1. INTRODUCTION:

The escalating prevalence of drug resistance in bacteria and fungi highlights an urgent demand for the development of innovative drug compounds¹. The misuse of drugs and the emergence of mutations in pathogens have further complicated the challenge of drug resistance². Candida yeasts, which are naturally occurring microorganisms in the human body, have witnessed a significant increase in infections, especially among individuals with compromised immune systems, over the past three decades³. Different kinds of Candida have been distinguished as causative specialists of contaminations, on-going epidemiological examinations recommend a shift towards drug safe Candida species, like Candida Para psilosis, Candida glabrata, Candida tropicalis, and, Candida krusei slowly supplanting Candida albicans in sickness⁴.

The raising pervasiveness of parasitic contaminations, combined with expanded utilization of antifungal medication, for example azoles is expected to promote an expansion in the opposition of Candida species to antifungal mixtures. Thusly, there is a dire requirement for the investigation, plan, and union of new anti-microbial mixtures inside the field of treatment.

oxadiazole subsidiaries are important organic compounds celebrated for their flexible exercises. The oxadiazole structure appears as a five-membered ring with one oxygen and two nitrogen atoms⁵. 1,3,4-Oxadiazoles are the heterocycles that defiantly standout during most recent twenty years as the likely anti-inflammatory^6-8, anticonvulsant⁹, antifungal and antibacterial^10-18, antiviral¹⁹, antioxidant^20-21, analgesic^22-23, anti-tubercular²⁴, anticancer²⁵, anti-HIV²⁶, and antidiabetic^27-28, activities demonstrated by the oxadiazole nucleus.

Pyrazine derivatives are another significant class of compounds, characterized by a two-nitrogen-containing six-membered ring²⁹. Pyrazine compounds have the ability to accommodate substituents at one or more of the four carbon atoms in the ring structure. Pyrazinamide, among all, stands out as a highly effective antitubercular drug³⁰, various derivatives of pyrazine, including analogues of pyrazinamide; demonstrate notable antibacterial³¹properties.

In answer to the above challenges, our research is focused on the plan, preparation, characterization, docking investigation, and biological assessment of a novel family of 5-(pyrazin-2-yl)-1,3,4-oxadiazol-2-amine derivatives. With a major focus on antifungal activity, the synthesized compounds work well as anti-tubercular agents.

In addition, molecular docking research is a part of our work. This allows us to see the interactions between these subsidiaries and their targets in biological applications. But through computational methods we hope to be able to predict binding affinity, interaction mechanisms and even how they may interact against contagious or mycobacterial targets. With this all-round approach, it can further help promote the production of useful antimicrobial agents with which to combat today's growing menace from drug Resistant pathogens.

2. MATERIALS AND METHODS:

2.1. Materials:

All reagents utilized in the synthesis were came from a commercial source and used without further purification. The structure of the synthesized compound is confirmed by ¹H NMR,¹³C NMR, IR, Mass Spectroscopy. NMR spectra recorded on Bruker 300Mz using CDCl₃ or DMSO-d₆ solvent, chemical shifts are reported in ppm with signals from TFA-d(δ=11.50ppm/164.2ppm), further δ explained by respective coupling constant(J). IR spectrum was recorded using Nicolaite 380 FTIR spectrophotometer as ATR (attenuated total reflection) from 4000-500cm^-1. Mass spectra recorded on a Shimadzu LCMS 2010 spectrophotometer. The melting points of targeted compounds were assessed using MEL-TEMP II. Moreover, the values are uncorrected

2.2. Methods:

2.2.1. Synthesis of 5-(Pyrazine-2-yl)1,3,4-oxadiazol-2-amine (3):

The two substances Pyrazine-2-carbohydrazides (2) (10 mmol) and Cyanogen bromide (10mmol) were dissolved in 10ml of 100% ethanol and refluxed for a duration of 12hours. Upon completion of the reaction, as confirmed by TLC (eluent 1:9 = Methanol: Methylene chloride), the resulting solution was cooled and subsequently neutralized using a saturated NaHCO₃solution (10ml). The precipitated solid product was isolated through filtration, subjected to thorough washing with saturated NaHCO₃ solution (10ml) and water (10ml). The final purification involved crystallization from ethanol, yielding the corresponding pure 5-(pyrazine 2-yl) 1,3,4-oxadiazole-2-amine in quantitative yields. (Scheme 1)

