Synthesis and antitumor activity of 4-cyclohexyl/aryl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones

Article abstract of DOI:10.1007/s00044-014-1216-5

The reaction of 2-isonicotinoyl-N-cyclohexyl/arylhydrazinecarbothioamide (2a-r) with sodium hydroxide, in each case, a single product was obtained. The structures of the compounds were confirmed on the basis of their elemental analysis and spectral data.

Full text of DOI:10.1007/s00044-014-1216-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1216-5
ORIGINAL RESEARCH
Synthesis and antitumor activity of 4-cyclohexyl/aryl-5-(pyridin-4-
yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones
•
Mashooq Ahmad Bhat Mohamed A. Al-Omar
•
•
Ahmed M. Naglah Mohamed M. Abdulla
•
Hoong-Kun Fun
Received: 4 March 2014 / Accepted: 1 August 2014
Ó Springer Science+Business Media New York 2014
Abstract The reaction of 2-isonicotinoyl-N-cyclohexyl/
arylhydrazinecarbothioamide (2a–r) with sodium hydrox-
ide, in each case, a single product was obtained. The
structures of the compounds were confirmed on the basis of
their elemental analysis and spectral data. The single
crystal X-ray analysis confirmed the structure of these
products as N-4-cyclohexyl/aryl-5-(pyridine-4-yl)-2,4-
dihydro-3H-1,2,4-triazole-3-thiones (3a–r). The in vitro
antitumor activity of compounds was screened against
three cell lines; BEL-7402, HUH-7 and HepG2 human
hepatoma using MTT assay. Sorafenib (50 lM) was used
as a positive control. The results of the MTT-dye reduction
assay indicated that most of the compounds exert potent
cytotoxic/antiproliferative effect in a time and dose-
dependent manner via induced apoptosis of HepG2 cells.
Results also showed that the tested compounds could sig-
nificantly enhance the activity of caspase-3 which plays a
very important role as the central effecter during apoptosis.
The effect of different substitutions on the aromatic portion
on the activity was found to be in the following order
CH3 [ OCH3 [ I [ SO2NH2 [ OC2H5 [ C2H5 [ NO2 [
Cl [ CH3CONH.
Keywords Isoniazid ꢀ 1,2,4-Triazole-3-thione ꢀ
Cytotoxic activity ꢀ MTT assay
Introduction
A number of five membered nitrogen-containing hetero-
cycles have turned out to be potential chemotherapeutic
agents. The biological profile of triazoles derivatives is
very extensive. Compounds bearing a symmetrical triazole
moiety are reported to show a broad spectrum of pharma-
cological activities such as antibacterial (Sztanke et al.,
2006; Prakash et al., 2004), antifungal (Liu et al., 2008;
Lebouvier et al., 2007), antimicrobial (Kaplancikli et al.,
2008), antimycobacterial (Kucukguzel et al., 2008), anti-
pyretic (Grossi et al., 2002), anticancer (Sztanke et al.,
2008; Holla et al., 2002), anticonvulsant (Almasirad et al.,
2004), and anti-inflammatory activities (Labanauskas et al.,
2004). Also there are known drugs containing 1,2,4-tria-
zole moiety, e.g., alprozolam (tranquilizer), benatraden
(diuretic), trapidil (hypotensive), trazodone (antidepres-
sant), anastrozole, letrozole, vorozole (antineoplastic),
ribavirin (antiviral) (Mathew et al., 2006), and antimycotic
ones such as fluconazole, itraconazole, voriconazole
(Haber, 2001). Pyridyl ring, a prominent scaffold present in
various bioactive molecules, has played a vital role in the
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1216-5) contains supplementary
material, which is available to authorized users.
M. A. Bhat (&) ꢀ M. A. Al-Omar ꢀ H.-K. Fun
Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, P.O. Box 2457, Riyadh 11451, Kingdom
of Saudi Arabia
e-mail: mashooqbhat@rediffmail.com
A. M. Naglah
Pharmaceutical Chemistry Department, Drug Exploration &
Development Chair, College of Pharmacy, King Saud
University, Riyadh 11451, Kingdom of Saudi Arabia
A. M. Naglah
Peptide Chemistry Department, Chemical Industries Research
Division, National Research Centre, Dokki, Cairo 12622, Egypt
M. M. Abdulla
Research Unit, Saico Pharm. Co., 6th October City 11632, Egypt
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Med Chem Res
4-Phenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-
development of different medicinal agents (Duan et al.,
2011; Luedtke et al., 2010; Packiarajan et al., 2011).
