Synthesis and antitumor evaluation of some 1,3,4-oxadiazole-2(3H)-thione and 1,2,4-triazole-5(1H)-thione derivatives

Article abstract of DOI:10.1007/s00044-010-9544-6

A series of 1,3,4-oxadiazole-2-thione and 1,2,4-triazole-5-thione derivatives have been successfully synthesized and studied for their antitumor activity. These compounds were prepared from carboxylic acids and chiral aminoacid esters. The prepared compounds were evaluated for their cytotoxicity against cancer in vitro using hydroxyurea (HU) as positive control. A fair number of compounds were found to have significant antitumor activity. To study the molecular basis of interaction and affinity of binding of the target molecules, all the compounds were docked into the ATPase domain of TP-II and tubulin using Schroedinger. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9544-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:315–320
DOI 10.1007/s00044-010-9544-6
ORIGINAL RESEARCH
Synthesis and antitumor evaluation of some 1,3,4-oxadiazole-
2(3H)-thione and 1,2,4-triazole-5(1H)-thione derivatives
•
ChengTao Feng LingDong Wang
•
•
YuGang Yan Jian Liu ShaoHua Li
•
Received: 28 July 2010 / Accepted: 15 December 2010 / Published online: 25 December 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of 1,3,4-oxadiazole-2-thione and 1,2,4-
triazole-5-thione derivatives have been successfully syn-
thesized and studied for their antitumor activity. These
compounds were prepared from carboxylic acids and chiral
aminoacid esters. The prepared compounds were evaluated
for their cytotoxicity against cancer in vitro using hydroxy-
urea (HU) as positive control. A fair number of compounds
were found to have significant antitumor activity. To study
the molecular basis of interaction and affinity of binding of
the target molecules, all the compounds were docked into the
Substituted 1,2,4-triazole-5-thiones and 1,3,4-oxadiaz-
oles are associated with many types of biological properties
(Omar et al., 1996; Khanum et al., 2005). Antitumor
activity data of these structures showed their considerable
antitumor activity (Holla et al., 2002; Al-Souda et al.,
2003; Loetchutinat et al., 2003; Aboraiaa et al., 2006;
Formagio et al., 2008). A large number of metallo-enzymes
incorporate Zn(II) or Cu(II) ions coordinated by one or
several –SH groups belonging to cysteine residues. The
inhibition of metallo-enzymes is largely due to the coordi-
nation of inhibitor with the catalytical metal ions of active
site. The combination of the exocyclic thione/thiol group
and the heterocyclic molecule, which may contain nitrogen,
oxygen, or sulfur, generates a group of molecules with
considerable coordination potential (Raper, 1997; Ferloni
et al., 2005). In addition to being the building blocks of
proteins and peptides, amino acids serve as precursors of
many kinds of small molecules that have important and
diverse biological roles, for example, 6-aminopenicillanic
acid (6-APA) and 7-aminocephalosporanic acid (7-ACA)
are derived from cysteine and valine. Some antitumor
agents, methotrexate and bactinnomycin D, contain amino
acids. Keeping the above facts in mind, it was of our interest
to synthesize some new 1,3,4-oxadiazole-2(3H)-thiones
and 1,2,4-triazole-5(1H)-thiones, which are synthesized
from different chiral aminoacid esters for their antitumor
properties.
¨
ATPase domain of TP-II and tubulin using Schrodinger.
Keywords 1,3,4-Oxadiazole-2(3H)-thione Á
1,2,4-Triazole-5(1H)-thione Á Synthesis Á
Antitumor activity Á Docking study
Introduction
Of the various human diseases, cancer has proven to be one
of the most intractable diseases to which humans are sub-
jected, and as yet no practical and generally effective drugs
or methods of control are available. Therefore, identifica-
tion of novel potent anticancer agents remains one of the
most pressing health problems.
