Med Chem Res (2010) 19:127–135

MEDICINAL
DOI 10.1007/s00044-009-9178-8
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH

Synthesis and evaluation of 4-amino-5-phenyl-4H-

[1,2,4]-triazole-3-thiol derivatives as antimicrobial
agents

Prasanta K. Sahoo Æ Rajesh Sharma Æ

Priyabrata Pattanayak

Received: 27 December 2008 / Accepted: 10 February 2009 / Published online: 17 March 2009
Ó Birkhäuser Boston 2009

Abstract A series of novel 1,2,4-triazole derivatives have been synthesized by

condensing the methyl benzoate and methyl salicylate with various substituents;
their structures were established on the basis of elemental analysis, Fourier trans-
form infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), and
mass spectral data. All title compounds were subjected to in vitro antibacterial and
antifungal screening against four different bacterial and fungal strains. Preliminary
results indicate that some of them exhibit promising activities and deserve further
consideration as potential antimicrobials.

Keywords 1,2,4-Traizole
Antibacterial screening
Antifungal screening

Introduction

The usage of most antimicrobial agents is limited, not only by the rapidly
developing drug resistance, but also by the unsatisfactory status of present
treatments of bacterial and fungal infections and drug side-effects (Fidler, 1998).
Therefore, the development of new and different antimicrobial drugs is a very
important objective and many research programs are directed towards the design of
new antimicrobial agents.
During the last few decades, considerable attention has been devoted to synthesis
of 1,2,4-triazole derivatives possessing such comprehensive bioactivities as
antibacterial, antifungal (Karabasanagouda et al., 2007; Sztanke et al., 2008),

P. K. Sahoo
R. Sharma
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshashila Campus,
Khandwa Road, Indore 452017, Madhya Pradesh, India

P. Pattanayak (&)
Jeypore College of Pharmacy, Jeypore(K), Orissa 764002, India
e-mail: priyabrata2005@gmail.com
128 Med Chem Res (2010) 19:127–135

antimycobacterial (Klimesová et al., 2004), anti-inflammatory (Mullican et al.,

1993), analgesic (Tozkoparan et al., 2005), anticancer (Demirba et al., 2002),
antihypertensive (Kakefuda et al., 2002), anticonvulsant (Küçükgüzel et al., 2004),
antiviral (El-Essawy et al., 2008), antidepressant (Kane et al., 1988), antiasthmatic
(Youichiro et al., 1996), diuretic (Mhasalkar et al., 1970) and hypoglycemic (Blank
et al., 1972) activities.
Regarding antimicrobial activity, triazole is structurally similar to imidazole
molecule. Although triazole and imidazole act by the same mechanism of action,
triazoles possess advantages over imidazoles, which have slow metabolic rate, oral
bioavailability, and less effect on human sterol synthesis. For these reasons
imidazoles are slowly being replaced by triazole molecules. It was reported that
incorporation of various halo substituents into the heterocyclic ring systems
augments biological activities considerably (Wang and Shi, 2001) and substitutions
at 3-position by chloro-substituted benzene and at 4-position by free -NH2 group
give a broad spectrum of antimicrobial activity due to the ability of halogen to act as
polar hydrogen or hydroxy mimic. Substitution of hydrogen by halogen has been a
strategy in designing molecules for biological studies (Clerici and Donato, 2001). In
view of the above findings, it was thought worthwhile to synthesize a series of eight
compounds containing the 1,2,4-triazole moiety with different halogenated and
nonhalogenated substituents and study their antimicrobial activities.

Materials and methods

Chemistry

Reaction sequence employed for the synthesis of title compounds is shown in

Schemes 1, 2.

Step 1 (Wang and Shi, 2001)

The esters of substituted aromatic acid (0.1 M) (1 and 5) were dissolved in 30 ml

ethanol, and hydrazine hydrate (0.1 M) was added dropwise to the mixture with
stirring. The resulting mixture was allowed to reflux for 6 h. Excess ethanol was
distilled out and the contents were allowed to cool. The crystals formed were
filtered, washed thoroughly with water, and dried. The completion of the reaction
was monitored by thin layer chromatography (TLC) by using silica gel-G coated
plates by using ethyl acetate and petroleum ether (1:1) as the eluent and observed
with ultraviolet (UV) light.

