Synthesis and Cytotoxic Evaluation of Novel 1,2,3-Triazole-4-Linked (2E,6E)-2-Benzylidene-6-(4-nitrobenzylidene)cyclohexanones

Article abstract of DOI:10.1002/hlca.201500210

This work describes the synthesis of novel 1,2,3-triazole-4-linked (2E,6E)-2-benzylidene-6-(4-nitrobenzylidene)cyclo-hexanones starting from cyclohexanone. 1-(Cyclohex-1-en-1-yl)piperidine, the enamine from cyclohexanone and piperidine, reacted with 4-nitrobenzaldehyde to obtain 2-(4-nitrobenzylidene)cyclohexanone. Condensation of the latter compound with (prop-2-yn-1-yloxy)benzaldehyde derivatives under acidic conditions gave (4-nitrobenzylidene)-[(prop-2-yn-1-yloxy)-benzylidene]cyclohexanones. Finally, 'click reaction' of these derivatives and various organic azides led to the title compounds. All compounds were examined by MTT assay for cytotoxic activity in one human breast cancer cell line, MDA-MB-231.

Full text of DOI:10.1002/hlca.201500210

Helv. Chim. Acta 2016, 99, 175 – 180
175
FULL PAPER
Synthesis and Cytotoxic Evaluation of Novel 1,2,3-Triazole-4-Linked (2E,6E)-2-
Benzylidene-6-(4-nitrobenzylidene)cyclohexanones
by Mohammad Mahdavia), Maryam Akhbarib), Mina Saeedic)d), Maryam Karimib), Niloufar Foroughib), Elahe Karimpour-
Razkenarid), Heshmatollah Alinezhade), Alireza Foroumadia), Abbas Shafieea), and Tahmineh Akbarzadeh*d)f)
a) Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran
University of Medical Sciences, Tehran, Iran
b) The Essential Oil Research Center, University of Kashan, Kashan, Iran
c) Faculty of Pharmacy, Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran
d) Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
(fax: +98-21-66461178; e-mail: akbarzad@tums.ac.ir)
e) Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
f) Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
This work describes the synthesis of novel 1,2,3-triazole-4-linked (2E,6E)-2-benzylidene-6-(4-nitrobenzylidene)cyclo-
hexanones starting from cyclohexanone. 1-(Cyclohex-1-en-1-yl)piperidine, the enamine from cyclohexanone and piperidine,
reacted with 4-nitrobenzaldehyde to obtain 2-(4-nitrobenzylidene)cyclohexanone. Condensation of the latter compound with
(prop-2-yn-1-yloxy)benzaldehyde derivatives under acidic conditions gave (4-nitrobenzylidene)-[(prop-2-yn-1-yloxy)-
benzylidene]cyclohexanones. Finally, ‘click reaction’ of these derivatives and various organic azides led to the title compounds.
All compounds were examined by MTT assay for cytotoxic activity in one human breast cancer cell line, MDA-MB-231.
Keywords: (E,E)-2-Benzylidene-6-(4-nitrobenzylidene)cyclohexanones, 1,2,3-Triazoles, Click chemistry, Cyclohexanone,
Cytotoxic activity.