C. Feng Á L. Wang Á Y. Yan Á J. Liu Á S. Li (&)
Department of Pharmacy, Nanchang University Medical
College, 603, Bayi Road, Nanchang 330006,
People’s Republic of China
Results and discussion
e-mail: lishaohua81@126.com
Chemistry
C. Feng
Department of Chemistry, Huainan Union University, Dongshan
West Road, Huainan 232001, Anhui, People’s Republic of China
The synthesis of the target compounds was carried out as
outlined in Scheme 1. Three amino acid methyl esters
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Med Chem Res (2012) 21:315–320
hydrochloride, 2a–c, were converted into their corre-
sponding amides 3a–h by a reaction with carboxylic acid in
an alkaline medium using standard methods. Treatment of
3a–h, with 80% hydrazine hydrate, furnished the corre-
sponding hydrazides 4a–h in good yields. Intramolecular
cyclization of 4a–h with carbon disulfide resulted
5-substituted-1,3,4-oxadiazole-2(3H)-thiones 5a–h. Com-
pounds 5a–c were further treated with hydrazine hydrate to
obtain compounds 4-amino-3-substituted-1,2,4-triazole-
5(1H)-thiones 6a–c.
the amide) of 5a–h and 6a–c were seen at about
8.67–9.38 ppm, and the NH2 protons of 6a–c were seen at
5.57–5.63 ppm.
It has been reported that the crystal structures of 5a–
h and 6a–c like compounds correspond to the thione form
(Horning and Muchowski, 1972), but they showed thiol–
thione tautomerism in solution. There is a evident thiol
1
peak at 14–15 ppm in H NMR spectra. In the solid state,
these compounds are present in thione (–C=S) form, as
indicated in their IR spectrum by the absence of absorption
band at 2500 cm-1 (–SH stretching) and the presence of
absorption bands at 1240–1142 cm-1, characteristic of the
–C=S group for this class of compounds.
All the newly synthesized compounds gave satisfactory
analyses for the proposed structures, which were confirmed
on the basis of their IR, 1H NMR, and MS. The C=O
stretching vibrations for the amide group were absorbed in
the range of 1655–1635 cm-1. The detection of strong C=S
and C=N stretching bands at about 1240–1142 and
1647–1597 cm-1, respectively, are evidences for ring
closure of 1,3,4-oxadiazoles-2(3H)-thiones 5a–h. In 1H
NMR spectra, all protons were seen according to the
expected chemical shift and integral values. Aromatic ring
protons were seen at 6.97–8.33 ppm. The NH protons (in
Antitumor activity
The anticancer activity of all synthesized 1,3,4-oxadiazole-
2(3H)-thiones and 1,2,4-triazole-5(1H)-thiones derivatives
was evaluated in vitro against human tumor cell line:
leukemia (K-562). IC50 were calculated for each compound
and the results were summarized in Table 1.
Scheme 1 Synthesis of the
target compounds
R2
O
R2
O
N2H4 H2O
CH3OH
DCC, HOBT
THF, NMM
O
O
+
H2N
R1
N
R1
1a-g
OH
H
O
O
HCl
2a-c
3a-h
O
R2
O
R2
R2
H
N
(1) CS2, KOH
(2) Dil. HCl
(1) N2H4 H2O, C2H5OH
(2) Dil. HCl
N
R1
N
NH
R1
R1
N
H
NH2
H
O
O
N
S
4a-h
5a-h
O
N
N
H
NH
S
H2N
6a-c
R1
R2
CH3
CH3
5a, 6a
5b, 6b
C6H5
4-ClC6H4
C6H5
5c, 6c
5d
CH(CH3)2
CH(CH3)2
CH3
4-NO2C6H4
3-pyridine
C6H5CH2
5e
5f
CH3
5g
Trans-4-CH3OC6H4CH=CH
Trans-C6H4CH=CH
CH3
5h
CH2OH
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Med Chem Res (2012) 21:315–320
317
Table 1 Docking score and IC50 values for compounds 5a–i, 6a–c
Compound
Glide (docking score)
K562
TP-II [PDB:
1ZXM]
Tubulin [PDB:
1SA0]
IC50
(lM)
5a
5b
5c
5d
5e
5f
-6.81
[-5
[-5
377.25
31.14
[-5
-6.87
-6.87
-6.66
[-5
[-5
244.83
43.55
[-5
-6.23
-6.36
[-5
738.84
1017.80
329.94
435.07
458.00
371.85
98.62
5g
5h
5i
[-5
Fig. 1 Docked 6c displaying H-bond interaction (dotted lines) in
ATPase domain of topoisomerase II
-6.63
-7.02
-7.63
-7.65
-7.76
No docking
-6.29
-6.48
[-5
6a
6b
6c
the 6c predicted to impede but not eliminate ATP binding
as the ATP binding pocket of TP-II consisting of amino
acid residues Asn46, Asp73, Ile78, Arg136, and Thr165
(Leroy et al., 2001) just above the binding site of 6c.
