Synthesis, characterization, and anticancer studies of S and N alkyl piperazine-substituted positional isomers of 1,2,4-triazole derivatives

Article abstract of DOI:10.1007/s00044-013-0757-3

A series of 3-[3-[4-(substituted)-1-cyclicamine] propyl]thio-5- substituted[1,2,4]triazoles (8a-m) and 2-[3-[4-(substituted)-1-cyclicamine] propyl]-5-(substituted)-2,4-dihydro-3H[1,2,4]triazole-3-thiones (9a-h) were synthesized with good yields starting from corresponding carboxylic acids using two different methods. The cytotoxicity studies of these derivatives were studied against five different human cancer cell lines. Six compounds had shown good anticancer activity. The triazole derivatives, 9d, 8j, and 8i were most potent particularly against U937 and HL-60 cells. The cytotoxic potency of the compounds varied between the cell lines suggesting that a structural property of these compounds as possible determinant of their biological activity. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0757-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1661–1671
DOI 10.1007/s00044-013-0757-3
ORIGINAL RESEARCH
Synthesis, characterization, and anticancer studies
of S and N alkyl piperazine-substituted positional
isomers of 1,2,4-triazole derivatives
•
M. S. R. Murty Kesur R. Ram B. Ramalingeswara Rao Rayudu Venkateswara Rao
•
•
•
•
Mohana Rao Katiki Janapala Venkateswara Rao R. Pamanji L. R. Velatooru
•
•
Received: 28 February 2013 / Accepted: 21 August 2013 / Published online: 8 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 3-[3-[4-(substituted)-1-cyclicamine]
propyl]thio-5-substituted[1,2,4]triazoles (8a–m) and 2-[3-
[4-(substituted)-1-cyclicamine]propyl]-5-(substituted)-2,4-
dihydro-3H[1,2,4]triazole-3-thiones (9a–h) were synthe-
sized with good yields starting from corresponding car-
boxylic acids using two different methods. The cytotoxicity
studies of these derivatives were studied against five dif-
ferent human cancer cell lines. Six compounds had shown
good anticancer activity. The triazole derivatives, 9d, 8j,
and 8i were most potent particularly against U937 and HL-
60 cells. The cytotoxic potency of the compounds varied
between the cell lines suggesting that a structural property
of these compounds as possible determinant of their bio-
logical activity.
compounds possess important pharmacological activities
such as, anti-inflammatory, CNS stimulants sedatives,
antianxiety, antimicrobial (Heindel and Reid, 1980; Holla
et al., 1994), and antimycotic agents such as fluconazole,
itraconazole, voriconazole (Haber, 2001). Also, there are
known drugs containing the 1,2,4-triazole group, e.g.,
Triazolam (Raslan and Khalil, 2003), Alprazolam (Coffen
and Fryer, 1974), Etizolam, and Furacylin.
Moreover, sulfur-containing heterocycles represent an
important group of compounds and these heterocycles, i.e.,
the S- and N-alkylated mercapto 1,2,4-triazole ring systems
exhibit biological activities, such as anticancer (Holla
et al., 2003; Duran et al., 2002), antitubercular (Mir et al.,
1970), diuretic (Yale and Piala, 1966), antibacterial (Burch
and Smith, 1966), antifungal (Rollas et al., 1993), anti-
mycobacterial (Rudnicka et al., 1986), and hypoglycemic
(Mhasalkar et al., 1970).
Keywords 3-Mercapto-1,2,4-triazoles Á Acid hydrazides Á
Piperazines Á Anticancer
Several piperazine derivatives were synthesized in the
last decade, as useful chemotherapeutic agents for various
diseases, such as crixivan (indinavir sulfate) and delavir-
dine (rescriptor), powerful inhibitors for the protease and
reverse transcriptase inhibitor of HIV enzymes, respec-
tively (Al-Masoudi et al., 2006). Some piperazinyl
[1,2,4]triazole derivatives were reported as 5-HT1A sero-
tonin receptor ligands (Sarva et al., 2002).
Introduction
The chemistry of 1,2,4-triazoles and their fused heterocy-
clic derivatives has received considerable attention in the
last few decades, owing to their synthetic and effective
biological importance (Shaker, 2006). The 1,2,4-triazole
Cancer drug discovery has traditionally focused on tar-
geting DNA synthesis, angiogenesis, and cell division (Do
et al., 2009). Many of the major classes of cytotoxic drugs
in current use owe their overall therapeutic effectiveness,
but lack of selectivity for tumor cells over normal cells can
lead to severe side effects (Atkins and Gershell, 2002).
Design and synthesis of novel small molecules which can
specifically block some targets in tumor cells are in per-
spective direction in modern medicinal chemistry. There-
fore, there is an urgent need to establish a process to assess
M. S. R. Murty (&) Á K. R. Ram Á B. R. Rao Á
R. V. Rao Á M. R. Katiki
Medicinal Chemistry & Pharmacology Division, Discovery
Laboratory, Indian Institute of Chemical Technology, Uppal
Road, Hyderabad 500007, India
e-mail: msrmurty@ymail.com; msrmurty@iict.res.in
J. V. Rao Á R. Pamanji Á L. R. Velatooru
Toxicology Unit, Biology Division, Indian Institute of Chemical
Technology, Uppal Road, Hyderabad 500007, India
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Med Chem Res (2014) 23:1661–1671
anticancer drug action, i.e., safety, efficacy, and mecha-
nism of action. Many synthetic heterocyclic small mole-
cules with cytotoxic activity have been reported and
several of them under gone for the clinical trials (Lesyk
et al., 2007).
stretching in IR spectra and molecular weight had con-
firmed the formation of thiosemicarbazides (4). Thiose-
micarbazides (4) were reacted with aqueous sodium
hydroxide to produce 5-substituted[1,2,4]triazole-3-thiones
(5) (Hoggarth, 1949). They can also exist in tautomeric
thiol form. Here we found that they mainly exist in thione
form which is also supported by some earlier reports
(Shaker, 2006). Some reports states that they mainly exist
in thione–thiol tautomeric forms in solution but the thione
structures dominate in the solid state (Katica et al., 2001).
It has been reported that the crystal structure of these types
of compounds corresponded to the thione form (Ozturk
et al., 2004). The product was confirmed by appearance of
characteristic broad peak for NH protons with chemical
shift 13–14 ppm. Appearance of characteristic triazole NH
stretching in IR spectra and molecular weight had further
confirmed the formation of 5-substituted[1,2,4]triazole-3-
thiones (5).
A major interest in our group is the design, synthesis, and
evaluation of new anti-proliferative compounds as new
drug molecules. In continuation of our research in this field
(Murty et al., 2012a, b), we described herein the synthesis of
3-[3-[4-(substituted)-1-cyclicamine]propyl]thio-5-substituted
[1,2,4]triazoles (8) and 2-[3-[4-(substituted)-1-cyclicamine]
propyl]-5-(substituted)-2,4-dihydro-3H[1,2,4]triazole-3-
thiones (9), the positional isomers of S- and N-alkylated
derivatives of mercapto-1,2,4-triazoles. The cytotoxic
activity of these derivatives was studied against five dif-
ferent types of human cancer cell lines, i.e., U937, THP-1,
Colo205, MCF7, and HL-60.
The, 1-(3-Chloropropyl)-4-substituted cyclicamines (7)
were prepared by the reaction of cyclicamines (6) with
1-bromo-3-chloropropane in presence of activated zinc in
THF at ambient temperature (Scheme 2) (Murty et al.,
2003).