Among a large spectrum of N-heterocyclic com-
pounds, 1,2,3-triazoles are largely attractive because of
Introduction
1,3-Diarylprop-2-en-1-ones, known as chalcones are the valuable biological properties such as cytotoxic [14], anti-
main component of various natural products and impor-
tant starting material for the synthesis of flavonoids and
HIV-1 [15], anti-influenza [16], antiplatelet [17], and anti-
tuberculosis [18] activities. Considering the fact that
isoflavonoids [1]. The presence of the a,b-unsaturated hybridization of two or more biologically active scaffolds
moiety in chalcones leads to the important biological
activities such as antimalarial [2], antibacterial [3], anti-
tumor [4], antifungal [5], antiamoebic [6], and antiplas-
modial [7] activities. The promising biological properties
of chalcones come back to the fact that they interact with
thiols instead of amino and OH groups of nucleic acids
avoiding the genotoxic problems [8]. Therefore, they have
been the center of attention for preparing anticancer
agents. It has been revealed that the electronic properties
of substituents on the benzylidene moiety play an impor-
tant role on the cytotoxicity and among them nitro group
has displayed excellent activity [9 – 12]. In this respect,
(E,E)-2-benzylidene-6-(4-nitrobenzylidene)cyclohexanones
possessing a,b-unsaturated moiety as versatile chalcone
analogs were found to have potential anticancer activity
has been a versatile tool for advanced drug discovery
research. Herein, in continuation of our research program
on the synthesis of novel and bioactive heterocycles
[19 – 26], we focused on the synthesis and cytotoxic evalua-
tion of novel 1,2,3-triazole-4-linked (2E,6E)-2-benzylidene-
6-(4-nitrobenzylidene)cyclohexanones 13 (Scheme).
Results and Discussion
Considering the efficiency of the NO2 group on the
bioactivity of chalcones, we specially focused on 1,2,3-tria-
zole-4-linked 2-benzylidene-6-(4-nitrobenzylidene)cyclo-
hexanones. Our investigation was started by the
preparation of 2-(4-nitrobenzylidene)cyclohexanone (5).
Usually, it was obtained by the reaction of cyclohexanone
[10]. Also, anti-inflammatory activity of nitrobenzylidene (2) and 4-nitrobenzaldehyde (4) [10]. In spite of the fact
chalcones has been successfully reported [13].
that we tried the similar procedure in the literature, the
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DOI: 10.1002/hlca.201500210
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Helv. Chim. Acta 2016, 99, 175 – 180
Scheme. Synthesis of novel 1,2,3-triazole-4-linked (2E,6E)-2-benzylidene-6-(4-nitrobenzylidene)cyclohexanones 13
corresponding yield was low and compound 5 was pre- them, possessing electron donating or electron withdraw-
pared through the reaction of 1-(cyclohex-1-en-1-yl)piper- ing groups as well as halogens, underwent ‘click reaction’
idine (3) and 4-nitrobenzaldehyde (4) in the presence of
to afford product 13. The structure of products 13 was
TsOH in refluxing EtOH to give the compound 5 in good confirmed using IR, 1H- and 13C-NMR spectroscopy as
yield (70%). It should be noted that compound 3 was well as by chemical analysis. According to the 1H-NMR
easily prepared from the reaction of piperidine (1) and spectra of the products, they were isomerically pure as
cyclohexanone (2) in refluxing toluene [27].
As outlined in the Scheme, (prop-2-yn-1-yloxy)benzal- of 7.00 – 8.00 ppm which is distinguishing for (E) isomers.
dehyde derivatives 8 were prepared by the reaction of It should be noted that the corresponding H-atoms
the absorptions of the olefinic H-atoms were in the region
HCꢀCCH2Br (6) and hydroxybenzaldehyde derivatives 7 related to (Z) configuration absorb at higher fields [29].
in the presence of K2CO3 in DMF at 80 °C. Next, the
reaction of compounds 5 and 8 in the presence of HCl
Then, the in vitro cytotoxic activity of all 1,2,3-tria-
zole-4-linked nitrochalcones 13 were evaluated against
(gas) led to the formation of products 9, possessing a MDA-MB-231, a human breast cancer cell line and com-
CꢀC bond, which were readily converted to the desired pared with curcumin. All results were summarized in the
product 13 through ‘click reaction’ [28]. For this purpose, Table. According to the IC50 values, compounds 13
various organic azides 12 were prepared by the reaction
of different benzyl chlorides/bromides 10 and NaN3 (11)
in the presence Et3N in the mixture of H2O/tBuOH at and 13i (IC50 = 8.0 lM) exhibited higher activity in com-
room temperature. Then, compound 9, sodium ascorbate, parison to curcumin (IC50 = 11.1 lM). It is clear that the
and catalytic amount of CuSO4 were added to the freshly presence of a MeO group at 3-position of the benzylidene
prepared azides 12 leading to the formation of different moiety plays an important role in the cytotoxicity.
showed good cytotoxic activity against MDA-MB-231.