Glide score (-6.57) revealed that 5b bound in tubulin
with high affinity. As shown in Fig. 2, the N–H of the
1,3,4-oxadiazole-2(3H)-thione group (5b) engages in
hydrogen bonding to the Val 238 C=O group. The phenyl
ring of 5b fits in the trough which is defined by Leu248,
Asn258, Met259, and Gln247 residues. The existence of
hydrophobic pocket containing Val238, Cys241, Leu242,
Leu248, Ala250, Leu255, Ala317, Val318, and Ala354 of
b-tubulin near to the disubstituted oxadiazole moiety of 5b
might allow for an improvement in the interactions
between oxadiazole derivatives and vacant regions of the
binding site (Fig. 2), depending on specific substituents.
Although the MTT screening protocol did not conclude
of any possible mechanisms for the observed anticancer
activity of the test compounds, activity of 5b may be
attributed to an antimitotic action, this is due to the structural
similarity to the antimitotic 1,3,4-oxadiazole derivatives
(Movrin and Maysinger, 1983) and the result of molecular
modeling studies. The activity of some compounds (6b, 6c)
[-5
-6.47
No docking
154.45
687.94
Hydroxyurea
(standard drug)
Most of the compounds have shown higher activity
compared to hydroxyurea (HU) against K-562. It is
worthwhile to note that compounds 5b, 5d, and 6b
exhibited significant cytotoxic activities, which might be
attributed to the presence of a free SH at oxadiazole ring or
triazole ring, as well as the NO2 and Cl substituents of the
benzyl group.
To firmly establish whether or not the phenyl group in
5a is the optimal R1 side-chain, some analogs of 5a were
synthesized. Replacement of the phenyl group with benzyl
(5f) led to a decrease in potency. Introduction of unsatu-
ration chain (5g, 5h) restored activity relative to the lead
5a. Replacement of the phenyl ring of 5a with heterocyclic
ring (pyridine) led to decreases in potency.
With respect to the R1 of 5 or 6, replacement of the
methyl group (5a, 6c) with isopropyl (5c, 6c) resulted in a
slightly increase in activity. When 1,3,4-oxadiazole-2(3H)-
thiones (5a–c) were converted to 1,2,4-triazole-5(1H)-thi-
ones (6a–c), there were not significant changes in potency.
Molecular modeling studies, with automated docking
simulations, were conduced to explore the interaction
between targets (TP-II and tubulin) and the test com-
pounds. The docking result of these ligands is shown in
Table 1.
Three analogs (5b, 5f, 5g) were found not to be good
binders with ATPase domain ([-5). Glide score \-6.63
revealed that major molecules bind in ATPase domain with
high affinity. The interaction of 6c with ATPase domain of
TP-II is shown in figure. The amino acids putatively
involved in the binding of 6c are Thr215, Asp94, and
Ser149 (Fig. 1), which could collectively contribute three
stabilizing hydrogen bonds to the ligands. The binding of
Fig. 2 Docked 5b displaying H-bond interaction (dotted line) in
tubulin
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Med Chem Res (2012) 21:315–320
General procedure for the synthesis of compounds 5a–h
(Khanum et al., 2005)
may be attributed to an inhibition of DNA topoisomerase II.
Moreover, it is possible that test compounds could act
through a different mechanism, such that further studies are
required to elucidate this mechanism of action.
To a solution of acid hydrazide (4a–h, 10 mmol) in abso-
lute EtOH (80 ml) was added CS2 (10 mmol), followed by
an addition of ethanol solution of KOH (10 ml, 5 mmol).
