J = 8.0 Hz), 7.68
(2H, d, J = 8.4 Hz), 7.63 (1H,s), 7.41 (2H, d, J = 8.0 Hz),
7.15 (2H, d, J = 8.4 Hz), 7.01 (2H, d, J = 8.4 Hz), 6.83
(2H, d, J = 8.0 Hz), 5.63 (2H, s), 5.53 (1H, dd, J = 4 and
11.6 Hz), 5.26 (2H, s), 3.76 (3H, s), 3.68 (1H, dd, J = 11.6
and 17.2 Hz), 3.11 (1H, dd, J = 4 and 17.2 Hz), 2.30 (3H,
s); Anal. Calcd for C28H26N6O5: C, 63.87; H, 4.98; N,
15.96. Found: C, 64.19; H, 4.75; N, 16.27. HRMS m/z:
549.1830 (M??Na).
1
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.24
(2H, d, J = 9.3 Hz), 7.68 (2H, d, J = 8.7 Hz), 7.6 (1H, s),
7.38 (2H, d, J = 8.4 Hz), 6.90–6.9 (3H, m), 6.70–6.75 (2H,
m), 5.61 (2H, s), 5.49 (1H, dd, J = 4.2 and 11.4 Hz), 5.25
(2H, s), 3.85 (3H, s), 3.80 (3H, s), 3.70 (1H, dd, J = 11.4
and 17.7 Hz), 3.10 (1H, dd, J = 4.2 and 17.7 Hz), 2.40
(3H, s); 13C NMR (CDCl3, 75.4 MHz): 168.12, 158.73,
147.13, 145.91, 139.19, 139.23, 129.67, 128.87, 126. 93.
124. 66, 122.14, 113.62, 65.31, 59.51, 55.31, 50.19, 29.16
Anal. Calcd for C29H28N6O6: C, 62.58; H, 5.07; N, 15.10.
Found: C, 62.47; H, 5.39; N, 14.88. HRMS m/z: 579.1957
(M??Na)
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-(4-
nitrophenyl)pyrazol-1-yl)ethanone (JGPT-5)
1-(3-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-4,5-dihydro-5-(3,4-
dimethoxyphenyl)pyrazol-1-yl)ethanone (JGPT-8)
% Yield: 68 %; mp: 65–68 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1130 (C–N, stretch); 1H NMR (CDCl3,
300 MHz, d, TMS = 0): 8.27 (2H, d, J = 8.7 Hz), 8.22
(2H, d, J = 8.1 Hz), 7.88 (1H, d, J = 8.4 Hz), 7.60 (1H,
s), 7.49 (1H, d, J = 8.4 Hz), 7.38 (2H, d, J = 8.7 Hz), 6.96
(1H, d, J = 8.1 Hz), 6.69–6.75 (2H, m), 5.61 (2H, s), 5.60
(1H, dd, J = 4.2 and 11.4 Hz), 5.25 (2H, s), 3.81 (3H, s),
3.70 (1H, dd, J = 11.4 and 17.7 Hz), 3.18 (1H, dd, J = 4.2
and 17.7 Hz), 2.40 (3H, s); Anal. Calcd for C28H25N7O7:
C, 58.84; H, 4.41; N, 17.15. Found: C, 59.23; H, 4.08; N,
17.49. HRMS m/z: 594.1010 (M??Na).
% Yield: 67 %; semisolid; I.R. (KBr, cm-1): 1660 (C=O,
stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1270 (C–O, stretch), 1130 (C–N,
1
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.23
(2H, d, J = 7.2 Hz), 7.70 (2H, d, J = 8.7 Hz), 7.61 (1H,
s), 7.53 (2H, d, J = 8.7 Hz), 7.08 (2H, d, J = 9.0 Hz),
6.65–6.85 (3H, m), 5.78 (2H, S), 5.46 (1H, dd, J = 4.2 and
11.4 Hz), 5.21 (2H, s), 3.77 (6H, s), 3.70 (1H, dd, J = 11.4
and 17.7 Hz), 3.10 (1H, dd, J = 4.2 and 17.7 Hz), 2.52
(3H, s); Anal. Calcd for C29H28N6O6: C, 62.58; H, 5.07; N,
15.10 Found: C, 62.32; H, 5.44; N, 14.75. HRMS m/z:
579.1231 (M??Na).