Results and discussion
Chemistry
5-Substituted [1,2,4]triazole-3-thiones (5) were synthesized
by following reported sequence of the reactions from cor-
responding aryl carboxylic acids (1) (Scheme 1). The aryl
carboxylic acids (1) were converted to corresponding
methyl esters (2) with catalytic amount of sulfuric acid in
methanol. This was confirmed by disappearance of broad
carboxylic acid peak in 1H NMR spectra and disappearance
of broad ‘‘OH’’ stretching of carboxylic acid in IR spectra
of the product. It was also supported by appearance of
By careful literature work we found the methods for the
selective N- and S-alkylation of 4-amino-5-aryl-1,2,4-tria-
zole-3-thiols (Collin et al., 2003; Sauleau and Sauleau,
2000). In the present work it was planned to prepare 2-[3-
[4-(N-substituted)-1-cyclicamine]propyl]-5-substituted-2,4-
dihydro-3H[1,2,4]triazole-3-thiones (9) in addition to 8 as
1
OCH3 peak in H NMR spectra of methyl esters (2). The
X
X
Zn
methyl esters (2) were converted to corresponding acid
hydrazides (3) using hydrazine hydrate in methanol (Car-
penter et al., 2010). The acid hydrazides (3) were reacted
with potassium thiocyanate and concentrated hydrochloric
acid to produce corresponding thiosemicarbazides (4)
(Boots and Cheng, 1967). Appearance of additional NH
Cl
Br
Cl
N
+
HN
THF, rt
7
6
X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyrimidyl, N-2-pyridyl, N-3-chlorophenyl
Scheme 2 Synthesis of 1-(3-chloropropyl)-4-substituted cyclicam-
ines (7)
Scheme 1 Synthesis of
5-substituted[1,2,4]triazole-3-
thiones (5)
(i)
(ii)
RCOOH
(iii)
RCOOCH3
RCONHNH2
3
2
H
N
N
N
N
(iv)
S
SH
RCONHNHCSNH2
N
H
N
R
R
H
4
5
R = 4-Cl-C6H4, 3-Cl-C6H4, 2-Cl-C6H4, 4-CH3-C6H4, 3-CH3-C6H4, 4-F-C6H4, 4-C4H9-C6H4,
4-OCH3-C6H4, C6H5CH2, 3,4-di OCH3-C6H3.
Reagents and conditions: (i) Cat. H2SO4, CH3OH, reflux, 4 h; (ii) NH2NH2.H2O, CH3OH, reflux, 2-3 h;
(iii) KSCN/ Conc. HCl, CH3OH, reflux, 1 h; (iv) 10% Aqueous NaOH, CH3OH, reflux, 5-6 h.
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Med Chem Res (2014) 23:1661–1671
1663
Scheme 3 Synthesis of 3-[3-[4-
(substituted)-1-
cyclicamine]propyl]thio-5-
substituted [1,2,4]triazoles
(8a–m)
H
N
N
N(C2H5)3 / TBAI
X
N N
S
N
+
Cl
N
N
X
R
S
C2H5OH, reflux, 3 h
H
R
N
H
8a-m
5
7
R = 4-Cl-C6H4, 3-Cl-C6H4, 2-Cl-C6H4, 4-CH3-C6H4, 3-CH3-C6H4, 4-F-C6H4, 4-C4H9-C6H4, C6H5CH2
X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyrimidyl, N-2-pyridyl, N-3-chlorophenyl
Table 1 3-[3-[4-(Substituted)-
1-cyclicamine]propyl]thio-5-
substituted[1,2,4]triazoles
(8a-m)
Entry
Compound
R
X
Yield (%)
1
8a
8b
8c
8d
8e
8f
4-C4H9–C6H4
4-C4H9–C6H4
4-CH3–C6H4
4-CH3–C6H4
4-Cl–C6H4
4-Cl–C6H4
4-F–C6H4
O
72
66
70
63
75
65
70
66
70
68
60
65
73
2
N-Phenyl
3
N-3-Chlorophenyl
N-Ethyl
4
5
N-3-Chlorophenyl
N-2-Pyrimidyl
N-Benzyl
6
7
8g
8h
8i
8
3-CH3–C6H4
3-CH3–C6H4
3-Cl–C6H4
2-Cl–C6H4
2-Cl–C6H4
C6H5CH2
N-Phenyl
9
N-2-Pyridyl
N-2-Pyrimidyl
N-Ethyl
10
11
12
13
8j
8k
8l
N-2-Pyridyl
N-2-Pyridyl
8m
Scheme 4 Synthesis of 2-[3-[4-
(substituted)-1-
cyclicamine]propyl]-5-
(substituted)-2,4-dihydro-
3H[1,2,4]triazole-3-thiones
(9a–h)
H
N
X
K2CO3/TBAI
CH3CN, reflux, 4 h
N
N N
N
+
S
Cl
N
R
N
S
X
R
N
H
H
7
5a-d and 5i,j
9a-h
R = 4-Cl-C6H4, 4-CH3-C6H4, 4-F-C6H4, 4-C4H9-C6H4, 4-OCH3-C6H4, 3,4-di OCH3-C6H3
X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyridyl, N-3-chlorophenyl
this may lead to some interesting results in biological
activity point of view.
The 5-substituted-1,2,4-triazole-3-thiones (5) and
1-(3-chloropropyl)-4-substituted cyclicamines (7) were
reacted in the presence of K2CO3 in acetonitrile with a
catalytic amount of TBAI. The 2-[3-[4-(N-substituted)-
1-cyclicamine]propyl]-5-substituted-2,4-dihydro-3H[1,2,4]
triazole-3-thiones (9) were formed, i.e., N-alkylated
products with the traces of 8 (Scheme 4). In the deriv-
atives 9, a triplet for the methylene group of the propyl
chain that connects the piperazine moiety and the tria-
zole part of the molecule is observed with a chemical
shift in the range of d 4.45–4.35, which is typical for
NCSN–CH2 connectivity (Sarva et al., 2002; Salerno
et al., 2004). Further the products 9a-h were confirmed
by mass and IR spectroscopy and the details are given in
Table 2.
When the 5-substituted[1,2,4]triazole-3-thiones (5) and
1-(3-chloropropyl)-4-substituted cyclicamines (7) were reac-
ted in the presence of triethyl amine in ethanol with a catalytic
amount of tetra butyl ammonium iodide (TBAI) exclusively
3-[3-[4-(substituted)-1-cyclicamine]propyl]thio-5-substituted
[1,2,4]triazoles (8) were formed, i.e., S-alkylated products
(Scheme 3). In the derivatives 8, a triplet for the methylene
groupofthepropylchainthatconnectsthecyclicaminemoiety
and the triazole part of the molecule is observed with a
chemical shift in the range of d 3.25–3.15, which is typical for
S–CH2 connectivity (Sarva et al., 2002; Salerno et al., 2004).
Further the products 8a–m were confirmed by mass and IR
spectroscopy and the details are given in Table 1.
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Cytotoxicity studies
Table 2 2-[3-[4-(Substituted)-1-cyclicamine]propyl]-5-substituted-
2,4-dihydro- 3H[1,2,4]triazole-3-thiones (9a–h)
The cytotoxicity of the newly synthesized compounds was
measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide] assay, according to the
method of Mossman (Mossman, 1983). The Triazole
derivatives 8a–m and 9a–h were tested for in vitro cyto-
toxic activity against U937, THP-1, Colo205, MCF7, and
HL-60 human cancer cell lines. IC50 values of the test
compound for 24 h on each cell line was calculated and
presented in Table 3. It is evident from the results that the
triazole derivatives were found to be more potent on U937
and HL-60 cells than other three cell lines of THP-1,
Colo205, and MCF-7. Some of the triazole derivatives had
shown significant decrease in cell viability in some of the
test cell lines on concentration dependent manner. Etopo-
Entry Compound R
X
Yield (%)