Among them, 13e (IC50 = 7.5 lM), 13f (IC50 = 5.8 lM),
1,2,3-triazole-4-linked nitrochalcones 13.
The scope of the reaction was studied by varying
azide derivatives 12 (Table). The results show that all of
Comparing our results of cytotoxicity with those obtained
by Dimmock et al. [10] revealed that the (2E,6E)-2-ben-
zylidene-6-(4-nitrobenzylidene)cyclohexanone derivative
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Helv. Chim. Acta 2016, 99, 175 – 180
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Table. Synthesis of 1,2,3-triazole-4-linked dibenzylidenecyclohexanones 13 and their in vitro cytotoxic activities (IC50 in lM) against MDA-
MB-231
Entry
X
Y
Product 13
Yield [%]a)
MDA-MB-231 IC50 [lM]
1
2
–
–
–
–
4-F
4-Cl
13a
13b
13c
13d
13e
13f
13g
13h
13i
60
65
60
70
65
60
62
55
60
12.0 ꢂ 0.3
13.2 ꢂ 0.1
12.6 ꢂ 0.5
13.5 ꢂ 0.5
7.5 ꢂ 0.7
5.8 ꢂ 0.9
14.0 ꢂ 0.4
14.5 ꢂ 0.1
8.0 ꢂ 0.4
11.1 ꢂ 0.1
3
2,3-Cl2
4-NO2
H
4
5
MeO
MeO
MeO
MeO
MeO
Curcumin
6
4-F
4-Cl
7
8
3,4-Cl2
4-MeO
9
10
a) Yield of isolated product.
possessing MeO groups showed higher activity as an Synthesis of (2E)-2-(4-Nitrobenzylidene)cyclohexanone
effective factor in our study. However, it should be noted (5) [10]. A mixture of 1-(cyclohex-1-en-1-yl)piperidine (3;
that their study was related to cytotoxicity on human
Molt/C8 and CEM T-lymphocytes as well as murine
L1210 cells.
0.16 g, 1 mmol), 4-nitrobenzaldehyde (4; 0.15 g, 1 mmol),
and TsOH (0.08 g, 0.5 mmol) in EtOH (15 ml) was
heated at reflux for 5 h. After completion of the reaction,
the mixture was cooled to r.t. and resulted crystals were
filtered off and used for the subsequent reaction.
Conclusions
Synthesis of (Prop-2-yn-1-yloxy)benzaldehyde Deriva-
tives 8. Typical Procedure. A mixture of HCꢀCCH2Br (6;
0.12 g, 1 mmol), hydroxybenzaldehyde derivative 7 (1
mmol), and K2CO3 (0.14 g, 1 mmol) in DMF (15 ml) was
In summary, we described an operationally procedure for
the synthesis of novel 1,2,3-triazole-4-linked nitrochal-
cones. Also, all compounds showed satisfactory cytotoxic-
ity in MDA-MB-231, human breast cancer cell line. Good stirred at 80° for 4 h. After completion of the reaction
yields of products (55 – 70%) and user-friendly steps
encourage organic and medicinal chemists to profit from
both synthetic and biological aspects.
(checked by TLC), the mixture was cooled to r.t. and
poured into crushed ice. The precipitated product was fil-
tered off and used for the next reaction.
Synthesis of (2E,6E)-(4-Nitrobenzylidene)-[(prop-2-
9.
