Background: 1,2,4-Triazole and its derivatives are important group of heterocyclic compounds characterized by a five-membered ring of two carbons and three nitrogen atoms and exhibited good pharmacological profile. A new class of 3,4,5-substituted-1,2,4-triazole scaffolds was synthesized and screened for its antimicrobial and analgesic activity.

Results and discussion: The synthesized 3,4,5-substituted-1,2,4-triazole derivatives were confirmed by 13C/1H-NMR and IR spectral data and evaluated its antimicrobial and analgesic activity. The synthesized derivatives showed good antimicrobial potential against Gram positive and Gram negative bacterial and fungal strains by tube dilution technique and analgesic activity by Tail immersion and Acetic acid induced writhing method and compared with standard drugs.

Conclusion: Especially compounds TA-11, TA-20 and TA-26 displayed more antimicrobial activity than standard drugs (cefadroxil (antibacterial) and fluconazole (antifungal) due to presence of electron withdrawing group. The compounds TA-13, TA-15 and TA-2, TA-15 exhibited good analgesic activity by Tail immersion and Acetic acid induced writhing method respectively due to presence of electron withdrawing group.

1,2,4-Triazoles; Antibacterial; Antifungal; Analgesic

The growing problem of antimicrobial resistance has induced additional urgency to design and develop novel types of antimicrobial agents with chemical structures different from the traditional drugs and improved the different types of biological activities. Heterocycles containing a 1,2,4-triazole or 1,3,4-thiadiazole moiety and the compounds consisting of 1,2,4-triazole and 1,3,4-thiadiazole condensed nucleus systems constitute a class of compounds possessing a wide spectrum of biological activity [1]. 1,2,4-Triazole and its derivatives are important group of heterocyclic compounds characterized by a five-membered ring of two carbons and three nitrogen atoms. 1,2,4-Triazole ring systems have been well studied and so far a variety of biological activities have been reported for a large number of their derivatives, such as anti-inflammatory, antianxiety, antimicrobial, antitubercular, antimycobacterial, anticancer, analgesic, diuretic and hypoglycemic properties [2].

The analgesic activity of opioids has been demonstrated in multiple species, including rodents and humans. In animal studies, the analgesic actions of these drugs are readily seen using a variety of analgesic assays, including the radiant heat tail-flick assay, which is one of the earliest paradigms used to assess analgesic drugs. Opioids produce their effects by activating specific opioid receptors. There are three major classes of opioid receptors, each with its own ligand selectivity. Most clinical drugs work through morphine-like receptors. Hydrocodone is a well-established opioid analgesic. Ibuprofen, a nonsteroidal anti-inflammatory drug is widely used for mild to moderate acute and chronic pain. Unlike opioids that have no ceiling effects on their analgesic activity, NSAID analgesia does display a ceiling effect and when given alone, has limited activity against severe pain. Opioids are often used in combination with NSAIDs and a number of products containing combinations are currently used widely in the management of pain. Preclinical studies have suggested interactions between NSAIDs in neuropathic and inflammatory pain models. In a model of chronic neuropathic pain, systemic ketorolac or piroxicam was found to synergize with spinal morphine. Others have observed interactions between opioids and NSAIDs in a range of inflammatory models where the NSAIDs would be expected to be active [3].

It was found that 1,2,4-triazole and its derivatives have diversified pharmacological activities, like synthesized 2,4-dihydro-1,2,4-triazol-3-one derivatives and found that it possesses antibacterial activity, synthesized 1-[(?-alkoxy-?-dialkylamino)propyl-1H-1,2,4-triazole and reported their antifungal activity, synthesized 5-phenyl-1-(3-pyridyl)-1H-1,2,4-triazole-3-carboxylic acid derivatives and found their anti-inflammatory activity, synthesized 3,4-disubstituted-5-mercapto-1,2,4-triazole derivatives and reported their analgesic activity, synthesized 4-benzylidenamino-4,5-dihydro-1H-1,2,4-triazole-5-ones and found their anti-oxidant activity, synthesized 3-[4-(3H)-quinazolinone-2-yl)thiomethyl]-1,2,4-triazole-5-thiols and reported their insecticidal activity, synthesized N`-[1-aryl-2-(1H-imidazol-1-yl and 1H-1,2,4-triazol-1-yl)-ethylidine]-pyridine-2-carboxami drazone derivatives and found their antitubercular activity [4-10].

