Synthesis and antioxidant and antimicrobial evaluation of novel 4-substituted-1H-1,2,4-triazole derivatives

Article abstract of DOI:10.3906/kim-1202-62

A series of 4-benzyl/phenyl-3-(1-methyl-1H -indole-2-yl)-1H-1,2,4-triazole- 5(4H)-thione (4a,b) and 2- {4-[benzyl/phenyl-5-(substitutedbenzylthio)]-4H -1,2,4-triazole-3-yl}-1-methyl-1H-indole derivatives (5a-p) were synthesized and evaluated for their in vitro scavenging of DPPH and superoxide radical, and lipid peroxidation inhibition effects as well as their antimicrobial properties. DPPH radical scavenging capacity was found to be equipotent with BHT and found in compounds containing 1,2,4-triazole-5(4H) -thione moiety (4a,b). With regard to antimicrobial properties, compound 5k showed slight antimicrobial activity against all the test microorganisms. TUeBITAK.

Full text of DOI:10.3906/kim-1202-62

Turk J Chem
36 (2012) , 867 – 884.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1202-62
Synthesis and antioxidant and antimicrobial evaluation
of novel 4-substituted-1H-1,2,4-triazole derivatives
1
2
3
Sultan BAYTAS¸1∗, Evin KAPC¸ AK , Tu¨lay C¸ OBAN , Hatice OZBILGE
¨
˙
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University,
06330, Ankara-TURKEY
e-mail: baytas@gazi.edu.tr
2Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ankara University,
06100, Ankara-TURKEY
3Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Erciyes University,
38039, Kayseri-TURKEY
Received: 27.02.2012
A series of 4-benzyl/phenyl-3-(1-methyl-1H -indole-2-yl)-1H -1,2,4-triazole-5(4H)-thione (4a,b) and 2-
{4-[benzyl/phenyl-5-(substitutedbenzylthio)]-4H -1,2,4-triazole-3-yl}-1-methyl-1H -indole derivatives (5a-p)
were synthesized and evaluated for their in vitro scavenging of DPPH and superoxide radical, and lipid per-
oxidation inhibition effects as well as their antimicrobial properties. DPPH radical scavenging capacity was
found to be equipotent with BHT and found in compounds containing 1,2,4-triazole-5(4H)-thione moiety
(4a,b). With regard to antimicrobial properties, compound 5k showed slight antimicrobial activity against
all the test microorganisms.
Key Words: Indole, 1,2,4-triazole, DPPH, superoxide, lipid peroxidation, antimicrobial activity
Introduction
Exogenous chemicals in food systems and endogenous metabolic processes in the human body produce highly
reactive free radicals, particularly oxygen-derived free radicals. Reactive oxygen species (ROS) is a collective
term that describes the chemical species that are formed upon incomplete reduction of oxygen and includes
the superoxide anion (O•2−), hydrogen peroxide (H2 O2), and the hydroxyl radical (HO). ROS are constantly
generated in the human body and are involved in various physiologically important biological reactions.1−3
∗Corresponding author
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Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
Under physiological conditions, there is a balance between the production of reactive oxygen and a biological
system’s ability to detoxify the reactive intermediates. Oxidative stress occurs when the generation of ROS
in a system exceeds the system’s ability to eliminate them. Excessive generation of ROS induced by various
stimuli leads to a variety of pathophysiological abnormalities such as inflammation, diabetes, genotoxicity, and
cancer.4−6 Treatment with antioxidants seemed to be a promising therapeutic approach for these conditions.
Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might
otherwise cause. Therefore, in recent years there has been a growing interest in developing new and efficient
antioxidant compounds.7,8
Indoles are very common in the body and the diet, and synthetic and natural products containing indole
moieties are an important class of therapeutic agents.9,10 In recent decades, antioxidant activity of synthetic
indole derivatives and their possible activity mechanisms have been studied. Indole structure, especially substi-
tution of 2-position of indole, influences the antioxidant efficacy in biological systems.11 N -Substituted indole-
2/3-carboxamide and also ester derivatives show potent antioxidant activity against superoxide radical.12−14
Inhibitory effect on lipid peroxidation of 2-phenyl indole derivatives was found as potent as butylatedhydrox-
ytoluene (BHT) and compounds bearing electron-withdrawing groups showed the highest reduction in lipid
peroxidation values.15
1,2,4-Triazole moiety has great versatility in fusing to various ring systems and possesses a broad spectrum
of biological activities.16−19 Antioxidant properties of 1,2,4-triazole derivatives have been investigated by
employing various in vitro systems, i.e. interaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH), scavenging of
superoxide radical, and microsomal NADPH-dependent inhibition of lipid peroxidation (LP).20−23 1,2,4-Triazole
derivatives have been found to have interactions with DPPH. Triazole-containing indole derivatives showed
protective activity against the oxidative injury of the ischemic myocardium and they scavenged superoxide
anion and hydroxyl radical.24
At the beginning of the twentieth century, infectious diseases were the leading cause of death worldwide.
However, not only the emergence of new sources of infection but also developing resistance to antibiotics of
existing infectious pathogens caused difficulties in the treatment of these diseases. 25
New antibacterial agents to treat infections caused by these gram-positive bacteria have recently been
introduced, including the semisynthetic streptogramins quinupristin/dalfopristin, daptomycin, the synthetic
oxazolidinone linezolid and tigecycline.26
Some indole-containing compounds are known to have antibacterial activities.27 Coumarin containing
indole derivatives exhibited good to excellent in vitro activities against Staphylococcus aureus and Enterococcus
faecium including drug-resistant gram-positive bacterial pathogens methicillin-resistant S. aureus (MRSA) and
vancomycin-resistant enterococci (VRE).28 Bis(indole) alkaloids were found to be important key structures for
the treatment of S. aureus infections via inhibition of sortase A (SrtA) activity.27 In addition, 1H -indole-4,7-
dione derivatives showed potent antifungal activity against Candida krusei, Candida neoformans, and Aspergillus
niger.29 Substituted pyrazino[1,2-a]indole was found to have antibacterial activity against pathogenic strains of
S. aureus, Salmonella typhi, Pseudomonas aeruginosa, Streptomyces thermonitrificans, and Escherichia coli.30
Moreover, indole-substituted 2,5-dihydro-1H -2,5-pyrrolediones were found to have antibacterial activity against
resistant strains of Mycobacterium smegmatis, S. aureus, and some other gram-positive bacteria. It was found
that the activity of compounds against S. aureus and M. smegmatis improves with increasing hydrophobic
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Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
properties as well as with hydrogen bond acceptors, depending on their distance from the indole-2-position. 31
Ethyl 6-bromo-5-hydroxy-1H -indole-3-carboxylate derivatives display a variety of biological effects, such as
antiviral effect, immunostimulative effect, and interferon-induced activity. In support of these findings, ethyl
1H-indole-3-carboxylate derivatives were examined for their anti-HCV activities and found to be promising
leads.32
The 1,2,4-triazole and its derivatives were reported to exhibit various pharmacological activities such as
antimicrobial, analgesic, anti-inflammatory, anticancer, and antioxidant properties.33,34 Some of the present
day drugs such as ribavirin (antiviral agent), rizatriptan (antimigraine agent), alprazolam (anxiolytic agent),
fluconazole, terconazole, and itraconazole (antifungal agents) are the best examples of potent molecules possess-
ing a triazole nucleus (Figure 1).35 Compounds containing indole and 1,2,4-triazole moieties as hybrid molecules
including different pharmacophores were found to have antibacterial activity against Bacillus subtilis (MIK =
250 μg/mL) (A in Figure).36 Moreover, some other indolic 1,2,4-triazole derivatives exhibited good to excellent
in vitro activities against Bacillus cereus (MIK = 125 μg/mL) (B in Figure).37
Figure. Examples of drugs used in clinical treatment based on triazole and indole structure.
869