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-(3,4,5-
trimethoxyphenyl)pyrazol-1-yl)ethanone (JGPT-6)
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-
phenylpyrazol-1-yl)ethanone (JGPT-9)
% Yield: 63 %, mp: 88–90 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1270 (C–O, stretch), 1130 (C–N,
% Yield: 77 %; mp: 64–66 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1270 (C–O, stretch), 1130 (C–N,
1
1
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 8.24
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 7.52
(2H, d, J = 8.4 Hz), 7.54 (3H, m), 7.04 (2H, s), 6.80 (3H,
m), 5.78 (2H, s), 5.50 (1H, dd, J = 4.2 and 11.4 Hz), 5.09
(2H, s), 3.84 (6H, s), 3.83 (3H, s), 3.79 (3H, s), 3.70 (1H,
dd, J = 11.4 and 17.7 Hz), 3.10 (1H, dd, J = 4.2 and
17.7 Hz), 2.32 (3H, s); Anal. Calcd for C31H32N6O8: C,
(4H, m), 7.93 (1H, s), 7.63 (2H, d, J = 8.7 Hz), 7.52 (2H,
d, J = 8.7 Hz), 7.01 (1H, d, J = 8.1 Hz), 6.80 (1H, s), 6.61
(1H, d, J = 8.4 Hz), 5.76 (2H, s), 5.48 (1H, dd, J = 4.2
and 11.4 Hz), 5.07 (2H, s), 4.02 (1H, dd, J = 11.4 and
17.7 Hz), 3.70 (3H, m), 3.10 (1H, dd, J = 4.2 and
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Med Chem Res (2013) 22:3160–3169
17.7 Hz), 2.41 (3H, s). 13C NMR (CDCl3, 75 MHz):
167.34, 158.23, 146.23, 146.71, 141. 21, 140.13, 128.67,
128.36, 127.93. 125.06, 123.54, 114.62, 63.23, 60.51,
53.21, 30.46. Anal. Calcd for C28H26N6O5: C, 63.87; H,
4.98; N, 15.96. Found: C, 64.19; H, 4.74; N, 16.22. HRMS
m/z: 549.0086 (M??Na).
Cell cultivation
Cells were cultured in culture medium in a 75 cm2 tissue
culture flask (BD Falcon). Cell density was always kept
under 1 9 106/ml to avoid mutations of the cells. Pas-
saging of the cells was done four times a week. Experi-
ments were performed with cells cultivated not longer
than 20 passages. Further passaging of immortalized cell
lines is possible but the risk of genotypic changes
increases. Cultivation and experimental incubation were
performed in a cell incubator at 37 °C, 90 % air humidity
and 5 % CO2. Cells grown in logarithmic phase were
treated with tested botanical dissolved in DMSO while the
untreated control cultures received only the vehicle
(DMSO, \0.5 % v/v).
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-3-(4-((1-(4-nitrobenzyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4,5-dihydropyrazol-
1-yl)ethanone (JGPT-10)
% Yield: 72 %; mp: 148–150 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2, sym-
metric stretch), 1270 (C–O, stretch), 1130 (C–N, stretch); 1H
NMR (CDCl3, 300 MHz, d, TMS = 0): 8.24 (4H, d,
J = 9.3 Hz), 7.68 (2H, d, J = 8.7 Hz), 7.6 (2H, s), 7.38 (4H,
d, J = 8.4 Hz), 6.90–6.94 (3H, m), 6.70–6.75 (2H, m), 5.61
(4H, S), 5.49 (1H, dd, J = 4.2 and 11.4 Hz), 5.25 (4H, s),
3.85 (3H, s), 3.70 (1H, dd, J = 11.4 and 17.7 Hz), 3.10 (1H,
dd, J = 4.2 and 17.7 Hz), 2.52 (3H, s); Anal. Calcd for
C38H34N10O8: C, 60.15; H, 4.52; N, 18.46. Found: C, 59.96;
H, 4.78; N, 18.08. HRMS m/z: 781.0087 (M??Na).