Encouraged by these reports, some 1,2,4-triazole deriva-
tives bearing pyridyl moiety were synthesized and screened
with the aim to achieve the compounds with better anti-
tumor activity.
thione (3a)
Yield: (70 %). m. p.: 270–272 °C. FT-IR (v, cm-1): 3000
(NH str.), 1600, 1494 (C=N str.), 1284 (C=S str.). 1H NMR
(500 MHz, DMSO-d6, d ppm): 7.69–7.40 (m, 7H, Ar–H),
8.98–8.75 (m, 2H, Ar–H), 14.42 (s, 1H, NH, D2O exch.).
13C NMR (125.76, DMSO-d6, d ppm): 169.19, 148.47
(triazole ring), 150.09, 134.21, 133.32, 129.83, 129.57,
128.70, 122.01 (Ar–C). MS (EI) m/z = 254.2 [M?]. Anal.:
Calcd. for C13H10N4S (254.3): C (61.40), H (3.96), N
(22.03), S (12.61). Found: C (61.30), H (3.95), N (22.04), S
(12.58) (Siddiqui and Azam, 2012). The single crystal
X-ray of compound 3a has been obtained (Fig. 1).
Chemistry
Experimental
All the solvents were obtained from Merck. The homoge-
neity of the compounds was checked by TLC performed on
Silica gel G coated plates (Merck). Iodine chamber was used
for visualization of TLC spots. The FT-IR spectra were
recorded in KBr pellets on a (Spectrum BX) Perkin Elmer
FT-IR spectrophotometer. Melting points were determined
on a Gallenkamp melting point apparatus, and thermometer
was uncorrected. NMR Spectra were scanned in DMSO-d6
on a Bruker NMR spectrophotometer operating at 500 MHz
4-Cyclohexyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3b)
Yield: (72 %). m. p.: 314–316 °C. FT-IR (v, cm-1): 3098
(arom. CH), 2941, 2854 (ali. CH), 1558 (C=N), 1284(C=S).
1H NMR (500 MHz, DMSO-d6, d ppm): 0.96 (d,
J = 12.9 Hz, 1H alip.), 1.19 (q, J = 12 Hz, 1H, Haliph.),
1.57 (d, J = 12.5 Hz, 1H, Haliph.), 1.72 (d, J = 12.9 Hz,
2H, Haliph.), 1.76 (d, J = 11.7 Hz, 2H, Haliph.), 2.13 (br,
1
for H and 125.76 MHz for 13C at the Research Center,
College of Pharmacy, King Saud University, Saudi Arabia.
Chemical shifts are expressed in d-values (ppm) relative to
TMS as an internal standard, and D2O was added to confirm
the exchangeable protons. Mass spectra (MS) were measured
on triple quadruple mass spectroscopy.
2H,
Haliph.), 4.25 (br, 1H, CH–N), 7.62 (d, 2H,
J = 4.6 Hz), 8.8 (d, J = 4.5, 2H, Ar–H), 14.08 (s, 1H,
–NH, D2O exchang.); MS (EI) m/z = 260.0 [M?]. Anal.:
Calcd. for C13H16N4S (260.3): C (59.97), H (6.19), N
(21.52), S (12.32). Found: C (59.87), H (6.2), N (21.56), S
(12.33). (Ebrahimi, 2010). The single crystal X-ray of
compound (3b) has been obtained (Fig. 2).
General procedure for the synthesis of: N-4-cyclohexyl/
aryl-2-(pyridin-4-ylcarbonyl) hydrazinecarbothioamide
(2a–r)
To a solution of appropriate substituted aniline (0.01 mol)
in absolute ethanol (20 ml) were added potassium
hydroxide (0.01 mol) and carbon disulphide (0.75 ml), and
the mixture was stirred at 0–5 °C for 1 h to form a
potassium salt of substituted phenyl dithiocarbamate. To
the stirred mixture of phenyl thiocarbamate salt was added
isoniazid, INH (0.01 mol) and the stirring was continued at
80 °C for 1 h and on adding crushed ice to obtain
N-(substituted)-2-isonicotinoylhydrazinocarbothioamides
(2a–r) (Sriram et al., 2009; Bhat et al., 2014).