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
In view of the above observations, in this study, a series of indole derivatives containing 1,2,4-triazole
moiety at the 2-position were prepared in order to evaluate their in vitro antioxidant properties by deter-
mining superoxide anion formation, DPPH free radical scavenging activity, and LP on rat liver homogenate.
Antimicrobial activities of the title compounds were also tested using microdilution.
Experimental
Chemistry
The chemical reagents used in synthesis were purchased from Sigma (Germany) and Aldrich (US). Thin-layer
chromatography (TLC) was performed on Merck 60F254 plates. Reactions were monitored by TLC on silica
gel, with detection by UV light (254 nm) or charring Dragendorff reagent. Melting points were determined
with an SMP-II Digital Melting Point Apparatus and are uncorrected. Elemental analyses were performed
with a LECO-932 (C, H, N, S-Elemental Analyzer) at the Faculty of Pharmacy, Ankara University. 1 H-NMR
spectra were recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400 MHz FT-NMR spectrometer using
tetramethylsilane as the internal standard at the NMR facility of the Faculty of Pharmacy, Ankara University.
All chemical shifts were recorded as δ (ppm). IR spectra were obtained using a Perkin Elmer Spectrum 400
FTIR/FTNIR spectrometer equipped with a Universal ATR Sampling Accessory. High resolution mass spectra
data (HRMS) were collected using a Waters LCT Premier XE Mass Spectrometer operating in ESI (+) mode,
also coupled to an AQUITY Ultra Performance Liquid Chromatography system.
Ethyl 1-methyl-1H-indole-2-carboxylate 1
Thionyl chloride (1.35 mL, 0.018 mol) was added dropwise with stirring to dry ethanol (25 mL) at –10 ◦ C;
indole-2-carboxylic acid 2.5 g (0.015 mol) was then added in one portion and the mixture was stirred for 30
min at 0 ◦ C. After stirring for a further 1 h at room temperature, the mixture was heated to reflux for 4 h and
evaporated. The white crystalline powder was dissolved in dichloromethane, extracted with 5% sodium hydrogen
carbonate solution and dried over sodium sulfate. The product, ethyl 1H -indole-2-carboxylate, was filtered off
and dried. Yield 2.77 g, (94.4%), mp 123 ◦ C (Lit. mp 121-123 ◦ C).38 Ethyl 1H -indole-2-carboxylate (2.57 g,
0.013 mol) was dissolved in 25 mL of N,N-dimethylformamide and then (0.48 g, 0.02 mol) sodium hydride was
added in an ice bath. Methyl iodide (125 μL, 0.01 mol) was added at room temperature and stirred for 1 h.
The reaction mixture was poured into ice-water. The crude product was filtered off and dried. Yield 2.11 g,
(76.4%), mp 61 ◦ C (Lit. mp 60-61 ◦ C).38
1-Methyl-1H -indol-2-carbohydrazide 2
Hydrazine hydrate (3 mL, 63 mmol) and ethyl 1-methyl-1H -indole-2-carboxylate (11.57 g, 8 mmol) in 20 mL
of ethanol were refluxed for 6 h. The reaction mixture was cooled to room temperature and poured into water.
The crude product was filtered off and dried. Yield 1.0 g, (68.5%), mp 158 ◦ C (Lit. mp 156.2-157 ◦ C).38
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Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
General procedure for the preparation of N -substituted-2-(1-methyl-1H-indole-2-carbonyl)hydra-
zinecarbothioamide derivatives 3a,b
1-Methyl-1H -indol-2-carbohydrazide 2 (6 mmol) in 35 mL of absolute ethanol and appropriate isothiocyanates
(8 mmol) were heated under reflux for 3 h. The reaction mixture was cooled to room temperature and the
precipitate formed was filtered off and recrystallized.
N -Benzyl-2-(1-methyl-1H-indole-2-carbonyl)hydrazinecarbothioamide 3a
Recrystallized from ethanol as white solid (yield 2.12 g, 85%). mp 185-186 ◦ C; IR (FTIR/FTNIR-ATR): 3174
(N-H), 2973 (C-H), 1671 (C=O), 1350 (C=S). 1 H-NMR (CDCl3); δ 8.86 (bs, 1H, NH), 8.68 (bs, 1H, NH), 7.61
(d, 1H, J = 8.4 Hz, indole H4), 7.37-7.13 (m, 9H, ArH, NH), 7.07 (s, 1H, indole H3), 4.80 (d, 2H, J = 8 Hz,
NH-CH2 -phenyl), 3.85 (s, 3H, CH3). HRMS C18 H18 N4 OS [M+H]+ Calc. 339.1280, Found m/z 339.1263.
N-Phenyl-2-(1-methyl-1H-indole-2-carbonyl)hydrazinecarbothioamide 3b
Recrystallized from ethanol as white solid (yield 1.68 g, 78.5%). mp 162-163 ◦ C; IR (FTIR/FTNIR-ATR):
3132 (N-H), 2955 (C-H), 1666 (C=O), 1351 (C=S). 1 H-NMR (CDCl3); δ 9.29 (bs, 1H, NH), 8.98 (bs, 1H,
NH), 8.28 (s, 1H, NH), 7.66 (d, 1H, J =8.4 Hz, indole H4), 7.41-7.15 (m, 8H, ArH), 7.14 (s, 1H, indole H3),
3.99 (s, 3H, CH3). HRMS C17 H16 N4 OS [M+H]+ Calc. 325.1123, Found m/z 325.1109. Anal. Calc. (%) for
C17 H15 N4 OS. C: 62.94 H: 4.97 N: 17.27, S: 9.72; Found C: 62.33 H: 5.17 N: 16.87 S: 9.74.
General procedure for the preparation of 4-Benzyl/phenyl-3-(1-methyl-1H-indole-2-yl)-1H-1,2,4-
triazole-5(4H )-thion derivatives 4a,b
Appropriate carbothioamides (3 mmol) (3a,b) in 20 mL of 2 N sodium hydroxide were refluxed for 7 h. The
reaction mixture was cooled and then acidified to pH 6 with 1 N hydrochloric acid. The precipitate was filtered,
washed with water, and recrystallized from ethanol.
4-Benzyl-3-(1-methyl-1H-indole-2-yl)-1H-1,2,4-triazole-5(4H )-thione 4a
White solid (yield 0.97 g, 96.6%). mp 236 ◦ C; IR (FTIR/FTNIR-ATR): 3086 (N-H), 2940 (C-H), 1589-1508
(C=C), 1341 (C=S); 1 H-NMR (CDCl3); δ 11.34 (s, 1H, N-H), 7.61 (d, 1H, J = 8 Hz, indole H4), 7.34-7.17 (m,
6H, ArH), 7.10-7.07 (m, 2H, ArH), 6.61 (s, 1H, indole H3), 5.38 (s, 2H, N-CH2 -phenyl), 3.55 (s, 3H, N-CH3);
HRMS C18 H16 N4 S [M+H]+ Calc. 321.1174; Found m/z 321.1178. Anal. Calc. (%) for C18 H15 N4 S. C: 67.47
H: 5.03 N: 17.49, S: 9.87; Found C: 67.09 H: 4.96 N: 17.25 S: 9.88.
4-Phenyl-3-(1-methyl-1H-indole-2-yl)-1H-1,2,4-triazole-5(4H)-thione 4b
White solid (yield 0.92 g, 88.2%). mp 280 ◦ C; IR (FTIR/FTNIR-ATR): 3062 (N-H), 2914 (C-H), 1587-1511
(C=C), 1350 (C=S); 1 H-NMR (CDCl3); δ 11.32 (bs, 1H, N-H), 7.56-7.53 (m, 3H, indole H4 , ArH), 7.42 (d,
1H, J = 8.4 Hz, indole H7), 7.38-7.27 (m, 4H, ArH), 7.08 (m, 1H, J = 7.6 Hz, ArH), 6.03 (s, 1H, indole H3),
3.96 (s, 3H, N-CH3); HRMS C17 H14 N4 S [M+H]+ Calc. 307.1017, Found m/z 307.1019. Anal. Calc. (%) for
C17 H13 N4 S. C: 66.04 H: 4.61 N: 18.29, S: 10.30; Found C: 65.75 H: 4.68 N: 18.05 S: 10.33.
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Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
General procedure for the preparation of 2-{4-[benzyl/phenyl-5-(substituted benzylthio)]-4H-
1,2,4-triazole-3-yl}-1-methyl-1H-indole derivatives 5a-p
4-Benzyl/phenyl-3-(1-methyl-1H -indole-2-yl)-1H-1,2,4-triazole-5(4H)-thione (0.8 mmol) in 20 mL of 2 N
ethanolic sodium hydroxide and appropriate benzyl bromide were heated under reflux for 7 h. The precipi-
tate formed was cooled to room temperature, filtered, and recrystallized from ethanol.
2-{4-[Benzyl-5-(benzylthio)]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5a
White solid (yield 115.3 mg, 37%). mp 159-160 ◦ C; IR (FTIR/FTNIR-ATR): 3027 (C-H), 2955 (C-H), 1589-
1494 (C=C); 1 H-NMR (CDCl3); δ 7.56 (d, 1H, J = 8.0 Hz, indole H4), 7.37 (d, 1H, J = 8.8 Hz, indole H7),
7.35-7.25 (m, 9H, ArH), 7.13 (m, 1H, ArH), 6.88-6.85 (m, 2H, ArH), 6.51 (s, 1H, indole H3), 5.05 (s, 2H, N-
CH2 -phenyl), 4.48 (s, 2H, S-CH2 -phenyl), 3.79 (s, 3H, N-CH3); HRMS C25 H22 N4 S [M+H]+ Calc. 411.1643,
Found m/z 411.1630; Anal. Calc. (%) for C25 H21 N4 S. C: 73.14 H: 5.40 N: 13.65, S: 7.63; Found C: 73.28 H:
5.19 N: 13.59 S: 7.84.
2-{4-Benzyl-5-[(4-methylbenzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5b
White solid (yield 100 mg, 40%). mp 159-160 ◦ C; IR (FTIR/FTNIR-ATR): 3023 (C-H), 1589-1512 (C=C);
1 H-NMR (CDCl3); δ 7.56 (d, 1H, J = 8.0 Hz, indole H4), 7.37 (d, 1H, J = 8.8 Hz, indole H7), 7.32-7.22 (m,
6H, ArH), 7.15-7.13 (m, 2H, ArH), 7.11 (t, 1H, J = 7.6 Hz, ArH), 6.87 (m, 2H, ArH), 6.52 (s, 1H, indole H3),
5.07 (s, 2H, N-CH2), 4.45 (s, 2H, S-CH2), 3.80 (s, 3H, N-CH3), 2.33 (s, 3H, CH3 -phenyl); HRMS C26 H24 N4 S
[M+H]+ Calc. 425.1800, Found m/z 425.1792; Anal. Calc. (%) for C26 H23 N4 S. C: 73.55 H: 5.70 N: 13.20, S:
7.23; Found C: 73.66 H: 5.80 N: 13.13 S: 7.54.
2-{4-Benzyl-5-[(2,6-dichlorobenzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5c
White solid (yield 154.3 mg, 41.2%). mp 129-130 ◦ C; IR (FTIR/FTNIR-ATR): 3063 (C-H), 2934 (C-H), 1582-
1562 (C=C); 1 H-NMR (CDCl3); δ 7.58 (d, 1H, J = 8.0 Hz, indole H4), 7.38 (d, 1H, J = 8.4 Hz, indole H7),
7.33-7.26 (m, 6H, ArH), 7.19 (m, 1H, ArH), 7.14 (m, 1H, ArH), 6.92-6.89 (m, 2H, ArH), 6.55 (s, 1H, indole
H3), 5.12 (s, 2H, N-CH2), 4.74 (s, 2H, S-CH2), 3.80 (s, 3H, N-CH3); HRMS C25 H20 Cl2 N4 S [M+H]+ Calc.
479.0864, Found m/z 479.0861; Anal. Calc. (%) for C25 H19 Cl2 N4 S. C: 62.63 H: 4.20 N: 11.69, S: 6.49; Found
C: 62.54 H: 4.28 N: 11.68 S: 6.73.
2-{4-Benzyl-5-[(4-bromobenzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5d
White solid (yield 144 mg, 37.7%). mp 148-149 ◦ C; IR (FTIR/FTNIR-ATR): 2935 (C-H), 1583-1495 (C=C);
1 H-NMR (CDCl3); δ 7.57 (d, 1H,J = 8.0 Hz, indole H4), 7.42 (d, 1H, J = 8.8 Hz, indole H7), 7.39-7.23 (m,
8H, ArH), 7.13 (m, 1H, ArH), 6.87-6.85 (m, 2H, ArH), 6.53 (s, 1H, indole H3), 5.12 (s, 2H, N-CH2), 4.43 (s,
2H, S-CH2), 3.81 (s, 3H, N-CH3); HRMS C25 H21 BrN4 S [M+H]+ Calc. 489.0749, Found m/z 489.0749; Anal.
Calc. (%) for C25 H20 BrN4 S. C: 61.35 H: 4.32 N: 11.45, S: 6.49; Found C: 61.47 H: 4.08 N: 11.38 S: 6.61.
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Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
2-{4-Benzyl-5-[(3-methoxybenzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5e
White solid (yield 183.7 mg, 53.5%). mp 89-90 ◦ C; IR (FTIR/FTNIR-ATR): 2935 (C-H), 1599-1584 (C=C),
1270 and 1040 (C-O-C); 1 H-NMR (CDCl3); δ 7.57 (d, 1H, J = 8.0 Hz, indole H4), 7.37 (d, 1H, J = 8.4 Hz,
indole H7), 7.32-7.20 (m, 6H, ArH), 7.15 (m, 1H, ArH), 6.93 (d, 1H, J = 7.6 Hz, ArH), 6.88 (m, 2H, ArH),
6.83 (m, 1H, ArH), 6.53 (s, 1H, indole H3), 5.06 (s, 2H, N-CH2 -phenyl), 4.45 (s, 2H, S-CH2 -phenyl), 3.79 (s,
3H, N-CH3), 3.77 (s, 3H, O-CH3). HRMS C26 H24 N4 OS [M+H]+ Calc. 441.1749, Found m/z 441.1743; Anal.
Calc. (%) for C26 H23 N4 OS. C: 70.88 H: 5.59 N: 12.72, S: 7.07; Found C: 70.60 H: 5.55 N: 12.69 S: 7.27.
2-{4-Benzyl-5-[(4-methoxybenzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5f
White solid (yield 245.4 mg, 71.4%). mp 125-126 ◦ C; IR (FTIR/FTNIR-ATR): 3033 (C-H), 2936 (C-H), 1607-
1583 (C=C), 1251 and 1033 (C-O-C); 1 H-NMR (CDCl3); δ 7.56 (d, 1H,J = 7.6 Hz, indole H4), 7.37 (d, 1H,
J = 8.4 Hz, indole H7), 7.32-7.26 (m, 6H, ArH), 7.13 (t, 1H, J = 7.6 Hz, ArH), 6.88 (m, 2H, ArH), 6.29 (d, 2H,
J = 8.8 Hz, ArH), 6.52 (s, 1H, indole H3), 5.09 (s, 2H, N-CH2), 4.45 (s, 2H, S-CH2), 3.80 (s, 3H, N-CH3),
3.79 (s, 3H, O-CH3). HRMS C26 H24 N4 OS [M+H]+ Calc. 441.1749, Found m/z 441.1760; Anal. Calc. (%)
for C26 H23 N4 OS. C: 70.88 H: 5.59 N: 12.72, S: 7.07; Found C: 70.76 H: 5.61 N: 12.61 S: 7.21.
2-{4-Benzyl-5-[(4-(trifluoromethyl)benzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5g
White solid (yield 339.5 mg, 91%). mp 136-137 ◦ C; IR (FTIR/FTNIR-ATR): 3033 (C-H), 2941 (C-H), 1615
and 1584 (C=C); 1 H-NMR (CDCl3); δ 7.56 (d, 1H,J = 8.0 Hz, indole H4), 7.55-7.49 (m, 4H, ArH), 7.38 (d,
1H, J = 8.8 Hz, indole H7), 7.33-7.25 (m, 4H, ArH), 7.14 (m, 1H, ArH), 6.88 (m, 2H, ArH), 6.54 (s, 1H, indole
H3), 5.14 (s, 2H, N-CH2), 4.53 (s, 2H, S-CH2), 3.82 (s, 3H, N-CH3); HRMS C26 H21 F3 N4 S [M+H]+ Calc.
479.1517, Found m/z 479.1525; Anal. Calc. (%) for C26 H20 F3 N4 S. C: 65.26 H: 4.42 N: 11.71, S: 6.49; Found
C: 65.09 H: 4.25 N: 11.63, S: 6.73.
2-{4-Benzyl-5-[(4-(trifluoromethoxy)benzyl)thio]-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5h
White solid (yield 340.6 mg, 88.39%). mp 123-124 ◦ C; IR (FTIR/FTNIR-ATR): 3037 (C-H), 2943 (C-H), 1596-
1504 (C=C), 1255 and 1086 (C-O-C); 1 H-NMR (CDCl3); δ 7.57 (d, 1H, J = 8.0 Hz, indole H4), 7.42-7.26 (m,
7H, ArH), 7.15 (m, 3H, ArH), 6.88 (m, 2H, ArH), 6.54 (s, 1H, indole H3), 5.13 (s, 2H, N-CH2 -phenyl), 4.48
(s, 2H, S-CH2 -phenyl), 3.82 (s, 3H, N-CH3); HRMS C26 H21 F3 N4 OS [M+H]+ Calc. 495.1466, Found m/z
495.1471; Anal. Calc. (%) for C26 H20 F3 N4 OS. C: 63.15 H: 4.28 N: 11.33, S: 6.39; Found C: 63.20 H: 4.13 N:
11.42, S: 6.62.
2-[5-(Benzylthio)-4-phenyl-4H-1,2,4-triazole-3-yl]-1-methyl-1H-indole 5i
White solid (yield 231.4 mg, 72.7%). mp 156 ◦ C; IR (FTIR/FTNIR-ATR): 3059 (C-H), 2949 (C-H), 1588-1578
(C=C); 1 H-NMR (CDCl3); δ 7.51-7.44 (m, 3H, indole H4 , ArH), 7.40-7.35 (m, 3H, indole H7 , ArH), 7.31-7.24
(m, 5H, ArH), 7.14 (d, 2H, J = 7.6 Hz, ArH), 7.05 (m, 1H, ArH), 5.92 (s, 1H, indole H3), 4.52 (s, 2H, S-CH2),
4.12 (s, 3H, N-CH3); HRMS C24 H20 N4 S [M+H]+ Calc. 397.1487, Found m/z 397.1485; Anal. Calc. (%) for
C24 H19 N4 S. C: 72.70 H: 5.08 N: 14.13, S: 8.02; Found C: 72.73 H: 4.97 N: 14.00, S: 8.13.
873