We gratefully acknowledge the financial support from
Research Council of Tehran University of Medical Sciences.
yn-1-yloxy)benzylidene]cyclohexanone
Derivatives
Typical Procedure. Dry HCl gas was passed through an
ice-cold soln. of (2E)-2-(4-nitrobenzylidene)cyclohexa-
none (5) (0.23 g, 1 mmol) and compound 8 (1 mmol) in
abs. EtOH (3 ml) for 5 min. The mixture was allowed to
stand at r.t. for further 5 min. The precipitated product
was filtered off, washed with cold EtOH, and used for the
next reaction.
Experimental Part
General
M.p.: Kofler hot stage apparatus; uncorrected. IR Spectra:
Nicolet Magna FTIR 550 spectrophotometer; in KBr; ṽ in
Synthesis of 1,2,3-Triazole-4-Linked (2E,6E)-2-Benzy-
cmꢁ1. H- and 13C-NMR spectra: Bruker FT-400 (400 and lidene-6-(4-nitrobenzylidene)cyclohexanones 13. Typical
100 MHz, resp.); d in ppm rel. to Me4Si as internal standard, Procedure. A soln. of benzyl chloride/bromide derivative
J in Hz. Elemental analyses: VarioEL CHNS mode (Ele- 10 (1.1 mmol), NaN3 (11; 0.06 g, 0.9 mmol), and Et3N
mentar Analysensysteme GmbH); in %. 1-(Cyclohex-1-en-1- (0.13 g, 1.3 mmol) in a mixture of H2O (4 ml)/tBuOH (4
1
yl)piperidine (3) was prepared according to [27].
ml) was stirred at r.t. for 1 h to obtain azide 12.
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Helv. Chim. Acta 2016, 99, 175 – 180
Subsequently, a mixture of 9 (1 mmol), sodium ascorbate
(0.2 g, 0.1 mmol), and CuSO4 (0.01 g, 7 mol %) was
added to the freshly prepared azide derivative 12, and the (d, J = 8.8, H–C(200,600)); 7.82 (d, J = 8.8, H–C(2,6)); 8.13
mixture was stirred at r.t. for 24 – 48 h. Upon completion (d, J = 8.8, H–C(300,500)); 8.24 (d, J = 8.8, H–C(3,5)); 8.29
5.22 (s, CH2); 5.81 (s, CH2); 7.10 – 7.15 (m, 3 H, H–C
(30,50), CH); 7.51 – 7.61 (m, 3 H, H–C(20,60), CH); 7.65
of the reaction, monitored by TLC, the mixture was
diluted with H2O, poured into crushed ice, extracted using
AcOEt (3 9 30 ml), and the solvent was evaporated under
vacuum. Finally, all products were recrystallized from pet-
roleum ether/AcOEt (1:1) to give pure products 13.
(s, 1 H, triazole). 13C-NMR: 24.1; 29.2; 34.5; 52.4; 61.6;
115.3; 123.4; 124.4; 129.5; 130.5; 131.7; 132.8; 132.9; 136.2;
136.7; 137.0; 137.8; 141.5; 140.1; 143.3; 143.8; 144.4; 159.5;
189.0. Anal. calc. for C30H25N5O6 (551.56): C 65.33, H
4.57, N 12.70; found: C 65.52, H 4.42, N 12.81.
(2E,6E)-2-(4-{[1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl]
methoxy}benzylidene)-6-(4-nitrobenzylidene)cyclohexa-
none (13a). Pale-yellow solid. Yield: 0.32 g (60%). M.p.
(2E,6E)-2-{2-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy]-
3-methoxybenzylidene}-6-(4-nitrobenzylidene)cyclohexa-
none (13e). Pale-yellow solid. Yield: 0.35 g (65%). M.p.
