Microwave Induced Synthesis of 1,2,4-triazole derivatives and Study of their Anthelmintic and Anti-microbial Activities

 

Krishna Chandra Panda*, B.V.V Ravi Kumar, Biswa Mohan Sahoo

Roland Institute of Pharmaceutical Sciences, Berhampur – 760010

Affiliated to Biju Patnaik University of Technology (BPUT), Rourkela, Odisha, India.

 

ABSTRACT:

Microwave-assisted chemical reactions are utilized as an emerging tool in drug synthesis as this technique is simple, economic, and efficient. So, a new series of 1,2,4-triazole derivatives are obtained via multi-component reactions under microwave irradiation. First of all, benzohydrazide was obtained by esterification of benzoyl chloride with methanol in presence of concentrated sulphuric acid followed by reaction with hydrazine hydrate. So, carbon disulfide was added dropwise to benzohydrazide in ethanolic potassium hydroxide solution to yield potassium dithiocarbazinate which was cyclized by reacting with hydrazine hydrate to afford 1,2,4-triazole-3-thiol. Further, various Schiff’s bases were prepared by reacting 1,2,4-triazole-3-thiol with different substituted benzaldehydes under microwave irradiations. The characterization of the newly synthesized triazole derivatives were carried out by spectral studies including FT-IR, ¹H-NMR, and LC-MS. The tested compounds exhibited significant in-vitro anthelmintic and antimicrobial activities as compared to the standard drugs.

 

 

INTRODUCTION:

Triazole derivatives are five-membered heterocyclic compounds that possess three nitrogen atoms present at 1, 2 and 4 positions (Fig. 1)^1,2. 1,2,4-triazole derivatives display significant bioological activities such as anti-fungal, antibacterial, anti-viral, anti-inflammatory, anticancer, anti-tubercular, antioxidant, anthelmintic, anti-diabetic and anti-epileptic, etc³.

 

Fig. 1: Structure of 1,2,4-triazole with centroid, plane and exclusion sphere.

 

The various antifungal drugs containing triazole ring includes fluconazole, and voriconazole etc. Similarly, compounds with triazole moieties are reported to be very effective anticancer agents that include letrozole, and anastrozole, etc. Whereas, ribavirin exhibits anti-viral activity^4-6 (Fig. 2).

 

Fig. 2: Clinically used drugs containing triazole scaffold.

 

Further, Schiff bases play a vital role in the development of compounds with diverse structures via cycloaddition, replacement, and ring closure reactions⁷. The presence of the imine (-C=N-) group in Schiff’s base plays a key role in imparting diverse biological activities. Schiff bases are generated by condensation reaction of primary amines and carbonyl compounds⁸. The reactions under microwave irradiation can be performed at a faster rate with improved product yields and high purity by minimizing the formation of unwanted byproduct^9-12. Due to diverse therapeutic potential activities of triazole scaffold, various triazole derivatives (4a-j) were synthesized by reacting 1,2,4-triazole-3-thiol with various substituted benzaldehydes under microwave irradiations for 5-10min^13-15. The structural confirmation of the synthesized compounds was demonstrated by spectral studies. Finally, the characterized compounds were evaluated for their anthelmintic and anti-microbial activities^16-20.

 

MATERIALS AND METHODS:

All chemicals, regents and solvents used during synthesis were of synthetic grade (S. D. Fine. Chem. Ltd. Mumbai, India) and used without further purification. The synthesis of 1,2,4-triazole derivatives was performed in a scientific microwave oven (Catalyst System, operating between power level of 140-700W). The chemical reactions were conducted at power level-2 with energy of 210 W. Simultaneously, the conventional heating method is also applied to check the reaction time, reaction condition, and yield of the product as compared to the MWI method. The purity of the synthesized compounds was confirmed by melting point determination and TLC analysis using mobile phase (chloroform, ethyl acetate 60:40). The synthesized compounds were characterized by using FT-IR, ¹H-NMR, LC-MS, and Elemental analysis^21-24. Infrared (IR) spectra were recorded (in KBr) on a Fourier-transform IR, model IR Affinity-1 (SHIMADZU), and the values are expressed in cm^-1. The ¹H NMR spectra were obtained on multinuclear FT-NMR Spectrometer, model Advance-II (Bruker), (400 MHz) using CDCl₃ as solvent, tetramethylsilane (TMS) as an internal standard. The chemical shift values are expressed as ppm (parts per million) units, downfield from TMS. The LC-MS spectra were recorded on Waters Micromass Q-Tof Micro (70 eV), a hybrid quadrupole time of flight mass spectrometer. The elemental analysis was performed on a Perkin Elmer Eal 240 elemental analyser (Perkin Elmer, USA).