General procedure for the synthesis of 4-cyclohexyl/aryl-5-
(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a–r)
A solution of thiocarbamate in ethanol was dissolved in
4 N NaOH (2 mL) and refluxed for 2 h. The resulting
solution was cooled to room temperature and filtered. The
filtrate was adjusted to pH 5–6 with dilute acetic acid and
kept aside for 1 h. The crystals produced were washed with
water, dried, and recrystallized from EtOH to give the
compound (Bayrak et al., 2009).
Fig. 1 ORTEP diagram of 3a at 50 % probability
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Med Chem Res
Fig. 2 ORTEP diagram of 3b
at 50 % probability
4-(4-Chlorophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
DMSO-d6, d ppm): 1.98 (s, 6H, CH3), 7.1–7.4 (m, 5H,
Ar–H), 8.5 (m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O
exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 168.2,
150.5, 147.4, 135.9, 132.5, 132.2, 130.2, 128.9, 120.0, 18.1,
17.4. MS (EI) m/z = 282.0 [M]?. Anal.: Calcd. for
C15H14N4S (282.3): C (63.80), H (5.00), N (19.84), S (11.36).
Found: C (63.71), H (4.98), N (19.86), S (11.38).The single
crystal X-ray of compound 3e has been obtained (Fig. 3).
triazole-3-thione (3c)
Yield: (75 %). m. p.: 310–315 °C. FT-IR (v, cm-1): 3028 (NH
str.), 1606, 1494 (C=N str.), 1288 (C=S str.). 1H NMR
(500 MHz, DMSO-d6, d ppm): 7.2–7.5 (m, 6H, Ar–H), 8.6 (m,
2H,Ar–H), 14.4(s, 1H, NH, D2O exchang.). 13C NMR (125.76,
DMSO-d6, d ppm): 169.1, 150.0, 148.0, 135.3, 133.1, 131.1,
130.0, 128.8, 127.6, 122.0. MS (EI) m/z = 288.5 [M?]. Anal.:
Calcd. for C13H9ClN4S (288.7): C (54.07), H (3.14), N (19.40),
S (11.10). Found: C (54.14), H (3.11), N (19.44), S (11.11).
4-(3-Ethylphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3f)
Yield: (70 %). m. p.:[310 °C. FT-IR (v, cm-1): 3449 (NH
str.), 1606, 1549 (C=N str.), 1280 (C=S str.). 1H NMR
(500 MHz, DMSO-d6, d ppm): 3.3 (s, 3H, CH3), 4.1 (2H, q,
CH2), 7.24–7.75 (m, 6H, Ar–H), 8.5 (m, 2H, Ar–H), 14.5 (s,
1H, NH, D2O exchang.). 13C NMR (125.76, DMSO-d6, d
ppm): 150.02, 145.3, 133.1, 129.3, 129.1, 127.7, 125.7, 121.8,
48.5, 27.7, 15.2. MS (EI) m/z = 282.0 [M]?. Anal.: Calcd. for
C15H14N4S (282.3): C (63.80), H (5.00), N (19.84), S (11.36).
Found: C (63.70), H (4.99), N (19.85), S (11.34).
4-(4-Methoxyphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (3d)
Yield: (75 %). m. p.:[320 °C. FT-IR (v, cm-1): 3433 (NH
str.), 1653, 1515 (C=N str.), 1290 (C=S str.), 1079 (C–O).
1H NMR (500 MHz, DMSO-d6, d ppm): 7.2–7.5 (m, 6H,
Ar–H), 8.6 (m, 2H, Ar–H), 14.4 (s, 1H, NH, D2O
exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 169.0,
150.0, 148.0, 135.1, 133.0, 131.1, 130.0, 128.1, 127.5,
122.0; MS (EI) m/z = 284.1 [M?]. Anal.: Calcd. for
C14H12N4OS (284.3): C (59.14), H (4.25), N (19.70), S
(11.28). Found: C (59.20), H (4.25), N (19.66), S (11.31).
