Advanced Studies in Biology, Vol. 5, 2013, no. 6, 303 - 318

HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2013.3419

Synthesis, Characterization and Evaluation of

Antibacterial Activity of Six Novel 1,2,4-Triazole

Derivatives against Standard and Medical Bacteria

Jacob H. Jacob*

Department of Biological Sciences, Faculty of Sciences

Al al-Bayt University, Al-Mafraq, Jordan

Fawzi I. Irshaid

Department of Biological Sciences, Faculty of Sciences

Al al-Bayt University, Al-Mafraq, Jordan

Yaseen A. Al-Soud

Department of Chemistry, Faculty of Sciences

Al al-Bayt University, Al-Mafraq, Jordan

Abdullah M. Al-Balushi

Department of Chemistry, Faculty of Sciences

Al al-Bayt University, Al-Mafraq, Jordan

Hamzeh R. Al-Arqan

Department of Biological Sciences, Faculty of Sciences

Al al-Bayt University, Al-Mafraq, Jordan

*Corresponding Author: Jacob H. Jacob, e-mail: jjacob@aabu.edu.jo

Copyright © 2013 Jacob H. Jacob et al. This is an open access article distributed under the

Creative Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

304 Jacob H. Jacob et al.

Abstract

1,2,4-triazoles have been reported to possess a wide range of biological activities,

including anticancer, antifungal, antiviral and antibacterial activities. Therefore,

the current investigation is intended to synthesize six novel 1,2,4-triazole

derivatives (designated as 2aa, 2ab, 2ac, 2bb, 2bd, and 2be). These derivatives

were screened for their antibacterial activity against two Gram-negative bacteria

isolated from clinical specimens (stool and ear exudates of infected patients) and

two Gram-positive standard bacteria (Staphylococcus aureus ATCC29213 and

Bacillus cereus ATCC11778) using both well diffusion and broth dilution

methods. The chemical structures of the new 1,2,4-triazole derivatives were

characterized by 1H and

13C-NMR spectra, in addition to elemental analysis. The

bacterial strains designated as GN1 and GN2 were identified by biochemical and

molecular methods and confirmed as new strains of Shigella sp., and

Pseudomonas aeruginosa, respectively. All triazole derivatives were found to be

active against bacteria with varying degrees. Some derivatives were found to have

the same antibacterial activity as penicillin G (the positive control) against certain

strains, for instance, 2ab, 2bd, and 2be against B. cereus ATCC11778.

Interestingly, some compounds were more active than penicillin G against certain

strains. The activity of 2aa against B. cereus ATCC11778 was higher than the

activity of penicillin G, whereas 2ab and 2ac were more active than penicillin G

against the clinical isolate P. aeruginosa. The variation seen in antimicrobial

activity could be attributed to the difference in the substituent groups attached to

the 1,2,4-triazole nucleus. In conclusion, these findings might hold promise for

development of a new class of a novel expanded spectrum antibiotics against

clinically important bacteria that are highly resistant to current antibacterial

agents and associated with serious and life-threatening infections.

Keywords: 1,2,4-triazole derivatives, antibacterial activity, medical bacteria.

1. Introduction

Infectious diseases caused by highly resistant bacteria are still the leading cause of

death in developing countries and among the chief causes of mortality in

developed countries. Worldwide, infectious diseases account for 23.5% of 58.7

million annual deaths [1]. Therefore, pathogenic microorganisms, particularly

emerging pathogens and drug- and multidrug- resistant pathogens, represent a

serious challenging problem in modern medicine. Consequently, more careful use

of currently available drugs and discovery of new drugs are needed to overcome

the nonstop problem of infectious diseases [2].

Scientists around the world have begun synthesizing and developing a wide range

of synthetic antimicrobial agents by the means of organic chemistry.

Sulfonamides, nalidixic acid, and quinolones are just few examples of the early

synthetic antimicrobial agents with chemotheraptic applications [3]. Recently,

Synthesis, characterization and evaluation 305

triazole compounds such as 1,2,4-triazoles represent an important group of such

synthetic antimicrobial compounds that are gaining more interest and access in the

field of microbial chemotherapy [4,5,6].

