INTRODUCTION:

Thiadiazole confined five membered di-substituted ring structure composed of two nitrogen atoms and one sulphur atom. Thiadiazole are in four isomeric forms 1,2,4-thiadiazole, 1,3,4-thiadiazole, 1,2,5-thiadiazole, and 1,2,3-thiadiazole; The 1,3,4-Thiadiazole and its derivatives possess a wide range of biological activities probably due to the presence of-N=C-S moiety^1,2. Whereas Nitrogen and Sulphur Show potential chemotherapeutic and pharmacotherapeutic agents^3,4. Thiadiazole is an important scaffold known to be associated with several biological activities of the compounds dependent on their molecular structures⁵ and various functional groups attached to the thiadiazole ring⁶. The basic ring, 1,3,4-Thiadiazole are the fused heterocyclic ring compound that has many biological activities^7-12. Piperazine is a heterocyclic-compound having two nitrogen atoms and four carbon atoms at 4 and 1 position. In particular, the structural analogues of piperazine derivatives present various pharmacological activities^13-15.

The scaffold of piperazine derivatives exhibits a wide range of pharmaceutical activities as antimicrobial^16-19, antifungal^20-22, antipsychotics²³ and cytotoxic²⁴.

The unremitting and general use of antimicrobial agents has resulted in the development of resistance to these drugs by pathogenic microorganisms hereafter there is a most requirement in an innovative class of drugs. Thus, deep determinations in antimicrobial drugs discovery are, still needed to develop more hopeful and effective antimicrobial agents for use in the field of clinical research. Prompted by the scope of the newer class of antimicrobial drugs and in continuation of research on biologically active heterocycles. In this view, the current work was proposed to prepare novel piperazine-1,3,4-thiadiazoles analogues and their biological studies assessment.

EXPERIMENTAL:

Materials and Methods:

All solutions are used in the current research were analytical grade. The synthesized compounds were confirmed by measuring their structural elucidation using ¹H-NMR (Bruker), their melting points (Fisher-Johns apparatus) and their retention times using thin layer chromatography. IR spectra were recorded on Perkin Elmer FT-IR spectrophotometer using KBr pellets and UV spectra were recorded on Perkin Elmer Spectrophotometer.

Synthesis of Piperazine-Thidiazole Analogues:

Preparation of 1-(4-chlorophenyl) piperazine:

Thionyl chloride (0.02moles) dissolved in chloroform (25mL) and diethanolamine (0.01moles) was added at room temperature under stirring. The mixture was refluxed for 2 hrs. After completion of the reaction mixture was cooled, filtered and recrystallized with ethanol. Resultant (Bis (2-chloroethyl) amine hydrochloride (0.02 moles)) and 4-Chloro aniline (0.05 mL) dissolved in 1-butanol (100mL), and refluxed for 24 hrs and subsequently the resultant was placed at 0^oC for 1 hr, to this, K₂CO₃ (0.02moles) was added and refluxed for 72 hrs. The reaction progress was monitored by TLC, then the reaction mixture was cooled to room temperature and the final product was filtered with suction pump followed by washed with 1-butanol.

Scheme-1

Preparation of Thiadiazole Derivatives:

To the substituted aromatic acid (0.01moles) added thiosemicarbazide (0.01moles) and concentrated sulphuric acid (0.01moles) and refluxed for 1hr, the product 5-(4-phenyl)-1, 3, 4-thiadizol-2-amine formed and it was poured into crushed ice and recrystallized with ethanol. Resultant 5-(4-phenyl)-1, 3, 4-thiadizol-2-amine (0.01moles), potassium carbonate (0.01moles), chloroacetyl chloride (0.01moles) and triethylamine (0.05moles) dissolved in chloroform (25mL) and refluxed for 6 hrs. An obtained product cooled at room temperature, later it was transferred into a crushed ice container. The precipitate was filtered and followed by dried. The final product was recrystallized with ethanol

Scheme-2

Preparation of piperazine-1, 3, 4-thiadiazole analogue

To the chloroform (25 mL), a mixture of substituted 2-chloro-N-(5-phenyl-1,3,4-thiadizol-2-yl) acetamide (0.04 moles), 1-(4-chlorophenyl)piperazine (0.05 moles), and N, N-Diisopropylethylamine (0.02 moles) added and refluxed for 3 hr. The progress of the reaction was monitored by TLC (TLC mobile phase hexane: ethyl acetate 8:2). The reaction mixture was cooled and the solid separated was collected by filtration.

