Synthesis and biological evaluation of heterocyclic 1,2,4-triazole scaffolds as promising pharmacological agents

Background Triazole is an important heterocyclic moiety that occupies a unique position in heterocyclic chemistry, due to its large number of biological activities. It exists in two isomeric forms i.e. 1,2,4-triazole and 1,2,3-triazole and is used as core molecule for the design and synthesis of many medicinal compounds. 1,2,4-Triazole possess broad spectrum of therapeutically interesting drug candidates such as analgesic, antiseptic, antimicrobial, antioxidant, anti-urease, anti-inflammatory, diuretics, anticancer, anticonvulsant, antidiabetic and antimigraine agents. Methods The structures of all synthesized compounds were characterized by physicochemical properties and spectral means (IR and NMR). The synthesized compounds were evaluated for their in vitro antimicrobial activity against Gram-positive (B. subtilis), Gram-negative (P. aeruginosa and E. coli) bacterial and fungal (C. albicans and A. niger) strains by tube dilution method using ciprofloxacin, amoxicillin and fluconazole as standards. In-vitro antioxidant and anti-urease screening was done by DPPH assay and indophenol method, respectively. The in-vitro anticancer evaluation was carried out against MCF-7 and HCT116 cancer cell lines using 5-FU as standards. Results, discussion and conclusion The biological screening results reveal that the compounds T5 (MICBS, EC = 24.7 µM, MICPA, CA = 12.3 µM) and T17 (MICAN = 27.1 µM) exhibited potent antimicrobial activity as comparable to standards ciprofloxacin, amoxicillin (MICCipro = 18.1 µM, MICAmo = 17.1 µM) and fluconazole (MICFlu = 20.4 µM), respectively. The antioxidant evaluation showed that compounds T2 (IC50 = 34.83 µg/ml) and T3 (IC50 = 34.38 µg/ml) showed significant antioxidant activity and comparable to ascorbic acid (IC50 = 35.44 µg/ml). Compounds T3 (IC50 = 54.01 µg/ml) was the most potent urease inhibitor amongst the synthesized compounds and compared to standard thiourea (IC50 = 54.25 µg/ml). The most potent anticancer activity was shown by compounds T2 (IC50 = 3.84 μM) and T7 (IC50 = 3.25 μM) against HCT116 cell lines as compared to standard 5-FU (IC50 = 25.36 μM).


Introduction
Triazole is an N-bridged aromatic heterocyclic compound that received a considerable attention in recent years due to their biological activities [1]. The name "triazole" was first use by Bladin in 1855 for describing the carbon-nitrogen ring system C 2 H 3 N 3 [2]. It is a white to pale yellow crystalline solid with a weak, characteristic odour, soluble in water and alcohol, melts at 120 °C and boils at 260 °C [3]. Triazole exists in two isomeric forms such as 1,2,4-triazole and 1,2,3-triazole [4]. The SAR studies of triazole derivative reveals that substitution on positions 3, 4 and 5 of triazole ring can be varied but the greatest changed in physicochemical Open Access BMC Chemistry *Correspondence: salonikakkar2007@gmail.com 1 Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India Full list of author information is available at the end of the article properties and biological profile is exerted by the groups attached to the nitrogen atom at the 4th position [3]. It favours the hydrogen bonding and is also stable for metabolic degradation, which could be favorable in increasing solubility as well as in binding bimolecular targets [5].
At present time, our medical field is suffering from the problem of antimicrobial resistance towards many microbial strains. Hence as prioritized by various health organizations, there is a need for the discovery or development of novel antimicrobial compounds possessing a broad spectrum activity exhibiting high effectiveness against those highly resistant Gram positive, Gram negative bacterial and fungal strains [14].
Human cells face threats everyday because the attack of various viruses, infections and free radicals damage the body cells and DNA. Scientists observed that the free radicals contribute to the ageing process and also contribute in diseases, like cancer, diabetes and heart disease. Antioxidants are the chemicals that stop or limit the damage caused by the free radicals and also boost our immunity [15].
Ureases relate to the class of Urea amidohydrolases enzymes containing two nickel(II) atoms. Ureases are mainly obtained from plants, algae, fungi and bacteria. Bacterial ureases are responsible for causing many diseases like pyelonephritis, hepatic coma, peptic ulceration, urinary stones and stomach cancer. Rationally, A category of antiurease or urease inhibitory drugs was developed for curing the urease caused disease by inhibiting urease enzymes. The two nickel(II) atoms present in active site of Ureases accelerate the hydrolysis of urea into ammonia and carbon dioxide gas. Both CO 2 and NH 3 are important virulence factor for the pathogenesis of many above given clinical conditions. Anti-urease compounds inhibit the hydrolysis of urea by antagonising urease enzyme [16]. This article also focuses on some new 1,2,4-triazole derivatives exhibiting anti-urease activity.
Colorectal cancer is the third most lethal cancer worldwide in both males and females with drug resistance and metastasis being the major challenge to effective treatments. Maximum deaths due to colon cancer are related with metastatic ailment. The growth of colorectal cancer is promoted by epigenetic factors, such as abnormal DNA methylation. Targeted therapy is a kind of chemotherapy that specifically targets the proteins that resist the development of some cancers [17].
Palmitic acid (common name) is categorized as saturated fatty acid with chemical formula CH 3 (CH 2 ) 14 COOH (IUPAC name: hexadecanoic acid). The main sources of palmitic acid are palm oil, olive oil, meats, cheese, cocoa butter, breast milk and dairy products [18]. Napalm, is a derivative of palmitic acid, synthesized by the combination of aluminium salts of palmitic acid and naphthenic acid and it was used as fuel during World War II [19].