4-(4-Nitrophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3g)
Yield: (72%). m. p.: 320–322 °C. FT-IR (v, cm-1): 3450 (NH
str.), 1605, 1549 (C=N str.), 1280 (C=S str.); 1H NMR
(500 MHz, DMSO-d6, d ppm): 6.7–7.5 (m, 6H, Ar–H), 7.8
(m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 172.0, 155.0, 136.5, 134.0, 125.0,
4-(2,6-Dimethylphenyl)-5-(pyridin-4-yl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (3e)
Yield: (85 %). m. p.: 310–312 °C. FT-IR (v, cm-1): 3028
(NH), 1494 (C=N), 1284 (C=S). 1H NMR (500 MHz,
123

Med Chem Res
(500 MHz, DMSO-d6, d ppm): 3.1 (s, 3H, CH3), 7.1–7.4 (m,
6H, Ar–H), 8.6 (m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O
exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 168.8,
149.9, 148.3, 138.9, 134.6, 133.6, 130.0, 128.8, 126.4, 125.5,
123.2, 121.4, 48.5, 21.3. MS (EI) m/z = 268.1 [M]?. Anal.:
Calcd. for C14H12N4S (268.3): C (62.66), H (4.51), N (20.88),
S (11.95). Found: C (62.57), H (4.49), N (20.90), S (11.91).
4-(2-Methylphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3k)
Yield: (75 %). m. p.:[320 °C. FT-IR (v, cm-1): 3200 (NH
str.), 1607, 1500 (C=N str.), 1319 (C=S str.). H NMR
1
(500 MHz, DMSO-d6, d ppm): 3.0 (s, 3H, CH3), 7.1–7.4
(m, 6H, Ar–H), 8.6 (m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O
exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 160.4,
155.0, 149.5, 139.2, 136.8, 129.4, 126.1, 124.7, 121.0,
15.8. MS (EI) m/z = 268.1 [M?]. Anal.: Calcd. for
C14H12N4S (268.33): C (62.66), H (4.51), N (20.88), S
(11.95). Found: C (26.57), H (4.53), N (20.90), S (11.93).
Fig. 3 ORTEP diagram of 3e at 50 % probability
120.9, 116.0. MS (EI) m/z = 299.0 [M?]. Anal.: Calcd. for
C13H9N5O2S (299.3): C (52.17), H (3.03), N (23.40), S
(10.71). Found: C (52.26), H (3.05), N (23.43), S (10.73).
4-(4-Sulfapyridinephenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-
4-(3-Chorophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
1,2,4-triazole-3-thione (3h)
triazole-3-thione (3l)
Yield: (60 %). m. p.:[320 °C. FT-IR (v, cm-1): 3425 (NH
str.), 1600, 1586 (C=N str.), 1290 (C=S str.), 1095 (SO2).
1H NMR (500 MHz, DMSO-d6, d ppm): 5.0 (s, 1H,
SO2NH, D2O exchang.), 7.2–7.6 (m, 7H, Ar–H), 8.7 (m,
4H, Ar–H), 14.4 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 169.3, 160.4, 157.9, 155.0,
149.5, 110.3, 126.8, 121.0, 110.3. MS (EI) m/z = 411.20
[M]?. Anal.: Calcd. for C17H13N7O2S2 (411.46): C (49.62),
H (3.18), N (23.83), S (15.59). Found: C (49.54), H (3.17),
N (23.81), S (15.60).
Yield: (70 %). m. p.:[320 °C. FT-IR (v, cm-1): 3481 (NH
str.), 1606, 1507 (C=N str.), 1298 (C=S str.). H NMR
1
(500 MHz, DMSO-d6, d ppm): 7.2–7.6 (m, 6H, Ar–H), 8.6
(m, 2H, Ar–H), 14.4 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 169.0, 150.1, 148.2, 135.3,
133.3, 131.0, 129.9, 128.8, 127.6, 122.0. MS (EI)
m/z = 288.5 [M?]. Anal.: Calcd. for C13H9ClN4S (288.7):
C (54.07), H (3.14), N (19.40), S (11.10). Found: C (54.15),
H (3.12), N (19.43), S (11.13).