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
2-{5-[(4-Methylbenzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5j
White solid (yield 262.2 mg, 79.6%). mp 160-162 ◦ C; IR (FTIR/FTNIR-ATR): 3048 (C-H), 1580-1512 (C=C);
1 H-NMR (CDCl3); δ 7.53-7.35 (m, 4H, indole H4 , H7 , ArH), 7.28-7.23 (m, 4H, ArH), 7.15 (d, 2H, J = 6.8
Hz, ArH), 7.10 (d, 2H, J = 7.6 Hz, ArH), 7.05 (t, 1H, J = 7.2 Hz, ArH), 5.92 (s, 1H, indole H3), 4.50 (s, 2H,
S-CH2), 4.12 (s, 3H, N-CH3), 2.31 (s, 3H, phenyl-CH3); HRMS C25 H22 N4 S [M+H]+ Calc. 411.1643, Found
m/z 411.1629; Anal. Calc. (%) for C25 H21 N4 S. C: 73.14 H: 5.40 N: 13.65, S: 7.58; Found C: 73.41 H: 5.26 N:
13.54, S: 7.85.
2-{5-[(2,6-dichlorobenzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5k
White solid (yield 191 mg, 51%). mp 153-154 ◦ C; IR (FTIR/FTNIR-ATR): 3092 and 3060 (C-H), 2958 (C-H),
1594-1582 (C=C); 1 H-NMR (CDCl3); δ 7.52-7.36 (m, 5H, indole H4 , ArH), 7.29-7.22 (m, 5H, indole H3 ,
ArH), 7.15 (t, 1H, J = 8.0 Hz, ArH), 7.06 (m, 1H, ArH), 5.96 (s, 1H, indole H3), 4.81 (s, 2H, S-CH2), 4.13
(s, 3H, N-CH3); HRMS C24 H18 Cl2 N4 S [M+H]+ Calc. 465.0707, Found m/z 465.0701; Anal. Calc. (%) for
C24 H17 Cl2 N4 S C: 61.04 H: 3.90 N: 12.04, S: 6.63; Found C: 61.24 H: 3.90 N: 12.84, S: 6.81.
2-{5-[(4-Bromobenzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5l
White solid (yield 292.3 mg, 76.5%). mp 185-186 ◦ C; IR (FTIR/FTNIR-ATR): 3060 (C-H), 2951 (C-H), 1587-
1578 (C=C); 1 H-NMR (CDCl3); δ 7.55-7.35 (m, 6H, indole H4 , H7 , ArH), 7.31-7.24 (m, 4H, ArH), 7.17 (d,
2H, J = 8.0 Hz, ArH), 7.05 (m, 1H, ArH), 5.92 (s, 1H, indole H3), 4.46 (s, 2H, S-CH2), 4.11 (s, 3H, N-CH3);
HRMS C24 H19 BrN4 S [M+H]+ Calc. 475.0592, Found m/z 475.0595; Anal. Calc. (%) for C24 H18 BrN4 S C:
60.63 H: 4.03 N: 11.79, S: 6.52; Found C: 60.36 H: 3.88 N: 11.61, S: 6.78.
2-{5-[(3-Methoxybenzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5m
White solid (yield 288.3 mg, 84.2%). mp 156-157 ◦ C; IR (FTIR/FTNIR-ATR): 3100 (C-H), 2945 (C-H), 1598-
1578 (C=C), 1271 and 1035 (C-O-C); 1 H-NMR (CDCl3); δ 7.51-7.45 (m, 3H, indole H4 , ArH), 7.40 (dd, 2H,
J = 12.8 Hz, J =13.2 Hz, indole H4 , ArH), 7.27 (m, 1H, indole H7), 7.20 (m, 1H, ArH), 7.14 (d, 2H, J =7.2
Hz, ArH), 7.05 (m, 1H, ArH), 6.93 (d, 1H, J =7.2 Hz, ArH), 6.90 (s, 1H, ArH), 6.81 (d, 1H, J = 8.4 Hz, ArH),
5.93 (s, 1H, indole H3), 4.49 (s, 2H, S-CH2), 4.11 (s, 3H, N-CH3), 3.77 (s, 3H, O-CH3); HRMS C25 H22 N4 OS
[M+H]+ Calc. 427.1593, Found m/z 427.1573; Anal. Calc. (%) for C25 H21 N4 OS C: 70.40 H: 5.20 N: 13.14 S:
7.43; Found C: 70.66 H: 5.12 N: 13.10 S: 7.59.
2-{5-[(4-Methoxybenzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5n
White solid (yield 201.2 mg, 58.8%). mp 146-147 ◦ C; IR (FTIR/FTNIR-ATR): 3100 (C-H), 2931 (C-H), 1608
and 1582 (C=C), 1247 and 1031 (C-O-C); 1 H-NMR (CDCl3); δ 7.51-7.44 (m, 3H, indole H4 , ArH), 7.38 (d,
2H, J = 12.0 Hz, ArH), 7.31 (d, 1H, J = 8.4 Hz, indole H7), 7.25 (m, 2H, ArH), 7.17 (d, 2H, J = 8.0 Hz, ArH),
7.05 (m, 1H, ArH), 6.82 (d, 2H, J = 8.4 Hz, ArH), 5.93 (s, 1H, indole H3), 4.49 (s, 2H, S-CH2), 4.12 (s, 3H,
N-CH3), 3.78 (s, 3H, O-CH3); HRMS C25 H22 N4 OS [M+H]+ Calc. 427.1593, Found m/z 427.1595; Anal.
Calc. (%) for C25 H21 N4 OS C: 70.40 H: 5.20 N: 13.14 S: 7.43; Found C: 70.28 H: 5.04 N: 13.03 S: 7.46.
874

