Synthesis of substituted-phenyl-1,2,4-triazol-3-thione analogues with modified d-glucopyranosyl residues and their antiproliferative activities

Article abstract of DOI:10.1016/j.ejmech.2009.05.030

A series of d-glucopyranosyl-1,2,4-triazole-3-thione derivatives 1a-1d were synthesized by the reaction of 1,2,4-triazole-3-thione Schiff bases 5a-5d with 2,3,4,6-tetra-O-acetyl-σ-d-glucopyranosyl bromide. We demonstrate the conversion of 2 to 5, without the necessity of purification of both oxadiazole and triazole intermediates to afford the compounds 5. Their structures were confirmed by standard studies of ¹H NMR, IR, MS and elemental analysis. Analogues 5 and 1 have shown cytotoxic activity against human MCF-7 and Bel-7402 malignant cell lines. Crown Copyright

Full text of DOI:10.1016/j.ejmech.2009.05.030

European Journal of Medicinal Chemistry 44 (2009) 4716–4720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of substituted-phenyl-1,2,4-triazol-3-thione analogues
with modified -glucopyranosyl residues and their antiproliferative activities
D
,
,
,
Zhizhang Li a 1, Zheng Gu b 1, Kai Yin b, Rong Zhang b, Qin Deng b, Jiannan Xiang b
*
a Department of Chemistry, Hunan University of Science and Engineering, Yongzhou 425100, PR China
b College of Chemistry and Chemical Engineering, Biomedical Engineering Center and State Key Laboratory of Chemo/Biosensing and Chemometrics,
Hunan University, Changsha 410082, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of
D
-glucopyranosyl-1,2,4-triazole-3-thione derivatives 1a–1d were synthesized by the reaction
-glucopyranosyl bromide.
Received 27 February 2009
Received in revised form
2 May 2009
Accepted 20 May 2009
Available online 6 June 2009
of 1,2,4-triazole-3-thione Schiff bases 5a–5d with 2,3,4,6-tetra-O-acetyl-
s-D
We demonstrate the conversion of 2 to 5, without the necessity of purification of both oxadiazole and
triazole intermediates to afford the compounds 5. Their structures were confirmed by standard studies of
1H NMR, IR, MS and elemental analysis. Analogues 5 and 1 have shown cytotoxic activity against human
MCF-7 and Bel-7402 malignant cell lines.
Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
1,2,4-Triazol-3-thiones
D
-Glucopyranosyl
Schiff base
Antiproliferative activity
1. Introduction
chemicals, and biologically active pharmaceuticals vital for
enhancing the quality of life [12].
1,2,4-Triazoles nucleus and their derivatives emerge rapidly
with the advance of modern heterocyclic chemistry, promising
a variety of medical applications such as antibacterial, antifungal,
anticancer, antitumor, anticonvulsant, anti-inflammatory, and
analgesic properties [1–6]. 1,2,4-Triazole nucleus has been incor-
porated into a wide variety of therapeutically interesting molecules
to transform them into better drugs [7–9].
Recently, for the rapid development of drug resistance, new
antiproliferative agents should be designed and synthesized with
chemical characteristics clearly differ from those of existing agents.
4-Amino-3-mercapto-1,2,4-triazol derivative is an ideal heterocyclic
by virtue of its vicinal nucleophiles amino and mercapto groups
constitutes a ready-made building block for construction of various
organic heterocycles [10]. It has been established that introduction of
4-methyl mercapto phenyl to different heterocycles has yielded
many biologically active compounds endowed with wide spectrum
of pharmacological and antimicrobial activities [11]. Nitrogen-con-
taining heterocyclic molecules constitute the largest portion of
chemical entities, which are part of many natural products, fine
Schiff bases of 1,2,4-triazoles find diverse applications and
extensive biological activity. Schiff bases derived from 3-substituted-
4-amino-5-mercapto-1,2,4-triazoles show analgesic, antimicrobial,
anti-inflammatory, and antidepressant activities [13]. The incorpo-
ration of the 1,2,4-triazole unit into Schiff base macrocycles is of
considerable current interest as complexes of 1,2,4-triazoles are
being developed for potential use in applications such as magnetic
materials and photochemically driven molecular devices [14].