The reaction mixture was stirred for 30 min and then
refluxed with stirring until the evolution of hydrogen sul-
fide ceased. Ethanol was distilled off under reduced pres-
sure and the residue was dissolved in water, washed with
ethyl acetate, and acidified with 1 N HCl to pH 2–3. The
solid (5a–h) obtained was filtered, washed with water, and
recrystallized from 60% aqueous EtOH. The physical and
analytical data of the synthesized title compounds are given
as follows.
In conclusion, we have described a straightforward
synthesis of new 1,3,4-oxadiazole-2(3H)-thiones and 1,2,4-
triazole-5(1H)-thiones derivatives. Our studies indicate that
compounds (5b, 5d, 6b) has antitumor effects, and it rep-
resents a promising lead for the generation of new analogs
with important biological properties.
Experimental
Chemistry
(S)-5-(1-benzamido-ethyl)-1,3,4-oxadiazole-2(3H)-
thione (5a)
Melting points were determined on a electrothermal cap-
illary melting-point apparatus and were uncorrected.
Infrared spectra were recorded on a Shimadzu FT-IR 8000,
using KBr disks. 1H NMR (400 MHz) spectra were
recorded on a Bruker NMR spectrophotometer, using tet-
ramethylsilane (TMS) as internal standard with d values
and coupling constants in Hz. Mass spectra were recorded
on Agilent 1100 series LC/MSD trap and Waters 2695
LC-ZQ 4000 system.
Yield: 86%; m.p.: 172–173°C; IR(KBr) cm-1: 3301, 3155,
3089, 1647, 1608, 1577, 1519, 1485, 1326, 1296, 1180,
1157, 1053; 1H NMR (DMSO-d6, 400 MHz): d (ppm)
14.56 (s, 1H, SH), 9.22 (s, 1H, NH), 7.50–7.89 (m, 5H,
ArH), 5.25 (m, 1H, CH), 1.53 (d, J = 7.0 Hz, 3H, CH3);
ES-API m/z: [M ? H]? 250.1.
(S)-5-(1-(4-chlorobenzamido)-ethyl)-1,3,4-oxadiazole-
2(3H)-thione (5b)
Preparation of amide esters 3a–h
Yield: 81%. m.p.: 178–180°C; IR (KBr) cm-1: 3371, 3175,
3082, 2939, 1643, 1597, 1570, 1535, 1485, 1303, 1265,
1145, 1087, 1056; 1H NMR (DMSO-d6, 400 MHz): d
(ppm) 14.59 (s, 1H, SH), 9.23 (s, 1H, NH), 7.97 (s, 2H,
ArH), 7.65 (s, 2H, ArH), 5.29 (s, 1H, CH), 1.60 (s, 3H,
CH3); ES-API m/z: [M ? H]? 284.0.
A mixture of carboxylic acid (1a–g, 20 mmol), 1-Hydroxy-
benzotriazole (1-HOBT, 20 mmol), and N,N-dichyclohex-
ylcarbodiimide (DCC, 22 mmol) in tetrahydrofuran (THF,
100 ml) was stirred at 0°C for 1 h. A slurry of amino acid
methyl ester hydrochloride (2a–c, 20 mmol) and triethyl-
amine (Et3N, 40 mmol) in THF (20 ml) was added to the
above mixture which was then stirred at25°C for anadditional
24 h and then filtrated. The filtrate is evaporated under vac-
uum to remove THF. The residue is dissolved in 150 ml of
ethyl acetate and the solution is washed successively with 5%
sodium bicarbonate (30 ml 9 3), dilute hydrochloric acid
(30 ml 9 3), and saturated sodium chloride (30 ml 9 3) and
the ethyl acetate phase is separated and dried over anhydrous
sodium sulfate. By filtration and evaporation under reduced
pressure the product (3a–h) is obtained.