2.2.2. Synthesis of Acylated N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) amine-(3 a-h) (General Procedure):

10mmol of 5-(pyrazine 2-yl) 1,3,4-oxadiazole-2-amine was dissolved in 10ml of pyridine, future 10ml of anhydride is added to the mixture. The resulting composition refluxed for 12 hours. After the reaction was finished, as confirmed by TLC (eluent 1:9 = Methanol: Methylene chloride), the solution was cooled, and pyridine was completely distilled. The resulting residue then treated with a saturated NaHCO₃solution (10ml). The precipitated solid product collected through filtration, subjected to thorough washing with saturated NaHCO₃ solution (10ml) and water (10ml). Finally, the product was crystallized from ethanol, resulting in the corresponding pure Acylated N-(5-(pyrazin-2-yl)-1,3,4-oxadiazol-2-yl) amines in quantitative yield. (Scheme 1)

2.2.3. Biological activity:

Pyrazine-1,3,4-oxadiazole derivatives exhibit an extensive array of biological properties, rendering them an intriguing subject of study in the quest for innovative pharmaceuticals. Therefore, the development of efficient techniques enabling the swift and late-stage modification of this molecular class is of significant interest for pharmaceutical research and optimization. As part of this exploration, we investigated the newly synthesized compound 5-(Pyrazine-2-yl) 1,3,4-oxadiazol-2-amine (referred as 3) and its various derivatives, denoted as 3(a-h), for their potential antibacterial and antifungal activities.

Anti-bacterial activity:

The Agar well-diffusion method was employed to investigate the Anti-bacterial activity of synthesized compounds. Four concentrations (25, 50, 75 and 100µl) were examined against various bacterial pathogens, such as of Staphylococcus aureus and E. coli. Following after 18-to24 hours of incubation at 37℃ the diameter of the inhibition zone (mm) is measured, and the activity index computed. The readings were taken in three distinct fixed directions in all three repetitions and the average values were calculated

Antifungal activity:

Using four different concentrations of test compound (25, 50, 75 and 100µl). The Anti-fungal activity was tested using well diffusion method. Utilizing the spread plates approach the candida albicans fungus spread on the prepared SDA culture plates. The plates then incubated at 37+2ºC for 48 hours for fungal activity. After 48hours, the plates were examined for development of zone around the wells, the zone of inhibition (mm) was measured, and the activity index is computed. The readings were taken in three distinct fixed directions in all three repetitions and the average values were calculated.

2.2.4. Molecular Docking Study:

Molecular docking studies were performed using the Auto Dock Vina 4.2³². Precius structures of the applied proteins were taken from the RCSB Protein Data Bank ³³. All proteins were first ready and cleaned using BIOVIA Discovery Studio 2020³⁴ and using Auto Dock tools³⁵. All the ligand structures are improved using Gaussian09 software package³⁶. The obtained docked presents examined and envisioned using BIOVIA Discovery Studio 2020.

3. RESULT AND DISCUSSION:

Numerous synthetic pathways have been devised to produce 1,3,4-oxadiazole compounds from a variety of source materials, as recently indicated in the literature ^37,38. Nonetheless, this research has introduced a unique approach to the synthesis, exploration of biological activities, and docking studies of a set of innovative compounds. The structural integrity of these compounds was verified through spectral data analysis. The 1,3,4-oxadiazole moiety is widely acknowledged for its crucial role in various bioactivities, such as anti-bacterial^39,40 and anti-fungal⁴¹ properties, anti-inflammatory⁴² effects, and antimalarial potential, often associated with the presence of pyrazine and oxadiazole rings⁴³.