 

EXPERIMENTAL:

Benzohydrazide reacts with carbon disulfide (CS₂) in presence of ethanolic potassium hydroxide solution to produce potassium dithiocarbazinate which further undergoes cyclization by reacting with hydrazine hydrate to produce 1,2,4-triazole-3-thiol. Finally, different substituted benzaldehyde (0.001m) was added to the solution of 1,2,4-triazole-3-thiol (0.001m, 1.9g) in ethanol. To this, glacial acetic acid was added dropwise and the reaction mixture was subjected to microwave irradiation at 210 W for 5-10min. Similarly, the reaction mixtures of different substituted benzaldehydes (0.001m) were added to the solution of 1,2,4-triazole-3-thiol (0.001m, 1.9g) in ethanol and were refluxed on a water bath at 80-100⁰C for 1-2h to obtain the substituted benzylidene amino-5-phenyl-4H-1,2,4-triazole-3-thiol. The completion of the each chemical reactions was verified by checking TLC. The solid products of 1,2,4-triazole derivatives (4a-j) were obtained by pouring ice-cold water into the reaction mixture (Fig. 3). The solid product was collected by filtration followed by recrystallization from ethanol.

 

Fig. 3: Synthetic route for 1,2,4-triazoles derivatives  (4a-j).

 

BIOLOGICAL EVALUATION:

Anthelmintic Activity:

Helminthiasis is considered as a parasitic disorder found in humanbeings due to infection by parasitic worms such as nematodes or cestodes which are present in the lungs, liver, lymph, muscles, eye, brain and skin, etc. The clinically available anthelmintic drugs are administered to remove parasitic worms from the body by destorying them without causing any major harm to the host cell. To study the anthelmintic activity, Indian adult earthworms (Pheritima Posthuma) were selected. The earthworms were obtained from the water-logged areas of soils. All the earthworms were washed with normal saline water to remove all the fecal matter and waste surrounding their body. For anthelmintic activity, suitable earthworms of 5-8cm in length, 0.2-0.3cm width, and weighing 0.8-3.04g were selected²⁵.

 

Procedure:

Gum acacia solution (1%) was prepared in normal saline. Test solutions (100mg/ml) were prepared by using this solution. Sample solutions were poured into Petri plates and the adult healthy earthworms (n=6) were placed in Petri plates. It was observed to determine the time spent to paralyze and time spent for the death of the organisms. Paralysis was considered to occur when the worms do not review even in normal saline. Similarly, death was considered when the earthworms lost their motility followed by fading away of the body color and the values are summarized in table 2 and presented in figure 4. Albendazole was used as a standard drug for the evaluation of the anthelmintic activity.

 

Antimicrobial Activity:

The titled compounds were evaluated for their in-vitro antimicrobial activity against different types of bacterial and fungal strains. The activity of tested compounds was determined by calculating the zone of inhibition in millimeters and compared with the standard drugs. The antibacterial activity was performed by using the cup plate method against Staphylococcus aureus (MTCC 87), Staphylococcus epidermidis (MTCC 2639), Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424). The sample solutions (100μg/ml) were prepared by using DMSO and evaluated by using a nutrient agar medium. The solvent DMSO was used as a control. The zones of inhibitions were measured after 24h of incubation at 37^oC. Further, the anti-fungal activity was determined by using fungal strains such as Candida albicans (MTCC 183) and Aspergillus niger (MTCC 281). Ampicillin and Ketoconazole were used as standard drugs for antibacterial and antifungal activities respectively²⁶.