1
2
9a
9b
4-C4H9–C6H4
4-C4H9–C6H4
N-Phenyl
60
68
N-3-
Chlorophenyl
3
4
9c
4-CH3–C6H4
4-Cl–C6H4
4-F–C6H4
N-3-
58
65
Chlorophenyl
9d
N-3-
Chlorophenyl
5
6
9e
9f
N-Benzyl
66
59
3,4-di OCH3–
C6H3
O
7
8
9g
9h
3,4-di OCH3–
C6H3
N-Ethyl
60
62
4-OCH3–C6H4
N-2-Pyridyl
Table 3 In vitro cytotoxicity of Triazole derivatives 8a–m and 9a–h
S. no.
Compound
ICa50 (lM)
U937
THP-1
COLO 205
MCF7
HL-60
1
8a
156.11 1.47
–
155.36 9.4
286.8 35.0
–
2
8b
8c
49.31 3.97
97.63 11.8
–
–
–
3
256.84 39.01
–
139.29 0.15
–
–
4
8d
8e
–
–
–
–
–
5
–
–
–
–
–
6
8f
102.24 5.59
–
–
–
105.06 5.49
7
8g
124.0.3 6.61
255.42 8.85
181.41 4.28
184.42 2.86
–
8
8h
8i
–
247.55 15.26
–
–
–
9
52.33 3.12
–
–
–
29.36 2.23
10
11
12
13
14
15
16
17
18
19
20
21
8j
49.13 2.86
–
–
–
18.51 1.16
8k
8l
–
–
–
–
–
–
–
–
–
–
8m
9a
163.14 13.85
–
–
–
–
88.94 7.86
146.57 3.95
–
–
–
9b
9c
–
–
–
–
–
–
–
–
–
–
9d
9e
28.19 1.140
–
–
–
6.67 0.55
–
159.53 1.36
110.99 16.6
128.46 0.65
–
9f
–
–
–
–
–
9g
–
–
–
–
–
9h
Etoposideb
–
–
–
–
114.95 6.00
1.84 0.20
10.43 2.0
3.66 0.25
12.3 2.14
29.49 2.22
Exponentially growing cells were treated with different concentrations of triazole derivatives for 24 h and cell growth inhibition was analyzed
through MTT assay. – Indicates IC50 value [300 lM
a
IC50 is defined as the concentration, which results in a 50 % decrease in cell number as compared with that of the control cultures in the
absence of an inhibitor and were calculated using the respective regression analysis. The values represent the mean SE of three individual
observations
b
Etoposide was employed as positive control
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Med Chem Res (2014) 23:1661–1671
1665
side was used as a positive control in these assays and the
IC50 values obtained on U937, THP-1, Colo205, MCF-7,
and HL-60 as 10.43, 3.66, 12.3, 29.49, and 1.84 lM,
respectively.
cytotoxic potency of the compounds varied between the
cell lines suggesting that a structural property of these
compounds as possible determinants of their biological
activity.
The triazole derivatives, 9d, 8j, 8i, and 8f were potent
anti-proliferative agents against U937 cells, with IC50
values of 28.19, 49.13, 52.33, and 102.24 lM, respectively.
Interestingly, similar trend was observed even in HL-60
cells, with IC50 values of 6.67, 18.51, 29.36, and
105.06 lM, respectively. The structural isomeric triazole
derivatives, 8b (S-alkylated) and 9a (N-alkylated) exhibited
similar trend with significant activity on U937 with IC50
values of 49.31 and 88.94 lM and for THP-1 cells with
IC50 values of 97.63 and 146.57 lM. While it was inter-
esting that S-alkylated isomer of 9d, i.e., 8e had not shown
anticancer activity. Very few of the triazole derivatives (8a,
8c, 8g, and 9e) were active against Colo205 and MCF-7.
On the structure point of view, the compounds with
chloro group at 3rd or 4th position of the phenyl ring of
triazole moiety had shown good cytotoxic activity (9d, 8j,
8f). Alternatively, the compounds with piperazine moiety
containing N-3-chlorophenyl, N-2-pyrimidyl, and N-2-
pyridyl groups had shown good cytotoxic activity (9d, 8j,
8i, 8f).
Experimental
Chemistry
Melting points were determined on a Buchi capillary
melting point apparatus. The 1H NMR (200 MHz) and 13
C
NMR (75 MHz) spectra were recorded on Varian Gemini
using TMS as an internal standard. J values are given in
Hz. The IR spectra were recorded on Perkin-Elmer Infra-
red-683 and the mass spectra were recorded on Agilent
Technologies LC/MSD SL single quadrupole (Agilent
ChemStation software). Elemental analyses were per-
formed on Elemental VARIO EL elemental analyzer.
General procedure for the preparation of methyl esters (2)
A catalytic amount of concentrated H2SO4 was added to a
solution of an appropriate aryl carboxylic acid 1 (0.15 mol)
in 150 mL of methanol and the mixture was refluxed for
4 h. It was allowed to cool. The saturated solution of
NaHCO3 was to the reaction mixture and was extracted
with methylene dichloride (CH2Cl2) (2 9 100 mL). The
combined organic layer was dried (Na2SO4) and concen-
trated to obtain pure methyl ester (2).
Overall it was observed from the results that cytotoxicity
depends on the particular substituents on the phenyl ring
and piperazine ring rather than S and N alkylation of tria-
zoles. These findings suggest that the newly synthesized
triazole have shown significant anti-proliferative activity
on U937 and HL-60 cells than other three cell lines of
THP-1, Colo205, and MCF-7. The cytotoxic potency of the
compounds varied between the cell lines suggesting that a
structural property of these compounds as possible deter-
minants of their biological activity.
General procedure for the preparation of acid hydrazides
(3) (Carpenter et al., 2010)
To a solution of an appropriate methyl ester 2 (0.1 mol) in
150 mL of methanol was added 95 % hydrazine hydrate
(0.2 mol), and the mixture was refluxed for 2–3 h up to
reaction completed (TLC). It was allowed to cool, and the
obtained solid was washed with methanol and dried
(Na2SO4). The crude compounds were recrystallized from
ethanol.
Conclusions
Synthesis of 3-[3-[4-(substituted)-1-cyclicamine]pro-
pyl]thio-5-substituted[1,2,4]triazoles (8a–m) and 2-[3-[4-
(substituted)-1-cyclicamine]propyl]-5-substituted-2,4-
dihydro-3H[1,2,4]triazole-3-thiones (9a–h) was achieved
with good yields starting from corresponding carboxylic
acids using two different methods. The anticancer activity
of these derivatives was studied. Among the compounds
(n = 21) tested for anticancer activity, six compounds have
exhibited good anticancer activity. The triazole derivatives,
9d, 8j and 8i were the most potent particularly against
U937 and HL-60 cells. On the structure point of view, the
compounds with chloro group at 3rd or 4th position of the
phenyl ring of triazole moiety and piperazine moiety
containing N-3-chlorophenyl, N-2-pyrimidyl, and N-2-
pyridyl groups had shown good cytotoxic activity. The
General procedure for the preparation
of thiosemicarbazides (4) (Boots and Cheng, 1967)
To a solution of an appropriate acid hydrazide 3 (0.02 mol)
in 50 mL of methanol was added a solution of KSCN
(0.03 mol) and 3 mL of HCl with constant stirring. The
mixture was immediately evaporated to dryness on a steam
bath and heated for an additional 1 h with another 50 mL
of methanol. The resulting solid was treated with distilled
water and with a small amount of ethanol. The crude thi-
osemicarbazides (4) were used as such for next step.
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General procedure for the preparation of 5-substituted
layer was dried (Na2SO4) and concentrated. The crude
product was subjected to flash column chromatograph to
obtain pure product.
[1,2,4]triazole-3-thiones (5a–j) (Hoggarth, 1949)
To a solution of an appropriate thiosemicarbazide 4
(0.01 mol) in 15 mL of methanol was added a solution of
10.0 % NaOH (20 mL), and the reaction mixture was
refluxed immediately for 5–6 h up to reaction was com-
pleted (TLC). The mixture was cooled and acidified with
dilute HCl at pH 5–6. The resulting solid was filtered,
washed with distilled water, and dried (Na2SO4). The crude
compounds were recrystallized from ethanol.