121 – 123°. IR: 2970, 2850, 1665, 1583, 1575, 1350. 1H- 168 – 170°. IR: 2925, 2850, 1660, 1620, 1575, 1350. 1H-
NMR: 1.71 – 1.98 (m, CH2); 2.73 – 2.87 (m, 2 CH2); 5.20 NMR: 1.75 – 1.88 (m, CH2); 2.89 – 2.94 (m, 2 CH2); 3.77
(s, CH2); 5.68 (s, CH2); 7.11 – 7.26 (m, 3 H, H–C(30,50), (s, MeO); 5.18 (s, CH2); 5.62 (s, CH2); 7.13 – 7.24 (m, 2
CH); 7.35 – 7.65 (m, 7 H, H–C(20,60,200,300,500,600), CH); H, H–C(40), CH); 7.33 – 7.38 (m, 6 H, Ph, CH); 7.60 –
7.76 (d, J = 8.0, H–C(2,6)); 8.25 (d, J = 8.0, H–C(3,5)); 7.66 (m, H–C(50,60)); 7.78 (d, J = 8.2, H–C(2,6)); 8.27 (d,
8.29 (s, 1 H, triazole). 13C-NMR: 24.1; 28.3; 34.5; 47.4;
J = 8.2, H–C(3,5)); 8.30 (s, 1 H, triazole). 13C-NMR:
61.6; 115.3 (d, J(C,F) = 4.9); 116.0; 123.3; 125.3 (d, J(C,F) 22.7; 28.2; 34.5; 53.3; 55.9; 62.0; 113.6; 114.8; 124.0;
= 3.2); 125.4; 128.5; 130.4; 131.2 (d, J(C,F) = 8.1); 131.6; 125.4; 128.4; 128.6; 128.9; 129.2; 130.4; 131.7; 133.1;
132.8; 132.9; 133.1; 133.8; 134.3; 141.5; 147.2; 159.2; 160.5 134.5; 136.4; 137.5; 140.1; 142.6; 143.1; 147.2; 148.8;
(d, J(C,F) = 245.3); 189.0. Anal. calc. for C30H25FN4O4 149.1; 189.0. MS: 536 (M+), 42, 404, 263, 215, 163, 135,
(524.55): C 68.69, H 4.80, N 10.68; found: C 68.51, H 4.68,
N 10.55.
106, 88. Anal. calc. for C31H28N4O5 (536.59): C 69.39, H
5.26, N 10.44; found: C 69.50, H 5.38, N 10.58.
(2E,6E)-2-(4-{[1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-
yl]methoxy}benzylidene)-6-(4-nitrobenzylidene)cyclohexa-
none (13b). Pale-yellow solid. Yield: 0.35 g (65%). M.p.
(2E,6E)-2-(2-{[1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl]-
methoxy}-3-methoxybenzylidene)-6-(4-nitrobenzylidene)
cyclohexanone (13f). Pale-yellow solid. Yield: 0.33 g
61 – 163°. IR: 2925, 2855, 1660, 1585, 1575, 1350. 1H- (60%). M.p. 144 – 146°. IR: 2925, 2850, 1660, 1625,
NMR: 1.70 – 1.95 (m, CH2); 2.75 – 2.87 (m, 2 CH2); 5.20 1575, 1350. 1H-NMR: 1.75 – 1.97 (m, CH2); 2.90 – 2.94
(s, CH2); 5.65 (s, CH2); 7.13 (d, J = 8.0, H–C(30,50)); 7.20 (m, 2 CH2); 3.78 (s, MeO); 5.18 (s, CH2); 5.62 (s, CH2);
– 7.25 (m, 3 H, H–C(200,600), CH); 7.36 – 7.55 (m, 3 H, H– 7.13 – 7.24 (m, 4 H, H–C(40,300,500), CH); 7.39 – 7.42 (m,
C(300,500), CH); 7.65 (d, J = 8.0, H–C(20,60)); 7.78 (d, J = 3 H, H–C(50,60), CH); 7.5 (d, J = 7.5, H–C(200,600)); 7.79
8.0, H–C(2,6)); 8.27 (d, J = 8.0, H–C(3,5)); 8.30 (s, 1 H, (d, J = 8.4, H–C(2,6)); 8.28 (d, J = 8.4, H–C(3,5)); 8.30
triazole). 13C-NMR: 24.1; 28.3; 34.5; 63.5; 61.6; 115.4; (s, 1 H, triazole). 13C-NMR: 22.7; 28.3; 34.9; 52.5; 55.9;
124.0; 125.6; 128.4; 128.8; 129.2; 129.3; 130.4; 131.7; 132.9; 60.0; 113.6; 114.8; 116.1 (d, J(C,F) = 21.3); 123.4; 124.0;
133.1; 133.8; 134.4; 138.8; 142.6; 142.9; 147.2; 159.1; 189.0. 125.3; 128.9; 130.8 (d, J(C,F) = 8.3); 131.7; 133.1; 134.5;
Anal. calc. for C30H25ClN4O4 (541.00): C 66.60, H 4.66, N
10.36; found: C 66.75, H 4.51, N 10.24.