Scheme-3

Table -1: Molecular information of the six compounds

Compound	Molecule Name	Molecular Formula	R	Yield (%)
PT1	2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide	C₂₀H₂₀ClN₅OS	C₆H₅-	75
PT2	N-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-(4-(4-chlorophenyl)piperazin-1-yl)acetamide	C₂₀H₁₉Cl₂N₅OS	o-Cl-C₆H₄-	60
PT3	N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)-2-(4-(4-chlorophenyl)piperazin-1-yl)acetamide	C₂₀H₁₉Cl₂N₅OS	p-Cl-C₆H₄-	65
PT4	2-4(4-(4-Chlorophenyl)piperazin-1yl)-N-(5-(2-Nitrophenyl)-1,3,4-thiadiazol-2yl)-acetamide	C₂₀H₁₉ClN₆O₃S	o-NO₂-C₆H₄-	65
PT5	2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide	C₂₀H₁₉ClN₆OS	p-NO₂-C₆H₄-	70
PT6	2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide	C₂₀H₂₀ClN₅O₂S	p-OH-C₆H₄-	60

Spectral Data:

2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-phenyl-1,3,4-thidiazol-2-yl) acetamide (PT1): Melting Point: 200°C, Mass (m/z): 414. (75% yield). IR (KBr Cm^-1) : 3374 (CO-NH), 2882 (Aromatic C-H),1563 (C=N), 1498 (C=C), 1032 (N=N) 668 (C-S-C), ¹H NMR (400 MHz, CDCl₃) Ar - H δ 7.45-7.99 (m, 5H), CON-H δ 9.21(s, 1H), COC-H δ 3.29 (s, 2H), Piperazine-CH2 δ 2.73, (t, 4H) δ 3.43 (t, 4H), Piperazine Ar-H δ 7.00 to 7.27 (m, 4H), ¹³C NMR(100 MHz, CDCl₃) δ 171.01, 161.66, 160.26, 148.92, 133.46, 130.90, 130.49, 128.62, 127.69, 126.82, 117.17, 60.61, 52.40, 48.21.

2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (PT2): Melting Point: 239 °C, Mass (m/z): 470 (60% yield). IR (KBr Cm^-1): 3256 (CO-NH), 2924 (Aromatic C-H),1591 (C=N), 1433 (C=C), 1012 (N=N) 654 (C-S-C), ¹H NMR (400 MHz, CDCl₃) 2-Chloro Ar -H δ 7.37-7.77 (m, 4H), CON-H δ-9.21 (s, 1H), COCH2 δ 3.41 (s, 2H), Piperazine-CH2 δ 2.85 (t,4H) δ 3.39 (t, 4H) Piperazine Ar -H δ 7.11 to 7.39 (m, 4 H), ¹³C NMR (100 MHz, CDCl₃) δ 171.01, 169.25, 167.82, 158.72, 144.02, 134.71, 133.96, 131.59, 130.84, 129.19, 124.60, 117.09, 113.27, 62.60, 53.32, 46.31.

2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (PT3):

Melting Point: 255 °C, Mass (m/z): 470, (65% yield). IR (KBr Cm^-1): 3422 (CO-NH), 2895 (Aromatic C-H),1546 (C=N), 1465 (C=C), 1026 (N=N) 672 (C-S-C),¹H NMR (400 MHz, CDCl₃) 4-Chloro Ar -H δ 7.44-7.89 (m, 4H), CON-H δ 9.21 (s, 1H), COCH2 δ 3.46 (s, 2H), Pipearzine-CH2 δ-2.74 (t, 4H) δ 3.49 (t, 4H) Piperazine Ar-H δ 7.10 to 7.24 (m, 4H),¹³C NMR (100 MHz,CDCl₃) δ 171.01, 162.07, 161.16, 158.90, 148.11, 137.09, 136.40, 127.19, 125.36, 121.60, 114.16, 61.62, 56.40, 43.25.