The name “triazole” was first used by Bladin in 1855 to describe the

carbon-nitrogen ring system C2H3N3 and its derivatives [5]. Later, several novel

triazole compounds were synthesized and the biological activities of some of them

were proved. The confirmed biological activities include antibacterial [5,7],

antifungal [8,9,5], antiviral [10], anti-inflammatory [11] and antioxidant activities

[4]. Fluconazole, itraconazole, and posaconazole are examples of triazole

derivatives that are already available in the pharmaceutical market and being used

to treat several types of mycoses [5].

Despite these achievements, many clinically important bacterial isolates still

present a real challenge for many physicians, especially among those isolates that

are highly resistant to current antibacterial agents and associated with serious and

life-threatening infections. As consequence, a combination therapy of two or three

antimicrobial agents has been sometimes practiced by physicians, especially in

patients infected with highly resistant pathogenic bacteria. However, a great

concern to the usage of combination therapy has been recently increased due to

risks of side effects of drug reactions and interactions with other drugs

administered to those type of patients. Therefore, the need for new class of

compounds possessing a broad spectrum of antibacterial activity that are highly

effective against those highly resistant Gram positive and negative bacteria is

increasing, and it is becoming the top priority of most government heath

institutions and the world health organizations.

In the light of these facts, this current study is aimed to synthesize some novel

1,2,4-triazole derivatives with hope that these 1,2,4-triazole derivatives might

possess a broad spectrum of antibacterial activity against some bacterial isolates

obtained from clinical specimens obtained from infected patients as well as

against some well-known bacterial strains from American type culture collection

(ATCC).

2. Materials and Methods

Synthesis of the new 1,2,4-triazole derivatives

The triazole-hydrazide derivatives 1 (Scheme 1) were synthesized from the

corresponding esters [12]. These derivatives were used as starting materials for

the synthesis of compounds 2, by reaction with the appropriate sulfonyl

chloride (4-nitrophenyl-, 2,5-dichlorophenyl-, 4,7-dimethyl-

2-oxo-2H-chromene-6-, 2-thiophene-, and 3-trifluoromethyl-benzene sulfonyl

chloride) in the presence of pyridine as catalyst in 65-79% yield (Scheme 1). The

structures of the newly prepared compounds were confirmed on the basis of their

306 Jacob H. Jacob et al.

1H- and

13C-NMR spectra in addition to elemental analysis.

SG

O

O

Cl

N

N N

HN

NH2

R2R1

a

N

N N

HN

NH

R2R1

S

2aa-ac

2bb, bd, be

O

O

OG

O

Scheme 1. Reagents and conditions: a. CHCl3, pyridine, r.t., 48 h

NO2

Cl

Cl

OMe O

Me

S

CF3

a

b

c

d

e

G

1a: R1= Me, R2= Et

1b: R1= Et, R2= Et

Chemical and analytical methods

Chemical names follow IUPAC nomenclature. Starting high purity materials were

purchased from Aldrich, Acros, Lancaster, Merck, or Fluka and were used as

received without purification. Melting points were measured on a Mettler FP1

melting point apparatus and are uncorrected. All new compounds were analyzed

for C, H, and N, and the observed results agreed with the calculated percentages

to within ±0.4%. 1H and

13C-NMR spectra were recorded on a Bruker DRX-500

instrument. Chemical shifts are given in parts per million (ppm), and

tetramethylsilane (TMS) was used as internal standard for spectra obtained in

CDCl3. All coupling constants (J) are given in Hertz.

General procedure for synthesis of compounds (2aa-ac, 2bb, 2bd, 2be)

Compound 1 ( 0.5 mmol), reacted with substituted carbonyl chlorides (0.6 mmol)

in chloroform (20 ml) in the presence of pyridine (5 drops) and stirred for 48 h at

room temperature. Then residue was evaporated to dryness and the residue was

partitioned between CHCl3 (40 mL) and water (3 x 40 mL). The organic phase

Synthesis, characterization and evaluation 307

was evaporated to dryness (Na2SO4). The product was then filtered and washed

with diethyl ether to afford compounds 2. Recrystallization from CH2Cl2 / ether.