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
2-{5-[(4-(Trifluoromethyl)benzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5o
White solid (yield 232.8 mg, 62.4%). mp 134-135 ◦ C; IR (FTIR/FTNIR-ATR): 3055 (C-H), 2946 (C-H), 1578-
1496 (C=C); 1 H-NMR (CDCl3); δ 7.55-7.45 (m, 6H, indole H4 , H7 , ArH), 7.38 (dd, 2H, J = 10.8 Hz, J =10.4
Hz, ArH), 7.25 (m, 2H, ArH), 7.17 (d, 2H, J = 8.0 Hz, ArH), 7.05 (t, 1H,J = 7.6 Hz, ArH), 5.92 (s, 1H, indole
H3), 4.55 (s, 2H, S-CH2), 4.12 (s, 3H, N-CH3); HRMS C25 H19 F3 N4 S [M+H]+ Calc. 465.1361, Found m/z
465.1345; Anal. Calc. (%) for C25 H18 F3 N4 S C: 64.64 H: 4.12 N: 12.06 S: 6.88; Found C: 64.61 H: 4.24 N:
12.01 S: 6.94.
2-{5-[(4-(Trifluoromethoxy)benzyl)thio]-4-phenyl-4H-1,2,4-triazole-3-yl}-1-methyl-1H-indole 5p
White solid (yield 287 mg, 74.4%). mp 117-118 ◦ C; IR (FTIR/FTNIR-ATR): 3062 (C-H), 1577-1509 (C=C),
1248 and 1100 (C-O-C); 1 H-NMR (CDCl3); δ 7.53-7.43 (m, 4H, indole H4 , H7 , ArH), 7.38 (dd, 2H, J = 11.2
Hz, J =11.6 Hz, ArH), 7.25 (m, 2H, ArH), 7.15 (m, 4H, ArH), 7.05 (t, 1H, J = 8.0 Hz, ArH), 5.93 (s, 1H, indole
H3), 4.55 (s, 2H, S-CH2), 4.12 (s, 3H, N-CH3); HRMS C25 H19 F3 N4 OS [M+H]+ Calc. 481.1310, Found m/z
481.1293; Anal. Calc. (%) for C25 H18 F3 N4 S C: 62.49 H: 3.99 N: 11.66 S: 6.36; Found C: 62.32 H: 4.13 N:
11.54 S: 6.69.
Antioxidant and radical scavenging properties
All synthesized indole 1,2,4-triazole derivatives were tested for DPPH, superoxide radical scavenging, and anti-
LP activities. All the results were compared with BHT.
Superoxide radical scavenging assay
Superoxide was generated by xanthine/xanthine oxidase and measured by the inhibition of cytochrome c
reduction as described by McCord and Fridovich with some modification.39 The incubation mixture contained
100 μL of 4 mM xanthine, 400 μL of cytochrome c, 50 mL of 50 mM phosphate buffer (pH 7.8, 1 mM EDTA),
and 10 mL of the test compounds were prepared in a 96-well plate, and then 40 μL of xanthine oxidase was
added to each well. The absorbance of each reaction mixture was monitored at 550 nm for 3 min. Each
experiment was triplicated.
Superoxide radical scavenging activity (%) = (Acontrol – Asample)/( Acontrol – Ablank) × 100
(A
= the absorbance of the control, A
= the absorbance of test sample A
= the
control
sample
blank
absorbance of blank)
DPPH radical scavenging assay
The DPPH assay was performed using test compounds, purified isolates as previously described.40 DPPH reacts
with antioxidant compounds. It is reduced, and then its deep violet color in methanol is bleached to yellow,
showing a significant absorption decrease at 517 nm. According to the literature, test samples were dissolved
in DMSO and mixed with methanol solutions of DPPH (100 mM) in 96-well plates. After a 30 min incubation
period at room temperature, the absorbance was read against a blank at 517 nm. For each test compound,
875