The 1,2,4-triazole ring is a main pharmacophoric group responsible for antimicrobial and analgesic activity. Literature review revealed that di-substitution at 4 and 5 positions of 1,2,4-triazole-3-thiones is responsible for various biological activity. The marketed drugs containing 1,2,4-triazole ring as nucleus exhibiting antimicrobial activity are fluconazole, isavuconazole, anastrozole, letrozole, voriconazole (Figure 1).

Keeping this observation in mind, this work has presented the synthesis, antimicrobial, and analgesic activity of novel 3,4,5-substituted-1,2,4-triazole scaffolds.

Chemistry

In the study, we have reported a simple synthetic method for the synthesize of new class of 3,4,5-substituted-1,2,4-triazole derivatives (Scheme-1).

Scheme 1 (a): Synthesis of 3,4,5-substituted-1,2,4-triazole derivatives (TA-1 to TA-24)

Scheme 1 (b): Synthesis of 3,4,5-substituted-1,2,4-triazole derivatives (TA-25 to TA-27)

The procedure steps of the synthesized compounds were showing in experimental section. The molecular structures of the newly synthesized compounds were confirmed by 13C/1H-NMR, FT-IR spectral, Mass and elemental analysis and physicochemical properties (Table 1).

Table 1: The physicochemical properties of synthesized 3,4,5-substituted-1,2,4-triazole derivatives.
*TLC mobile phase: benzene; Chloroform:nHexene

The elemental analysis results of the newly synthesized compounds were within ± 0.39% of the theoretical values.

The chemical structures of the synthesized 3,4,5-substituted-1,2,4-triazole derivatives (TA-1 to TA-27) were confirmed by FT-IR (KBr pellets, cm-1) and 1H/13C-NMR (CDCl3, ? ppm) spectral, mass and elemental studies. The appearance of IR absorption band at 3467-3331 cm-1 in the spectral data of synthesized derivatives (TA-3 toTA-6, TA-9, TA-12, TA-15, TA-18, TA-21, TA-24 and TA-27) displayed the presence of -OH group. The existence of Ar-NO2 group symmetric and asymmetric Ar-NO2 stretches in the scale of 1388-1334 cm-1 and 1519-1511 cm-1 respectively. The impression of IR stretching vibration at 3162-3120 cm-1 and 1573-1511 cm-1 in the spectral data of synthesized compounds specified the existence of C-H and C=C group respectively. The appearance of IR stretching 1596-1511 cm-1 and 1384-1314 cm-1 in the spectral data of all synthesized compounds specified the existence of C=N and C-N groups respectively. The IR absorption band in the scale of 646 cm-1 to 621 cm-1 corresponds to the C-Br stretching of aromatic-bromo compounds (TA-2, TA-5, TA-8, TA-11, TA-14, TA-17, TA-20, TA-23 and TA-26). The impression of IR stretching vibration at 867-811 cm-1 and 1846-1723 cm-1 in the spectral data of synthesized compounds specified the existence of C-S and C=O groups respectively. The IR absorption band in the scale of 741 cm-1 to 719 cm-1 corresponds to the C-Cl stretching of aromatic-Chloro compounds (TA-7 to TA-11, TA-13 toTA-15 and TA-25 to TA-27). The IR absorption band in the scale of 634 cm-1 to 623 cm-1 corresponds to the C-I stretching of aromatic-Iodo compounds (TA-19 to TA-21). The impression of IR stretching vibration at 2887-2816 cm-1 in the spectral data of synthesized compounds specified the existence of C-H group of -CH2. The multiplet signals between 6.23-8.91 ? ppm in 1H-NMR spectra is indicative of aromatic proton of synthesized derivatives. The synthesized derivatives (TA-3, TA-6, TA-9, TA-12, TA-15, TA-18, TA-21, TA-24 and TA-27) showed singlet at 9.01-11.21 ? ppm due to the existence of OH group of COOH. Compounds TA-4, TA-5 and TA-6 showed singlet at 5.20-5.36 ? ppm due to the existence of Ar-OH group. All compounds showed singlet at 3.87-4.19 ? ppm due to the existence of CH2. Compounds, TA-1, TA-4, TA-7, TA-10, TA-13, TA-16, TA-19, TA-22 and TA-25 showed singlet at 2.45-2.67 ? ppm due to existence of -CH3. The elemental screened studies of the 3,4,5-substituted-1,2,4-triazole derivatives were found within ± 0.39 % of the theoretical results.