4-Nitrobenzene sulfonic acid N'[2-(1-ethyl-5-methyl-1H-[1,2,4]triazole -3-yl)-

acetyl]-hydrazide (2aa)

The title compound was prepared from compound 1a (0.092 g, 0.5 mmol), and

4-nitrophenyl sulfonyl chloride (0.133 g, 0.6 mmol) in chloroform (20 ml)

according to the procedure described above. The mixture extracted several times

with water. Then product washed with diethyl ether. Yield: 0.129 g (70 %) as

light brown crystals. m.p. 114-116 °C (dec). 1H-NMR (DMSO): δ 10.34 (bs, 1H,

NH); δ 10.33 (bs, 1H, NH); δ 8.31 (d, J= 8.7 Hz, 2H, Ar); δ 8.04 (d, J= 8.7 Hz,

2H, Ar); δ 4.02 (q, J= 7.2 Hz, 2H, CH2CH3); δ 3.31 (s, 2H, -CH2); δ 2,34 (s, 3H,

-CH3); δ 1.26 (t, 3H, CH2CH3). 13

C- NMR (DMSO): δ 167.6, 156.3, 152.1, 150.2,

145.3, 129.8, 124.4 (Ar-C); 42.8 (CH2CH3); 33.8 (-CH2); 15.2 (CH2CH3); 11.6

(-CH3); Anal. Calcd for C13H17N5O3S (368.4): C, 42.39; H,4.38; N, 22.81.

Found: C, 42.37; H, 4.62; N, 22.68.

2,5-Dichlorobenzene sulfonic acid N'[2-(1-ethyl-5-methyl-1H-[1,2,4]triazole

-3-yl)-acetyl]-hydrazide (2ab)

The title compound was prepared from compound 1a (0.092 g, 0.5 mmol), and

2,5-dichlorophenyl sulfonyl chloride (0.147 g, 0.6 mmol) in chloroform (20 ml)

according to the procedure described above. The mixture extracted several times

with water. Then product washed with diethyl ether. Yield: 0.141 g (72 %) as

milky crystals. m.p. 207-209 °C (dec). 1H-NMR (DMSO): δ 10.27 (bs, 2H,

2×NH); δ 7.91 (bs, 1H, , Ar); δ 7.72-7.63 (m, 2H, Ar); δ 4.01 (q, J= 7.1 Hz, 2H,

CH2CH3); δ 3.31 (s, 2H, -CH2); δ 2,32 (s, 3H, -CH3); δ 1.26 (t, 3H, CH2CH3). 13

C- NMR (DMSO): δ 168.0, 156.2, 152.0, 139.2, 134.3, 133.8, 131.9, 131.2,

130.8 (Ar-C); 42.8 (CH2CH3); 33.7 (-CH2); 15.2 (CH2CH3); 11.6 (-CH3); Anal.

Calcd for C13H15Cl2N5O3S (392.3): C, 39.03; H, 3.85; N, 17.85. Found: C, 39.26;

H, 3.77; N, 17.55.

4,7-Dimethyl-2-oxo-2H-chromene-6-sulfonic acid N'[2-(1-ethyl-5-methyl-1H-

[1,2,4]triazole-3-yl)-acetyl]-hydrazide (2ac)

The title compound was prepared from compound 1a (0.092 g, 0.5 mmol), and

4,7-dimethyl-2-oxo-2H-chromene-6-sulfonyl chloride (0.164 g, 0.6 mmol) in

chloroform (20 ml) according to the procedure described above. The mixture

extracted several times with water. Then product washed with diethyl ether. Yield:

0.136 g (65 %) as milky crystals. m.p. 204-206 °C (dec). 1H-NMR (DMSO): δ

10.21 (bs, 1H, NH); δ 10.04 (bs, 1H, NH); δ 8.13 (s, 1H, Ar); δ 7,39 (s, 1H, Ar);

δ 7.47 (s, 1H, Ar); δ 3.95 (q, J= 7.2 Hz, 2H, CH2CH3); δ 3.27 (s, 2H, -CH2); δ

2,73 (s, 3H); δ 2,45 (s, 3H); δ 2,25 (s, 3H); δ 1.23 (t, 3H, CH2CH3). 13

C- NMR

(DMSO): δ 167.3, 159.2, 155.7, 155.1, 152.7, 151.4, 142.9, 133.0, 127.2, 119.8,

116.9, 114.4 (Ar-C); 42.8 (CH2CH3); 33.0 (-CH2); 20.2; 17.8; 14.7 (CH2CH3);

11.0. Anal. Calcd for C18H21N5O5S (419.5): C, 51.54; H, 5.05; N, 16.70.

308 Jacob H. Jacob et al.

Found: C, 51.45; H, 5.26; N, 16.43.