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
different concentrations were tested. The antioxidant capacity of each test compound was expressed as an IC50
value
SD, i.e. the concentration in mM that inhibits DPPH absorption by 50%, and was calculated by linear
regression analysis. Tests were carried out in triplicate. Free radical DPPH inhibition as a percentage was
calculated as follows:
Free radical DPPH inhibition (%) = [(A0 – A1 /A0)× 100]
(A0 = the absorbance of the control; A1 = the absorbance of the sample)
Assay of lipid peroxidation
The lipid peroxidation assay was performed according to the method of Mihara et al.41 with some modification.
Throughout the experiments, the animals were treated under the audit of Gazi University’s Commission of
Animal Ethics according to the suggested international ethical guidelines for the care of laboratory animals
(Permission No: 10.046). Wistar rats (200-225 g) were fed standard laboratory rat chow and tap water ad
libitum. The animals were fasted for 24 h and then sacrificed by decapitation under anesthesia. Their livers
were removed, washed in ice-cold distilled water, and homogenized straight away with a Teflon homogenizer.
LP was measured spectrophotometrically by estimation of thiobarbituric acid reactive substances (TBARS).
Then 0.05 mL of different concentrations of synthesized compounds were incubated for 1 h at 37 ◦ C in an
assay mixture that contained 0.5 mL of liver homogenate, 0.1 mL of Tris-HCl buffer (pH 7.2), 0.05 mL of 0.1
mM ascorbic acid, and 0.05 mL of 4 mM FeCl2 . After incubation, 3.0 mL of H3 PO4 and 1 mL of 0.6% TBA
were added to the assay mixture. The prepared mixture was shaken vigorously, boiled for 30 min, and then
cooled. n-Butanol was added and then the absorbance of the supernatant was read at 532 nm against a blank,
which contained all reagents except liver homogenate. The difference was regarded as the TBA value. Lipid
peroxidation inhibitory activity (%) is expressed as follows:
Lipid peroxidation inhibitory activity (%) = (Acontrol – Asample)/(Acontrol – Ablank)× 100
(Acontrol = the absorbance of the control; Asample = the absorbance of the sample; Ablank = the
absorbance of the blank)
Microbiology
Mueller Hinton Agar (MHA) (Merck), Mueller Hinton Broth (MHB) (Merck), Sabouraud Dextrose Agar
(SDA) (Merck), RPMI-1640 medium with L-glutamine (Sigma), 3-[N -morpholino]-propansulfonic acid (MOPS)
(Sigma), 96-well microplates (Falcon), transfer pipette (Socorex), gentamicin (Sigma), fluconazole (Nobel), and
dimethylsulfoxide (DMSO) (Riedel de Haen) were used in the antimicrobial activity testing.
P. aeruginosa ATCC 27853 (American Type Culture Collection), E. coli ATCC 25922, S. aureus ATCC
25923, Enterococcus faecalis ATCC 29212, Candida albicans ATCC 90028, and Candida glabrata RSKK 90018
(Refik Saydam Culture Collection) were included in the study.
Standard powders of gentamicin and fluconazole were obtained from the manufacturers. Stock solutions
were dissolved in distilled water (gentamicin and fluconazole). All bacterial isolates were subcultured in MHA
plates and incubated overnight at 37 ◦ C and all Candida isolates were subcultured in SDA plates at 35 ◦ C for
24-48 h. The microorganisms were passaged at least twice to ensure purity and viability.
876

