ORIGINAL RESEARCH

Synthesis, characterization, and anticancer studiesof S and N alkyl piperazine-substituted positionalisomers 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

� 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.

Keywords 3-Mercapto-1,2,4-triazoles � Acid hydrazides �Piperazines � Anticancer

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

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).

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).

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 (&) � K. R. Ram � B. R. Rao �R. V. Rao � 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 � R. Pamanji � L. R. Velatooru

Toxicology Unit, Biology Division, Indian Institute of Chemical

Technology, Uppal Road, Hyderabad 500007, India

123

Med Chem Res (2014) 23:1661–1671

DOI 10.1007/s00044-013-0757-3

MEDICINALCHEMISTRYRESEARCH

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).

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.

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

OCH3 peak in 1H NMR spectra of methyl esters (2). The

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

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).

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).

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

RCOOH RCOOCH3 RCONHNH2

R NH

NH

NS

RCONHNHCSNH2R N

H

NNSH

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.

2 3

45

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.

(i) (ii)

(iii) (iv)

Scheme 1 Synthesis of

5-substituted[1,2,4]triazole-3-

thiones (5)

Cl BrNH

X Zn

THF, rt

Cl N

X

+

X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyrimidyl, N-2-pyridyl, N-3-chlorophenyl

7 6

Scheme 2 Synthesis of 1-(3-chloropropyl)-4-substituted cyclicam-

ines (7)

1662 Med Chem Res (2014) 23:1661–1671

123

this may lead to some interesting results in biological

activity point of view.

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

group of the propyl chain that connects the cyclicamine moiety

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.

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.

Cl N

X+

R NH

NH

NS

N(C2H5)3 / TBAI

C2H5OH, reflux, 3 h8a-m5 7

X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyrimidyl, N-2-pyridyl, N-3-chlorophenyl

R = 4-Cl-C6H4, 3-Cl-C6H4, 2-Cl-C6H4, 4-CH3-C6H4, 3-CH3-C6H4, 4-F-C6H4, 4-C4H9-C6H4, C6H5CH2

NH

NN

R SN

X

Scheme 3 Synthesis of 3-[3-[4-

(substituted)-1-

cyclicamine]propyl]thio-5-

substituted [1,2,4]triazoles

(8a–m)

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 4-C4H9–C6H4 O 72

2 8b 4-C4H9–C6H4 N-Phenyl 66

3 8c 4-CH3–C6H4 N-3-Chlorophenyl 70

4 8d 4-CH3–C6H4 N-Ethyl 63

5 8e 4-Cl–C6H4 N-3-Chlorophenyl 75

6 8f 4-Cl–C6H4 N-2-Pyrimidyl 65

7 8g 4-F–C6H4 N-Benzyl 70

8 8h 3-CH3–C6H4 N-Phenyl 66

9 8i 3-CH3–C6H4 N-2-Pyridyl 70

10 8j 3-Cl–C6H4 N-2-Pyrimidyl 68

11 8k 2-Cl–C6H4 N-Ethyl 60

12 8l 2-Cl–C6H4 N-2-Pyridyl 65

13 8m C6H5CH2 N-2-Pyridyl 73

Cl N

X K2CO3/TBAI

CH3CN, reflux, 4 h

+R N

H

NS

NH

9a-h5a-d and 5i,j 7

X= O, N-ethyl, N-phenyl, N-benzyl, N-2-pyridyl, N-3-chlorophenyl

R = 4-Cl-C6H4, 4-CH3-C6H4, 4-F-C6H4, 4-C4H9-C6H4, 4-OCH3-C6H4, 3,4-di OCH3-C6H3

N

R NH

NN

S X

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)

Med Chem Res (2014) 23:1661–1671 1663

123

Cytotoxicity studies

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-

Table 2 2-[3-[4-(Substituted)-1-cyclicamine]propyl]-5-substituted-

2,4-dihydro- 3H[1,2,4]triazole-3-thiones (9a–h)

Entry Compound R X Yield (%)

1 9a 4-C4H9–C6H4 N-Phenyl 60

2 9b 4-C4H9–C6H4 N-3-

Chlorophenyl

68

3 9c 4-CH3–C6H4 N-3-

Chlorophenyl

58

4 9d 4-Cl–C6H4 N-3-

Chlorophenyl

65

5 9e 4-F–C6H4 N-Benzyl 66

6 9f 3,4-di OCH3–

C6H3

O 59

7 9g 3,4-di OCH3–

C6H3

N-Ethyl 60

8 9h 4-OCH3–C6H4 N-2-Pyridyl 62

Table 3 In vitro cytotoxicity of Triazole derivatives 8a–m and 9a–h

S. no. Compound IC50a (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 49.31 ± 3.97 97.63 ± 11.8 – – –

3 8c 256.84 ± 39.01 – 139.29 ± 0.15 – –

4 8d – – – – –

5 8e – – – – –

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 – 247.55 ± 15.26 – – –

9 8i 52.33 ± 3.12 – – – 29.36 ± 2.23

10 8j 49.13 ± 2.86 – – – 18.51 ± 1.16

11 8k – – – – –

12 8l – – – – –

13 8m 163.14 ± 13.85 – – – –

14 9a 88.94 ± 7.86 146.57 ± 3.95 – – –

15 9b – – – – –

16 9c – – – – –

17 9d 28.19 ± 1.140 – – – 6.67 ± 0.55

18 9e – 159.53 ± 1.36 110.99 ± 16.6 128.46 ± 0.65 –

19 9f – – – – –

20 9g – – – – –

21 9h – – – – 114.95 ± 6.00

Etoposideb 10.43 ± 2.0 3.66 ± 0.25 12.3 ± 2.14 29.49 ± 2.22 1.84 ± 0.20

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 lMa 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

observationsb Etoposide was employed as positive control

1664 Med Chem Res (2014) 23:1661–1671

123

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.