4-(2-Methoxyphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-
4-(4-Methylphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
1,2,4-triazole-3-thione (3m)
triazole-3-thione (3i)
Yield: (70 %). m. p.:[320 °C. FT-IR (v, cm-1): 3481 (NH
str.), 1606, 1507 (C=N str.), 1298 (C=S str.). H NMR
Yield: (75 %). m. p.:[320 °C. FT-IR (v, cm-1): 3306 (NH
str.), 1652, 1558 (C=N str.), 1309 (C=S str.). H NMR
1
1
(500 MHz, DMSO-d6, d ppm): 7.2–7.6 (m, 6H, Ar–H), 8.6
(m, 2H, Ar–H), 14.4 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 169.0, 150.1, 148.2, 135.3,
133.3, 131.0, 129.9, 128.8, 127.6, 122.0. MS (EI)
m/z = 284.2 [M?]. Anal.: Calcd. for C14H12N4OS (284.3):
C (59.14), H (4.25), N (19.70), S (11.28). Found: C (59.22),
H (4.26), N (19.68), S (11.30).
(500 MHz, DMSO-d6, d ppm): 3.3 (s, 3H, CH3), 6.96–7.3
(m, 6H, Ar–H), 8.6 (m, 2H, Ar–H), 9.8 (s, 1H, NH, D2O
exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 179.5,
152.6, 137.2, 133.4, 130.3, 128.9, 123.8, 118.1, 48.5, 20.4.
MS (EI) m/z = 268.2 [M]?. Anal.: Calcd. for C14H12N4S
(268.3): C (62.66), H (4.51), N (20.88), S (11.95). Found: C
(62.56), H (4.50), N (20.92), S (11.93).
4-(3-Methoxyphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-
4-(3-Methylphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
1,2,4-triazole-3-thione (3n)
triazole-3-thione (3j)
Yield: (65 %). m. p.:[320 °C. FT-IR (v, cm-1): 3400 (NH
str.), 1600, 1500 (C=N str.), 1200 (C=S str.), 1100 (C–O). 1H
NMR (500 MHz, DMSO-d6, d ppm): 4.1(s, 3H, –OCH3),
Yield: (70 %). m. p.:[320 °C. FT-IR (v, cm-1): 3306 (NH
str.), 1652, 1558 (C=N str.), 1309 (C=S str.). 1H NMR
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Scheme 1 Synthetic protocol
of the compounds (3a–r)
H
H
H
N
H
N
S
N
O
N
R
RNCS/ C2H5OH
N
H
N
H
N
O
(1)
(2a-r)
NaOH
N
NH
N
S
N
R
(3a-r)
Compound
R
3a
3b
3c
3d
3e
3f
Phenyl
Cyclohexyl
4-Chloro phenyl
4-Methoxy phenyl
2,6-Dimethyl phenyl
3-Ethyl phenyl
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4-Nitro phenyl
4-Sulphapyrimidine phenyl
4-Methyl phenyl
3-Methyl phenyl
2-Methyl phenyl
3-Chloro phenyl
2-Methoxy phenyl
3-Methoxy phenyl
4-Acetamido phenyl
4-Ethoxy phenyl
4-Iodo phenyl
2-Nitro phenyl
4-(4-Ethoxyphenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
6.9–7.4 (m, 6H, Ar–H), 8.5 (m, 2H, Ar–H), 14.5 (s, 1H, NH,
D2O exchang.). 13C NMR (125.76, DMSO-d6, d ppm): 169.0,
162.3, 159.7, 150.0, 133.1, 130.3, 121.7, 120.5, 115.2, 114.6,
55.4, 48.5, 35.7. MS (EI) m/z = 284.1 [M?]. Anal.: Calcd. for
C14H12N4OS (284.3): C (59.14), H (4.25), N (19.70), S
(11.28). Found: C (59.04), H (4.23), N (19.72), S (11.30).
triazole-3-thione (3p)
Yield: (60 %). m. p.: 280–282 °C. FT-IR (v, cm-1): 3296
(NH str.), 1636, 1511 (C=N str.), 1246 (C=S str.), 1048
1
(C–O). H NMR (500 MHz, DMSO-d6, d ppm): 1.37 (s,
3H, CH3), 4.0 (s, 2H, OCH2), 6.8–7.3 (m, 6H, Ar–H), 8.6
(m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 160.4, 155.0, 149.5, 136.7,
127.1, 125.6, 121.0, 114.7, 64.7, 14.8. MS (EI)
m/z = 298.2 [M?]. Anal.: Calcd. for C15H14N4OS
(298.36): C (60.38), H (4.73), N (18.78), S (10.75). Found:
C (60.28), H (4.74), N (18.75), S (10.73).