Cell proliferation by MTT assay
The growth inhibitory effect of different samples towards
the HL-60, PC-3, MCF-7 and A549 cells was measured by
means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay. The cleavage and conversion
of the soluble yellowish MTT to the insoluble purple for-
mazan by active mitochondrial dehydrogenase of living
cells has been used to develop an assay system for mea-
surement of cell proliferation. HL-60 cells 15,000/200 ll,
PC-3, MCF-7 and A549 8,000 cells/well 200 ll media in
96-well culture plates were treated with various concen-
trations [(10, 30,100) lg] of samples for 48 h. 20 ll of
MTT solution (2.5 mg/ml) was added to each well and
incubated at 37 °C for 3 h before the termination of assay.
The plates were centrifuged and the supernatant was dis-
carded while the MTT-formazon crystals were dissolved in
100 ll of DMSO. The OD measured at 570 nm with ref-
erence wavelength of 620 nm.
1-(5-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)-3-methoxyphenyl)-4,5-dihydro-3-
(naphthalene-3-yl)pyrazol-1-yl)ethanone (JGPT-11)
% Yield: 73 %; mp: 137–140 °C; I.R. (KBr, cm-1): 1660
(C=O, stretch), 1635 (C=N, stretch), 1595 (C=C, aromatic
stretch), 1565 (NO2, asymmetric stretch), 1380 (NO2,
symmetric stretch), 1270 (C–O, stretch), 1130 (C–N,
1
stretch); H NMR (CDCl3, 300 MHz, d, TMS = 0): 9.19
(1H, d, J = 8.7 Hz), 8.19–8.30 (5H, m), 7.02 (2H, m),
7.49–7.79 (6H, m), 7.04 (1H, d, J = 8.1 Hz), 5.76 (2H, s),
5.48 (1H, dd, J = 4.2 and 11.4 Hz), 5.07 (2H, s), 4.02 (1H,
dd, J = 11.4 and 17.7 Hz), 3.70 (3H, m), 3.10 (1H, dd,
J = 4.2 and 17.7 Hz), 2.41 (3H, s); Anal. Calcd for
C32H28N6O5: C, 66.66; H, 4.89; N, 14.58. Found: C, 66.91;
H, 4.55; N, 14.83. HRMS m/z: 599.0041 (M??Na).
Results and discussion
Synthesis
Pharmacological evaluation
The synthesis of the hybrid (NAP–PNB) started from diaryl
propenones which were synthesized by acid catalyzed
Claisen Schmidt condensation of differently substituted
acetophenones and benzaldehydes. The method is attrac-
tive as it selectively yield E isomers. From 1H NMR
spectrum all diaryl propenones are geometrically pure with
trans configuration. These compounds underwent a trans-
position reaction with hydrazine hydrate and simultaneous
acetylation with acetic acid to yield N-acetyl pyrazole
(NAP). The N-acetyl pyrazoles (NAP) were propargylated
Human leukemia (HL-60), prostate cancer (PC-3), breast
cancer (MCF-7) and lung cancer (A549) cell line was
procured from National Cancer Institute, Frederick, U.S.A.
The cells were grown in RPMI-1640 medium supple-
mented with 10 % heat inactivated fetal bovine
serum, pencillin (100 units/ml), streptomycin (100 lg/ml),
L-glutamine(0.3 mg/ml), pyruvic acid, 0.37 % NaHCO3
and 50 lM of 2-mercaptoethanol at 37 °C in an atmo-
sphere of 95 % air and 5 % CO2 with 90 % humidity.