Step 2

KOH (0.15 M) was dissolved in absolute ethanol (200 ml). To the above solution,
aryl acid hydrazide (0.1 M) (2 and 6) was added and the solution cooled on ice. To
this, carbon disulfide (0.15 M) was added in small portions with constant stirring.
The reaction mixture was agitated continuously for a period of 15 h. It was then
Med Chem Res (2010) 19:127–135 129

O
O
C N2H4.H2O
O C NHNH2
CH3 Step -1
2
1

KOH/C2H5OH CS2

Step -2

N N
SH N2H4.H2O O S
N C NHNH C SK
NH2 Step -3
3
4

Step -4
PS-01
Glacial RCl KOH 85%, H2O
Acetic acid
Step - 5 Acetic anhydride

N N
N N SR
SH N
N NH2
NH
COCH3
PS-02, PS-03, PS-04, PS-05 & PS-08
PS-07

Scheme 1 Synthesis of substituted 1,2,4-triazole (PS01, PS02, PS03, PS04, PS05, PS07, and 08)

O
C O
O N2H4.H2O
C NHNH2
CH
OH 3 Step -1
OH
5 6

KOH/C2H5OH CS2

Step- 2

N N
SH N2H4.H2O O S
N C NHNH C SK
NH2 Step -3
OH OH
8 7
PS-06

Scheme 2 Synthesis of 2-(4-amino-5-mercapto-4H-[1,2,4]triazol-3-yl)-phenol (PS06)

diluted with anhydrous ether. The precipitated potassium dithiocarbazinate was

collected by filtration. The precipitate was further washed with anhydrous ether
(100 ml) and dried under vacuum. The potassium salt thus obtained was in
quantitative yield and was used in the next step without further purification.
130 Med Chem Res (2010) 19:127–135

Step 3

A suspension of potassium dithiocarbazinate (0.1 M) (3 and 7) in water (5 ml) and

hydrazine hydrate (15 ml, 0.3 M) was refluxed for 30 min with occasional shaking.
The color of the reaction mixture changed to green with the evolution of hydrogen
sulfide gas (lead acetate paper and odor). A homogeneous reaction mixture was
obtained during the reaction process. The reaction mixture was cooled to room
temperature and diluted with water (100 ml). On acidification with concentrated
hydrochloric acid, the required triazole 4 (PS01) and 8 (PS06) was precipitated out.
It was filtered, washed thoroughly with cold water, and recrystallized from ethanol.
The completion of the reaction was monitored with TLC by using silica gel-G
coated plates by using ethyl acetate and petroleum ether (1:1) as eluent and
observed with UV light.

Step 4

The appropriate (Clerici and Donato, 2001) triazole (0.1 mol) (PS01) was
suspended in water (9 ml), and 0.1 mol of KOH (85% solution) was added under
stirring at room temperature. After a few minutes (5–10 min), the solution was
brought to 0°C in an ice bath, and halo-substituted aromatic compound (0.1 mol)
was dropped in with vigorous stirring. The reaction mixture was checked by TLC
(Et2O as eluant). When reaction was complete, concentrated sulfuric acid (10 ml)
was added, and from the crude mixture a white precipitate was formed slowly
(PS02, PS03, PS04, PS05, and PS08). This was filtered, washed with water, and
crystallized from ethanol.