2,5-Dichlorobenzene sulfonic acidN'[2-(1,5-diethyl-1H-[1,2,4] triazole-3-yl)-

acetyl]-hydrazide (2bb)

The title compound was prepared from compound 1b (0.099 g, 0.5 mmol), and 2,

5-dichlorophenyl sulfonyl chloride (0.147 g, 0.6 mmol) in chloroform (20 ml)

according to the procedure described above. The mixture extracted several times

with water. Then product washed with diethyl ether. Yield: 0.159 g (78 %) as

light yellow crystals. m.p. 222-224 °C (dec). 1H-NMR (DMSO): δ 10.33 (bs,

2H, 2×NH); δ 7.90-7.89 (bs, 1H, Ar); δ 7.72-7.64 (m, 2H, Ar); δ 4.01 (q, J= 7.2

Hz, 2H, N-CH2CH3); δ 3.32 (s, 2H, -CH2); δ 2,67 (q, J= 7.5 Hz, 2H, C-CH2CH3);

δ 1.28 (t, 3H, N-CH2CH3); δ 1.16 (t, 3H, C-CH2CH3). 13

C- NMR (DMSO): δ

168.0, 156.5, 156.3, 139.2, 134.4, 133.8, 131.9, 131.2, 130.7 (Ar-C); 42.6

(N-CH2CH3); 33.8 (-CH2); 18.7 (C-CH2CH3); 15.4 (C-CH2CH3); 12.2

(N-CH2CH3); Anal. Calcd for C14H17Cl2N5O3S (406.3): C, 41.39; H, 4.22; N,

17.24. Found: C, 41.56; H, 4.07; N, 17.04.

2-Thiophene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2,4]triazole-3-yl)-acetyl]

-hydrazide (2bd)

The title compound was prepared from compound 1b (0.099 g, 0.5 mmol), and

2-thiophene sulfonyl chloride (0.110 g, 0.6 mmol) in chloroform (20 ml)

according to the procedure described above. The mixture extracted several times

with water. Then product washed with diethyl ether. Yield: 0.136 g (79 %) as

white crystals. m.p. 220-222 °C (dec). 1H-NMR (DMSO): δ 10.31 (bs, 1H, NH);

δ 10.07 (bs, 1H, NH); δ 7.95-7.08 (m, 3H, Ar); δ 4.03 (q, J= 7.2 Hz, 2H,

N-CH2CH3); δ 3.34 (s, 2H, -CH2); δ 2,69 (q, J= 7.5 Hz, 2H, C-CH2CH3); δ 1.27

(t, 3H, N-CH2CH3); δ 1.18 (t, 3H, C-CH2CH3). 13

C- NMR (DMSO): δ 167.4,

156.5, 156.4, 134.3, 133.8, 128.0 (Ar-C); 42.6 (N-CH2CH3); 33.9 (-CH2); 18.7

(C-CH2CH3); 15.5 (C-CH2CH3); 12.2 (N-CH2CH3); Anal. Calcd for

C12H17N5O3S2 (343.4): C, 41.97; H, 4.99; N, 20.39. Found: C, 42.24; H, 5.26; N,

20.10.

3-Trifluoromethyl-benzene sulfonic acid N'[2-(1,5-diethyl-1H-[1,2,4]triazole

-3-yl)-acetyl] -hydrazide (2be)

The title compound was prepared from compound 1b (0.099 g, 0.5 mmol), and

3-trifluoromethyl-benzenesulfonyl chloride (0.174 g, 0.6 mmol) in chloroform (20

ml) according to the procedure described above. The mixture extracted several

times with water. Then product washed with diethyl ether. Yield: 0.156 g (77 %)

as white crystals. m.p. 193-196 °C (dec). 1H-NMR (DMSO): δ 10.38 (bs, 1H,

NH); δ 10.29 (bs, 1H, NH); 8.10-8.01 (m, 3H, Ar); δ 7.73 (t, 1H, Ar); δ 4.01 (q,

J= 7.2 Hz, 2H, N-CH2CH3); δ 3.30 (s, 2H, -CH2); δ 2,68 (q, J= 7.5 Hz, 2H,

C-CH2CH3); δ 1.26 (t, 3H, N-CH2CH3); δ 1.18 (t, 3H, C-CH2CH3). 13

C- NMR

(DMSO): δ 167.5, 156.5, 156.3, 141.0, 132.1, 130.7, 130.1, 130.0, 124..5, 124.4

(Ar-C); 42.6 (N-CH2CH3); 33.9 (-CH2); 18.7 (C-CH2CH3); 15.4 (C-CH2CH3);