It was known that carbohydrates and their derivatives have
a significant biological role and occur widely in all living matter and
function even appear to be essential to the process of infection by
certain pathogenic species [15]. High levels of glycosylation are one
of the many molecular changes that accompany malignant trans-
formations. These changes are characteristic for cancer cells and
can protect them from immune surveillance and chemotherapeutic
agents, and enhance their metastatic capacity [16]. The carbohy-
drate moiety can be expected to play the role of drug carrier and
improve the selectivity of compounds for cancerous cell lines. For
example, vaccination using synthetic tumor-associated antigens
such as carbohydrate antigens, holds promise for generating
a specific antitumor response by targeting the immune system to
cancer cells [17]. The galectin family of carbohydrate receptors
represents a promising target for drug delivery. Galectin-3 is
selected as a model receptor for targeting of glycoside-bearing drug
* Corresponding author. Tel./fax: þ86 731 8821740.
E-mail address: jnxiang@hnu.cn (J. Xiang).
1
These authors contributed equally to this work.
0223-5234/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.030

Z. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4716–4720
4717
carriers and some reports describe the upregulation of galectin-3 in the
progression of colorectal cancer [18]. The incorporation of a carbohy-
drate moiety on a 1,2,4-triazoles scaffold unleashes the potential to
access a new dimension of structural diversity to complement the vast
structural diversity already inherent to 1,2,4-triazoles.
The aim of the present study was to modify the bioactivities of
1,2,4-triazole Schiff bases and gain the relative derivatives with
better curing effect, optimization of hydrophilic/lipophilic char-
acter, and improve the bioavailability by inducing glycosyl groups.
During the course of this work, we prepared some 1,2,4-triazole-3-
conveniently prepared using Koenigs–Knorr reaction, phase-transfer
catalysis, etc. [22–24]. Recently, phase-transfer catalysis has proven
to be a very useful method for various synthetic transformations. This
methodology allows synthetic modifications under mild conditions
that were heretofore reserved to very drastic reagents and reaction
media [25]. Then, the compounds 5 were treated with 2,3,4,6-tetra-
O-acetyl-s-D-glucopyranosyl bromide and Bu4NBr in ethanol,
producing the desired series of 1,2,4-triazole-3-thione derivatives
(1a–1d).
The chemical structures of the compounds were confirmed by
1H NMR, IR, MS spectra and elemental analysis and the results are
presented in Section 5.
thione Schiff bases and the related D-glucopyranosyl derivatives.
The newly synthesized compounds were evaluated for their cyto-
toxic activity against MCF-7 and Bel-7402 cell lines.
3. Biological evaluation
2. Chemistry
The newly synthesized 1,2,4-triazole-3-thione derivatives 5 and
1 were evaluated for their antiproliferative activity against estrogen
receptor positive breast adenocarcinoma MCF-7 and human liver
cancer cell lines Bel-7402. The MTT assay was widely applied to
examine in vitro cytotoxicity of the drug molecule after 24 h cell
treatment [26]. The results, including the IC50 values of compounds
5, 1 and the reference compound 5-fluorouracil [27], are summa-
rized in Table 1.
The synthesis of the 1,2,4-triazoles Schiff base and 2,3,4,6-tetra-
O-acetyl-s-D-glucopyranosyl-1,2,4-triazole-3-thione derivatives is
illustrated and outlined in Scheme 1.
We prepared 5-phenyl-1,3,4-oxadiazole-2(3H)-thione (3) from
benzohydrazide (2) and carbon disulfide in boiling absolute ethanol
with a yield of 90% [19]. Thione derivative (3) readily reacted with
hydrazine in absolute ethanol to produce the sulphhydryl deriva-
tive of 1,2,4-triazole (4) in 68% yield [20].
Table 1 shows that all the compounds were active in mM range
1,2,4-Triazole-3-thione Schiff bases (5a–5d) were synthesized by
the reaction of 4-amino-5-phenyl-2H-1,2,4-triazole-3(4H)-thione (4)
with aromatic aldehydes in glacial acetic acid medium. In literature,
synthesis of 1,2,4-triazole Schiff bases was catalyzed with acid or base
in ethanol medium [21]. The reaction requires several hours. And, the
product must be purified by crystallization or column chromatog-
raphy. Here we brought up a better approach: we used glacial acetic
acid as catalyst and solvent; and the reaction took only about 10 min.