Step 5

Acetic anhydride (0.19 ml, 0.002 mole) was added (Clerici and Donato, 2001; Afaf
et al., 2000) to a boiling solution of 4-amino-5-phenyl-4H-[1,2,4]-triazole-3-thiol
(0.001 mole) (PS01) in glacial acetic acid (3 ml). After refluxing for 2 h, the
reaction mixture was cooled and poured into ice-cold water. The formed precipitate
was filtered off and recrystallized from ethanol to give the title compound (PS07).
TLC was used to reach the completion of reaction and purity of the compounds
synthesized. Melting points were determined in open glass capillary tubes using
Thiels tube containing liquid paraffin and were uncorrected. IR spectra were
obtained in KBr discs on a Shimadzu-8400S FTIR spectrophotometer. Elemental
analysis was performed using EL 11/carlo Ebra 1108 analyzer. 1H NMR spectra
were recorded on a FT NMR (500 MHz); MeOD was used as a solvent. Chemical
shifts are reported as d (ppm). The fast atom bombardment (FAB) mass spectra were
recorded on a JEOL SX 102/DA-6000 mass spectrometer/data system using argon/
xenon (6 kV, 10 mA) as the FAB gas. Elemental analysis was within ± 0.4% of
theoretical values. Substitution pattern and characterization data of the synthesized
compounds are reported in Tables 1, 2, respectively. The spectral data are presented
in Table 3.
Med Chem Res (2010) 19:127–135 131

Table 1 Substitutions on the 1,2,4-triazole ring of compounds PS01–08

N N

R1 R3
N

R2

Compd. R1 R2 R3

PS01 –C6H5 –NH2 –SH

PS02 –C6H5 –NH2 –S(C6H4)4–SO2Cl
PS03 –C6H5 –NH2 –S(C6H4)4–Br
PS04 –C6H5 –NH2 –S(C6H4)4–CCl3
PS05 –C6H5 –NH2 –S(C6H4)4–Cl
PS06 –(C6H4)2–OH –NH2 –SH
PS07 –C6H5 –NHCOCH3 –SH
PS08 –C6H5 –NH2 –(C6H3)–2,4–(NO2)2

Table 2 Characterization of the title compounds (PS01–08)

Compd No. Formula (m. wt.) M. P. (°C) Yield (%) % Analysis (calculated)/found
C

PS01 C8H5N4S (189.19) 198–200 72 (49.98) 49.94

PS02 C14H11ClN4O2S2 (366.85) 230–232 67 (45.84) 45.74
PS03 C14H11BrN4S (347.24) 208–210 64 (48.43) 48.40
PS04 C15H11Cl3N4S (385.70) 122–124 68 (46.71) 46.68
PS05 C14H11ClN4S (302.78) 223–225 64 (55.53) 55.50
PS06 C8H8N4OS (208.22) 202–205 74 (46.14) 46.05
PS07 C10H10N4OS (234.28) 192–196 72 (51.27) 51.25
PS08 C14H10N6O4S (358.34) 234–236 69 (46.93) 46.86

Biological activities

Antibacterial activities

The newly synthesized compounds were screened for their antibacterial activity
against Escherichia coli DH5a, Bacillus subtilis ATCC 6633, Pseudomonas
aeruginosa MTCC 3363, and Pseudomonas fluoroscens ATCC 3458 (recultured)
bacterial strains by paper disc diffusion method (Chandrasekharan et al., 2005).
Muller–Hinton agar medium (38 g/l) was sterilized by autoclave at 121°C for
15 min and then poured into a Petri disc to a depth of 3–4 mm and allowed to
solidify. Plates were dried and 0.1 ml standardized inoculum suspension of the
microorganism [105 colony-forming units (CFU) /ml] was poured and uniformly
132 Med Chem Res (2010) 19:127–135