(S)-5-(1-benzamido-2-methylpropyl)-1,3,4-oxadiazole-
2(3H)-thione (5c)
Yield: 72%; m.p.: 146–147°C; IR (KBr) cm-1: 3267, 3162,
3085, 2962, 2935, 1647, 1603, 1577, 1527, 1492, 1469,
1
1292, 1257, 1142, 1057; H NMR (DMSO-d6, 400 MHz):
d (ppm) 14.57 (s, 1H, SH), 9.02 (d, J = 8.0 Hz, 1H, NH),
7.86 (d, J = 7.6 Hz, 2H, ArH), 7.46–7.57 (m, 3H, ArH),
4.90 (t, J = 8.3 Hz, 1H, CHNH), 2.27–2.32 (m, 1H,
CH(CH3)2), 1.00 (d, J = 6.6 Hz, 3H, CH3), 0.91 (d,
J = 6.6 Hz, 3H, CH3); ES-API m/z: [M ? H]? 278.2.
General procedure for preparation of compounds 4a–h
A mixture of ester (3a–h, 15 mmol) and 80% hydrazine
monohydrate (30 mmol) in absolute methanol (80 ml)
was heated under reflux over night. The reaction mixture
was cooled, a white solid separated, which on recrystal-
lization with ethanol gave acid hydrazide (4a–h) as white
solid.
(S)-5-(2-methyl-1-(4-nitrobenzamido)propyl)-1,3,4-
oxadiazole-2(3H)-thione (5d)
Yield: 77%; m.p.: 174–175°C; IR (KBr) cm-1: 3207, 3180,
3085, 2962, 2878, 2788, 1654, 1600, 1504, 1346, 1299,
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Med Chem Res (2012) 21:315–320
319
1165, 1107, 1057; 1H NMR (DMSO-d6, 400 MHz): d
(ppm) 14.60 (s, 1H, SH), 9.38 (d, J = 8.0 Hz, 1H, NH),
8.33 (d, J = 8.4 Hz, 2H, ArH), 8.09 (d, J = 8.4 Hz, 2H,
ArH), 4.92–4.96 (m, 1H, CHNH), 2.28–2.33 (m, 1H,
CH(CH3)2), 1.01 (d, J = 6.6 Hz, 3H, CH3), 0.93 (d,
J = 6.6 Hz, 3H, CH3); ES-API m/z: [M ? H]? 323.1.
General procedure for preparation of compounds 6a–c
To a mixture of 1,3,4-oxadiazole-2(3H)-thione derivatives
(5a–c, 5 mmol) in ethanol (10 ml), 80% hydrazine hydrate
(10 mmol) was added dropwise and the mixture was
refluxed for 5 h. After cooling water was added and the
mixture was acidified by excess of 3 N HCl, the separated
solid was filtered off, washed with water and crystallized
from ethanol to give 4-amino-1,2,4-triazole-5(1H)-thione
derivatives (6a–c).
(S)-5-(1-(nicotinamido)ethyl)-1,3,4-oxadiazole-2(3H)-
thione (5e)
Yield: 85%; m.p.: 194–196°C; IR (KBr) cm-1: 3279, 3062,
2986, 2959, 1828, 1643, 1597, 1543, 1481, 1450, 1377,
1326, 1292, 1226, 1195, 1168, 1118, 1045; 1H NMR
(DMSO-d6, 400 MHz): d (ppm) 14.54 (s, 1H, SH), 9.27 (d,
J = 7.5 Hz, 1H, NH), 9.01 (s, 1H, 2-H of pyridine), 8.73
(d, J = 4.6 Hz, 1H, 6-H of pyridine), 8.20 (d, J = 7.9 Hz,
1H, 4-H of pyridine), 7.53 (dd, J1 = 4.9 Hz, J2 = 7.8 Hz,
1H, 5-H of pyridine), 5.21–5.28 (m, 1H, CH), 1.53 (d,
J = 7.0 Hz, 3H, CH3); ES-API m/z: [M ? H]? 251.1.
(S)-4-amino-3-(1-benzamidoethyl)-1,2,4-triazole-
5(1H)-thione (6a)
Yield: 67%; m.p.: 173–174°C; IR (KBr) cm-1: 3279, 3216,
3109, 3043, 2947, 1635, 1569, 1527, 1485, 1350, 1272,
1157, 1118; 1H NMR (DMSO-d6, 400 MHz): d (ppm)
13.57 (s, 1H, SH), 8.85 (s, 1H, NH), 7.45–7.85 (m, 5H,
ArH), 5.57 (d, J = 5.36 Hz, 2H, NH2); 5.24 (d,
J = 4.04 Hz, 1H, CHNH), 3.01 (s, 2H, CH2), 1.49 (s, 3H,
CH3); ES-API m/z: [M ? H]? 264.1.