Furthermore, the products 5 can be purified easily by washing with
ethanol. The conversion of 2 to 5a–5d can be performed to achieve an
overall yield of 50% in three step, without the necessityof purification
of either oxadiazole or triazole intermediates. Sugar esters were
against the MCF-7 and Bel-7402 malignant cell lines. The glycosyl
esters 1a–1d showed similar activity against MCF-7 and Bel-7402
cells when compared to 5-fluorouracil. Meanwhile, 1a–1d showed
higher antiproliferative activity than Schiff bases 5a–5d, demon-
strating that our strategy of modifying these molecules was
successful. Perhaps, both bioactive component group and structure
were shown to improve the bioactivity of 1,2,4-triazole-3-thiones.
Interestingly,1c showed more potent cytotoxic activity against MCF-
7 cells being 2.7-fold more potent than the reference compound 5c.
This analogue was also active against Bel-7402 cells, but it was
almost 2.3-fold less potent with respect to the parent molecule 5c.
Compound 1b was approximately 2-fold and 1.4-fold more active
O
C
a
b
N
NH
N
NH
NHNH2
N
H2N
O
S
S
3
4
2
c
d
AcO
N
NH
N
N
O
N
N
OAc
OAc
N
N
AcO
R
R
S
S
1
5
5a R = H
1a R = H
5b R = 4-Cl
1b R = 4-Cl
5c R = 3-OCH3-4-OH
5d R = 4-OH
1c R = 3-OCH3-4-OH
1d R = 4-OH
Scheme 1. Synthesis of the 1,2,4-triazoles Schiff base and D-glucopyranosyl-1,2,4-triazole-3-thione derivatives. Reagents and conditions: (a) CS2, KOH, EtOH, 80 ꢀC, 8 h, 90% of 3; (b)
NH2NH2, EtOH, 90 ꢀC, 8 h, 68% of 4; (c) Aromatic aldehydes, CH3COOH, 120 ꢀC, 10 min, 82% of 5a, 86% of 5b, 78% of 5c, 79% of 5d; (d) 2,3,4,6-tetra-O-acetyl-
bromide, NaHCO3, Bu4NBr, rt, 24 h, 61% of 1a, 67% of 1b, 63% of 1c, 62% of 1d.
s-D-glucopyranosyl

4718
Z. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4716–4720
Table 1
were obtained on a Varian INOVA-400 Spectrometer, using CDCl3 as
a solvent; TMS ( 0.00 ppm) and trifluoroacetic acid were used as
internal standard. All NMR chemical shifts are reported as values
Cytotoxicity of the target compounds against MCF-7 and Bel-7402 cells in vitro.
d
a,b
d
Compound
IC50
(mM)
in parts per million (ppm) and coupling constants (J) are given in
hertz (Hz). The splitting pattern abbreviations are as follows: s,
singlet; d, doublet; br, broad peak; and m, multiplet. Mass spectra
were carried out on a ZAB-HS mass spectrometer. Elemental anal-
yses were recorded with Vario-EL-III Spectrometer.
MCF-7c
Bel-7402
5a
5b
5c
5d
1a
1b
1c
1d
31.8 ꢂ 2.2
30.5 ꢂ 2.1
26.3 ꢂ 2.3
29.9 ꢂ 2.0
18.0 ꢂ 1.2
14.1 ꢂ 1.1
9.7 ꢂ 0.6
22.5 ꢂ 1.8
24.0 ꢂ 1.5
19.6 ꢂ 1.3
25.2 ꢂ 1.7
10.7 ꢂ 0.8
17.4 ꢂ 1.0
8.6 ꢂ 0.6
2,3,4,6-Tetra-O-acetyl-s-D-glucopyranosyl bromide was synthe-
sized by our laboratory [16]. 4-Dimethylamino pyridine (DMAP) was
purchased from ACROS. Absolute ethanol (CH3CH2OH) and
dichloromethane (CH2Cl2) were dried and purified using standard
technique. Other reagents were of the highest commercially avail-
able quality.