Spectroscopic data for the synthesized compounds
5-[4-(Tert-butyl)phenyl]-4H-1,2,4-triazol-3-yl
(3-morpholinopropyl) sulfide (8a)
1
Yield: 72 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 14.12 (br s, 1H, NH), 7.90 (d, J = 8.6, 2H,
2XAr–H); 7.42 (d, J = 8.6, 2H, 2XAr–H); 3.69–3.57 (m,
4H, 2XO–CH2); 3.17 (t, J = 7.0 Hz, 2H, S–CH2);
2.51–2.32 (m, 6H, 3XN–CH2); 1.91 (quintet, J = 7.0 Hz,
2H, CH2CH2CH2); 1.34 (s, 9H, C(CH3)3). 13C NMR
(75 MHz, CDCl3) d: 158.6, 157.7 (2C=N), 150.3, 127.9,
127.3, 125.8 (6Aromatic C), 66.4 (2OCH2), 53.6 (2NCH2),
53.4 (NCH2), 40.4 (C(CH3)3), 34.3 (SCH2), 31.6
(C(CH3)3), 28.2 (CH2CH2CH2). IR (KBr, cm-1): 3152 (N–
H), 2959, 2862, 2812 (–C–H), 1615 (C=C), 1456, 1329 (C–
N), 1267, 1117 (C–O), 847, 756 (=C–H ben). ESI–MS (m/
z): 361 (M?1)?. Anal. calcd. for C19H28N4OS (360): C,
63.30; H, 7.83; N, 15.54. Found: C, 63.32; H, 7.82; N,
15.57.
General procedure for the preparation of 1-(3-
chloropropyl)-4-substituted cyclicamines (7a–g) (Murty
et al., 2003)
The activated zinc powder (0.002 mol) was added to a
solution of cyclicamine 6 (0.002 mol) and 1-bromo-3-
chloropropane (0.002 mol) in THF (20 mL) and stirred at
room temperature for 2 h. After completion of the reaction,
the mixture was filtered and the solid was washed with sol-
vent ether (3 9 20 mL). The combined filtrate was treated
with 10 % NaHCO3 (20 mL), water (2 9 20 mL), dried
(Na2SO4), and evaporated to give the crude product. The
crude product was purified by flash column chromatography.
5-[4-Tert-butyl)phenyl]-4H-1,2,4-triazol-3-yl [3-(4-
General procedure for the preparation of 3-[3-[4-
(substituted)-1-cyclicamine]propyl]thio-5-
substituted[1,2,4]triazoles (8a–m)
phenylpiperazino)propyl] sulfide (8b)
Yield: 66 %. White solid, mp 138–140 °C. 1H NMR
(200 MHz, CDCl3) d: 14.27 (br s, 1H, NH), 7.90 (d,
J = 8.3 Hz, 2H, 2XAr–H); 7.35 (d, J = 8.3 Hz, 2H,
2XAr–H); 7.16 (t, J = 7.9 Hz, 2H, 2XAr–H); 6.83–6.75
(m, 3H, 3XAr–H); 3.26–3.16 (m, 4H, 2XN–CH2); 3.13 (t,
J = 6.8 Hz, 2H, S–CH2); 2.76–2.68 (m, 4H, 2XN–CH2);
2.64 (t, J = 6.6 Hz, 2H, N–CH2); 1.99–1.94 (m, 2H,
CH2CH2CH2); 1.29 (s, 9H, C(CH3)3). 13C NMR (75 MHz,
CDCl3) d: 158.8, 157.8 (2C=N), 150.2, 149.6, 129.7, 127.4,
127.0, 125.4, 118.5, 114.4 (12Aromatic C), 53.2 (NCH2),
52.6 (2NCH2), 49.7 (2NCH2), 40.5 (C(CH3)3), 34.2
(SCH2), 31.5 (C(CH3)3), 28.1 (CH2CH2CH2). IR (KBr,
cm-1): 3448 (N–H), 2956 (–C–H), 1639 (C=C), 1495,
1420, 1225 (C–N), 768 (=C–H ben). ESI–MS (m/z): 436
(M?1)?. Anal. calcd. for C25H33N5S (435): C, 68.93; H,
7.64; N, 16.08. Found: C, 68.94; H, 7.64; N, 16.10.
5-Substituted [1,2,4]triazole-3-thione 5 (0.0005 mol) was
refluxedwithtriethyl amine (0.001 mol)inethanol (8 mL) for
15 min. 1-(3-Chloropropyl)-4-substituted cyclicamine 7
(0.0006 mol) and tetra butyl ammonium iodide (TBAI)
(10 mg) in ethanol (3 mL) were added to above mixture
carefully and was refluxed for 4 h. After the reaction was
completed, water (10 mL) was added and extracted with ethyl
acetate (3 9 10 mL). The combined organic layer was dried
(Na2SO4) and concentrated. The crude product was subjected
to flash column chromatograph to obtain pure product.
General procedure for the preparation of 2-[3-[4-
(substituted)-1-cyclicamine]propyl]-5-(substituted)-2,4-
dihydro-3H[1,2,4]triazole-3-thiones (9a–h)
5-Substituted [1,2,4]triazole-3-thione 5 (0.0005 mol) was
refluxed with K2CO3 (0.00075 mol) in acetonitrile (8 mL)
for 15 min. 1-(3-Chloropropyl)-4-substituted cyclicamine 7
(0.0006 mol) and tetra butyl ammonium iodide (TBAI)
(10 mg) in acetonitrile (3 mL) were added to above mix-
ture carefully and was refluxed for 4 h. After the reaction
was completed, water (10 mL) was added and extracted
with ethyl acetate (3 9 10 mL). The combined organic
3-[4-(3-Chlorophenyl)piperazino]propyl[5-(4-
methylphenyl)-4H-1,2,4-triazol-3-yl] sulfide (8c)
Yield: 70 %. Yellow solid, mp 85–86 °C. 1H NMR
(200 MHz, CDCl3) d: 14.25 (br s, 1H, NH), 7.88 (d,
J = 7.8 Hz, 2H, 2XAr–H); 7.21 (d, J = 7.8 Hz, 2H,
2XAr–H); 7.12 (t, J = 8.6 Hz, 1H, Ar–H); 6.85–6.67 (m,
3H, 3XAr–H); 3.28–3.04 (m, 6H, S–CH2– & 2XN–CH2);
123

Med Chem Res (2014) 23:1661–1671
1667
2.67–2.45 (m, 6H, 3XN–CH2); 2.40 (s, 3H, Ar–CH3); 1.97
(quintet, J = 7.0 Hz, 2H, CH2CH2CH2). 13C NMR
(75 MHz, CDCl3) d: 158.7, 157.6 (2C=N), 151.3, 138.4,
135.2, 131.1, 129.5, 127.8, 127.4, 118.3, 114.8, 112.6
(12Aromatic C), 53.1 (NCH2), 52.4 (2NCH2), 50.2
(2NCH2), 34.4 (SCH2), 27.8 (CH2CH2CH2), 24.2 (ArCH3).
IR (KBr, cm-1): 2947, 2826 (–C–H), 1594 (C=C), 1486,
1328, 1274 (C–N), 834, 760 (=C–H ben). ESI–MS (m/z):
428 (M?). Anal. calcd. for C22H26ClN5S (428): C, 61.74;
H, 6.12; N, 16.36. Found: C, 61.71; H, 6.14; N, 16.37.
J = 5.1 Hz, 2H, 2XAr–H); 7.99 (d, J = 8.9 Hz, 2H, 2XAr–
H); 7.40 (d, J = 8.9 Hz, 2H, 2XAr–H); 6.47 (t, J = 5.1 Hz,
1H, Ar–H); 3.85–3.74 (m, 4H, 2XN–CH2); 3.23 (t,
J = 7.4 Hz, 2H, S–CH2);2.57–2.30(m, 6H, 3XN–CH2);1.97
(quintet, J = 7.4 Hz, 2H, CH2CH2CH2). 13C NMR (75 MHz,
CDCl3) d: 162.7, 158.9, 157.9, 157.7 (5C=N), 134.4, 129.5,
128.7, 128.5, 110.4 (7Aromatic C), 53.1 (NCH2), 52.7
(2NCH2), 50.0 (2NCH2), 34.3 (SCH2), 27.9 (CH2CH2CH2).