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
The solutions of the newly synthesized compounds were prepared at 1256, 625, 312.5, 156.25, 78.125,
39.06, 19.5, and 9.76 μg/mL concentrations by diluting in MHB.
Bacterial susceptibility testing was performed according to the guidelines of the Clinical and Laboratory
Standards Institute (CLSI) M100-S16.42 The bacterial suspensions used for inoculation were prepared at 105
CFU/mL by diluting fresh cultures at McFarland 0.5 density (107 CFU/mL). Suspensions of the bacteria at
105 CFU/mL concentration were inoculated to the 2-fold diluted solution of the compounds. There were 104
CFU/mL bacteria in the wells after inoculations. MHB was used for diluting the bacterial suspension and for
2-fold dilution of the compound. DMSO, standard antimicrobials, pure microorganisms, and pure media were
used as control wells. Bacterial inoculum was added to each well of the microdilution trays. The trays were
incubated at 37 ◦ C in a humid chamber and MIC endpoints were read after 24 h of incubation. All organisms
were tested in triplicate in each run of experiments. The lowest concentration of the compound that completely
inhibited macroscopic growth was determined and minimum inhibitory concentrations (MICs) are noted in
Table 3.
All Candida isolates were subcultured in SDA plates, incubated at 35 ◦ C for 24-48 h prior to antifungal
susceptibility testing, and passaged at least twice to ensure purity and viability. Susceptibility testing was
performed in RPMI-1640 medium with L-glutamine buffered pH 7 with MOPS and culture suspensions were
prepared through the guideline of CLSI M27-A.43 The yeast suspensions used for inoculation were prepared at
104 cfu/mL by diluting fresh cultures at McFarland 0.5 density (106 CFU/mL). Suspensions of the yeast at 104
CFU/mL concentration were inoculated to 2-fold diluted solution of the compounds. There were 103 CFU/mL
bacteria in the wells after inoculations. Yeast inoculum was added to each well of the microdilution trays. The
trays were incubated at 35 ◦ C in a humid chamber and MIC endpoints were read after 48 h of incubation. All
organisms were tested in triplicate in each run of experiments. The lowest concentration of the compound that
completely inhibited macroscopic growth was determined and MICs are reported in Table 3.
Results and discussion
Chemistry
The synthetic route for the newly synthesized compounds, 1,2,4-triazolylindole derivatives (4a,b; 5a-p), is
illustrated and outlined in the Scheme. Ethyl 1-methyl-1H -indole-2-carboxylate 1 was synthesized according
to the literature.44 Ester 1 was converted to the corresponding acid hydrazides 2 by refluxing with hydrazine
hydrate (80%) in ethanol.45−48 Hydrazinecarbothioamide derivatives 3a,b were synthesized by the condensation
of acid hydrazide 2 and benzyl/phenylisothiocyanates in ethanol. These compounds were used as the key
intermediates for the synthesis of 1,2,4-triazole derivatives. The 4-substituted-3-(1-methyl-1H -indole-2-yl)-1H-
1,2,4-triazole-5(4H)-thions 4a,b were synthesized by intramolecular dehydrative cyclization of carbothioamides
3a,b refluxed in 4 N sodium hydroxide solution, followed by neutralization with concentrated HCl.49 3-Alkylthio
derivatives of 4a,b were synthesized in yields ranging between 37% and 91% by the action of several benzyl
chlorides on 4-substituted-3-(1-methyl-1H -indole-2-yl)-1H-1,2,4-triazole-5(4H)-thione 4a,b in the presence of
strong alkali. All the reactions were monitored by TLC. The structure elucidations of the newly synthesized
compounds were carried out by spectroscopic techniques like IR and 1 H-NMR (Table 1). Further confirmations
of the compounds were carried out by mass spectrometry and microanalysis.
877