12.8 ꢂ 1.0
4.3 ꢂ 0.7
11.3 ꢂ 0.9
4.7 ꢂ 0.4
5-Fluorouracil
a
IC50 is the concentration of compound required to inhibit the cell growth by 50%
compared to an untreated control.
b
Values are expressed as mean ꢂ SD (n ¼ 3).
c
MCF-7, human breast cancer cells; Bel-7402, human hepatocellular cancer cell.
5.1.1. General procedure for compounds 3 and 4
Compound 2 (0.27 g, 2.0 mmol) and KOH (0.17 g, 3.0 mmol)
were dissolved in 50 mL absolute ethanol. CS2 (0.18 mL, 3.0 mmol)
was added dropwise. The mixture was refluxed for 8 h, and then
distilled under reduced pressure. The residue was added into
distilled water and the solid formed was filtered. The filtrate was
acidified and set to pH ¼ 1 with 2 M HCl solution and turned to
slurry. The product was obtained by filtration and dissolved in ethyl
acetate. The solution dried with MgSO4 and evaporated by vacuum.
The compound 3 was obtained.
The mixture of compound 3 (0.36 g, 2.0 mmol), 80% hydrate
hydrazine aqueous solution (0.4 mL, 2.5 mmol) and absolute
ethanol (20 mL) was refluxed at 90 ꢀC for 8 h. Then the mixture was
cooled and evaporated by vacuum. Crystallization of the product
from ethanol gave pink solid of compound 4.
than compound 5b against MCF-7 andBel-7402 cells, respectively. As
shown in Table 1, we have demonstrated that the modified
compound series 1a–1d performed more effectively than the refer-
ence compounds 5a–5d to kill the cancer cells (MCF-7 and Bel-7402).
We are interested in testing these compounds on a broader range of
cancer cell lines.
Due to such cytotoxicity against certain malignant cell lines, and
their nontoxicity towards the normal MCF-7 and Bel-7402 cells, the
analogues 1a–1d may serve as important leads in the synthesis of
more potent and selective anticancer agents. In order to search the
relationships between biological activity and structure, it should be
performed the synthesis and a more detailed biological evaluation
of a series of 1,2,4-triazole-3-thione analogues bearing different
sugar groups in their amide moiety. Further investigations of the
structure–toxicity and structure–activity relationship of an
expanded library of 1,2,4-triazole-3-thione derivatives are currently
underway in our group.
5.1.1.1. 5-Phenyl-1,3,4-oxadiazole-2(3H)-thione(3). White solid,
yield: 90%, m.p. 199–201 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 7.50–7.54
(m, 2H, C6H5); 7.57–7.61 (m, 1H, C6H5); 7.93–7.96 (m, 2H, C6H5);
10.69 (s,1H, NH). Anal. calcd for C8H6N2OS: C 53.92, H 3.39, N 15.72;
found C 53.84, H 3.35, N 15.79.
4. Conclusion
In summary, we have accomplished the synthesis of various
1,2,4-triazole-3-thione derivatives bearing Schiff bases 5a–5d and
2,3,4,6-tetra-O-acetyl-s-D-glucopyranosyl-1,2,4-triazole-3-thione
5.1.1.2. 4-Amino-5-phenyl-2H-1,2,4-triazole-3(4H)-thione (4). Pink
solid, yield: 68%, m.p.192–193 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 4.86
(s, 2H, NH2); 7.51–7.53 (m, 2H, C6H5); 7.78 (d, 1H, J ¼ 7.6 Hz, C6H5);
8.08–8.10 (m, 2H, C6H5); 11.17 (s, 1H, NH). Anal. calcd for C8H8N4S: C
49.98, H 4.19, N 29.14; found C 49.92, H 4.15, N 29.18.
derivatives (1a–1d). The conversion of 2 to 5a–5d was carried out
without purification of both oxadiazole and triazole intermediates
to afford the compounds 5 about 50% overall yield (three steps),
respectively.