12.2 (N-CH2CH3); Anal. Calcd for C15H18F3N5O3S (405.4): C, 44.44; H, 4.48; N,

17.28. Found: C, 44.43; H, 4.40; N, 17.50.

Synthesis, characterization and evaluation 309

Antibacterial activity testing

Bacterial strains

The bacterial strains used in this study originate from two sources: ATCC and

medical samples. ATCC strains include two Gram-positive bacteria

Staphylococcus aureus ATCC29213 and Bacillus cereus ATCC11778. Those

standard strains were kindly offered by Dr. Emad Malkawi from the department

of biological sciences at Yarmouk University, Jordan. The medical strains were

isolated from the stool and the ear exudates of infected persons. Isolates were

obtained from the clinical microbiology laboratories at Al-Mafraq hospital in

Al-Mafraq city, Jordan. These two bacterial strains were designated as GN1 and

GN2. The two isolates were first identified by Gram staining and they were then

subjected to further identification using the biochemical and molecular methods.

Briefly, isolated bacteria were tested for oxidase [13]. Oxidase test is a prerequisite

to conduct the biochemical identification using RapID ONE system and ERIC

software (Remel, USA) which were used to identify the isolated Gram-negative,

oxidase-negative strains to the species level. GN1 strain was identified by the

aforementioned method, while GN2 could not be identified by the previously

mentioned approach due to positive oxidase test. Therefore, the GN2 strain was

identified by the molecular methods, namely, 16S rDNA sequencing and analysis.

In this method, DNA was extracted from the isolate and subjected for 16S rDNA

sequencing as previously described [14]. 16S rDNA sequencing was carried out

by Macrogen sequencing facility (MacrogenInc, Seoul, South Korea) following

the procedure mentioned previously by Jacob and Irshaid, 2012 [14]. The

resulting nucleotide sequences were then analyzed by the Basic Local Alignment

Search Tool (BLAST) search to identify the closest relatives of our strains.

Agar well diffusion and broth dilution methods

Ten milligrams of each newly synthesized triazole derivative were dissolved in 1

mL of dimethyl sulfoxide (DMSO). DMSO was used as negative control, whereas

penicillin G (10 mg mL-1

) was used as a positive control. All test chemicals and

controls were stored in dark at room temperature until use.

Antibacterial activity of the new compounds was first evaluated by well diffusion

method [15]. Briefly, Mueller-Hinton agar (MHA) medium was used to prepare

MHA agar plates. MHA plates were inoculated with the tested bacterium. Six

millimeter holes were punched in the MHA plates. Then, each well was filled

with 50 μL of one of each synthesized triazole (10 mg mL-1

). Penicillin G (10 mg

mL-1

) was used as positive control and DMSO was used as negative control. After

incubation, the average diameter of inhibition zone around each well was

measured to the nearest millimeter. Each experiment was done in triplicate. To

determine the MIC values of each active compound, mixtures of nutrient broth,

inoculums, and test compounds were prepared in a microwell plate to yield the

following final concentrations: 3000, 2000, 1000, 500, and 250 µg mL-1

.

310 Jacob H. Jacob et al.

Penicillin G and DMSO were used as positive control and negative control,

respectively. The MIC value is the lowest dilution of the test compound which

inhibit the growth judged by lack of turbidity in the well. Each experiment was

done in triplicate.

3. Results

Synthesis and identification of the new 1,2,4-triazole derivatives

Six new 1,2,4-triazole derivatives have been synthesized using substituted

carbonyl chlorides in chloroform in the presence of pyridine and stirred for 48 h at

room temperature. These six 1,2,4-triazole compounds differ in their side group

substituent's on the triazole ring. The structures of these six 1,2,4-triazole

compounds were confirmed by 1H-NMR, 13C-NMR, and elemental analysis

(Figure 1). Then, these compounds were investigated for their potential

antibacterial activity.

Figure 1: Chemical structure and IUPAC names of the newly synthesized

1,2,4-triazole derivatives.