Antimicrobial activity

Antimicrobial activity results indicated that in the case of Gram +ve bacterial study, compound TA-20 was found to be most potent against S. aureus with MIC value = 0.45 µM/mL and compound TA-26 was found to be most potent against B. subtilis with MIC value = 0.23 µM/mL. In the case of Gram-ve bacterial study, compound TA-20 (MICec = 0.22 µM/mL) was the most active one against E. coli. The antifungal activity results indicated that compounds TA-11 (MICca = 0.26 µM/mL) and compound TA-20 (MICan= 0.45 µM/mL) were found to be most effective ones against C. albicans and A. niger respectively. The antimicrobial activity result of synthesized compounds is comparable to the reference drugs, ciprofloxacin (antibacterial) and fluconazole (antifungal) studies.

Analgesic activity

Analgesic activity by Tail immersion method (for central action)

The study was carried out on healthy male Wister albino rats weighing between 70-120 g. The animals were divided into number of groups, each groups contain six animals. The animals were subjected to fasting overnight before administration. Ibuprofen used as standard drug: dose of 20 mg/kg body weight of rats was made in acacia suspension. Selected 1,2,4-triazole derivatives: dose of test compounds was 20 mg/kg body weights of rats. Control acacia suspension: 3.0 % w/v suspension was made in distilled water. Routes of administration: the test compounds and standard drugs were suspended in 3.0 % w/v acacia suspension was given by oral route by intubation. The Rats were divided into 11 groups, containing six animals in each group. These animals were fasted overnight, prior to the experiment. Animals of group-1 were considered as control and administered with 3 % acacia suspension. Animals of group-2 were treated with standard drug, i.e., Ibuprofen (20 mg/kg), which were considered as standard group. Animals of group 3-11 were treated with nine different synthesized novel drugs (20 mg/kg) respectively. The tolerance latency of each rat were recorded at time interval of 15 min, 30 min, 45 min, 60 min, 90 min and 120 min after the administration of test drugs by using tail immersion in hot water which was maintained at 51 oC. The percent analgesic activity (PAA) was calculated by the following formula: PAA = (T2- T1/T1) × 100

T1 is the basal latency (i.e. before any drug treatment) and T2 is the tolerance latency (i.e. after drug treatment) [12].

Statistical analysis

All the results were expressed as mean ± standard error of mean (SEM). Data was analyzed using student t-test and critical range for significance difference between two groups of observations was taken as p< 0.05, p<0.01 and P<0.001.

There was no increase in percent analgesic activity in control group. There was significant increase in percent analgesic activity till 60 min after that there was decrease in percent analgesic activity in standard drug group. There was increase in percent analgesic activity after 15 min, the group showed good activity after 45 min there after there was decrease in percent analgesic activity for a group administered with compound TA-1. The presented results are indicated in (Table 3)

Table 3: Percent analgesic activity by Tail immersion method

The group-4 administered with compound TA-2, showed good activity at 45 min after there was decrease in percent analgesic activity for a group. The onset of action was significantly seen after 15 min and drug action was remaining till 45 min, after that there was decrease in percent analgesic activity for compound TA-4. There was significantly increase in percent analgesic activity after till 45 min there after there was decrease in percent analgesic activity for a group administered with compound TA-7. The moderate increase in percent analgesic activity was seen with compound TA-10. Compound TA-13 showed good analgesic effect and after 1 h analgesic effect was started to decrease. The moderate increase in percent analgesic activity was seen with compound TA-15, TA-17 and TA-18.