 

RESULTS AND DISCUSSION:

New series of 1,2,4-triazole derivatives were synthesized by reaction of 1,2,4-triazole-3-thiol with different benzaldehydes.  The yield of the product was improved (64-84%) by using the microwave irradiation technique (Table 1). The IR spectrum of 1,2,4-triazole derivatives exhibited absorption bands at 3150-3000 cm^-1 (Ar-CH), 3000-2850 cm^-1 (C-H aliphatic), 1536 cm^-1 (Ar-NO₂), 1585-1601 cm^-1 (C=N  Str.),  2241-2734 cm^-1 (–SH Str.), 3056 cm^-1 (Ar-OH). The triazole derivatives substituted with halogens exhibited the IR absorption bands in the region 1400-1000cm^-1, 800-600 cm^-1 which corresponds to C-F str., C-Cl str. respectively. In the ¹H-NMR spectrum, the protons were observed as a singlet at δ11.26, 10.52, δ8.92, δ4.76 for -SH, =CH, -OH, -OCH₃, respectively. Similarly, the aromatic protons were observed as a multiplet at δ6.86-8.56. The mass spectra of the 1,2,4-triazole derivative exhibited a molecular ion peak that corresponds to their molecular formula. Compound 4b, 4c, and 4e showed molecular ion peaks at m/z 298.34, 296.35, and 325.06 respectively. Chlorinated compounds like 4f, 4g exhibited molecular ion peak (M+) and M+2 peak at m/z 314.04 (100.0%) and 316.04(36.9%) respectively due to the presence of chlorine atoms (³⁵Cl or ³⁷Cl). In case of elemental analysis, the calculated/(found) % for compound 4a was found to be 64.26(64.22), 4.31(4.28), 19.98(19.90), 11.44(11.40) towards C, H and N respectively. Similarly for compound 4b, it was found to be 65.73(65.70), 4.14(4.12), 19.16(19.14), 19.16(19.14), 6.37(6.35) towards C, H, N and F respectively.

 

The screening results of the evaluated compounds demonstrated that compounds 4b, 4e, 4f, 4g, 4i, and 4j displayed significant anthelmintic activity as compared to the standard drug (Albendazole) as presented in table 2. Compound 4g made paralysis and death of parasitic worms within 20 and 21 minutes respectively as compared to the standard drug Albendazole. Whereas the compound 4d takes 26 minutes to initiate paralysis and death of the parasitic worms. The anti-microbial activity of tested compounds was evaluated by using the cup-plate method against various gram-positive, gram-negative bacteria, and fungal strains (Table 3). Among the evaluated compounds, it was observed that compound 4c, 4e, 4f, 4g, 4i, and 4j exhibited significant anti-bacterial activity against both gram-positive and gram-negative bacteria as compared to the standard drug Ampicillin. Similarly, compounds 4f, 4g, 4i displayed significant antifungal activity as compared to standard drug Ketoconazole.

 

Table 1: Characterization and Optimization study on yield and reaction time.

Comp. code	Molecular Formula	X	Rf value	M.P (0C)	Conventional		MWI
					R.T (min)	Yield (%)	R.T (min)	Yield (%)
4a	C15H12N4S	H	0.55	198-200	60	67	10	72
4b	C15H11FN4S	-4F	0.63	220-223	90	56	9	66
4c	C15H12N4OS	-2OH	0.62	221-223	100	58	5	64
4d	C17H17N5S	4-(CH3)2-N-	0.65	210-212	120	67	6	72
4e	C15H11N5O2S	-4NO2	0.64	165-168	90	78	8	84
4f	C15H11ClN4S	-4Cl	0.61	211-213	120	56	9	68
4g	C15H11ClN4S	-2Cl	0.63	168-171	80	62	10	76
4h	C16H14N4OS	-4OCH3	0.62	202-204	90	67	7	74
4i	C15H11N5O2S	-2NO2	0.64	174-178	60	63	8	69
4j	C15H12N4OS	-4OH	0.62	218-220	80	66	10	73

 

Table 1: Cont…….