Chemistry
The multistep synthetic process of 1,2,4-triazole derivatives (T 1 -T 20 ) was depicted in Scheme 1. Initially, ethylpalmitate (Int-i) was synthesized by the reaction of palmitic acid, ethanol and sulphuric acid. Palmitohydrazide (Int-ii) was synthesized from ethanolic solution of ethylpalmitate (Int-i) followed by addition of hydrazine hydrate. 5-Pentadecyl-1,3,4-oxadiazole-2(3H)-thione (Int-iii) was synthesized using Int-ii in alc. potassium hydroxide solution followed by the addition of carbon disulfide and then followed by addition of hydrazine hydrate to Int-iii yielded 4-amino-5-pentadecyl-4H-1,2,4-triazole-3-thiol (Int-iv). Finally, the Int-iv on reaction with different substituted aromatic aldehydes in ethanol yielded the title compounds (T 1 -T 20 ). The physicochemical properties of the synthesized compounds are depicted in Table 1. The synthesized derivatives of 1,2,4-triazole were confirmed by Infrared (IR) and Nuclear Magnetic Resonance ( 1 H/ 13 CNMR). The spectro-analytical data has been depicted in Table 2. The presence of aliphatic -CH-stretch in all compounds was confirmed at 2990-2879 cm −1 . The intermediates (Int-ii, iii and iv) exhibited the -NH stretch in range of 3424-3319 cm −1 . The presence of -CONH-group in Int-ii was indicated by appearance of -CONH-stretch at 1630 cm −1 . The peak range 1677-1589 cm −1 in Int-iii, iv and compounds T 1 -T 20 indicated the presence of -C=N stretch. The presence of -SH stretching vibrations in Intiv and compounds T 1 -T 20 were indicated in a scale of 2593-2505 cm −1 . The compounds T 4 , T 5 and T 6 showed the -OCH 3 stretching vibrations in the range of 2860-2848 cm −1 . The presence of phenolic group in compounds T 6 , T 7 , T 8 and T 18 was indicated by peaks in the range of 3483-3400 cm −1 . The peak range 701-699 cm −1 of compounds T 13 and T 14 was indicated the presence of Ar-Br group. The compounds T 15 , T 16 and T 17 showed the Ar-NO 2 stretching vibrations in the range of 1545-1424 cm −1 . The presence of Ar-Cl group in compounds T 10 , T 11 and T 12 was confirmed by the appearance of peaks in the range of 767-750 cm −1 . The presence of tertiary amine in compound T 9 was confirmed by the appearance of peak at 3431 cm −1 . The presence of aromatic ring in compounds T 1 -T 20 was indicated by the appearance of peak in the range of 1796-1719 cm −1 . DMSO was used as solvent for the analysis of compounds by 1 HNMR spectra. The presence of singlet signal at 1.22-2.47 δ ppm and 0.82-0.84 δ ppm indicated the presence of protons of -CH 2 and -CH 3 groups in Intii, iii and iv, respectively. Singlet at 2.25 δ ppm and 8.87 δ ppm showed the presence of protons of NH 2 and NH groups in Int-ii, iii and iv, respectively. The presence of proton of SH group was indicated by appearance of singlet at 3.30 in Int-iv. The findings of elemental analysis of synthesized derivatives were recorded within theoretical results of ± 0.4%. Mass spectra of the synthesized derivatives reflected the characteristic molecular ion peaks.

Structure activity relationship (SAR) studies
In the synthesized compounds, the substitution on m-and p-position of the aromatic ring with methoxy group improved the antimicrobial activity (compound respectively. The p-substitution of nitro (compound T 17 , MIC AN = 27.1 µM) group improved the antifungal activity against A. niger. The substituent methyl at p-position of ring (compound T 3 , IC 50 = 54.01 µg/ml) enhanced the anti-urease activity. The antioxidant activity has been improved by p-substituents i.e. aldehyde (compound T 2 , IC 50 = 34.83 µg/ml) and methyl groups (compound T 3 , IC 50 = 34.38 µg/ml). The most potent anticancer activity showed by compounds T 2 (IC 50 = 3.84 μM) and T 7 (IC 50 = 3.25 μM) against HCT116 cell lines as compared to standard 5-FU (IC 50 = 25.36 μM). From the analysis of antimicrobial activity, it may be concluded that the substitution of methoxy group increase the antibacterial activity whereas introduction of nitro as electron withdrawing groups at p-position may enhance the antifungal activity of synthesized compounds. The introduction of methyl substituent as electron donating groups at p-position of aromatic ring may increase the anti-urease as well as antioxidant activity. The substitution of of p-aldehyde and o-hydroxy group on the aromatic ring may enhance the anticancer activity against HCT116 cells (Fig. 3).