4-(4-Acetamidophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (3o)
Yield: (60 %). m. p.:[320 °C. FT-IR (v, cm-1): 3326 (NH
str.), 1680, 1518 (C=N str.), 1335 (C=S str.). H NMR
1
(500 MHz, DMSO-d6, d ppm): 4.1 (s, 3H, –COCH3), 5.5
(s, 1H, NH, D2O exchang.), 6.6–7.2 (m, 6H, Ar–H), 8.6 (m,
2H, Ar–H), 14.5 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 150.0, 149.8, 128.8, 121.7,
113.7, 48.5. MS (EI) m/z = 311.1 [M?]. Anal.: Calcd. for
C15H13N5OS (311.36): C (57.86), H (4.21), N (22.49), S
(10.30). Found: C (57.76), H (4.22), N (22.50), S (10.27).
4-(4-Iodophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3q)
Yield: (65 %). m. p.:[320 °C. FT-IR (v, cm-1): 3100 (NH
str.), 1600, 1500 (C=N str.), 1200 (C=S str.). H NMR
1
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Table 2 The apoptosis ratio of cells treated after treatment and
incubation for 24 h values of compounds (3c–q)
Table 3 The Protein Breaks % of cells treated after treatment and
incubation for 24 h values of compounds (3c–q)
Compd. The apoptosis ratio of cells treated after treatment and
incubation for 24 h in HepG2
Compd. The DNA Breaks % of cells treated after treatment and
incubation for 24 h values of compounds
50 lmol/L
100 lmol/L
150 lmol/L
50 lmol/L
100 lmol/L
150 lmol/L
3c
3d
3e
3f
14.23 %
22.07 %
23.18 %
17.29 %
15.69 %
19.07 %
24.33 %
25.55 %
16.47 %
14.94 %
20.02 %
12.33 %
18.16 %
21.02 %
0.57 %
47.95 %
74.39 %
78.11 %
58.29 %
52.87 %
64.26 %
82.02 %
86.12 %
55.51 %
50.35 %
67.485 %
34.49 %
61.20 %
70.85 %
1.22 %
40.54 %
74.54 %
79.75 %
53.14 %
46.42 %
60.84 %
85.34 %
91.31 %
49.67 %
43.385 %
65.10 %
45.16 %
56.86 %
69.66 %
3.65 %
3c
3d
3e
3f
5.94 %
9.75 %
9.94 %
7.10 %
6.56 %
7.84 %
10.14 %
10.35 %
6.83 %
6.18 %
8.16 %
5.60 %
7.54 %
9.56 %
0.20 %
6.28 %
9.56 %
20.01 %
20.61 %
12.47 %
11.08 %
14.45 %
21.23 %
21.86 %
11.75 %
10.14 %
15.33 %
8.74 %
14.38 %
38.33 %
39.86 %
20.46 %
17.49 %
24.90 %
41.46 %
43.11 %
18.92 %
15.55 %
26.93 %
12.78 %
23.02 %
36.85 %
0.75 %
3g
3h
3i
3g
3h
3i
3j
3j
3k
3l
3k
3l
3m
3o
3p
3q
aC
bC
3m
3o
3p
3q
aC
bC
13.62 %
19.42 %
0.44 %
23.78 %
35.66 %
60.13 %
20.34 %
45.89 %
Data are expressed as mean and evaluated by one-way analysis of
variance (ANOVA); results are representative of three replicates
(* P \ 0.01)
Data are expressed as mean and evaluated by one-way analysis of
variance (ANOVA)
Results are representative of three replicates (* P \ 0.01)
a
a
C = negative control
C = negative control
b
b
C = positive control, sorafenib
C = positive control, sorafenib
(500 MHz, DMSO-d6, d ppm): 6.8–7.4 (m, 6H, Ar–H), 8.0
(m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 160.4, 155.0, 149.5, 137.9,
136.7, 132.9, 128.1, 121.0, 90.0. MS (EI) m/z = 380.0
[M?]. Anal.: Calcd. for C13H9N4SI (380.20): C (41.07), H
(2.39), N (14.74), S (8.43). Found: C (41.15), H (2.40), N
(14.73), S (8.40).