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).

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.

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

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.

Experimental

Chemistry

Melting points were determined on a Buchi capillary

melting point apparatus. The 1H NMR (200 MHz) and 13C

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).

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.

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.

Med Chem Res (2014) 23:1661–1671 1665

123

General procedure for the preparation of 5-substituted

[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.

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.

General procedure for the preparation of 3-[3-[4-

(substituted)-1-cyclicamine]propyl]thio-5-

substituted[1,2,4]triazoles (8a–m)

5-Substituted [1,2,4]triazole-3-thione 5 (0.0005 mol) was

refluxed with triethyl amine (0.001 mol) in ethanol (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

layer was dried (Na2SO4) and concentrated. The crude

product was subjected to flash column chromatograph to

obtain pure product.

Spectroscopic data for the synthesized compounds

5-[4-(Tert-butyl)phenyl]-4H-1,2,4-triazol-3-yl

(3-morpholinopropyl) sulfide (8a)

Yield: 72 %. Brown gummy liquid. 1H 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.

5-[4-Tert-butyl)phenyl]-4H-1,2,4-triazol-3-yl [3-(4-

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.

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);

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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.

3-(4-Ethylpiperazino)propyl [5-(4-methylphenyl)-4H-

1,2,4-triazol-3-yl] sulfide (8d)

Yield: 63 %. Brown gummy liquid. 1H NMR (200 MHz,

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.

3-[4-(3-Chlorophenyl)piperazino]propyl [5-(4-

chlorophenyl)-4H-1,2,4-triazol-3-yl] sulfide (8e)

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.

5-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-

pyrimidinyl)piperazino]propyl sulfide (8f)

Yield: 65 %. White solid, mp 128–129 �C. 1H NMR

(200 MHz, CDCl3) d: 14.16 (br s, 1H, NH), 8.24 (d,

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-Benzylpiperazino)propyl [5-(4-fluorophenyl)-4H-

1,2,4-triazol-3-yl] sulfide (8g)

Yield: 70 %. Brown gummy liquid. 1H 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–H ben). 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.

5-(3-Methylphenyl)-4H-1,2,4-triazol-3-yl [3-(4-

phenylpiperazino)propyl] sulfide (8h)

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-(3-Methylphenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-

pyridyl)piperazino]propyl sulfide (8i)

Yield: 70 %. White solid, mp 109–101 �C. 1H NMR

(200 MHz, CDCl3) d: 14.25 (br s, 1H, NH), 8.14 (dd,

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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.

5-(3-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-

pyrimidinyl)piperazino]propyl sulfide (8j)

Yield: 68 %. White solid, mp 115–117 �C. 1H NMR

(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.

5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl [3-(4-

ethylpiperazino)propyl] sulfide (8k)

Yield: 60 %. Brown gummy liquid. 1H 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.

5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl 3-[4-(2-

pyridyl)piperazino]propyl sulfide (8l)

Yield: 65 %. Brown gummy liquid. 1H 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-Benzyl-4H-1,2,4-triazol-3-yl 3-[4-(2-

pyridyl)piperazino]propyl sulfide (8m)

Yield: 73 %. Yellow gummy liquid. 1H 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.

3-[4-(Tert-butyl)phenyl]-1-[3-(4-

phenylpiperazino)propyl]-4,5-dihydro-1H-1,2,4-triazole-5-

thione (9a)

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

1668 Med Chem Res (2014) 23:1661–1671

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(=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.

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-(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-(4-Chlorophenyl)-1-3-[4-(3-

chlorophenyl)piperazino]propyl-4,5-dihydro-1H-1,2,4-

triazole-5-thione (9d)

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,

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.

1-[3-(4-Benzylpiperazino)propyl]-3-(4-fluorophenyl)-4,5-

dihydro-1H-1,2,4-triazole-5-thione (9e)

Yield: 66 %. Brown gummy liquid. 1H 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.

3-(3,4-Dimethoxyphenyl)-1-(3-morpholinopropyl)-4,5-

dihydro-1H-1,2,4-triazole-5-thione (9f)

Yield: 59 %. Brown gummy liquid. 1H 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-(3,4-Dimethoxyphenyl)-1-[3-(4-ethylpiperazino)propyl]-

4,5-dihydro-1H-1,2,4-triazole-5-thione (9g)

Yield: 60 %. Brown gummy liquid. 1H 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,

Med Chem Res (2014) 23:1661–1671 1669

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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.

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

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.

Test concentrations

Initially, stock solutions of each test substances were pre-

pared in 100 % Dimethyl Sulfoxide (DMSO, Sigma

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.

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).

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