Procedure for synthesized 1,2,4-triazole derivatives (T 1 -T 20 )
Step A: synthesis of Int-i A mixture of palmitic acid (2.6 g, 0.01 mol), absolute ethanol (50 ml) and few drops of conc. sulphuric acid (0.5 ml) was refluxed for 10 h in a round bottom flask and then cooled to 5 °C. The liquid product was separated from reaction mixture by using ether on the basis of density and then purified [26].

Step B: synthesis of Int-ii
To a solution of ethyl palmitate (Int-i, 2.8 g, 0.01 mol) in absolute ethanol (30 ml), hydrazine hydrate (0.64 g, 0.02 mol) was added and refluxed for 6 h and then left to cool. The solid product was collected by filtration and recrystallized from ethanol [26].

Step C: synthesis of Int-iii
Palmitohydrazide (Int-ii, 3.12 g, 0.01 mol) dissolved in the solution of potassium hydroxide (1.12 g, 0.02 mol) in ethanol (30 ml) and then (0.76 g, 0.01 mol) carbon disulfide was added slowly in the reaction mixture. The reaction mixture was refluxed for 10-12 hand then cooled at room temperature followed by addition of    hydrochloric acid for neutralization of product. The precipitated solid was filtered, washed with ethanol, dried and recrystallized from ethanol [27].

Step E: synthesis of 1,2,4-triazole derivatives (T 1 -T 20 )
The reaction mixture of 4-amino-5-pentadecyl-4H-1,2,4triazole-3-thiol (Int-iv, 3.26 g, 0.01 mol) and different substituted aldehydes (0.01 mol) in ethanol followed by addition of few drops of sulphuric acid was refluxed for an appropriate time. The reaction was monitored by thin layer chromatography. After completion of reaction, the product was poured in ice and filtered, then wash and finally solid products were collected and recrystallized from ethanol [27].

Biological studies Antimicrobial evaluation
The in vitro antimicrobial screening of the synthesized 1,2,4-triazole derivatives (T 1 -T 20 ) in μM was determined against Gram-positive Bacillus subtilis, Pseudomonas aeruginosa, Gram-negative Escherichia coli bacterium and fungal strains Candida albicans and Aspergillus niger by tube dilution method using ciprofloxacin, amoxycillin (antibacterial) and fluconazole (antifungal) as reference drugs. DMSO was used to dissolve the reference and sample derivatives (T 1 -T 20  [17].

In vitro antioxidant evaluation
In the DPPH free radical scavenging activity, compounds (T 1 -T 20 ) were evaluated for their free radical scavenging activity with ascorbic acid as standard compound. The IC 50 was calculated for each compound as well as ascorbic acid as standard and summarized in (2021) 15:5  where A control is the absorbance of the control, A sample is the absorbance of the test compound.

Urease inhibition evaluation
Urease inhibitory potential for each synthesisized compound (T 1 -T 20 ) was evaluated using Jack Bean Urease by Indophenol method ( Absorbance of reaction mixture was recorded by ELISA at 570 nm. Ammonium carbonate increased the pH of phosphate buffer from 6.8 to 7.7 which was produced from urea by urease enzyme and the end peak was measured by the colour of phenol red indicator [16]. The percentage inhibition of urease enzyme was calculated by using following formula:  where A control is the absorbance of the control; A sample is the absorbance of the test compound.

Anticancer evaluation
HCT116 (human colon cancer cells) were seeded at 2500 cells/well (96 well plate), allowed to attach overnight, exposed to the respective compounds for 72 h and subjected to SRB assay (570 nm). Data represent mean IC 50 of at least triplicates. The compounds were all dissolved in DMSO as stock of 100 mg/ml. DMSO of < 1.5% did not result in cell kill. The highest concentration of each compound tested (100 μg/ml) contained only 0.1% DMSO. Compounds T 2 (IC 50 = 3.84 μM) and T 7 (IC 50 = 3.25 μM) exhibited the most potent anticancer activity against HCT116 cell lines as compared to standard 5-FU (IC 50 = 25.36 μM) given in Table 6 and Figs. 12, 13, 14.

Table 6 Anticancer screening results of the synthesized compounds (T 1 -T 20 )
Italics signifies the most active compound in comparison to the standard compound