(2E,6E)-2-(4-{[1-(2,3-Dichlorobenzyl)-1H-1,2,3-triazol-
4-yl]methoxy}benzylidene)-6-(4-nitrobenzylidene)cyclo-
137.5; 140.1; 143.2; 147.2; 148.8; 149.1; 160.1 (d, J(C,F) =
245.0); 189.0. Anal. calc. for C31H27FN4O5 (554.58): C
67.14, H 4.91, N 10.10; found: C 67.32, H 5.11, N 10.21.
(2E,6E)-2-(2-{[1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl]-
hexanone (13c). Pale-yellow solid. Yield: 0.34 g (60%). methoxy}-3-methoxybenzylidene)-6-(4-nitrobenzylidene)-
M.p. 158 – 160°. IR: 2920, 2850, 1665, 1588, 1572, 1350. cyclohexanone (13g). Pale-yellow solid. Yield: 0.35 g
1H-NMR: 1.72 – 1.89 (m, CH2); 2.85 – 2.90 (m, 2 CH2); (62%). M.p. 178 – 180°. IR: 2933, 2850, 1660, 1595, 1575,
5.22 (s, CH2); 5.65 (s, CH2); 7.10 – 7.14 (m, 3 H, H–C 1350. 1H-NMR: 1.75 – 1.88 (m, CH2); 2.90 – 2.92 (m, 2
(30,50), CH); 7.29 – 7.32 (m, H–C(500,600)); 7.50 – 7.67 (m, 4 CH2); 3.75 (s, MeO); 5.20 (s, CH2); 5.57 (s, CH2); 7.11 –
H, H–C(20,60,400), CH); 7.78 (d, J = 8.8, H–C(2,6)); 8.27 (d, 7.25 (m, 4 H, H–C(40,200,600), CH); 7.37 – 7.41 (m, 3 H,
J = 8.8, H–C(3,5)); 8.36 (s, 1 H, triazole). 13C-NMR: 24.1; H–C(50,60), CH); 7.47 (d, J = 8.0, H–C(300,500)); 7.79 (d, J
28.3; 34.5; 51.9; 61.6; 115.4; 123.4; 124.0; 125.5; 128.9;
= 8.5, H–C(2,6)); 8.16 (d, J = 8.5, H–C(3,5)); 8.28 (s, 1
130.5; 130.6; 131.5; 131.7; 132.8; 132.9; 133.1; 134.4; 136.4; H, triazole). 13C-NMR: 22.7; 28.3; 34.5; 55.9; 62.0; 66.3;
137.0; 137.4; 143.3; 144.4; 147.2; 159.1; 189.0. Anal. calc.
for C30H24Cl2N4O4 (575.45): C 62.62, H 4.20, N 9.74;
found: C 62.79, H 4.38, N 9.58.
(2E,6E)-2-(4-Nitrobenzylidene)-6-(4-{[1-(4-nitroben-
zyl)-1H-1,2,3-triazol-4-yl]methoxy}benzylidene)cyclohexa-
none (13d). Pale-yellow solid. Yield: 0.38 g (70%). M.p.