Isolation and identification of new medical bacterial strains

Two new bacterial strains were collected from the stool and the ear exudates of

infected human. These two strains were isolated and identified by biochemical

and molecular methods and used in this study. These isolates were assigned as

GN1 and GN2. GN1 are characterized by being Gram-negative and

oxidase-negative. GN1 was further identified based on biochemical analysis as

Shigella sp. The other main biochemical properties of strain GN1 are shown in

Table 1. On the other hand, strains GN2 was identified only after sequencing the

16S rDNA. The 16S rDNA sequence of the newly isolated strains GN2 is shown

in Figure 2. The closest relatives of the isolated strains is Pseudomonas

aeruginosa with 99% identity according to BLAST search.

GGGGAAACGTTATCGGAATACTGGGGCGTAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGC

GTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGG

AGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCG

CACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCA

GAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAA

ACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTA

CAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG

TCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCGCAAGGGGGACGGTTACCACGGAGTGATTCATGA

CTGGGGTGAATCTAAGGGGGGGGGCCCGCATAAAAAAAAAAAAAAAATCCGGGGGGTTTT

Synthesis, characterization and evaluation 311

Table 1: The qualitative results of biochemical analysis of strain GN1 (identified

as Shigella sp.) by RapID ONE system and ERIC software (probability: 91.26%).

Abbreviations of chemical tests, URE: hydrolysis of urea, ADH, hydrolysis of arginine, ODS:

hydrolysis of ornithine, LDC: hydrolysis of lysine, TET: utilization of aliphatic thiol, LIP:

hydrolysis of the fatty acid ester, KSF: utilization of sugar aldehyde, SBL: utilization sorbitol,

GUR: hydrolysis of ρ-Nitrophenyl-β,D-glucoronide, ONPG: hydrolysis of

ρ-Nitrophenyl-β,D-galactoside, BGLU: hydrolysis of ρ-Nitrophenyl-β,D-glucoside, BXYL:

ρ-Nitrophenyl-β,D-xyloside, NAG: ρ-Nitrophenyl-nacetyl-β,D-glucoseaminide, MAL: utilization

of malonate, PRO: hydrolysis of proline-β-naphthylamide, GGT: hydrolysis of

γ-glutamyl-β-naphthylamide, PYR: hydrolysis of pyrrolidonyl-β-naphthylamide, ADON:

utilization of adonitol, and IND: utilization of tryptophane.

Figure 2: Partial 16S rRNA gene sequence obtained from strain GN2 identified as

Pseudomonas aeruginosa with 99% identity according to BLAST search.

Antibacterial activity of the new 1,2,4-traizole derivatives

The new 1,2,4-traizole derivatives (2aa, 2ab, 2ac, 2bb, 2bd, and 2be) were tested

for their antibacterial activity by two methods: agar well diffusion method and

broth dilution method. Figure 3 shows the results obtained from agar well

diffusion method. As shown in the figure, the compound 2aa was active against

two strains: Shigella sp., and B. cereus. The highest activity was observed against

B. cereus (Inhibition zone: 15 mm). Additionally, 2ab was active against three

strains. The susceptible strains are: P. aeruginosa, S. aureus, and B. cereus.

Shigella sp. was resistant to this compound. It was also found that 2ac is active

Test

UR

E

AD

H

OD

S

LD

C

TE

T

LIP

KS

F

SB

L

GU

R

ON

PG

BG

LU

BX

YL

NA

G

MA

L

PR

O

GG

T

PY

R

AD

ON

IND

Strain GN1

- - - - - - - + - + - - - - + - - - -

0

5

10

15

20

25

30

35

40

S. aureus ATCC29213

B. cereus ATCC11778

P. aeruginosa(GN2)

Shigella sp.(GN1)

Inh

ibiy

ion

Zo

ne

(m

m)

Bacterial Strain

2aa

2ab

2ac

2bb

2bd

2be

Penicllin G

312 Jacob H. Jacob et al.

against three bacterial strains Shigella sp., B. cereus, and the clinical isolate P.

aeruginosa. The compound was most active against P. aeruginosa (Inhibition

zone: 16 mm). 2bb was active against B. cereus but not active against P.

aeruginosa, S. aureus, and Shigella sp. Regarding 2bd, the compound was

active against two strains: P. aeruginosa and B. cereus. S. aureus and Shigella

sp. were resistant to this compound. 2be was active against B. cereus but not

against P. aeruginosa, S. aureus, and Shigella sp. (Figure 3).

The activity of penicillin G (the positive control) against the test strains is also

shown in Figure 3. The positive control was not active against P. aeruginosa, but

active against B. cereus (Inhibition zone: 10 mm), S. aureus (Inhibition zone: 27

mm), and Shigella sp. (Inhibition zone: 35 mm).