Analogues 5 and 1 have shown a potent cytotoxic activity against
human malignant cell lines (MCF-7 and Bel-7402). The preliminary
results obtained in this structure–activity relationship study have
shown that in both series, the introduction of 2,3,4,6-tetra-O-acetyl-
5.1.2. General procedure for the synthesis of 4-(arylmethyl-
ideneamino)-5-phenyl-2H-1,2,4-triazole-3(4H)-thiones (5a–5d)
Aromatic aldehydes (2.0 mmol) were dissolved in glacial acetic
acid (4.0 mL) and compound 4 (0.38 g, 2.0 mmol) was added. The
reaction mixture was refluxed for 10 min. Then the mixture was
cooled and the solid separated was filtered and washed with
ethanol. After drying, compounds 5 were obtained.
s-D-glucopyranosyl bromide into 1,2,4-triazole-3-thione system is
potentially of interest to improve the cytotoxic activity of this series.
All these findings support the need for further investigations to
clarify the features underlying the antiproliferative potential of
these new 1,2,4-triazole derivatives. It may be concluded that the
fusion of 1,2,4-triazole nuclei in the case of the examined carbo-
hydrates might result in bioactive molecules of potency, particu-
larly if the substituents are designed with optimum toxophoric
requirements.
5.1.2.1. (E)-4-(Benzylideneamino)-5-phenyl-2H-1,2,4-triazole-3(4H)-
thione (5a). White solid, yield: 82%, m.p. 180–182 ꢀC; 1H NMR
(400 MHz, CDCl3)
d: 7.47–7.52 (m, 5H, C6H5); 7.52–7.57 (m, 1H,
C6H5); 7.88–7.90 (m, 2H, C6H5); 7.95–7.97 (m, 2H, C6H5); 10.07 (s,
1H, CH]N); 11.21 (s, 1H, NH). IR (KBr), n
/cmꢁ1: 3028, 1603, 1543,
1502, 1363, 1277. MS: m/z ¼ 280.7 ([M þ 1]þ); Anal. calcd for
C15H12N4S: C 64.26, H 4.31, N 19.98; found C 64.23, H 4.38, N
19.92.
5. Experimental protocols
5.1. Chemistry
5.1.2.2. (E)-4-(4-Chlorobenzylideneamino)-5-phenyl-2H-1,2,4-triazole-
3(4H)-thione (5b). Yellow solid, yield: 78%, m.p. 218–220 ꢀC; 1H
Melting points were measured on an XRC-1 melting point
apparatus, and are uncorrected. FT Infrared (IR) spectra were
recorded in KBr disks using FD-5DX spectrometer. 1H NMR spectra
NMR (400 MHz, CDCl3)
J ¼ 7.6 Hz, C6H4), 7.92 (d, 2H, J ¼ 7.2 Hz, C6H4), 10.16 (s, 1H, CH]N),
10.94 (s, 1H, NH). IR (KBr),
/cmꢁ1: 3030, 1599, 1539, 1500, 1356,
d: 7.46–7.54 (m, 5H, C6H5), 7.82 (d, 2H,
n

Z. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4716–4720
4719
1281. MS: m/z ¼ 314.9 ([M þ 1]þ); Anal. calcd for C15H11ClN4S: C
([M þ H]þ), 667.0 ([M þ Na]þ); Anal. calcd for C29H29ClN4O9S: C
57.23, H 3.52, N 17.80; found C 57.15, H 3.58, N 17.83.
53.99, H 4.53, N 8.69; found C 53.92, H 4.52, N 8.72.
5.1.2.3. (E)-4-(4-Hydroxy-3-methoxybenzylideneamino)-5-phenyl-
2H-1,2,4-triazole-3(4H)-thione (5c). Yellow solid, yield: 80%, m.p.