(S)-5-(1-(2-phenylacetamido)ethyl)-1,3,4-oxadiazole-
2(3H)-thione (5f)
(S)-4-amino-3-(1-(4-chlorobenzamido)-ethyl)-1,2,4-
Yield: 70%; m.p.: 119–121°C; IR (KBr) cm-1: 3394, 3186,
3028, 2889, 1643, 1523, 1469, 1380, 1330, 1288, 1257,
triazole-5(1H)-thione (6b)
Yield: 50%; m.p.: 240–242°C; IR (KBr) cm-1: 3298, 3174,
3109, 3039, 2947, 1639, 1593, 1566, 1535, 1485, 1380,
1346, 1303, 1269, 1157, 1118, 1091, 1014; 1H NMR
(DMSO-d6, 400 MHz): d (ppm) 13.60 (s, 1H, SH), 8.96 (d,
J = 7.2 Hz, 1H, NH), 7.89–7.52 (m, 4H, ArH), 5.57 (s, 2H,
NH2); 5.23 (t, J = 6.8 Hz, 1H, CHNH), 1.49 (d,
J = 6.8 Hz, 3H, CH3); ES-API m/z: [M ? H]? 292.1.
1
1226, 1164, 1110, 1049; H NMR (DMSO-d6, 400 MHz):
d (ppm) 14.56 (s, 1H, SH), 8.89 (s, 1H, NH), 7.32 (d,
J = 3.2 Hz, 5H, ArH), 5.04 (s, 1H, CHNH), 3.01 (s, 2H,
CH2), 1.48 (s, 3H, CH3); ES-API m/z: [M ? H]? 264.0.
(R,E)-5-(1-(3-(4-methoxyphenyl)acrylamido)ethyl)-
1,3,4-oxadiazole-2(3H)-thione (5g)
Yield: 76%; m.p.: 190–191°C; IR (KBr) cm-1: 3302, 3074,
2931, 1655, 1605, 1523, 1504, 1454, 1303, 1257, 1230,
1176, 1110, 1022; 1H NMR (DMSO-d6, 400 MHz): d
(ppm) 14.55 (s, 1H, SH), 8.73 (d, J = 7.2 Hz, 1H, NH),
7.52 (d, J = 7.6 Hz, 2H, ArH), 7.42 (d, J = 16 Hz, 1H,
CHAr), 6.97 (d, J = 8.4 Hz, 2H, ArH), 6.46 (d,
J = 15.6 Hz, 1H, CHCO), 5.09 (s, 1H, CHNH), 3.77 (s,
3H, OCH3), 1.44 (d, J = 6.8 Hz, 3H, CHCH3); ES-API m/
z: [M ? H]? 306.1.
(S)-4-amino-3-(1-benzamido-2-methylpropyl)-1,2,4-
triazole-5(1H)-thione (6c)
Yield: 74%; m.p.: 162–164°C; IR (KBr) cm-1: 3275, 3225,
3058, 2958, 2927, 1674, 1636, 1578, 1527, 1488, 1465,
1380, 1334, 1261, 1218, 1153, 1076, 1037; 1H NMR
(DMSO-d6, 400 MHz): d (ppm) 13.67 (s, 1H, SH), 8.67 (d,
J = 8.4 Hz, 1H, NH), 7.44–7.86 (m, 5H, ArH), 5.63 (s, 2H,
NH2), 5.08 (t, J = 8.4 Hz, 1H, CHNH), 2.42–2.33 (m, 1H,
CH(CH3)2), 1.00 (d, 3H, CH3), 0.91 (d, 6H, CH3); ES-API
m/z: [M ? H]? 292.1.