Table 3 Spectral data for the newly synthesized compounds

Compound Spectral data

PS01 IR: (KBr, cm-1): 3412, 3070, 2667, 1640; 1H NMR (MeOD): d5.6 (s, 2H, NH2) and
7.25–7.6 (m, 5H, Ar–H and SH); MS: m/z 193 (M??1).
PS02 IR: (KBr, cm-1): 3450, 3070, 1640, 648; 1H NMR (MeOD): d5.6(s, 2H, NH2), 7.1–7.8
(m, 9H, Ar–H); MS: m/z 366 (M?), 368 (M??2).
PS03 IR: (KBr, cm-1): 3490, 3050, 760, 560; 1H NMR (MeOD): d6.9(s, 2H, NH2); MS: m/z 346
(M?), 348 (M??2).
PS04 IR: (KBr, cm-1): 3445, 3070, 1630, 1014, 627; 1H NMR (MeOD): d7.2–7.75 (m, 11H, Ar–H
and NH2); MS: m/z 384 (M?), 386 (M??2), 388 (M??4), 390 (M??6).
PS05 IR: (KBr, cm-1): 3490, 1014, 760; 1H NMR (MeOD): d6.85 (s, 2H, NH2), 7.38 (m, 9H,
Ar–H); MS: m/z 304 (M??2).
PS06 IR: (KBr, cm-1): 3400, 3312, 2667; 1H NMR (MeOD): d7.25–7.6 (m, 5H, Ar–H and SH),
6.85 (s, 2H, NH2), 9.5 (s, 1H, OH); MS: m/z 208 (M?).
PS07 IR: (KBr, cm-1): 3210, 2667; 1H NMR (MeOD): d2.2 (s, 3H, COCH3), 3.3 (bs, 1H, NH),
7.2–8.5 (m, 6H, Ar–H and SH); MS: m/z 234 (M?).
PS08 IR: (KBr, cm-1): 3312, 3047, 1511, 1325, 630; 1H NMR (MeOD): d7.15 (s, 2H, NH2),
7.6–8.4 (m, 8H, Ar–H); MS: m/z 358 (M?).

spread. Then the paper impregnated with the test and standard compound was
placed on the solidified medium. The plates were preincubated for 1 h at room
temperature and incubated at 30°C for 24 h. Then, after this incubation, the
minimum inhibitory concentration (MIC) was noted by observing the lowest
concentration of the drug at which there was no visible growth. A number of
antibacterial discs were placed on the agar for the sole purpose of producing zones
of inhibition in the bacterial lawn. Antibacterial activity was determined by
measuring the diameter of inhibition zone. Activity of each compound was
compared with ceftriaxone as standard. Zone of inhibition were determined for
PS01–08 and the results are summarized in Table 4.

Antifungal activities

Newly prepared compounds were screened for antifungal activity against Asper-
gillus niger ATCC 4578, Sacharomyces cerevisiae 180, Trichoderma sp. ATCC
5248, and Fusarium monaliforme NCIM 1276 by the same method as in the
antibacterial screening but the medium was replaced by potato dextrose agar
medium (39 g/l). Antifungal activity was determined by measuring the diameter of
the inhibition zone. Activity of each compound was compared with ketoconazole as
standard. Zones of inhibition were determined for PS01–08 and the results are
summarized in Table 5.

Results and discussion

The investigation of antibacterial and antifungal screening data revealed that all the
tested compounds (PS01–08) showed moderate to good inhibition at 5–20 lg/ml in
Med Chem Res (2010) 19:127–135 133

Table 4 Antibacterial activity of compounds PS01–08

Compd. MIC in lg/ml and zone of inhibition (mm)

E. coli B. subtilis P. aeruginosa P. fluoroscens

PS01 12.5 (16–20) 12.5 (16–20) 12.5 (16–20) 12.5 (16–20)

PS02 15.0 (\10) 15.0 (\10) 15.0 (\10) 15.0 (\10)
PS03 10.0 (16–20) 10.0 (16–20) 10.0 (16–20) 10.0 (16–20)
PS04 5.0 (14–20) 5.0 (14–20) 5.0 (16–22) 5.0 (14–18)
PS05 10.0 (16–20) 10.0 (16–20) 10.0 (16–20) 10.0 (16–20)
PS06 12.5 (16–20) 12.5 (16–20) 12.5 (16–24) 12.5 (16–20)
PS07 15.0 (\10) 15.0 (\10) 15.0 (\10) 15.0 (\10)
PS08 15.0 (\10) 15.0 (\10) 15.0 (\10) 15.0 (\10)
Ceftriaxone 5.0 (16–20) 5.0 (16–20) 5.0 (18–25) 5.0 (16–20)