2-4(4-(4-Chlorophenyl)piperazin-1yl)-N-(5-(2-Nitrophenyl)-1,3,4-thiadiazol-2yl)acetamide (PT4):

Melting Point: 175 °C, Mass (m/z): 397. (65% yield). IR (KBr Cm^-1): 3387 (CO-NH), 2896 (Aromatic C-H),1534 (C=N), 1453 (C=C), 1035 (N=N) 674 (C-S-C),¹H NMR (400 MHz,CDCl₃) 2-Nitro Ar -H δ 7.67-7.92 (m, 4H), CON-H δ-9.29 (s, 1H), COCH2 δ 3.61 (s, 2H), Piperazine-CH2 δ 2.76 (t, 4H) δ 3.33 (t, 4H) Piperazine Ar-H δ 7.09 to 7.32 (m, 4H), ¹³C NMR (100 MHz, CDCl₃) δ 171.01, 155.80, 152.75, 144.62, 141.31, 138.90, 135.49, 128.50, 126.09, 123.24, 121.67, 119.75, 115.57, 64.62, 50.43, 47.31.

2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (PT5):

Melting Point: 272 °C, Mass (m/z): 497, (70% yield). IR (KBr Cm^-1): 3320 (CO-NH), 2921 (Aromatic C-H),1519 (C=N), 1439 (C=C), 1054 (N=N) 649 (C-S-C),¹H NMR (400 MHz, CDCl₃) 4-Nitro Ar-H δ 8.17- 8.36 (m,4H), CON-H δ -9.21 (s,1H), COCH2δ 3.36 (s,2H), Piperazine-CH2 δ-2.73 (t, 4H) δ 3.47 (t, 4H) Piperazine Ar-H δ 7.13 to 7.29 (m, 4H),¹³C NMR (100 MHz, CDCl₃) δ 171.01, 165.16, 163.29, 158.12, 147.61, 135.25, 132.09, 129.39, 128.41, 121.08, 116.37, 62.91, 55.40, 46.34.

2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide (PT6) :

Melting Point: 171 °C, Mass (m/z): 468, (60% yield). IR (KBr) (cm^-1): 3349 (CO-NH), 2897 (Aromatic C-H),1540 (C=N), 1489 (C=C), 1062 (N=N) 663 (C-S-C),¹H NMR (400 MHz, CDCl₃) 4-Hydroxy Ar-H δ 6.94-7.85 (m, 4H),O-H δ 7.60 (s,1H), CON-H δ-9.29(s, 1H), COCH2 δ 3.33 (s, 2H), Piperazine-CH2 δ 2.67 (t, 4H) δ-3.34 (t, 4H) Piperazine Ar-H δ 7.12 to 7.34 (m, 4H),¹³C NMR (100 MHz, CDCl₃) δ 171.01, 167.42, 160.86, 155.33, 147.90, 135.45, 129.71, 125.62, 122.65, 114.07, 112.30, 69.01, 51.43, 41.11.

Anti-Microbial Activity:

Organisms:

Two micro-organisms, Bacillus subtilis MTCC211 and Vibrio cholera MTCC3904 were used in the present study collected from MTCC, Chandigarh, India.

Media:

Mueller Hinton Agar medium to be used for routine susceptibility testing of bacteria due to its acceptable reproducibility, satisfactory growth of most pathogens^25.

Agar-well diffusion testing:

The antibacterial activity was measured for the targeted compounds utilizing agar well dissemination method²⁶. The technique is fundamentally on the dissemination of the ideal drug in a vertical cylinder well in an agar petri plate, which is pre-defined with the testing bacterial strain. The activity of the compound will be estimated utilizing the arrangement of zones around the wells^27,²⁸. In the present investigation, Muller-Hinton agar was utilized to culture the test microorganisms scale life forms on petri dishes. After hardening of agar, a cotton swab was utilized to spread the testing of bacterial strains and afterward 6mm wells were set on agar plate with sterile steel borer. At that point, 25 µL of synthesized drugs (PT1-PT6) were tried on chosen microscopic organisms utilizing vancomycin as standard medication (30µg) and dimetylsulphoxide (DMSO) as the vehicle. Subsequent to, placing the test compounds, standard, the vehicle in wells placed the petri dishes aside for 1hr without unsettling influence for dispersion of compounds in wells. In Specific, plates were incubated for 24hrs at 37°C. After completion of incubation, the plates were used to measure the compounds zones of inhibition around the wells using a well reader (scale), the investigation was repeated thrice and the outcomes were communicated as normal in mm. Which compounds are unfit to display inhibition zone (inhibition zone measurement under 7 mm) were considered non-active.