(R,E)-5-(1-cinnamamido-2-hydroxyethyl)-1,3,4-
oxadiazole-2(3H)-thione (5h)
In vitro cytotoxicity screening
Yield: 59%; m.p.: 152–154°C; IR (KBr) cm-1: 3437, 3275,
3063, 2947, 1655, 1628, 1535, 1497, 1342, 1312, 1219,
1153, 1068; 1H NMR (DMSO-d6, 400 MHz): d (ppm)
14.50 (s, 1H, SH), 8.81 (d, J = 7.2 Hz, 1H, NH), 7.16–7.55
(m, 4H, ArH), 7.41 (m, 1H, CHAr), 6.73 (d, J = 15.6, 1H,
CHCO), 5.39 (s, 1H, OH), 5.03 (s, 1H, CHNH), 3.75 (s,
2H, CH2OH); ESI-MS m/z: [M - 1]? 289.78.
The anticancer activities of compounds 5a–i and 6a–c were
evaluated in vitro on the leukemia human myelogenous
leukemia K562 by measuring cell viability by the MTT
method, with HU as the positive control. The cells were
seeded in RPM I 1640 medium (100 ll) in a 96-well plate
at a concentration of 4,000 cells per well. After culturing
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Med Chem Res (2012) 21:315–320
for 12 h at 37°C and 5% CO2, cells were incubated with
samples for 24 h using five concentrations at 10-fold
dilutions (10-3–10-7 mol/ml) for each test compound.
MTT was added at a terminal concentration of 5 lg/ml and
incubated with the cells for 4 h. The formazan crystals
were dissolved in DMSO (100 ll) in each well and the
optical density was measured at 570 nm (for the absor-
bance of MTT formazan) (Mai et al., 2010). The IC50 was
calculated using the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software.
molecular docking of the test compounds in a similar
docking procedures (Mooberry et al. 2007).
Acknowledgments This work was supported by the Natural Science
Foundation of Nanchang, Jiangxi, China (2008). We greatly appreciate
ShiTang Ma (the Institute of Radiation Medicine, Chinese Academy of
Medicine Science) for Molecular Modeling and Docking Studies.
References
Aboraiaa AS, Abdel-Rahman HM, Mahfouza NM, El-Gendy MA
(2006) Novel 5-(2-hydroxyphenyl)-3-substituted-2, 3-dihydro-
1,3,4-oxadiazole-2-thione derivatives: promising anticancer
agents. Bioorg Med Chem 14(4):1236–1246. doi:10.1016/j.bmc.
2005.09.053
Molecular modeling and docking studies
Al-Souda YA, Al-Masoudi NA, Ferwanah AES (2003) Synthesis and
properties of new substituted 1, 2, 4-triazoles: potential antitu-
mor agents. Bioorg Med Chem 11(8):1701–1708. doi:10.1016/
S0968-0896(03)00043-9
Each structure was assigned an appropriate bond order using
¨
ligprep script shipped by Schrodinger. The target molecules
were converted to MAE format (for Glide docking) using
´
¨
maestro (Schrodinger Inc.) and optimized by means of the
Ferloni E, Lanfranchi M, Marchio L, Metta G, Pellinghelli MA,
Tegoni M (2005) Structure, solution equilibria and magnetic
properties of copper(II) complexes with new sulfurated triazoline
derivatives of a-amino acids. Inorg Chim Acta 358(1):147–160.
doi:10.1016/j.ica.2004.08.016
OPLS_2005 force field using a default setting.
The starting coordinates of the human topoisomerase-II
ATPase (TPII)-AMP-PNP complex [PDB: 1ZXM] was
taken from the Protein Data Bank (www.rcsb.org) and
further modified for docking calculations. For Glide
Formagio ASN, Tonin LTD, Foglio MA, Madjarof C, de Carvalho JE,
Costa WF, Cardoso FP, Sarragiotto MH (2008) Synthesis and
antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-5-yl)
and 3-(5-substituted-1,2,4-triazol-3-yl) b-carboline derivatives. Bioorg
Med Chem 16(22):9660–9667. doi:10.1016/j.bmc.2008.10.008
Holla BS, Poojary KN, Rao BS, Shivananda MK (2002) New bis-
aminomercaptotriazoles and bis-triazolothiadiazoles as possible
anticancer agents. Eur J Med Chem 37(6):511–517. doi:10.1016/
S0223-5234(02)01358-2
Horning DE, Muchowski JM (1972) Five-membered heterocyclic thiones.