5.1.3.3. N-2-2,3,4,6-tetra-O-acetyl-s-D-glucopyranosyl-(E)-4-(4-
hydroxy-3-methoxy benzylideneamino)-5-phenyl-2H-1,2,4-triazole-
171–173 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.95 (s, 3H, OCH3), 7.03 (d,
3(4H)-thione (1c). White solid, yield: 63%, m.p. 201–203 ꢀC; 1H
1H, J ¼ 8.0 Hz, C6H5), 7.20 (dd, 1H, J ¼ 1.6 Hz, J ¼ 8.4 Hz, C6H3), 7.45
NMR (400 MHz, CDCl3) d: 1.95 (s, 3H, COCH3); 2.05 (s, 3H, COCH3);
(d, 2H, J ¼ 7.2 Hz, C6H3), 7.50 (d, 2H, J ¼ 1.6 Hz, C6H5), 7.88–7.90 (m,
2.08 (s, 3H, COCH3); 2.09 (s, 3H, COCH3); 3.98 (s, 3H, OCH3); 4.04
(dd, 1H, J ¼ 4.8 Hz, J ¼ 2.4 Hz); 4.20 (dd, 1H, J ¼ 12.4 Hz, J ¼ 2.0 Hz);
4.33 (dd, 1H, J ¼ 12.4 Hz, J ¼ 4.8 Hz); 5.29 (t, 1H, J ¼ 9.6 Hz); 5.45 (t,
1H, J ¼ 9.6 Hz); 5.96 (t, 1H, J ¼ 9.6 Hz); 6.31 (d, 1H, J ¼ 9.6 Hz); 7.01
(d, 1H, J ¼ 8.0 Hz, C6H5); 7.19 (dd, 1H, J ¼ 1.6 Hz, J ¼ 8.4 Hz, C6H3);
7.43 (d, 2H, J ¼ 7.2 Hz, C6H3); 7.51 (d, 2H, J ¼ 1.6 Hz, C6H5); 7.88–7.90
2H, C6H5), 9.70 (s, 1H, CH]N), 11.17 (s, 1H, NH). IR (KBr), n :
/cmꢁ1
3070, 1593, 1514, 1383, 1292. MS: m/z ¼ 327.4 ([M þ 1]þ); Anal.
calcd for C16H14N4O2S: C 58.88, H 4.32, N 17.17; found C 58.94, H
4.28, N 17.15.
5.1.2.4. (E)-4-(4-Hydroxybenzylideneamino)-5-phenyl-2H-1,2,4-tri-
azole-3(4H)-thione (5d). White solid, yield: 84%, m.p. 227–229 ꢀC;
(m, 2H, C6H5); 9.89 (s, 1H, CH]N). IR (KBr), n
/cmꢁ1: 3428, 3066,
2927, 1750, 1603, 1515, 1442, 1372, 1227, 1070, 1038, 959, 871, 769,
695, 653, 577. MS: m/z ¼ 656.9 ([M þ H]þ), 679.0 ([M þ Na]þ); Anal.
calcd for C30H32N4O11S: C 54.87, H 4.91, N 8.53; found C 54.92, H
4.86, N 8.55.
1H NMR (400 MHz, DMSO-d6)
d
: 6.94 (d, 2H, J ¼ 8.8 Hz, C6H4), 7.52–
7.54 (m, 3H, C6H5), 7.75 (d, 2H, J ¼ 9.2 Hz, C6H4), 7.86–7.89 (m, 2H,
C6H5), 9.37 (s, 1H, CH]N), 10.42 (s, 1H, OH), 14.16 (s, 1H, NH). IR
(KBr),
n
/cmꢁ1: 3030, 1606, 1568, 1512, 1356, 1284. MS: m/z ¼ 297.3
([M þ 1]þ); Anal. calcd for C15H12N4OS: C 60.79, H 4.08, N 18.91;
5.1.3.4. N-2-2,3,4,6-tetra-O-acetyl-s-D-glucopyranosyl-(E)-4-(4-
found C 60.85, H 4.02, N 18.87.