The culture was maintained at 37 °C with a gas mixture of
5 % CO2/95 % air. The medium was changed every 2 days
and the cells were sub cultured every 3 days.
Cell viability assay Cell viability was determined using
the MTT assay. Briefly, the cells were collected and
resuspended in RPMI-1640 medium at 4 9 104 cells/mL,
100 lL aliquots were added to each well of 96-well flat-
bottomed micro titer plates, followed by addition of
100 lL of the complexes one and two. Three replicate
wells were used for each data point in the experiments.
After incubation for the indicated intervals, 20 lL of
MTT (5 mg/mL in PBS) solution was added to each well
and plates were then incubated for 4 h at 37 °C. The
medium with MTT was removed from the wells. Intra-
cellular formazan crystals were dissolved by adding
150 lL of DMSO to each well, and the plates were
shaken for 10 min. The absorbance was read at 490 nm
with a microplate reader. Percentage of survival was
calculated as a fraction of the negative control (medium
only). The half-maximal inhibitory concentration (IC50)
was obtained from the dose-response curve with an ori-
ginal 6.0 software.
4-(2-Nitrophenyl-5-(pyridin-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione 3r
Yield: (70 %). m. p.:[320 °C. FT-IR (v, cm-1): 3100 (NH
str.), 1600, 1494 (C=N str.), 1200 (C=S str.). 1H NMR
(500 MHz, DMSO-d6, d ppm): 6.7–7.4 (m, 6H, Ar–H), 7.8
(m, 2H, Ar–H), 14.5 (s, 1H, NH, D2O exchang.). 13C NMR
(125.76, DMSO-d6, d ppm): 172.2, 155.9, 136.8, 134.7, 125.3,
120.8, 116.3. MS (EI) m/z = 299.0 [M?]. Anal.: Calcd. for
C13H9N5O2S (299.3): C (52.17), H (3.03), N (23.40), S
(10.71). Found: C (52.27), H (3.04), N (23.44), S (10.70).
In vitro antitumor activity
Cell culture The cells were routinely cultured in RPMI-
1640 medium, supplemented with 10 % fetal calf serum.
123

Med Chem Res
Flow cytometry analysis
NMR spectra of the compounds (3a–r) that are taken in
DMSO-d6, D2O exchangeable NH proton was seen as
singlet at 9.8–14.5 ppm. The aromatic protons appear as
multiplet at 6.6–8.7 ppm.MS of the compounds showed
molecular ion peaks [M]?, in agreement with their
molecular formula. In the 13C-NMR spectra of all com-
pounds (3a–r), all aromatic and aliphatic carbons were
observed at expected regions. All compounds gave satis-
factory elemental analysis.
Cells were seeded into 100 mL cell culture bottles at
12 9 106 cells 24 h before treatment. Then cells were
treated according to the aforementioned method and
incubated for 24 h. After wards, cells were collected,
combined into single cell suspension, and centrifuged at
8,000 rpm for 5 min. The supernatant was discarded and
the cells were washed three times with cooled PBS and
fixed for 24 h with cold alcohol at 4 °C. One milliliter cell
suspension (106/mL) was washed three times with the cool
PBS, treated with RNase for 30 min at 37 °C, stained it
with PI for 30 min at 37 °C in a dark environment, and
taken for flow cytometry analysis.