MIC values were evaluated using concentration range 5–20 lg/ml

Escherichia coli (E. coli) DH5a, Bacillus subtilis (B. subtilis) ATCC 6633, Pseudomonas aeruginosa
(P. aeruginosa) MTCC 3363, Pseudomonas fluoroscens (P. fluoroscens) ATCC 3458

Table 5 Antifungal activity of compounds PS01–08

Compound MIC in lg/ml and zone of inhibition (mm)

A. niger S. cerevisiae T. sp. F. monaliforme

PS01 12.5 (16–22) 12.5 (16–22) 12.5 (16–22) 12.5 (16–22)

PS02 15.0 (\12) 15.0 (\12) 15.0 (\12) 15.0 (\12)
PS03 10.0 (16–22) 10.0 (16–22) 10.0 (16–22) 10.0 (16–22)
PS04 5.0 (14–18) 5.0 (16–20) 5.0 (22–26) 5.0 (18–22)
PS05 10.0 (16–22) 10.0 (16–22) 10.0 (16–22) 10.0 (16–22)
PS06 12.5 (16–22) 12.5 (16–22) 12.5 (16–22) 12.5 (16–22)
PS07 15.0 (\12) 15.0 (\12) 15.0 (\12) 15.0 (\12)
PS08 15.0 (\12) 15.0 (\12) 15.0 (\12) 15.0 (\12)
Ketoconazole 5.0 (16–20) 5.0 (18–24) 5.0 (25–32) 5.0 (20–26)

MIC values were evaluated using concentration range 5–20 lg/ml

Aspergillus niger (A. niger) ATCC 4578, Sacharomyces cerevisiae (S. cerevisiae) 180, Trichoderma sp.
(T. sp.) ATCC 5248, Fusarium monaliforme (F. monaliforme) NCIM 1276

ethanol. The screening results indicate that all of the compounds tested exhibited
significant antibacterial and antifungal activities when compared with the reference
drugs. It was observed that the compound containing chloro-substituted group in 3-
position with free NH2 group in 4-position of 1,2,4-triazole (PS04) shows maximum
bactericidal as well as fungicidal activity as compared with other compounds,
whereas PS03 and PS05 showed good activity. This good activity is attributed to
presence of pharmacologically active 4-chloro, 4-bromo, and 4-trichloromethyl
groups attached to phenyl ring of the triazole ring. The compounds with free NH2
groups at the 4-position (PS01 and PS06) showed moderate activity as compared
134 Med Chem Res (2010) 19:127–135

with PS04. The other compounds (PS02, PS07, and PS08) showed lower fungicidal
effects compared with their bactericidal effects.

Conclusions

We report successful synthesis and antimicrobial activity of a new 1,2,4-triazole

moiety. The antimicrobial activity study revealed that all the tested compounds
showed moderate to good antibacterial and antifungal activities against pathogenic
strains. Structure and biological activity relationship of title compounds showed that
presence of 4-chloro, 4-bromo, and 4-carbon trichloride groups attached to phenyl
ring to the triazole ring of the title compounds is responsible for good antimicrobial
activity.
The field is further open for study of these compounds with respect to toxicity,
chronic toxicity, pharmacokinetics, and clinical studies to establish these molecules
as drugs in the market.

Acknowledgments The authors are grateful to the Head of the School of Pharmacy, Devi Ahilya
Vishwavidyalay, Indore, Madhya Pradesh, India and Sophisticated Analytical Instrument Facility, Central
Drug Research Institute, Lucknow, India for elemental analysis, 1H NMR, and mass spectral data of the
synthesized compounds reported herein. One of the authors, Mr. Priyabrata Pattanayak, is grateful to the
All India Council of Technical Education (AICTE) for providing financial aid.

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