IR (KBr, cm-1): 3171 (N–H), 2930, 2852 (–C–H), 1586, 1493
(C=C), 1448, 1260 (C–N), 752 (=C–H ben). ESI–MS (m/z):
417 (M?1)?. Anal. calcd. for C19H22ClN7S (416): C, 54.87;
H, 5.33; N, 23.57. Found: C, 54.89; H, 5.30; N, 23.61.
3-(4-Ethylpiperazino)propyl [5-(4-methylphenyl)-4H-
1,2,4-triazol-3-yl] sulfide (8d)
3-(4-Benzylpiperazino)propyl [5-(4-fluorophenyl)-4H-
1
Yield: 63 %. Brown gummy liquid. H NMR (200 MHz,
1,2,4-triazol-3-yl] sulfide (8g)
CDCl3) d: 14.29 (br s, 1H, NH), 7.90 (d, J = 7.6 Hz, 2H,
2XAr–H); 7.16 (d, J = 8.3 Hz, 2H, 2XAr–H); 3.15 (t,
J = 6.8 Hz, 2H, S–CH2); 2.72–2.40 (m, 12H, 6XN–CH2);
2.37 (s, 3H, Ar–CH3); 2.02–1.93 (m, 2H, CH2CH2CH2);
1.26 (t, J = 6.8 Hz, 3H, CH3CH2). 13C NMR (75 MHz,
CDCl3) d: 158.9, 157.9 (2C=N), 138.3, 129.7, 127.8, 127.3
(6Aromatic C), 53.2 (NCH2), 53.0 (2NCH2), 52.8 (2NCH2),
49.6 (NCH2CH3), 34.2 (SCH2), 28.1 (CH2CH2CH2), 24.5
(ArCH3), 13.6 (CH2CH3). IR (KBr, cm-1): 3406 (N–H),
2930, 2823 (–C–H), 1615 (C=C), 1450, 1327, 1267 (C–N),
830, 752 (=C–H ben). ESI–MS (m/z): 346 (M?1)?. Anal.
calcd. for C18H27N5S (345): C, 62.57; H, 7.88; N, 20.27.
Found: C, 62.58; H, 7.90; N, 20.24.
1
Yield: 70 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 14.22 (br s, 1H, NH), 8.08–7.94 (m, 2H, 2XAr–H);
7.33–7.16(m,5H,5XAr–H);7.11–6.97(m,2H,2XAr–H);3.54
(s, 2H, Ph–CH2); 3.12 (t, J = 6.0 Hz, 2H, S–CH2); 2.72–2.43
(m, 10H, 5XN–CH2); 1.97 (quintet, J = 6.0 Hz, 2H,
CH2CH2CH2).13CNMR(75 MHz,CDCl3)d:162.9(F–C=C),
158.7, 157.7(2C=N), 135.6, 129.3, 128.9, 128.6, 127.5, 126.4,
115.8 (11Aromatic C), 60.2(NCH2Ph), 53.1 (NCH2), 52.7
(2NCH2),52.3(2NCH2),34.3(SCH2),28.2(CH2CH2CH2).IR
(KBr, cm-1): 3405 (N–H), 2932, 2830 (–C–H), 1606 (C=C),
1455,1326,1226(C–N),847,752(=C–Hben).ESI–MS(m/z):
412 (M?1)?. Anal. calcd. for C22H26FN5S (411): C, 64.21; H,
6.37;N,17.02.Found:C,64.18;H,6.39;N,17.00.
3-[4-(3-Chlorophenyl)piperazino]propyl [5-(4-
chlorophenyl)-4H-1,2,4-triazol-3-yl] sulfide (8e)
5-(3-Methylphenyl)-4H-1,2,4-triazol-3-yl [3-(4-
phenylpiperazino)propyl] sulfide (8h)
Yield: 75 %. White solid, mp 72–74 °C. 1H NMR
(200 MHz, CDCl3) d: 14.19 (br s, 1H, NH), 7.96 (d,
J = 8.8 Hz, 2H, 2XAr–H); 7.33 (d, J = 8.1 Hz, 2H,
2XAr–H); 7.18–7.04 (m, 1H, Ar–H); 6.94–6.62 (m, 3H,
3XAr–H); 3.44–3.04 (m, 6H, S–CH2–& 2XN–CH2);
2.77–2.45 (m, 6H, 3XN–CH2); 2.00 (quintet, J = 7.3 Hz,
2H, CH2CH2CH2). 13C NMR (75 MHz, CDCl3) d: 158.6,
157.8 (2C=N), 151.2, 135.4, 134.2, 131.1, 129.4, 128.8,
128.6, 118.4, 114.8, 112.5 (12Aromatic C), 53.2 (NCH2),
52.6 (2NCH2), 50.1 (2NCH2), 34.2 (SCH2), 28.1
(CH2CH2CH2). IR (KBr, cm-1): 3077 (C–N), 2942, 2825
(–C–H), 1594 (C=C), 1486, 1327, 1239 (C–N), 839, 755
(=C–H ben). ESI–MS (m/z): 448 (M?). Anal. calcd. for
C21H23Cl2N5S (448): C, 56.25; H, 5.17; N, 15.62. Found:
C, 56.27; H, 5.13; N, 15.63.
Yield: 66 %. White solid, mp 75–77 °C. 1H NMR (200 MHz,
CDCl3) d: 14.18 (br s, 1H, NH), 7.88–7.74 (m, 2H, 2XAr–H);
7.37–7.10 (m, 4H, 4XAr–H); 6.89–6.70 (m, 3H, 3XAr–H);
3.27–3.05 (m, 6H, S–CH2–& 2XN–CH2); 2.62–2.45 (m, 6H,
3XN–CH2); 2.40 (s, 3H, Ar–CH3); 1.95 (quintet, J = 7.0 Hz,
2H, CH2CH2CH2). 13C NMR (75 MHz, CDCl3) d: 158.9,
157.6(2C=N), 149.8, 138.6, 130.7, 130.5, 129.8, 129.6, 129.1,
124.7, 118.4, 114.6 (12Aromatic C), 53.1 (NCH2), 52.6
(2NCH2), 50.2 (2NCH2), 34.2 (SCH2), 28.3 (CH2CH2CH2),
24.6 (ArCH3). IR (KBr, cm-1): 3421 (N–H), 2921, 2852 (–C–
H), 1599 (C=C), 1456, 1328, 1234 (C–N), 741, 685 (=C–H
ben). ESI–MS (m/z): 394 (M?1)?. Anal. calcd. for
C22H27N5S (393): C, 67.14; H, 6.92; N, 17.79. Found: C,
67.18; H, 6.91; N, 17.82.
5-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-
5-(3-Methylphenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-
pyrimidinyl)piperazino]propyl sulfide (8f)
pyridyl)piperazino]propyl sulfide (8i)
Yield: 65 %. White solid, mp 128–129 °C. 1H NMR
(200 MHz, CDCl3) d: 14.16 (br s, 1H, NH), 8.24 (d,
Yield: 70 %. White solid, mp 109–101 °C. 1H NMR
(200 MHz, CDCl3) d: 14.25 (br s, 1H, NH), 8.14 (dd,
123

1668
Med Chem Res (2014) 23:1661–1671
5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-
pyridyl)piperazino]propyl sulfide (8l)
J = 3.8, 1.5 Hz, 1H, Ar–H); 7.94–7.74 (m, 2H, 2XAr–H);
7.42 (t, J = 7.6 Hz, 1H, Ar–H); 7.22 (t, J = 7.6 Hz, 1H,
Ar–H); 7.12 (d, J = 6.8 Hz, 1H, Ar–H); 6.61–6.54 (m, 2H,
2XAr–H); 3.665–3.57 (m, 4H, 2XN–CH2); 3.17 (t,
J = 6.8 Hz, 2H, S–CH2); 2.62–2.52 (m, 6H, 3XN–CH2);
2.34 (s, 3H, Ar–CH3); 1.97 (quintet, J = 6.0 Hz, 2H,
CH2CH2CH2). 13C NMR (75 MHz, CDCl3) d: 158.8,
157.6, 154.2, 148.3 (4C=N), 138.7, 138.5, 130.6, 130.5,
129.4, 129.1, 124.7, 113.4, 109.8 (9Aromatic C), 53.3
(NCH2), 52.4 (2NCH2), 50.1 (2NCH2), 34.3 (SCH2), 28.3
(CH2CH2CH2), 24.4 (ArCH3). IR (KBr, cm-1): 3067 (N–
H), 2926, 2828 (–C–H), 1595 (C=C), 1483, 1247 (C–N),
772 (=C–H ben). ESI–MS (m/z): 395 (M?1)?. Anal. calcd.
for C21H26N6S (394): C, 63.93; H, 6.64; N, 21.30. Found:
C, 63.95; H, 6.61; N, 21.34.