178 – 180°. IR: 2918, 2850, 1660, 1608, 1570, 1350.
1H-NMR: 1.72 – 1.89 (m, CH2); 2.85 – 2.92 (m, 2 CH2);
113.9; 114.8; 123.4; 124.1; 125.7; 128.4; 128.8; 129.2; 129.3;
130.4; 131.7; 133.5; 138.1; 138.8; 141.6; 142.9; 146.7; 147.2;
148.8; 149.2; 189.0. Anal. calc. for C31H27ClN4O5
(571.03): C 65.21, H 4.77, N 9.81; found: C 65.36, H 4.61,
N 9.66.
(2E,6E)-2-(2-{[1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-
4-yl]methoxy}-3-methoxybenzylidene)-6-(4-nitrobenzyli-
dene)cyclohexanone (13h). Pale-yellow solid, Yield:
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Helv. Chim. Acta 2016, 99, 175 – 180
179
0.33 g (55%). M.p. 166 – 168°. IR: 2933, 2850, 1654,
1592, 1570, 1350. H-NMR: 1.75 – 1.88 (m, CH2); 2.75 –
overnight incubation, 5 ll of the media containing various
concentrations of the compounds was added per well in
1
2.92 (m, 2 CH2); 3.77 (s, MeO); 5.16 (s, CH2); 5.73 (s, triplicate (final concentration 1, 5, 10, and 20 lg/ml). The
CH2); 7.12 – 7.22 (m, 2 H, H–C(40), CH); 7.34 – 7.66
plates were incubated for further 72 h. The final concen-
(m, 6 H, H–C(50,60,200,500,600), CH); 7.80 (d, J = 8.2, H–C tration of DMSO was 0.1%. In each plate, there were
(2,6)); 8.15 (d, J = 8.2, H–C(3,5)); 8.28 (s, 1 H, triazole). three control wells (cells without test compounds) and
13C-NMR: 22.9; 28.3; 34.5; 55.9; 60.0; 62.3; 113.6; 114.8; three blank wells (the medium with 0.1% DMSO) for cell
123.4; 124.0; 124.1; 125.4; 125.6; 128.5; 128.1; 129.1; viability. Curcumin was used as positive control for cyto-
129.3; 130.1; 131.6; 132.5; 133.1; 138.1; 141.5; 142.7; toxicity. After treatment, the medium was removed and
146.8; 147.1; 148.9; 149.0; 189.0. Anal. calc. for 200 ll phenol red-free medium containing MTT (1 mg/
C31H26Cl2N4O5 (605.47): C 61.50, H 4.33, N 9.25; found:
C 61.35, H 4.41, N 9.39.
ml) was added to the wells, followed by 4 h incubation.
After incubation, the culture medium was replaced with
100 ll of DMSO and the absorbance of each well was
measured by using a microplate reader at 492 nm. For
each compound, the concentration causing 50% cell
(2E,6E)-2-(3-Methoxy-2-{[1-(4-methoxybenzyl)-1H-1,2,
3-triazol-4-yl]methoxy}benzylidene)-6-(4-nitrobenzylidene)-
cyclohexanone (13i). Pale-yellow solid. Yield: 0.34
g
(60%). M.p. 138 – 140°. IR: 2932, 2850, 1650, 1600, 1570, growth inhibition (IC50) compared with the control was
1350. 1H-NMR: 1.73 – 1.93 (m, CH2); 2.90 – 2.94 (m, 2
calculated from concentration response curves by nonlin-
CH2); 3.74 (s, MeO); 3.77 (s, MeO); 5.17 (s, CH2); 5.53 (s, ear regression analysis.
CH2); 6.93 (d, J = 8.4, H–C(300,500)); 7.15 – 7.17 (m, 2 H,
H–C(40), CH); 7.31 (d, J = 8.4, H–C(200,600)); 7.63 – 7.66
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Received August 9, 2015
Accepted November 30, 2015
€
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