Figure 3: Antibacterial activity of the newly synthesized triazole compounds and

the positive control (penicillin G) against Gram positive and Gram negative

bacteria. Antibacterial activity expressed as inhibition zone diameters. Values are

given as means ± SE (n = 3).

Regarding the MIC values of the active compounds, it was found that the MIC

value of 2aa compound against the sensitive strains B. cereus and P. aeruginosa is

750 µg mL-1

(Figure 4). The highest MIC value of this compound was 1250 µg

mL-1

against Shigella sp. 2ab was active against P. aeruginosa and B. cereus with

MIC value of 500 µg mL-1

, and the compound has lower activity against S. aureus

with higher MIC value (2000 µg mL-1

) (Figure 4). The MIC values of 2ac

compound against the sensitive strains were also determined. MIC value of the

compound against the clinical isolate P. aeruginosa was found to be 750 µg mL-1

.

The MIC value against B. cereus was 1000 µg mL-1

. The MIC value against

Shigella sp. was 1000 µg mL-1

. The MIC value of 2bb against B. cereus was also

0

500

1000

1500

2000

2500

S. aureus ATCC29213

B. cereus ATCC11778

P. aeruginosa(GN2)

Shigella sp.(GN1)

MIC

(µ

g/m

l)

Bacterial Strain

2aa

2ab

2ac

2bb

2bd

2be

Penicllin G

Synthesis, characterization and evaluation 313

high as compared to other values with 2000 µg mL-1

. The activity of 2bd against

P. aeruginosa and B. cereus was high with 500 µg mL-1

as MIC value. Finally,

2be was intermediate in antibacterial activity with 1000 µg mL-1

against B. cereus.

Figure 4: The comparative MIC values (µg mL-1

) of the newly synthesized

triazole compounds and (penicillin G) against the against Gram positive and Gram

negative bacteria.

Some compounds were found to have the same antibacterial activity of penicillin

G against certain strains. For instance, 2ab, 2bd, and 2be created the same

inhibition zone as penicillin G against B. cereus (Inhibition zone of 10 mm).

Interestingly, some compounds were more active than penicillin G against certain

strains. For instance, the activity of 2aa against B. cereus was higher than the

activity of penicillin G (Figure 2). Also, 2ab, 2ac, and 2bd were more active than

penicillin G when tested against the clinical isolate P. aeruginosa (Figure 2). The

inhibition zones of 2ab was 13 mm, and in case of 2ac was 16 mm, and in case of

2bd was 15 mm. However, no inhibition zone was detected in case of penicillin G

was against P. aeruginosa.

4. Discussion

The presence of highly resistant pathogenic bacteria that are resistant to

commercial available antibacterial agents led scientists to look for more potent or

effective antibacterial agents. Therefore, this study was undertaking to synthesize

six 1,2,4-triazole compounds with hope that these derivatives may exhibit potent

antibacterial activity against some bacterial isolates obtained from clinical

314 Jacob H. Jacob et al.

specimens and some bacterial strains obtained from American type culture

collection.

In this study, six 1,2,4-triazole derivatives were newly synthesized and tested for

their antibacterial activity against different Gram-positive and Gram-negative

bacterial strains. The bacterial strains used in this study include standard bacterial

strains as well as clinical bacterial strains. Bacteria from clinical samples were

newly isolated from stool and ear exudate of infected persons. They were

identified using biochemical and molecular identification techniques, and their

susceptibility to the new 1,2,4-triazole derivatives were then evaluated.

Two different types of bacterial strains (i.e., standard and clinical strains) were

included in this study to include pathogenic bacteria of medical importance. The

first medical bacterial isolate represents a new strain in the genus Shigella.

Shigella sp. is the etiologic agent of human shigellosis [16]. It has been estimated

that Shigella sp. can causes about 90 million cases of serious dysentery with at

least 100 thousands of these resulting in death each year, particularly among

children in the developing world [17].

The second medical isolate represents a new strain of P. aeruginosa which is an

opportunistic bacterium that can cause serious infections in human [18]. P.

aeruginosa is a well known as opportunistic pathogen of human being that can

cause a variety of infections, including urinary tract infections, respiratory system

infections, gastrointestinal infections, bacteremia, and other systemic infections

[19,20]. P. aeruginosa infection can be very serious or sometimes life threatening

to human, particularly in patients with severe burns, cancer, cystic fibrosis, AIDS

as well as immunosuppressed patients.