hydroxybenzylidene amino)-5-phenyl-2H-1,2,4-triazole-3(4H)-thi-
one (1d). White solid, yield: 62%, m.p. 213–215 ꢀC; 1H NMR
5.1.3. General procedure for the synthesis of N-2-2,3,4,6-tetra-O-
(400 MHz, CDCl3) d: 1.95 (s, 3H, COCH3); 2.05 (s, 3H, COCH3); 2.08
acetyl-
s
-
D
-glucopyranosyl-4-(arylmethylideneamino)-5-phenyl-
(s, 3H, COCH3); 2.09 (s, 3H, COCH3); 4.04 (dd, 1H, J ¼ 4.8 Hz,
J ¼ 2.4 Hz); 4.20 (dd, 1H, J ¼ 12.4 Hz, J ¼ 2.0 Hz); 4.33 (dd, 1H,
J ¼ 12.4 Hz, J ¼ 4.8 Hz); 5.30 (t, 1H, J ¼ 9.6 Hz); 5.45 (t, 1H,
J ¼ 9.6 Hz); 5.97 (t, 1H, J ¼ 9.6 Hz); 6.32 (d, 1H, J ¼ 9.2 Hz); 6.91 (d,
2H, J ¼ 8.8 Hz, C6H4); 7.43–7.49 (m, 3H, C6H5); 7.73–7.75 (m, 2H,
C6H5); 7.93 (d, 2H, J ¼ 8.8 Hz, C6H4); 9.65 (s, 1H, CH]N); 10.50 (s,
2H-1,2,4-triazole-3(4H)-thiones (1a–1d)
Compounds 5 (5a–5d, 2.2 mmol) and 4-dimethylamino pyridine
(DMAP, 0.13 g, 1.0 mmol) were suspended in a mixture of 10 mL
dichloromethane and 4% aqueous KOH (3.0 mL, 2.4 mmol). The
suspension was slowly heated to completely dissolve with vigor-
ously stirring. A solution of 2,3,4,6-tetra-O-acetyl-
s
-D-glucopyr-
1H, OH). IR (KBr), n
/cmꢁ1: 3421, 3290, 3064, 2927, 1751, 1604, 1516,
anosyl bromide (0.82 g, 2.0 mmol) in dichloromethane (10 mL) was
added dropwise carefully. The reaction solution was heated to
reflux and stirred for 5 h under nitrogen atmosphere. The solution
was washed with 4% aqueous NaOH (15 mL) and distilled water
(20 mL ꢃ 3). The organic layer was dried over with anhydrous
Na2SO4, and the filtrate was distilled under reduced pressure to
remove the solvent. The residue was purified by column chroma-
tography (silica gel 60, mesh size 200–300, ethyl acetate/petroleum
ether, v/v) to giveproducts 1 (1a–1d).
1439, 1371, 1223, 1063, 1038, 924, 850, 771, 692, 604. MS:
m/z ¼ 626.9 ([M þ H]þ), 649.0 ([M þ Na]þ); Anal. calcd for
C29H30N4O10S: C 55.58, H 4.83, N 8.94; found C 55.52, H 4.87, N 8.96.
5.2. In vitro cytotoxicity assay
The antiproliferative activities of 5a–5d and 1a–1d were
assessed by use of the MTT assay. The MTT assay is a simple
nonradioactive colorimetric assay to measure cell cytotoxicity,
proliferation, or viability. MTT is a yellow, water-soluble, tetrazo-
lium salt. Metabolically active cells are able to convert this dye into
a water-insoluble dark blue formazan by reductive cleavage of the
tetrazolium ring. Formazan crystals then can be dissolved and
quantified by measuring the absorbance of the solution at 570 nm,
and the resultant value is related to the number of living cells.