Analgesic activity by acetic acid induced writhing method (for peripheral action): 

Figure 2: Percent analgesic activity by acetic acid-induced writhing method

when compared with standard. Compounds TA-1, TA-4 and TA-7 were found to have moderate analgesic activity, while compounds, TA-2 and TA-15 were found to be good analgesic activity as compared to standard drugs. All the compounds showed appreciable analgesic activity.

SAR (Structure Activity Relationship) studies

From the antimicrobial and analgesic activities results of the synthesized 3,4,5-substituted-1,2,4-triazole analogues, the subsequent structure activity relationship can be derived in (Figure 3).

Figure 3: Structure activity relationship of the synthesized 3,4,5-substituted-1,2,4-triazole analogues

1. Presence of 1-(4-bromophenyl)propan-1-one group (Compounds TA-11, TA-20 and TA-26) enhanced the antimicrobial potential of the synthesized compounds against C. albicans, B. subtilis, S. aureus, A. niger and E. coli..

2. Presence of electron withdrawing group (-Cl, Compounds TA-11 and TA-26) on benzylidene portion improved the antimicrobial activity of the synthesized compounds against C. albicans and B. subtilis respectively and (-I, Compound TA-20) on benzylidene portion improved the antimicrobial activity of the synthesized compounds against S. aureus, A. niger and E. coli.

3. Presence of electron withdrawing groups (-Br, Compound TA-2 and –Cl, Compound 15) at para position of benzylidene portion improved the analgesic activity by acetic acid induced writhing method and (-Cl, Compound TA-2 and Compound TA-15) at para position of benzylidene portion improved the analgesic activity by Tail immersion method 

All the chemicals and reagents employed for the synthetic work were purchased from Loba Chemie Lab. Pvt Lmt, Mumbai; HiMedia Laboratories Pvt Ltd, Mumbai; Sisco Research Laboratory, Mumbai and Central Drug House, Pvt Ltd, Mumbai; Thomas Baker (Chemicals) Pvt. Ltd, Mumbai. Melting points were determined by open capillary method with a Lab Tech-Melting Point Apparatus and were uncorrected. IR spectra study of the compounds was recorded on a Shimadzu FT-IR-8300 spectrophotometer. The Mass spectral data were confirmed by Waters Micromass Q-ToF Micro instrument. 1H-NMR/13C-NMR spectra study of the compounds was recorded on an Agilent NMR 4300 MHz spectrometer using DMSO solvent. Chemical shift values are given in d scales. Elemental analyses were performed on a Flash 2000 series CHNS–O analyzer. The completion of the reaction was checked by thin layer chromatography (TLC) on silica gel coated aluminum sheets (Silica Gel 60 F254).

Procedure of 3,4,5-Substituted-1,2,4-triazole derivatives (TA-1 to TA-27)

Step i: Synthesis of ethyl substituted benzoate/ethyl 2-(2,4-dichlorophenoxy)acetate

A mixture of 0.1 mole of pure substituted benzoic acid/ 2-(2,4-dichlorophenoxy)acetic acid, 0.13 mole of conc. sulphuric acid, 0.4 mole of absolute ethanol were taken in a 500 mL of round bottom flask and refluxed on heating mantle at temperature 60oC for 4-5 h and checked by TLC and then allowed to cool after that poured the residue into about 250 mL of water in a separating funnel and rinsed the flask with a few mL of water which were also poured into separating funnel and shook the mixture in the funnel vigorously; upon standing the ethyl ester of aryl carboxylic acid separated sharply and rapidly at the bottom of the separating funnel. Lower layer of ester was collected at the bottom and upper aqueous layer was rejected, returned the ethyl ester of aryl carboxylic acid to the beaker and shook with a strong solution of potassium hydrogen carbonate until all free acid was neutralized and no further evolution of carbon dioxide occurred. Washed once with water, and dried by pouring into a dry china dish containing about 2 gm of magnesium sulphate. 