1
Yield: 65 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 14.21 (br s, 1H, NH), 7.99–7.91 (m, 1H, Ar–H);
7.46–7.39 (m, 1H, Ar–H); 7.34–7.24 (m, 2H, 2XAr–H);
3.17 (t, J = 6.8 Hz, 2H, S–CH2); 2.68–2.24 (m, 12H, 6XN–
CH2); 1.99 (quintet, 3.57–3.46 (m, 4H, 2XN–CH2); 3.16 (t,
J = 6.8 Hz, 2H, S–CH2); 2.58–2.45 (m, 6H, 3XN–CH2);
1.96 (quintet, J = 6.8 Hz, 2H, CH2CH2CH2). 13C NMR
(75 MHz, CDCl3) d: 158.6, 157.7, 154.2, 148.1 (4C=N),
138.6, 138.3, 132.3, 130.2, 129.4, 128.6, 127.5, 113.5, 110.0
(9Aromatic C), 53.2 (NCH2), 52.4 (2NCH2), 49.8 (2NCH2),
34.3 (SCH2), 28.1 (CH2CH2CH2). IR (KBr, cm-1): 3068
(N–H), 2926, 2831 (–C–H), 1596 (C=C), 1437, 1316, 1246
(C–N), 770, 745 (=C–H ben). ESI–MS (m/z): 415 (M?).
Anal. calcd. for C20H23ClN6S (415): C, 57.89; H, 5.59; N,
20.25. Found: C, 57.91; H, 5.56; N, 20.29.
5-(3-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-
pyrimidinyl)piperazino]propyl sulfide (8j)
5-Benzyl-4H-1,2,4-triazol-3-yl 3-[4-(2-
°
Yield: 68 %. White solid, mp 115–117 C. 1H NMR
pyridyl)piperazino]propyl sulfide (8m)
(200 MHz, CDCl3) d: 14.17 (br s, 1H, NH), 8.24 (d,
J = 4.7 Hz, 2H, 2XAr–H); 8.06–8.00 (m, 1H, Ar–H);
7.97–7.89 (m, 1H, Ar–H); 7.42–7.30 (m, 2H, 2XAr–H);
6.45 (t, J = 4.7 Hz, 1H, Ar–H); 3.88–3.75 (m, 4H, 2XN–
CH2); 3.25 (t, J = 7.0 Hz, 2H, S–CH2); 2.59–2.34 (m, 6H,
3XN–CH2); 1.98 (quintet, J = 7.0 Hz, 2H, CH2CH2CH2).
13C NMR (75 MHz, CDCl3) d: 162.8, 158.7, 157.7, 157.4
(5C=N), 134.6, 132.2, 130.6, 128.9, 127.6, 125.7, 110.4
(7Aromatic C), 53.1 (NCH2), 52.5 (2NCH2), 49.9
(2NCH2), 34.2 (SCH2), 28.1 (CH2CH2CH2). IR (KBr,
cm-1): 3448 (N–H), 2924, 2846 (–C–H), 1587 (C=C),
1485, 1255 (C–N), 795 (=C–H ben). ESI–MS (m/z): 417
(M?1)?. Anal. calcd. for C19H22ClN7S (416): C, 54.87; H,
5.33; N, 23.57. Found: C, 54.89; H, 5.29; N, 23.60.
1
Yield: 73 %. Yellow gummy liquid. H NMR (200 MHz,
CDCl3) d: 14.14 (br s, 1H, NH), 8.15–8.09 (m, 1H, Ar–H);
7.46–7.37 (m, 1H, Ar–H); 7.33–7.10 (m, 5H, 5XAr–H);
6.61–6.53 (m, 2H, 2XAr–H);3.97(s, 2H, Ph–CH2);3.58–3.44
(m, 4H, 2XN–CH2); 3.08 (t, J = 6.8 Hz, 2H, S–CH2);
2.55–2.41 (m, 6H, 3XN–CH2); 1.91(quintet, J = 6.8 Hz, 2H,
CH2CH2CH2). 13C NMR (75 MHz, CDCl3) d: 158.8, 154.6,
154.2, 148.3(4C=N), 138.6, 136.4, 129.1, 128.6, 125.9, 113.3,
109.7 (9Aromatic C), 53.1 (NCH2), 52.6 (2NCH2), 50.0
(2NCH2), 35.8 (CH2Ph), 34.1 (SCH2), 28.2 (CH2CH2CH2).
IR(KBr,cm-1):2932, 2825(–C–H), 1595, 1483(C=C),1438,
1313, 1247 (C–N), 757 (=C–H ben). ESI–MS (m/z): 395
(M?1)?. Anal. calcd. for C21H26N6S (394): C, 63.93; H, 6.64;
N, 21.30. Found: C, 63.89; H, 6.64; N, 21.32.
5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl [3-(4-
3-[4-(Tert-butyl)phenyl]-1-[3-(4-
phenylpiperazino)propyl]-4,5-dihydro-1H-1,2,4-triazole-5-
thione (9a)
ethylpiperazino)propyl] sulfide (8k)
1
Yield: 60 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 14.16 (br s, 1H, NH), 7.99–7.91 (m, 1H, Ar–H);
7.46–7.39 (m, 1H, Ar–H); 7.34–7.24 (m, 2H, 2XAr–H);
3.17 (t, J = 6.8 Hz, 2H, S–CH2); 2.68–2.24 (m, 12H,
6XN–CH2); 1.99 (quintet, J = 6.8 Hz, 2H, CH2CH2CH2);
1.08 (t, J = 7.6 Hz, 3H, CH3CH2). 13C NMR (75 MHz,
CDCl3) d: 158.7, 157.6 (2C=N), 138.3, 132.3, 130.4, 129.5,
128.7, 127.3 (6Aromatic C), 53.2 (NCH2), 53.0 (2NCH2),
52.4 (2NCH2), 49.4 (NCH2CH3), 34.2 (SCH2), 28.2
(CH2CH2CH2), 13.5 (CH2CH3). IR (KBr, cm-1): 3065 (N–
H), 2939, 2818 (–C–H), 1600 (C=C), 1450, 1323, 1266 (C–
N), 750 (=C–H ben). ESI–MS (m/z): 366 (M?). Anal.
calcd. for C17H24ClN5S (366): C, 55.80; H, 6.61; N, 19.14.
Found: C, 55.83; H, 6.63; N, 19.10.
Yield: 60 %. White solid, mp 152–154 °C. 1H NMR
(200 MHz, CDCl3) d: 13.68 (br s, 1H, NH), 7.92 (d,
J = 7.3 Hz, 2H, 2XAr–H); 7.41 (d, J = 8.8 Hz, 2H,
2XAr–H); 7.17 (t, J = 7.3 Hz, 2H, 2XAr–H); 6.89–6.71
(m, 3H, 3XAr–H); 4.39–4.28 (m, 2H, N–CH2); 3.21–3.10
(m, 4H, 2XN–CH2); 2.62–2.44 (m, 6H, 3XN–CH2);
2.05–1.88 (m, 2H, CH2CH2CH2); 1.34 (s, 9H, C(CH3)3).