Interestingly, P. aeruginosa was already resistant to pencillin G (the positive

control) but sensitive to three of the newly synthesized triazole compounds

(compounds 2ab, 2ac, and 2bd). In facts, numerous epidemiological studies

reported that antibiotic resistance is increasing among various bacterial species

especially in clinical isolates. This justifies the need to include newly isolated

clinical bacterial strains in addition to standards strains. The failure of known

antibiotics to control pathogens can be attributed to several factors. For instance,

the emergence of novel pathogens in human populations [21], re-emergence of

pathogens [22], acquisition of new virulence traits as a result of adaptation to new

hosts or acquisition of plasmids, conjugative transposons and integrons containing

various resistance genes [22].

Previous studies showed that 1,2,4-triazole derivatives are important group in the

field of medicinal chemistry due to their diverse biological properties [23]. The

biological activity of triazoles is highly dependent on the chemical side groups

that may enhance their activity. In this study, six 1,2,4-traizole derivatives were

newly synthesized: 2aa, 2ab, 2ac, 2bb, 2bd, and 2be. These compound differ in

their side groups. In the next section, we will further discuss the effect of side

groups on triazole activity.

Synthesis, characterization and evaluation 315

The 2aa compound has a nitrobenzene sulfonic acid as substituent group (Figure

1). 2aa showed antibacterial activity against one Gram negative bacteria (Shigella

sp.) and one Gram positive bacterium (B. cereus). Presence of nitrobenzene in

triazoles seem to enhance the antibacterial activity of trizaoles as documented in

previous studies [24].

The 2ab compound was almost the broadest in its activity spectrum. It was active

against three bacterial strains out of four, and it was more active than pencillin G

in case of P. aeruginosa. 2ab contains dichlorobenzene sulfonic acid. Presence of

chlorine in the structure is the most distinguished feature when comparing 2ab to

2aa. Presence of chlorine is expected to enhance the antibacterial activity because

due to its moderately oxidative effect and can react with various components of

the bacterial cell [25].

The distinguished substituent group in compound 2ac is chromene which is a

polycyclic organic structure that results from fusion of a benzene ring to a

heterocyclic pyran ring. Previous studies showed that heterocyclic compounds

containing chromene have an antimicrobial activity against both Gram positive

and negative bacteria [26]. This conclusion agrees with our results which showed

that the compound 2ac was active against two Gram negative bacteria, namely,

Shigella sp. and P. aeruginosa, as well as one Gram positive bacterium, B. cereus.

2bb contains a dichlorobenzene sulfonic acid as in 2ab. However, contrary to 2ab

which contains two ethyl group bonded to triazole ring, 2bb contains one methyl

and one ethyl group attached to triazole ring. Presence of ethyl group instead of

methyl group dramatically decreases the antibacterial activity of the triazole

compound when 2bb and 2ab are compared. 2be contains triflouromethyl benzene.

The distinguished feature in 2be is the presence of fluoride. The inhibitory effect

of fluoride on bacteria is well established [27]. Fluoride can inhibit the

metabolism of bacteria through different mechanisms like enzyme inhibitor or

increases the acid sensitivity of bacteria [27].

In conclusion, the 2aa triazole derivative was more active than penicillin G when

tested against B. cereus, and 2ab and 2ac triazole compounds were also more

active than penicillin G when tested against P. aeruginosa. Thus, it is possible to

anticipate that these novel triazole compounds are highly efficient against most

clinically important species of Gram negative and positive bacteria that are highly

resistant to current antibacterial agents and associated with serious and

life-threatening infections. These findings might hold promise for development of

a new class of a novel expanded spectrum antibiotic particularly from 2aa, 2ab

and 2ac triazole compounds. It is also possible to suggest that these three triazole

compounds appear to be good candidates for further clinical investigations in vivo.

Therefore, we recommend that clinical trials might be carried out to evaluate the

possibility of using them as an alternative to penicillin G for the treatment of

infections caused by these two microorganisms as well as other bacterial species.

In addition, their pharmacodynamic and pharmacokinetic properties must also be

studied.

316 Jacob H. Jacob et al.

Acknowledgments

The authors would like to thank the deanship of academic research and the

deanship of graduate studies at Al al-Bayt University, Jordan, for financial

support.

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Received: March 15, 2013