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
The IR spectra of N -benzyl/phenyl-2-(1-methyl-1H -indole-2-carbonyl) hydrazinecarbothioamide deriva-
tives 3a,b exhibited characteristic strong absorption bands for carbonyl group at 1671-1666 cm−1 , and C=S
stretching bands at 1351-1350 cm−1 . The C=S stretching bands of compounds 4a,b appeared in the 1350-1341
cm1 region. These bands were not detectable for compounds 5a-p, demonstrating the disappearance of this
group.
The 1 H-NMR spectra of compounds 3a,b displayed 3 singlets due to 3 different –NH groups in the δ
9.29-8.28 ppm range each showing the integration for 1 proton. In the 1 H-NMR spectra of compounds 4a,b,
signals of NH of the 1,2,4-triazole-5(4H)-thione derivatives were observed in the δ 11.34-11.32 ppm range as
singlets. In the 1 H-NMR spectra of 5a-p, the lack of signals arising from triazole CS-NH function at 11.34-
11.32 ppm, observed with 4a,b, provided the evidence for the formation of thioether function. Further support
for the structures of these compounds was obtained through the presence of resonances arising from benzylic
protons at 4.81-4.45 ppm. Further spectroscopic details of these compounds are presented in the experimental
part.
a
b
COOH
CONHNH2
COOC2H5
N
H
N
CH3
N
CH3
2
1
c
O
N
N
N
d
e
N
NH
N
CH3
HN NH
N
N
CH3
N
R
S
S
NH
R
CH3 R
R1
S
4a,b
3a,b
5a-p
Scheme. Reagent and conditions a) i. SOCl2 , EtOH, –10 ◦ C; ii. CH3 I, NaH, DMF, 0 ◦ C - rt; b) NH2 NH2 , EtOH,
reflux, 2 h; c) RNCS, EtOH; d) NaOH, H2 O, reflux; e) Ar-CH2 -Cl, 2N NaOH, EtOH, reflux, 7 h.
Pharmacology
Antioxidant activities
The compounds reported herein were tested for their scavenging effects of DPPH and superoxide radical, and
their ability to inhibit lipid peroxidation (Table 2). Each method is related to the generation of a different
radical acting through a variety of mechanisms.
878

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
Table 1. Synthesized indolic compounds containing 1,2,4-triazole-5(4H )-thione moiety.
N
N
NH
N
N
CH3
4a,b
N
R
N
CH3
5a-p
N
R
S
S
Ar
Compound
R
Ar
benzyl
phenyl
4a
4b
benzyl
5a
benzyl
5b
benzyl
5c
benzyl
benzyl
5d
5e
benzyl
benzyl
benzyl
phenyl
phenyl
5f
5g
5h
5i
5j
phenyl
5k
phenyl
phenyl
5l
5m
phenyl
phenyl
phenyl
5n
5o
5p
879