123

Med Chem Res (2013) 22:3160–3169
3165
O
O
Scheme 1 Reagents and
conditions: a CH3COOH,
H2SO4, 5 °C, stirring, 48 h;
b NH2NH2ꢀH2O, CH3COOH,
2 h, reflux; c propargyl
bromide, K2CO3, DMF, stirring,
24 h
O
C
OCH3
OH
OCH3
OH
a
CH3
H
+
R
R
b
O
O
CH3
CH3
N
N
N
N
R1
R2
OCH3
OCH3
c
O
OH
R
R3
R1 = R2 = R3 = H
R1 = R3 = H, R2 = NO2
R1 = R2 = R3 = OCH3
R =
R1 = R3 = H, R2 = Br
R1 = R3 = H, R2 = Cl
R1 = R3 = H, R2 = OCH3
O
Scheme 2 Reagents and
conditions: a CH3COOH,
H2SO4, 5 °C, stirring, 48 h;
b NH2NH2ꢀH2O, CH3COOH,
2 h, reflux; c propargyl
bromide, K2CO3, DMF, stirring,
24 h
O
O
C
R1
R2
R1
R2
a
CH3
H
+
HO
HO
R3
R3
b
O
CH3
O
N
N
N
CH3
R1
R2
N
c
R1
R2
O
R3
HO
R3
R1 = R2 = R3 = H
1 = R3 = H, R2 = OCH3
R1 = H, R2 = R3 = OCH3
R
d
Br
N3
The azide–alkyne Huisgen cycloaddition is a 1,3 dipolar
cycloaddition between azide and terminal alkyne to give
1,2,3-triazole. The conventional cycloaddition is known to
afford a mixture of regio isomers, i.e., 1,4 and 1,5 regio
isomers. However, the method employed in the present
study involves copper catalysis which yield single regio-
isomer, i.e., 1,4 regio isomer and thus copper catalysis
provides regioselectivity to the reaction. The reaction was
performed using a mixture of copper(II) sulfate and a
reducing agent (sodium ascorbate) to produce cop-
per(I) in situ (Scheme 4). The structures of all the hybrids
have been shown in Fig. 3.
NO2
NO2
Scheme 3 Reagents and conditions: d NaN3, DMF, stirring, 2 h
to yield acetylenic N-acetyl pyrazole which were further
utilized for the synthesis of desired hybrids (Schemes 1, 2).
The target compounds were synthesized by utilizing
azide–alkyne cycloaddition reaction of the acetylenic
N-acetyl pyrazoles and the azides. The azides were
synthesized by the treatment of p-nitro benzyl bromide
with sodium azide in dimethyl formamide (Scheme 3).
123

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Med Chem Res (2013) 22:3160–3169
O
Scheme 4 Reagents and
conditions: e CuSO4ꢀ5H2O,
sodium ascorbate, DMF,
stirring, 2 h
O
CH3
N
N
CH3
OCH3
O
N
N
OCH3
O
e
N
R
N
N
R
NO2
R1 = R2 = R3 = H
R1 = R3 = H, R2 = NO2
R1 = R2 = R3 = OCH3
R =
R1 = R3 = H, R2 = Br
1 = R3 = H, R2 = Cl
R
R1 = R3 = H, R2 = OCH3
O
N
CH3
O
N
N
CH3
N
R
e
O
R
N
O
N
N
O2N
R1 = R2 = R3 = H
R1 = R3 = H, R2 = OCH3
R1 = H, R2, R3 = OCH3
Table 1 The concentration-dependent cytotoxicity against human
cancer cell lines
Cytotoxicity studies
Cell line type
Tissue type
A-549 THP
COLO-205
The synthesized hybrids were evaluated for their anticancer
activity against three human cancer cell lines viz A-549
(lung); THP (leukemia); COLO-205 (colon) using sul-
forohodamine B assay. The concentration dependent
cytotoxicity of these scaffolds against human cancer cell
lines is shown in Table 1, Fig. 4 and Fig. 5.