Part I. 1,3,4-oxadiazole-2-thione. Can J Chem 50:3079–3082
Khanum SA, Shashikanth S, Umesha S, Kavitha R (2005) Synthesis
and antimicrobial study of novel heterocyclic compounds from
hydroxybenzophenones. Eur J Med Chem 40(11):1156–1162.
doi:10.1016/j.ejmech.2005.04.005
¨
(Schrodinger) calculations, TP-II complex was imported to
Maestro, the co-crystallized ligands were identified and
removed from the structure and the protein was minimized
¨
using the protein preparation wizard (shipped by Schro-
dinger) by applying OPLS_2005 force field by the appli-
cation of autoref.pl script. Progressively weaker restraints
(tethering force constants 3, 1, 0.3, and 0.1) were applied to
non-hydrogen atoms only. Water molecules were removed
and H atoms were added to the structure. The most likely
positions of hydroxyl and thiol hydrogen atoms, proton-
ation states and tautomers of His residues, and Chi ‘‘flip’’
assignments for Asn, Gln, and His residues were selected
Leroy D, Kajava AV, Frei C, Gasser SM (2001) Analysis of etoposide
binding to subdomains of human DNA topoisomerase IIa in the
absence of DNA. Biochemistry 40(6):1624–1634. doi:10.1021/
bi0019141
¨
by the protein assignment script shipped by Schrodinger.
Minimizations were performed until the average root mean
˚
square deviation of the non-hydrogen atoms reached 0.3 A.
Loetchutinat C, Chau F, Mankhetkorn S (2003) Synthesis and
evaluation of 5-aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2-
(3H)-thiones as P-glycoprotein inhibitors. Chem Pharm Bull
51:728–730. doi:10.1248/cpb.51.728
Mai X, Lu X, Xia H, Cao Y, Liaoand Y, Lv X (2010) Synthesis,
antitumor evaluation and crystal structure of hydroxyurea deriv-
atives. Chem Pharm Bull 58:94–97. doi:10.1248/cpb.58.94
Mooberry SL, Weiderhold KN, Dakshanamurthy S, Hamel E, Banner
EJ, Kharlamova A, Hempel J, Gupton JT, Brown ML (2007)
Identification and characterization of a new tubulin-binding
tetrasubstituted brominated pyrrole. Mol Pharmacol 72(1):
132–140. doi:10.1124/mol.107.034876
Movrin M, Maysinger D (1983) Synthesis and biological activity of
some N-dialkylaminomethyloxadiazolones and oxadiazolthi-
ones. Pharmazie 38(8):561
Omar FA, Mahfouz NM, Rahman MA (1996) Design, synthesis and
anti-inflammatory activity of some 1,3,4-oxadiazole derivatives.
Eur J Med Chem 31(10):819–825. doi:10.1016/0223-5234(96)
83976-6
After ensuring that the protein and ligands are in correct
form for docking, the receptor-grid files were generated
using a grid-receptor generation program. van der Waal
radii of receptor atoms was scaled by 1.00/0.80. A grid box
was generated by centroid of the binding site of ATPase
domain consisting of residues Asn91, Asn95, Asn 120,
Lys123, Gly124, Pro126, Ile141, Ser149, Glu155, and
Arg184 (Leroy et al. 2001) and the size of ligands to be
docked was selected form the workspace. The ligands were
docked with the binding site using the ‘‘standard precision’’
Glide algorithm. The final energy evaluation is done with
GlideScore and a single best pose is generated as the output
for a particular ligand.
The X-ray crystal structure of two a,b-tubulin hetero-
dimers complexed with DMA-colchicine and a stathmin
fragment [PDB: 1SA0] was used for the automated
Raper ES (1997) Complexes of heterocyclic thionates part 2:
complexes of bridging ligands. Coord Chem Rev 165:475–567.
doi:10.1016/S0010-8545(97)90167-3
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