RESULTS AND DISCUSSION:

The compounds were successfully synthesized with a fine percentage of yields and the reaction processes were showed in scheme-1. The synthesized compounds were confirmed by their analytical and spectral data (Table 1). IR; peaks at 2882 cm^-1corresponds to C-H of aromatic and 3374 cm^-1corresponds to N-H amide functional group. 1-phenylpiperazine structure by the presence of tertiary amine C-N stretch frequency at 1563 cm^-1. ¹H NMR spectral value 2.73-2.43 δ corresponds to CH₂ peaks, thidiazole connected benzene protons gives a multiplet at 7.45-7.99 δ. Piperazine connected phenyl with four protons gives a multiplet at 7.00 δ and 7.27 δ, whereas the other proton appears singlet at 9.21 δ corresponds to N-H peak are the commonly identified peaks of precursors

Antibacterial activity of synthesis compounds on bacteria:

Agar well-diffusion method was used to screen the antibacterial activity of synthesized compounds (PT1-PT6) at 50 and 100µg and the compounds showed concentration-dependent activity (Table 2). Antibacterial activity of the six selected compounds was varied with the microorganism tested in this study [Bacillus subtilis (Gram positive) and Vibro cholera (Gram negative)]. The results of the current study describe that tested compounds were more active against Gram positive bacteria compared to Gram negative bacteria. At 100µg concentration PT6 showed highest (24mm and 14mm zone of inhibition) antibacterial activity against Bacillus subtilis as well as Vibro cholera. On the other hand, standard antibiotic vancomycine at 30µg showed 30mm and 28mm zone of inhibition against B. subtilis and V. cholera. Whereas PT5 also showed results (11mm and 9mm zone of inhibition) against two pathogens, B. subtilis and V. cholera. The other compounds are moderately active. PT4 didn’t have activity at 50µg exhibited 9mm zone against B. subtilis at 50µg and PT3 showed 10mm to 8 mm zone of clearance against pathogen B. subtilis and V. cholera. PT2 exhibited 9mm to 10mm zone of clearance against the two bacteria. PT1 didn’t have activity at 100µg but showed 9mm inhibition zone against V. cholera at 100µg and PT1 showed 10mm and 13mm against B. subtilis and at 100µg and 50µg respectively.

Table-2: Zone of inhibition antibacterial activity of six compounds.

S. No	Compound Name	Inhibition zone (mm)
		Gram negative		Gram positive
		*(Vibrio cholera)*		*(Bacillus subtilis)*
		50 µg	100 µg	50 µg	100 µg
1	PT 1	0	9	10	13
2	PT 2	8	10	8	9
3	PT 3	0	8	9	10
4	PT 4	10	12	0	9
5	PT 5	8	9	10	11
6	PT 6	10	14	18	24
	Vancomycin (30 µg)	28		30

DISCUSSION:

The Piperazine-1,3,4-Thiadizole analogues were synthesized and categorized on the basis of ¹H NMR, ¹³C NMR and IR, conventional their structures. The results of the current study confirm that compounds PT6 had more active against the tested bacterial strains and that activity was equivalent to standard drug. These results reveal that hydroxyl substituted Piperazine-1,3,4-Thiadizole compound had better antibacterial activity.

CONCLUSION:

A novel series of Piperazine-1, 3, 4-thiadizaole analogues were synthesized with simple and efficient methods and were screened for antibacterial activity. The maximum activity was found in hydroxyl substituted derivative possess better antibacterial activity against grampositive bacteria and gramnegative bacteria. Piperazine-1, 3, 4-thiadizaole compounds could be advance screened for the antibacterial activities against Gram-positive and Gram-negative bacteria to complete their wide-ranging spectrum profile. It is possible that structural modifications of synthesized compounds will increase the antibacterial activity which helps the screening of bioactive compounds further. Further molecular modifications of these compounds will pave a way for finding the valuable antimicrobial agents.

The area of the synthesis of Piperazine-1, 3, 4-thiadiazole rings continues to grow, and the organic chemistry will provide better methods for the synthesis of this interesting heterocycles, allowing the increased spectrum of antimicrobial activity.

CONFLICTS OF INTEREST:

No conflicts of interest.

ACKNOWLEDGEMENT:

Author acknowledge to thanks research supervisor, Dr. P. Uma Devi, Professor, Department of Chemistry, GIS, Gandhi Institute of Technology and Management (GITAM) (Deemed to be University), for her valuable guidance.

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Received on 31.07.2020 Modified on 07.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4710-4714.

DOI: 10.52711/0974-360X.2021.00819