Lung Leukemia Colon
Compound no. Concentration (lM) Percentage inhibition
JGPT-1
JGPT-2
JGPT-3
JGPT-4
JGPT-5
JGPT-6
JGPT-7
JGPT-8
JGPT-9
JGPT-10
JGPT-11
10
100
10
15
80
0
0
22
1
18
55
26
47
0
All the hybrids showed poor inhibition against THP
(leukemia) cell lines, except JGPT-11. JGPT-11 was found
to have significant activity against A-549 in comparison to
the standard (paclitaxel), followed by JGPT-6. JGPT-6 was
found to be most active against COLO-205 among the
hybrids. JGPT-5 also showed significant cytotoxicity against
COLO-205 cell lines in comparison to the other hybrids
(Table 1). Table 1 reveals interesting structure activity
relationship and substituent effect on the cytotoxic potential.
The compounds with napthyl as ring A (JGPT-11) showed
better cytotoxic activity than all other hybrids against A-549
cancer cell lines, followed by hybrid with trimethoxy phenyl
ring as ring A (JGPT-6). Thus, increased electron density at
the periphery seems to be essential for activity. Deactivating
groups such as Br, Cl (JGPT-2, 3) on ring A remarkably
decreases the cytotoxicity except NO2 (JGPT-5) which
showed moderate growth inhibition at 100 lM against
A-549 and COLO-205 cancer cell lines (Fig. 5). The
enhanced cytotoxicity by the substitution of methoxy groups
on ring A is clearly evident from the Table 1 (JGPT-4 and
JGPT-6). Careful analysis of the in vitro cytotoxic evaluation
also indicates that the linkage of the triazole to ring B is
beneficial for the activity as scompared to its linkage to ring
A (Fig. 6) (compare JGPT-1 with JGPT-9).
100
10
17
0
23
17
37
8
100
10
0
10
10
75
33
85
60
99
4
43
84
39
72
68
85
26
33
11
36
0
100
10
14
7
100
10
59
29
43
15
26
8
100
10
100
10
37
9
100
10
9
38
7
0
100
10
11
29
42
89
95
59
–
10
0
11
27
51
46
72
–
100
10
15
10
79
62
65
–
100
10
Paclitaxel
Mitomycin
Adriamycin
10
–
1
–
72
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Med Chem Res (2013) 22:3160–3169
3167
O
N
CH3
O
N
CH3
N
N
R1
R2
O
O
N
R3
N
N
N
N
N
O2N
O2N
JGPT-1
JGPT-7 R1 = R3 = H, R2 = OCH3
JGPT-8 R1 = H, R2 = OCH3, R3 = OCH3
O
O
CH3
CH3
N
N
N
N
OCH3
O
R1
R2
OCH3
O
N
N
N
N
R3
N
N
NO2
NO2
JGPT-9
JGPT-2 R1 = R3 = H, R2 = Br
JGPT-3 R1 = R3 = H, R2 = Cl
JGPT-4 R1 = R3 = H, R2 = OCH3
JGPT-5 R1 = R3 = H, R2 = NO2
JGPT-6 R1 = R2 = R3 = OCH3
O
O
CH3
N
N
CH3
N
N
OCH3
O
OCH3
O
O
N
N
N
N
N
N
N
N
N
NO2
O2N
NO2
JGPT-10
JGPT-11
Fig. 3 The chemical structures of bifunctional hybrids
Conclusion
were evaluated for in vitro cytotoxic evaluation against three
human cancer cell lines using sulforhodamine B assay. The
preliminary cytotoxic evaluation indicates a marked
dependence of cytotoxicity on the electronic factors. The
trimethoxy phenyl (JGPT-6) and naphthyl (ring A) (JGPT-
11) based hybrids showed significant cytotoxicity against
In continuation of our drug discovery programme
and continuous search for new anticancer molecules,
NAP–PNB bifunctional hybrids were synthesized by a
cycloaddition reaction via click chemistry. The hybrids
123

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Med Chem Res (2013) 22:3160–3169
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