In our pursuit of developing new 5-(Pyrazine-2-yl) 1,3,4-oxadiazol-2-amine derivatives with broad pharmaceutical applications, we embarked on the synthesis of these novel compounds, denoted as 3 and 3(a-h). The structural confirmation of the synthesized compounds, were done using the various analytical methods like ¹H NMR (Nuclear Magnetic Resonance), ¹³C NMR (Carbon-13 Nuclear magnetic resonance) Mass spectrometry, FTIR (Fourier-transform infrared spectroscopy),

The new compound 3 shows a unique -NH₂ proton signal in the ¹H NMR spectra as it was detected as a singlet at δ7.59 ppm and it disappeared on the addition of deuterium oxide (D₂O). In addition, the –NH– proton of the prepared derivatives 3(a-h), observed as a broad peak at δ11.46 ppm and which vanish on addition of D₂O. The ¹³C NMR data revels, signals from 155.58 ppm to 164.64 ppm which confirms presence of two asymmetric carbons at 2^nd and 5^th position in the 1,3,4-oxadiazole ring. furthermore, the formation of 1,3,4-oxadiazole ring was attributed with absorption bands at approximately at 1700cm^-1 to 1720 cm−¹ for C-O stretching and 1610cm^-1 to 1660 cm−¹ for C=N stretching vibrations. At 1182.74 cm−¹ and 1216.54 cm−¹, the distinctive C-O-C vibrations identified, which confirms the presence of functional groups–NH₂ and –NH–.

5-(Pyrazine-2-yl)1,3,4-oxadiazol-2-amine (3):

Chemical Formula: C₆H₅N₅O, Colour: Grey, Yield: 78%, M.wt: 163.1, M.P: 239⁰C. IR (KBr, v cm−¹): 3044.6, 3086.88 (-NH₂), 1649.84 (C=N) 1113.5 (C-O). ¹H NMR (400 M.Hz, DMSO-d6, δ ppm):7.59(s, 2H,-NH₂, D₂O exchangeable) 8.74 (d,d, 2H, Aromatic-H), 9.18(s,1H, Aromatic-H). ¹³C NMR (400 MHz, DMSO-d₆) δ: 139.71, 142.28, 144.65, 145.36, 155.58, 164.8, (38.87-40.13-DMSO-d₆-Solvent peaks). LCMS: m/z 164.2(M+1)⁺.

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Propionamide (3-a):

Chemical Formula: C₉H₉N₅O_2, Colour: Black, Yield 68%; M.wt: 219.2, M.P: 215⁰C.¹H-NMR (400 M.Hz,DMSO-d₆, δ ppm): 1.09 (s,3H,-CH₃), 2.50(t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic- H),11.94(s, H,-NH-,)LCMS: m/z 220.3 (M+1)⁺.

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Butyramide (3-b):

Chemical Formula: C₁₀H₁₁N₅O₂, Colour: Grey, Yield 72%; M.wt: 233.2, M.P: 210⁰C.¹H-NMR (400 M.Hz,DMSO-d₆, δ ppm): 0.92(s,3H,-CH3), 1.61(q,2H), 2.50(t,2H) 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic-H),11.94 (s, H,-NH-,), LCMS: m/z 243.3(M +1)⁺

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Isobutyramide (3-c):

Chemical Formula: C₁₀H₁₁N₅O₂, Colour: light black, Yield 79%; M.wt: 233.2, M.P: 208⁰C. ¹H-NMR (400 M.Hz, DMSO-d₆, δ ppm): 1.14(s,3H,-CH3),2.50(t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic-H),11.93(s, H,-NH-,)LCMS: m/z 234.3(M+1)⁺

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Pentanamide (3-d):

Chemical Formula: C₁₁H₁₃N₅O₂, Colour: light black, Yield 80%; M.wt: 247.2, M.P: 215⁰C.¹H-NMR (400 M.Hz, DMSO-d₆, δ ppm): 0.90(s,3H,-CH3),1.34(m,2H)1.56(m,2H), 2.50(t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic-H),11.9(s, H,-NH-,), LCMS: m/z 248.4(M+1)⁺

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Pivalamide (3-e):

Chemical Formula: C₁₁H₁₃N₅O₂, Colour: Black, Yield 72%; M.wt: 247.2, M.P 258⁰C. IR (KBr, v cm−1): 3044.54 (-NH-),1649.84(-C=N) 1113.5 (-C-O-).¹H-NMR (400 M.Hz,DMSO-d6, δ ppm): 1.25(s, 9H, (-CH3)3,) 8.85 (d,d, 2H, Aromatic-H), 9.31(s,1H, Aromatic-H),11.46 (s, H,-NH-, D2O exchangeable),¹³C NMR (400 MHz, DMSO-d₆) δ: 39.50, 39.7, 143.20, 145.05, 146.74, 158.82, 159.11,176.27 (38.87-40.13-DMSO-d6-Solvent peak) LCMS: m/z 248.4 (M+1)⁺.