In vitro antitumor studies
The compounds were evaluated for their ability to inhibit the
growth of BEL-7402, HUH-7 and HepG2 human hepatoma
cell lines using MTT assay. The inhibition was expressed as
cell viability relative to control without compound treat-
ments. In the present study, BEL-7402, HUH-7 and HepG2
human hepatoma cells were used which have been recently
characterized as a suitable model for in vitro assessment of
hepatoma toxicity (Banik et al., 2011; Johnstone et al.,
2002). Sorafenib (50 lM) was used as a positive control,
which has been used extensively as an efficient anticancer
drug (Sonntag et al., 2014). After treated for 24, 48 and 72 h
on the selected three cell lines, the cells viability with their
the IC50 values was calculated (Table 1). The synthesized
compounds exhibit antiproliferative effect to human
hepatoma cells BEL-7402, HUH-7 and HepG2 in a time and
dose-dependent manner with increasing the concentra-
tions of compounds in the following order of activity 3j [
3i [ 3e [ 3d [ 3q [ 3m [ 3h [ 3p [ 3f [ 3k [ 3g [
3l [ 3c [ 3o. The results of the MTT-dye reduction assay
unambiguously indicate that the compounds exert potent
cytotoxic/antiproliferative effect. Flow cytometric analyses
of apoptosis to further examine the effects of the com-
pounds on apoptosis; flow cytometry was used to quantify
the apoptotic state (Table 2). After treatment and incuba-
tion for 24 h, the apoptosis ratio of cells treated with 50,
100, and 150 lmol/L concentration of the compounds. The
results also supported the notion that the tested compounds
induced apoptosis of HepG2 cells in a concentra-
tion-dependent manner in the following order of activity 3j [
3i [ 3e [ 3d [ 3q [ 3m [ 3h [ 3p [ 3f [ 3k [ 3g [
3l [3c [ 3o.After treatment with the compounds (50, 100
and 150 lmol/L, respectively) for 24 h, the caspase-3
zymogen protein bands became thinner. Studies have
proved that the unactivated caspase-3 will trigger apoptosis
when it is activated and play a very important role as the
central effector of apoptosis when cells start apoptosis. Our
results showed that the compounds could significantly
enhance the activity of caspase-3 (Table 3) in the following
order of activity 3j [ 3i [ 3e [ 3d [ 3q [ 3m [ 3h [
3p [ 3f [ 3k [ 3g [ 3l [ 3c [ 3o.
Western blotting
The cells were taken in the logarithmic growth phase,
treated according to the aforementioned method, and
incubated for 24 h. After fragmentation on ice for 20 min,
the lysates were centrifuged at 15,000 rpm for 10 min at
4 °C, the protein was collected, quantitated with the BCA
method, electrophoresed and isolated by the SDS-PAGE
(10 %) using the electrotransfer method, blocked and
hybridized on the cellulose nitrate film. The protein
expression of cells was detected using the ECL Western
blotting method. The densities of protein bands were cal-
culated using the Quantity One software.
Statistical analysis Data were expressed as the mean SD
from these independent experiments. Statistic analysis was
performed using the SPSS 13.0 for Windows. Comparisons
between two groups were performed by unpaired test.
Multiple comparisons between more than two groups were
performed by one-way analysis of variance (ANOVA).
Significance was accepted at value lower than 0.05.
Result and discussion
Chemistry
In the present work, eighteen compounds (3a–r) were
synthesized. (Scheme 1) illustrates the way used for the
preparation of target compounds. The reaction of thiose-
micarbazides (2a–r) with sodium hydroxide, in each case,
a single product was obtained. The FT IR, H NMR, 13C
1
NMR, MS, and single crystal X-ray crystallography ana-
lysis confirmed the structure of these products as N-4-
cyclohexyl/aryl-5-(pyridine-4-yl)-2,4-dihydro-3H-1,2,4-
triazole-3-thiones (3a–r). In IR spectra of all compounds
NH were observed at about 3,000–3,481 cm-1. In the H
1
123

Med Chem Res
Acknowledgments The authors would like to extend their sincere
appreciation to the Deanship of Scientific Research at King Saud
University for funding this research group No. (RG 1435-006).
Structure activity relationship (SAR)
Careful investigation of the relation between structure and
the data of tested activities revealed the following
assumption about SAR.
a) The effect of different substitutions on the aromatic
portion on the activity was in the following order CH3 [
OCH3 [ I [ SO2NH2 [ OC2H5 [ C2H5 [ NO2 [ Cl
[ CH3CONH.
b) Concerning the alkyl substitutions on aromatic part,
the methyl group greatly increases the activity rather
than the more bulky ethyl one, and also its position
and numbers greatly influence the activity where as the
number of alkyl substitutions increases, the activity
decreases and its presence in the para position also
decreases the activity.
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