The effect of all compounds on the cells‘ proliferation efficiency
was determined after 24 h incubation with cells. To determine cell
proliferation, the MCF-7 cell lines and Bel-7402 cell lines were
individually plated at a density of 1 ꢃ104 cells/well in 96-well
plates at 37 ꢀC in 5% CO2 atmosphere. After 24 h of culture, the
medium in the wells was replaced with the fresh medium con-
taining compounds of varying concentrations respectively. In the
microplate, five wells were tested in parallel for each concentration
5.1.3.1. N-2-2,3,4,6-tetra-O-acetyl-
lideneamino)-5-phenyl-2H-1,2,4-triazole-3(4H)-thione (1a). White
solid, yield: 61%, m.p. 209–211 ꢀC; 1H NMR (400 MHz, CDCl3)
: 1.95
s-D-glucopyranosyl-(E)-4-(benzy-
d
(s, 3H, COCH3); 2.05 (s, 3H, COCH3); 2.07 (s, 3H, COCH3); 2.09 (s, 3H,
COCH3); 4.03 (dd, 1H, J ¼ 4.8 Hz, J ¼ 2.0 Hz); 4.19 (dd, 1H,
J ¼ 12.4 Hz, J ¼ 2.0 Hz); 4.32 (dd, 1H, J ¼ 12.8 Hz, J ¼ 4.8 Hz); 5.29 (t,
1H, J ¼ 9.6 Hz); 5.45 (t, 1H, J ¼ 9.6 Hz); 5.96 (t, 1H, J ¼ 9.6 Hz); 6.31
(d, 1H, J ¼ 9.2 Hz); 7.45–7.52 (m, 5H, C6H5); 7.55–7.57 (m, 1H, C6H5);
7.86–7.89 (m, 2H, C6H5); 7.93–7.96 (m, 2H, C6H5); 10.03 (s, 1H,
CH]N). IR (KBr), n
/cmꢁ1: 3477, 3062, 2958, 1751, 1064, 1493, 1429,
1369, 1223, 1072, 1036, 924, 847, 758, 692, 607. MS: m/z ¼ 611.0
([M þ H]þ), 633.0 ([M þ Na]þ); Anal. calcd for C29H30N4O9S: C 57.04,
H 4.95, N 9.18; found C 57.12, H 4.90, N 9.13.
of the synthesized compounds. After 24 h, 10 mL of MTT dye solu-
5.1.3.2. N-2-2,3,4,6-tetra-O-acetyl-
s
-
D
-glucopyranosyl-(E)-4-(4-
tion (5 mg/mL in phosphate buffer pH 7.4) was added to each well
and incubated for 4 h at 37 ꢀC and 5% CO2 for exponentially growing
cells and 10 min for steady-state confluent cells. The formazan
chlorobenzylidene amino)-5-phenyl-2H-1,2,4-triazole-3(4H)-thione
(1b). White solid, yield: 67%, m.p. 185–187 ꢀC; 1H NMR (400 MHz,
CDCl3)
d: 1.95 (s, 3H, COCH3); 2.05 (s, 3H, COCH3); 2.07 (s, 3H,
crystals were solubilized with 100 mL of DMSO and the solution was
COCH3); 2.08 (s, 3H, COCH3); 4.03 (dd, 1H, J ¼ 10.0 Hz, J ¼ 2.4 Hz);
4.19 (dd, 1H, J ¼ 12.4 Hz, J ¼ 2.0 Hz); 4.32 (dd, 1H, J ¼ 12.4 Hz,
J ¼ 5.2 Hz); 5.28 (t,1H, J ¼ 9.6 Hz); 5.45 (t,1H, J ¼ 9.6 Hz); 5.95 (t,1H,
J ¼ 9.6 Hz); 6.30 (d, 1H, J ¼ 9.6 Hz); 7.46–7.52 (m, 5H, C6H5); 7.80 (d,
2H, J ¼ 8.4 Hz, C6H4); 7.91 (d, 2H, J ¼ 8.4 Hz, C6H4); 10.12 (s, 1H,
vigorously mixed to dissolve the reacted dye. The absorbance of
each well was read on a microplate reader (DYNATECH MR7000
instruments) at 570 nm. The spectrophotometer was calibrated to
zero using culture medium without cells. We selected inhibitory
effect to evaluate side effects of the silica-coated fluorescent
compounds to cells proliferation. The inhibitory effect of all
compounds was calculated as percentage inhibition in comparison
CH]N). IR (KBr), n
/cmꢁ1: 3458, 2970, 1747, 1597, 1491, 1423, 1371,
1227, 1072, 1036, 931, 850, 775, 694, 607. MS: m/z ¼ 645.0

4720
Z. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4716–4720
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We are grateful for the project (07JJ3019) supported by the
Natural Science Foundation of Hunan, project (20060532022)
supported by Doctoral Fund of Ministry of Education of China,
project (200520) supported by the State Key Laboratory of Chem/
Biosensing and Chemometrics of Hunan University and Key project
(200610) supported by the Science and Technology Bureau of
Yongzhou, China.
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