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
The scavenging effects of synthesized compounds on DPPH radical are presented in Table 2. Preliminary
screenings of the title compounds were performed at different concentrations to determine the scavenging effects
on the DPPH. Compounds 4a (IC50 90 μM) and 4b (IC50 65 μM), derivatives with a free 1,2,4-triazole-5(4H)-
thione ring, were found to be good scavengers of DPPH radical. In these conditions the reference drug BHT
showed better scavenging effects on the DPPH. Compound 5h bearing p-OCF3 substitution on the S-benzyl
ring exhibited a moderate effect on DPPH radical with an IC50 value of 0.91 mM. Although the scavenging
rates were not very pronounced, the compounds 5i, 5k, 5m, and 5n demonstrated a scavenging effect in the
range of 5%-20%. The rest of the compounds had no effect on DPPH radical (Table 2).
The superoxide radical scavenging activities of the compounds were also investigated by using the
xhanthine/xhantine oxidase system, and there was no significant activity pattern obtained from this experiment
(data are not shown).
Table 2. Effects of the compounds on LP levels and DPPH radical scavenging capacity. a
DPPH radical
Lipid peroxidation
inhibition %
(1 mM)
Compound
scavenging capacity
Inhibition% (1 mM)
(IC50 mM)
4a
4b
5a
5b
5c
87
88
3
4
0.09
0.05
12
22
2.4
2.3
0.065
0.019
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5d
5e
5f
5g
5h
5i
61
4.5
NA
4
0.91
0.1
11
1.5
1
NA
NA
NA
NA
5j
5k
5l
5.0
0.5
NA
10
20
NA
NA
98
5m
5n
5o
5p
BHT
1
2
8
1.2
2.1
NA
NA
65 1.4
17
2
0.054 0.005
aEach value represents mean SD of 2-4 independent experiments. NA: not active
The inhibitory effect of the compounds on LP levels was examined by measuring the formation of 2-
thiobarbituric acid reactive substance. As can be seen from Table 2, the BHT inhibited LP levels by about
65% at 1 mM concentration. The compounds 4a, 4b, 5h, 5m, and 5n showed inhibitory properties on lipid
880

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
peroxidation level at the same concentration. Nonsubstituted 1,2,4-triazole-5(4H)-thione ring (compounds 4a
and 4b) improved inhibitory action. The most active compound is N-phenyl-3-(1-methyl-1H-indole-2-yl)-1H-
1,2,4-triazole-5(4H)-thion 4b; it caused 22% inhibition on LP. Compounds 5h, 5m, and 5n showed weak
inhibitory effect in the range of 11%-17%. The other compounds had no inhibitory effect on LP levels.
Many studies have established beneficial effects of antioxidants that reduce the myocardial infarct size by
interventions, which either attenuate the generation or reduce the effects of reactive oxygen species.50,51 An-
dreadou et al. have reported on the synthesis and pharmacological properties of some indolic compounds bear-
ing triazole moiety. They found that 3-[(1H -1-indolyl)methyl]-4-amino-4,5-dihydro-1H,1,2,4-triazole-5-thione
has shown significant antioxidant properties by inhibiting in vitro non-enzymatic rat hepatic microsomal LP
levels.52 In our study, 4-benzyl/phenyl-3-(1-methyl-1H -indole-2-yl)-1H-1,2,4-triazole-5(4H)-thion derivatives
4a and 4b showed better antioxidant properties than their S-alkylated derivatives. The introduction of benzyl
and phenyl rings in the 4th position of the 1,2,4-triazole did not significantly alter the activity. Therefore, the
important structural feature for DPPH radical scavenging is the presence of the free 1,2,4-triazole-5(4H)-thione
ring in the tested indolic compounds.
Table 3. Antimicrobial activities of the title compounds (μg/mL).
Compound
Pa
625 625 NI
625 NI NI
625 625 625 625 NI
625 625 NI 625 625 625
Ec
Sa
Ef
NI 625 625
NI NI 625
NI
Ca Cg
4a
4b
5a
5b
5c
625 625 NI
625 NI NI
NI 625 625
NI 625 625
5d
5e
625 625 NI 625 NI
NI
5f
625 625 NI 625 625 NI
625 625 625 625 625 NI
5g
5h
625 625 625 625 NI
625 625 NI 625 NI 625
625 NI NI NI 625 625
625 625 625 625 625 625
625 625 NI NI 625 625
NI
5i
5j
5k
5l
5m
625 625 625 625 NI 625
625 625 625 625 NI 625
5n
5o
625 625 NI 625 NI
625 625 625 625 NI
NI
NI
—
64
5p
Gentamicin
Fluconazole
2
2
2
2
—
64
—
—
—
—
Abbreviations: Pa, P. aeruginosa ATCC 27853; Ec, E. coli ATCC 25922; Sa, S. aureus ATCC 25923; Ef, E. faecalis
ATCC 29212; Ca, C. albicans ATCC 90028; Cg, Candida glabrata RSKK 90018; NI: no inhibition
881

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
The relationships between the activities, the type of substituents, and their position in the title compounds
were evaluated. Introducing benzyl moiety to the triazole portion of the title compounds resulted in inactive
antioxidant compound. On the other hand, the presence of a methoxy group (compound 5m and 5n) in the
aromatic ring of S-benzylated derivatives showed a more positive effect on the inhibition of LP than the methyl
or halogen substituted compounds. It was found that p-OCF3 substitution on the S-benzyl ring (compound
5h) had a positive effect on DPPH radical scavenging capacity and the inhibition of LP.
Microbiology
All compounds were evaluated for their antimicrobial properties. MICs were recorded as the minimum concen-
tration of compound that inhibits the growth of the tested microorganisms. The MIC values were determined
by a microdilution method in MHB and RPMI-1640 medium for the antibacterial and antifungal assays, re-
spectively. According to the results, the compounds showed poor activity compared with gentamicin and
fluconazole. According to the data (Table 3), all tested compounds were weakly effective against gram-negative
P. aeruginosa. Compounds 4a and 4b were more effective against gram-negative bacteria and fungi than against
gram-positive bacteria. Compounds 5a, 5h, and 5p showed better activity against the tested bacteria than
fungi. Compound 5k showed slight antimicrobial activity against all the test microorganisms.
Conclusions
We studied the antioxidative capacity of the synthesized compounds by determining superoxide (O•2−) and
DPPH radical scavenging capacity and the inhibition of LP. The results of the biological evaluation revealed
that especially compounds 4a,b exhibited good scavenging effects on DPPH radical. Evaluation of activity
results for scavenger effect on DPPH and inhibition of LP suggested that the free 1,2,4-triazole-5(4H)-thione
ring might be important for the activity. We think that the preliminary in vitro activity results of this
class of compounds might lead to further studies to develop better candidates possessing antioxidant activity.
Antimicrobial evaluations of some of the presented structures against bacteria and fungi are encouraging since
compound 5k showed slight antimicrobial activity against all the test microorganisms. It is considered that
the solubility problem caused the generation of ineffective compounds. Designing more polar compounds may
overcome solubility problems, and might achieve more active compounds. Further studies with these types of
compounds are under investigation in our laboratory.
Acknowledgements
This investigation was partially supported by Scientific Research Grant 02/2010-43, awarded by Gazi University
BAP, and a part of this work was done as Evin Kapc¸ak’s master thesis at the Department of Pharmaceutical
Chemistry at the Institute of Health Sciences, Gazi University.
882

Synthesis and antioxidant and antimicrobial evaluation of novel..., S. BAYTAS¸, et al.
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