Comp. code	Molecular Formula	Elemental Analysis                Calc. / (Found) %
		C	H	N	S	Cl	O	F
4a	C15H12N4S	64.26 (64.22)	4.31 (4.28)	19.98 (19.90)	11.44 (11.40)	-	-	-
4b	C15H11FN4S	65.73 (65.70)	4.14 (4.12)	19.16 (19.14)	10.19 (10.18)	--	--	6.37 (6.35)
4c	C15H12N4OS	60.79 (60.75)	4.08 (4.06)	18.91 (18.88)	10.82 (10.78)	--	5.40 (5.39)	--
4d	C17H17N5S	63.13 (63.10)	5.30 (5.28)	21.65 (21.61)	9.91 (9.89)	--	--	--
4e	C15H11N5O2S	55.38 (55.36)	3.14 (3.12)	21.53 (21.50)	9.86 (9.83)	--	9.84 (9.82)	--
4f	C15H11ClN4S	57.23 (57.20)	3.52 (3.50)	17.80 (17.79)	10.19 (10.17)	11.26 (11.24)	--	--
4g	C15H11ClN4S	60.79 (60.77)	4.08 (4.05)	18.91 (18.89)	10.82 (10.80)	11.26 (11.24)	--	--
4h	C16H14N4OS	61.92 (61.90)	4.55 (4.51)	18.05 (18.01)	10.33 (10.30)	--	5.15 (5.12)	--
4i	C15H11N5O2S	55.38 (55.36)	3.41 (3.38)	21.53 (21.48)	9.86 (9.83)	--	9.84 (9.82)	--
4j	C15H12N4OS	60.79 (60.74)	4.08 (4.04)	18.91 (18.90)	10.82 (10.81)	--	5.40 (5.38)	--

 

Table 2: Anthelmintic activity of 1,2,4-triazole derivatives (4a-j)

Treatment groups	Dose	Paralysis time (minutes)	Death time  (minutes)
4a	100mg/ml	25	28
4b	100mg/ml	22	22
4c	100mg/ml	24	21
4d	100mg/ml	26	26
4e	100mg/ml	22	24
4f	100mg/ml	21	23
4g	100mg/ml	20	21
4h	100mg/ml	23	25
4i	100mg/ml	22	23
4j	100mg/ml	21	22
Control	Normal saline	-	-
Albendazole	100mg/ml	18	20

 

Table 3: Antimicrobial activity of 1,2,4-triazole derivatives (4a-j).

Compounds (100mg/mL)	Zone of Inhibition (mm)
	Antibacterial activity				Anti-fungal activity
	Gram-positive		Gram-negative		C. albicans	A. niger
	S. aureus	S. epidermidis	P. aeruginosa	E. coli
4a	11	13	12	14	9	8
4b	14	16	15	17	10	10
4c	16	14	17	18	11	11
4d	13	12	14	15	9	12
4e	17	18	16	16	10	10
4f	15	17	15	19	12	11
4g	17	16	17	22	15	13
4h	13	18	16	20	13	10
4i	18	17	14	19	14	11
4j	16	15	18	21	11	12
Control	-	-	-	-	-	-
Standard	22	20	25	26	16	18

 

 

Fig. 4: Anthelmintic activity (paralysis time) of titled compounds.

 

CONCLUSION:

In the present study, a new series of substituted benzylidene amino-5-phenyl-4H-1,2,4-triazole-3-thiol were synthesized based on the green protocol of microwave irradiated synthesis. With the help of microwave heating technology, the yield of the product was enhanced in a short period time with cleaner reaction conditions. This technique provides simple operation, selectivity, eco-friendly process in comparison with the conventional synthetic methods. The anthelmintic and antimicrobial evaluation of the tested compounds exhibited promising activities as compared to standard drugs. It was observed that the presence of electron-withdrawing substituents (nitro, chloro, methoxy, hydroxy) on the triazole ring exhibited better activity as compared to other derivatives. Further, the optimization of structure-activity relationship study is required to improve the therapeutic potential of triazoles.

 

CONFLICT OF INTEREST:

The authors confirm that the article content has no conflicts of interest.

 

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Accepted on 02.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5746-5750.