13C NMR (75 MHz, CDCl3) d: 176.3 (C=S), 155.6 (C=N),
151.6, 149.6, 129.6, 125.6, 125.3, 125.0, 118.5, 114.4
(12Aromatic C), 52.5 (2NCH2), 51.6 (NCH2), 49.8
(2NCH2), 43.1 (NCH2), 40.6 (C(CH3)3), 31.3 (C(CH3)3),
22.4 (CH2CH2CH2). IR (KBr, cm-1): 3422 (N–H), 3060
123

Med Chem Res (2014) 23:1661–1671
1669
(=C–H), 2956, 2819 (–C–H), 1599, 1499 (C=C), 1334,
1234 (C–N), 841, 755 (=C–H ben). ESI–MS (m/z): 435
(M?). Anal. calcd. for C25H33N5S (435): C, 68.93; H, 7.64;
N, 16.08. Found: C, 68.97; H, 7.61; N, 16.11.
3H, 3XAr–H); 4.54–4.37 (m, 2H, N–CH2); 3.30–3.13 (m,
4H, 2XN–CH2); 2.64–2.45 (m, 6H, 3XN–CH2); 1.97
(quintet, J = 6.6 Hz, 2H, CH2CH2CH2). 13C NMR
(75 MHz, CDCl3) d: 176.7 (C=S), 155.5 (C=N), 151.1,
135.8, 135.1, 131.1, 129.0, 127.4, 126.6, 118.4, 114.8,
112.5 (12Aromatic C), 52.6 (2NCH2), 51.5 (NCH2), 50.1
(2NCH2), 43.2 (NCH2), 22.7 (CH2CH2CH2). IR (KBr,
cm-1): 3075 (N–H), 2924, 2825 (–C–H), 1594, 1485
(C=C), 1449, 1237 (C–N), 837 (=C–H ben). ESI–MS (m/z):
448 (M?). Anal. calcd. for C21H23Cl2N5S (448): C, 56.25;
H, 5.17; N, 15.62. Found: C, 56.23; H, 5.18; N, 15.66.
3-[4-(Tert-butyl)phenyl]-1-3-[4-(3-
chlorophenyl)piperazino]propyl-4,5-dihydro-1H-1,2,4-
triazole-5-thione (9b)
Yield: 68 %. White solid, mp 160–162 °C. 1H NMR
(200 MHz, CDCl3) d: 13.70 (br s, 1H, NH), 7.91 (d,
J = 8.8 Hz, 2H, 2XAr–H); 7.39 (d, J = 8.0 Hz, 2H, 2XAr–
H); 7.11(t, J = 7.3 Hz, 1H, Ar–H); 6.83–6.67 (m, 3H, 3XAr–
H); 3.99–3.79 (m, 2H, N–CH2); 3.23–3.11 (m, 4H, 2XN–
CH2); 2.62–2.45 (m, 6H, 3XN–CH2); 2.06–1.87 (m, 2H,
CH2CH2CH2); 1.34 (s, 9H, C(CH3)3). 13C NMR (75 MHz,
CDCl3) d: 176.4 (C=S), 155.8 (C=N), 151.5, 151.0, 135.4,
131.3, 125.5, 125.3, 124.9, 118.5, 114.5, 112.5 (12Aromatic
C), 52.6 (2NCH2), 51.7 (NCH2), 49.7 (2NCH2), 43.2 (NCH2),
40.8 (C(CH3)3), 31.5 (C(CH3)3), 22.5 (CH2CH2CH2). IR
(KBr, cm-1): 3083 (N–H), 2957, 2835 (–C–H), 1593 (C=C),
1440, 1330, 1241 (C–N), 838, 773 (=C–H ben). ESI–MS (m/
z): 470 (M?). Anal. calcd. for C25H32ClN5S (470): C, 63.88;
H, 6.86; N, 14.90. Found: C, 63.84; H, 6.89; N, 14.91.
1-[3-(4-Benzylpiperazino)propyl]-3-(4-fluorophenyl)-4,5-
dihydro-1H-1,2,4-triazole-5-thione (9e)
1
Yield: 66 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 13.72 (br s, 1H, NH), 8.12–7.98 (m, 2H, 2XAr–H);
7.34–7.03 (m, 7H, 7XAr–H); 4.30 (t, J = 6.8 Hz, 2H, N–
CH2); 3.48 (s, 2H, Ph–CH2); 2.61–2.25 (m, 10H, 5XN–CH2);
1.96 (quintet, J = 6.8 Hz, 2H, CH2CH2CH2). 13C NMR
(75 MHz, CDCl3) d: 176.7 (C=S), 164.5 (F–C=C), 155.5
(C=N), 135.7, 128.7, 128.4, 127.8, 127.2, 124.2, 115.6
(11Aromatic C), 60.3 (NCH2Ph), 52.8 (2NCH2), 52.5
(NCH2), 51.7 (2NCH2), 43.2 (NCH2), 22.7 (CH2CH2CH2).
IR ((KBr, cm-1): 3028 (=C–H), 2936, 2811 (–C–H), 1606
(C=C), 1462, 1326, 1223 (C–N), 844, 746 (=C–H ben). EI–
MS (m/z): 411 (M?). Anal. calcd. for C22H26FN5S (411): C,
64.21; H, 6.37; N, 17.02. Found: C, 64.24; H, 6.35; N, 16.98.
1-3-[4-(3-Chlorophenyl)piperazino]propyl-3-(4-
methylphenyl)-4,5-dihydro-1H-1,2,4-triazole-5-thione (9c)
Yield: 58 %. Yellow solid, mp 73–75 °C. 1H NMR
(200 MHz, CDCl3) d: 13.64 (br s, 1H, NH), 7.90 (d,
J = 8.3 Hz, 2H, 2XAr–H); 7.20 (d, J = 7.9 Hz, 2H, 2XAr–
H); 7.10 (t, J = 7.9 Hz, 1H, Ar–H); 6.81 (t, J = 2.0 Hz, 1H,
Ar–H); 6.78–6.69 (m, 2H, 2XAr–H); 4.36 (t, J = 6.4 Hz,
2H, N–CH2); 3.20–3.15 (m, 4H, 2XN–CH2); 2.62–2.56 (m,
4H, 2XN–CH2); 2.53 (t, J = 7.2 Hz, 2H, N–CH2); 2.42 (s,
3H, Ar–CH3); 1.97 (quintet, J = 7.0 Hz, 2H, CH2CH2CH2).
13C NMR (75 MHz, CDCl3) d: 176.5 (C=S), 155.6 (C=N),
151.2, 139.8, 135.2, 131.0, 129.2, 126.2, 125.4, 118.4, 114.9,
112.4 (12Aromatic C), 52.5 (2NCH2), 51.6 (NCH2), 50.0
(2NCH2), 43.1 (NCH2), 24.5 (ArCH3), 22.5 (CH2CH2CH2).
IR (KBr, cm-1): 2951, 2822 (–C–H), 1595 (C=C), 1451,
1274 (C–N), 840 (=C–H ben). ESI–MS (m/z): 428 (M?).
Anal. calcd. for C22H26ClN5S (428): C, 61.74; H, 6.12; N,
16.36. Found: C, 61.77; H, 6.16; N, 16.34.
3-(3,4-Dimethoxyphenyl)-1-(3-morpholinopropyl)-4,5-
dihydro-1H-1,2,4-triazole-5-thione (9f)
1
Yield: 59 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 13.69 (br s, 1H, NH), 7.57 (dd, J = 6.8, 2.3 Hz,
1H, Ar–H); 7.43 (d, J = 1.5 Hz, 1H, Ar–H); 6.88 (dd,
J = 6.8 & 2.3 Hz, 1H, Ar–H); 4.29 (t, J = 6.8 Hz, 2H, N–
CH2); 3.87 (s, 6H, 2XOCH3); 3.64–3.56 (m, 4H, 2XO–CH2);
2.49–2.34 (m, 6H, 3XN–CH2); 1.89 (quintet, J = 6.8 Hz,
2H, CH2CH2CH2). 13C NMR (75 MHz, CDCl3) d: 176.6
(C=S), 155.7 (C=N), 151.3, 149.8, 121.7, 119.6, 115.2, 111.4
(6Aromatic C), 66.6 (2OCH2), 56.0 (2OCH3), 53.8
(2NCH2), 52.3 (NCH2), 43.1 (NCH2), 22.5 (CH2CH2CH2).