Step ii: Synthesis of substituted benzohydrazide/2-(2,4-dichlorophenoxy)acetohydrazide

A mixture of 0.1 mole of ethyl substituted benzoate/ethyl 2-(2,4-dichlorophenoxy) acetate and 0.116 moles of hydrazine hydrate was refluxed with 25.5 mL of absolute ethanol for 6-7 h and checked by TLC. Then excess of ethanol was distilled off, the reaction mixture was cooled and then substituted benzohydrazide/2-(2,4-dichlorophenoxy)acetohydrazide was purified by dissolving in small quantity of ethanol after that the excess of ethanol was distilled off and the product was dried and recrystallized with ethanol. 

Step iii: Synthesis of 1-(substituted benzoyl)thiosemicarbazide/1-(2-(2,4-dichlorophenoxy) acetyl)thiosemicarbazide

A mixture of 0.1 mole of substituted benzohydrazide/2-(2,4-dichlorophenoxy) acetohydrazide and 0.12 moles of potassium thiocyanate was taken in a 500 mL of round bottom flask. 20 mL of 10% hydrochloric acid solution was added drop wise to this mixture and refluxed for 3-4 h and checked by TLC. The solid crude product was separated out by filtration and washed with sufficient quantity of water and then dried. 

Step iv: Synthesis of 5-(substituted phenyl)-4H-1,2,4-triazole-3-thiol/5-((2,4-dichloro phenoxy)methyl)-4H-1,2,4-triazole-3-thiol

5 mL solution of 2 M sodium hydroxide (8% solution) was added portion wise to 0.01 mole of solid 1-(substituted benzoyl)thiosemicarbazide/1-(2-(2,4-dichlorophenoxy)acetyl) thiosemicarbazide and stirred well. Then refluxed for 4-5 h and checked by TLC. After the completion of reaction, the reaction mixture was treated with activated charcoal and filtered. Then filtrate was acidified with hydrochloric acid for pH 6 and the precipitate was filtered and recrystallized with ethanol/water. Then the product was dried. 

Step v: Synthesis of final 3,4,5-substituted-1,2,4-triazole derivatives (1-27)

The 0.01 mole of 5-(substituted phenyl)-4H-1,2,4-triazole-3-thiol/5-((2,4-dichloro phenoxy)methyl)-4H-1,2,4-triazole-3-thiol was dissolved in 0.01 mole of potassium carbonate solution. Then 0.02 mole of substituted acetophenone was added to this mixture. Then this mixture was refluxed for 5-6 h and reaction was checked by TLC. The hot reaction mixture was treated with activated charcoal, and then filtered and filtrate was acidified with dilute HCl solution till pH 6. The product was thus precipitated out, filtered, washed with water and recrystallized by ethanol/water (1:1). 

Spectral data of the synthesized compounds 

From our present work we conclude that the synthesized 3,4,5-substituted-1,2,4-triazole derivatives showed good antimicrobial and analgesic properties by different technique. Among the series, compounds TA-11, TA-20 and TA-26 displayed more antimicrobial activity than standard drugs cefadroxil (antibacterial) and fluconazole (antifungal) due to presence of electron withdrawing groups. Analgesic study demonstrated that compounds TA-13, TA-15 and TA-2, TA-15 exhibited good analgesic activity by Tail immersion and Acetic acid induced writhing method respectively. The electron withdrawing group plays an important role to enhance the antimicrobial and analgesic activities of the synthesized compounds and these compounds may be used as a lead for discover a new biological agents. 

Conflict Of Interest: The author(s) confirms that this article content has no conflicts of interest.

Author's Contributions

PKV- designed and finalized the scheme; DP- performed research work; GS-performed analgesic activity and DP analyzed the spectral and biological data and wrote the paper. All authors read and approved the final manuscript.

Acknowledgement

Thanks to Prof. Munish Garg, Head, Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak for kind support for providing chemicals etc. 

Permission by Institutional Animal Ethics Committee of MDU, Rohtak.

 All the protocol of the animal activity has been approved by Institutional Animal Ethics Committee of M. D. University, Rohtak and some of the compounds synthesized were evaluated for analgesic activity. All the animals were maintained under standard conditions and had access to pelleted animal feed and water.

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*Corresponding Author: Dr. Prabhakar Kumar Verma, Associate Professor, Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.

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