IR (KBr, cm-1): 3085 (N–H), 2956, 2851 (–C–H), 1601,
1514 (C=C), 1345, 1271 (C–N), 866, 764 (=C–H ben). ESI–
MS (m/z): 364 (M?). Anal. calcd. for C17H24N4O3S (364): C,
56.02; H, 6.64; N, 15.37. Found: C, 56.04; H, 6.61; N, 15.38.
3-(4-Chlorophenyl)-1-3-[4-(3-
chlorophenyl)piperazino]propyl-4,5-dihydro-1H-1,2,4-
triazole-5-thione (9d)
3-(3,4-Dimethoxyphenyl)-1-[3-(4-ethylpiperazino)propyl]-
4,5-dihydro-1H-1,2,4-triazole-5-thione (9g)
Yield: 65 %. White solid, mp 88–90 °C. 1H NMR
(200 MHz, CDCl3) d: 13.76 (br s, 1H, NH), 8.00 (d,
J = 8.8 Hz, 2H, 2XAr–H); 7.40 (d, J = 8.8 Hz, 2H,
2XAr–H); 7.13 (t, J = 8.1 Hz, 1H, Ar–H); 6.85–6.70 (m,
1
Yield: 60 %. Brown gummy liquid. H NMR (200 MHz,
CDCl3) d: 13.74 (br s, 1H, NH), 7.59 (dd, J = 6.3, 2.3 Hz,
1H, Ar–H); 7.45 (d, J = 1.6 Hz, 1H, Ar–H); 6.87 (d,
123

1670
Med Chem Res (2014) 23:1661–1671
J = 8.6 Hz, 1H, Ar–H); 4.29 (t, J = 7.0 Hz, 2H, N–CH2);
3.89 (s, 6H, 2XOCH3); 2.57–2.25 (m, 12H, 6XN–CH2);
1.91 (quintet, J = 7.0 Hz, 2H, CH2CH2CH2); 1.05 (t,
J = 7.0 Hz, 3H, CH3CH2). 13C NMR (75 MHz, CDCl3) d:
176.6 (C=S), 155.7 (C=N), 151.3, 149.7, 121.8, 119.6,
115.1, 111.3 (6Aromatic C), 56.1 (2OCH3), 52.8 (2NCH2),
52.5 (2NCH2), 51.8 (NCH2), 49.3 (NCH2CH3), 43.1
(NCH2), 22.6 (CH2CH2CH2), 13.4 (CH2CH3). IR (KBr,
cm-1): 3411 (N–H), 2939, 2811 (C–H), 1598, 1514 (C=C),
1345, 1271 (C–N), 765 (=C–H ben). ESI–MS (m/z): 391
(M?). Anal. calcd. for C19H29N5O2S (391): C, 58.29; H,
7.47; N, 17.89. Found: C, 58.25; H, 7.49; N, 17.92.
Chemical Co., St. Louis, MO) with a final concentration of
8 mg/ml. Exactly 150 ll of stock was diluted to 1 ml in
culture medium to obtain experimental stock concentration
of 1,200 lg/ml. This solution was further serially diluted
with media to generate a dilution series of 20–600 lg/ml.
Exactly 100 ll of each diluent was added to 100 ll of cell
suspension (total assay volume of 200 ll) and incubated
for 24 h at 37 °C in 5 % CO2.
Cytotoxicity
Cytotoxicity was measured using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
assay, according to the method of Mossman (Mossman,
1983). Briefly, the cells (2 9 104) were seeded in each well
containing 0.1 ml of medium in 96 well plates. After
overnight incubation at 37 °C in 5 % CO2, the cells were
treated with 100 ll of different test concentrations of test
compounds (2–60 lg) at identical conditions with five
replicates each. The final test concentrations were equiva-
lent to (10–300 lg/ml) or (10–300 ppm). The cell viability
was assessed after 24 h, by adding 10 ll of MTT (5 mg/
ml) per well. The plates were incubated at 37 °C for
additional 3 h. The medium was discarded and the for-
mazan blue, which formed in the cells, was dissolved with
100 ll of DMSO. The rate of color formation was mea-
sured at 570 nm in a spectrophotometer (Spectra MAX
Plus; Molecular Devices; supported by SOFTmax PRO-
5.4). The percent inhibition of cell viability was determined
with reference to the control values (without test com-
pound). The data were subjected to linear regression ana-
lysis and the regression lines were plotted for the best
straight-line fit. The IC50 (inhibition of cell viability)
concentrations were calculated using the respective
regression equation.
3-(4-Methoxyphenyl)-1-3-[4-(2-pyridyl)piperazino]propyl-
4,5-dihydro-1H-1,2,4-triazole-5-thione (9h)
Yield: 62 %. Yellow solid, mp 110–112 °C. 1H NMR
(200 MHz, CDCl3) d: 13.68 (br s, 1H, NH), 8.13 (dd,
J = 3.0, 1.5 Hz, 1H, Ar–H); 7.96 (d, J = 8.3 Hz, 2H,
2XAr–H); 7.47–7.37 (m, 1H, Ar–H); 6.88 (d, J = 8.3 Hz,
2H, 2XAr–H); 6.60–6.54 (m, 2H, 2XAr–H); 4.35 (t,
J = 6.8 Hz, 2H, N–CH2); 3.85 (s, 3H, OCH3); 3.57–3.49
(m, 4H, 2XN–CH2); 2.60–2.50 (m, 6H, 3XN–CH2); 1.98
(quintet, J = 7.6 Hz, 2H, CH2CH2CH2). 13C NMR
(75 MHz, CDCl3) d: 176.5 (C=S), 162.2 (OC=C), 155.7,
154.3, 148.3 (3C=N), 138.3, 127.1, 121.0, 114.3, 113.5,
110.0 (8Aromatic C), 55.9 (OCH3), 52.6 (2NCH2), 51.7
(NCH2), 49.8 (2NCH2), 43.2 (NCH2), 22.7 (CH2CH2CH2).
IR (KBr, cm-1): 3449 (N–H), 2928, 2842 (–C–H), 1597
(C=C), 1481, 1253 (C–N), 772 (=C–H ben). ESI–MS (m/z):
411 (M?1)?. Anal. calcd. for C21H26N6OS (410): C, 61.44;
H, 6.38; N, 20.47. Found: C, 61.41; H, 6.39; N, 20.44.
Cytotoxicity studies
Cell lines and cell culture
Acknowledgments The authors are grateful to the Director, Indian
Institute of Chemical Technology, Hyderabad, India for the encour-
agement. KRR, BR, RVR, MRK, RP, and LRV are thankful to the
Council of Scientific & Industrial Research (CSIR), New Delhi, India
for the award of fellowships. We thank CSIR for financial support
under the 12th Five Year plan project ‘‘Affordable Cancer Thera-
peutics (ACT)’’ (CSC 0301).
The cell lines U937 (human leukemic monocytic lymphoma),
THP-1 human acute monocytic leukemia), Colo205 (human
colorectal cancer), MCF7 (human breast adenocarcinoma),
and HL-60 (human myeloid leukemia) cell lines were
obtained from the National Centre for Cellular Sciences,
Pune, India. Cells were cultured either in RPMI-1640 (U937,
THP-1, Colo205, and HL-60) or MEM (MCF7) media, sup-
plemented with 10 % heat-inactivated fetal bovine serum,
1 mM NaHCO3, 2 mM glutamine, 100 units/ml penicillin,
and 100 lg/ml streptomycin. All cell lines were maintained in
culture at 37° C in an atmosphere of 5 % CO2.
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