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Hexanamide (3-f):

Chemical Formula: C₁₂H₁₅N₅O₂, Colour: Grey, Yield 66%; M.wt: 261.2, M.P: 205⁰C.1H-NMR (400 M.Hz, DMSO-d₆, δ ppm): 0.88 (s,3H,-CH3),1.29 (m,2H)1.31(m,2H)1.60 (m,2H), 2.46(t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29 (s,1H, Aromatic-H),11.94(s, H,-NH-,), LCMS: m/z 262.5 (M+1)⁺.

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl) Heptanamide (3-g):

Chemical Formula: C₁₃H₁₇N₅O₂, Colour: Grey, Yield 69%; M.wt: 275.3, M.P:210⁰C.1H-NMR (400 M.Hz, DMSO-d₆, δ ppm): 0.87(s,3H,-CH3), 1.28(t,2H)1.31(m,2H)1.59(m,2H), 2.46(t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic-H),11.94(s, H,-NH-,),LCMS: m/z 276.5 (M+1)⁺.

N-(5-(Pyrazin-2-yl)-1,3,4-Oxadiazol-2-yl)Octanamide (3-h):

Chemical Formula: C₁₄H₁₉N₅O₂, Colour: Grey, Yield 73%; M.wt: 289.3, M.P: 208⁰C.1H-NMR (400 M.Hz, DMSO-d₆, δ ppm): 0.86 (s,3H,-CH3),1.28 (t,2H)1.31(m,2H)1.59 (m,2H), 2.50 (t,2H), 8.84 (d,d, 2H, Aromatic-H), 9.29(s,1H, Aromatic-H),11.93(s, H,-NH-,), LCMS: m/z 290.5 (M+1)⁺.

3.1 Biological activity:

3.1.1 Anti-bacterial activity

Table 1: The antibacterial effectiveness of the compounds 3 and (3 a-h) against Staphylococcus aureus and E. coli bacterial strains with reference to (Ampicillin)

S No	Compound	Concentration (µg) / Zone of Inhibition (mm)
		Staphylococcus aureus				E coli
		25	50	75	100	25	50	75	100
1	3	0	0	0	0	0	0	0	6
2	3a	0	0	0	0	0	0	0	0
3	3b	0	0	0	0	0	0	0	0
4	3c	0	0	0	0	0	0	0	0
5	3d	0	0	0	0	0	0	0	0
6	3e	0	0	7	0	0	0	0	0
7	3f	0	0	0	0	0	0	5	8
8	3g	0	0	0	8	0	0	0	0
9	3h	0	0	0	0	0	0	0	5
10	Ampicillin	17	19	23	20	16	18	20	26

The antibacterial viability of the integrated mixtures mentioned as 3 and 3(a-h) was explored against both gram-positive and gram-negative i.e. (S. aureus) (E. coli) bacterial strains. Notably, compound 3f exhibited commendable antibacterial activity at concentrations of 75 µg/ml and 100µg/ml against E. coli, producing zones of inhibition measuring 5 mm and 8 mm, respectively. Following closely, compounds 3 and 3h displayed zones of 6mm and 5mm at a concentration of 100 µg/ml. Compound 3(g) demonstrated notable antibacterial efficacy at 100 µg/ml against Staphylococcus aureus, resulting in an 8 mm inhibition zone, while compound 3(e) produced a 7 mm zone at 75 µg/ml. unfortunately, the leftover compounds showed no antibacterial movement (as displayed fig.1, table 1) against both S. aureus and E. coli strains.

3.1.2 Antifungal activity:

The antifungal efficacy of compounds 3 and its derivatives (3a-h) was assessed against the Candida albicans fungal strain, with Fluconazole serving as the standard reference. All newly synthesized compounds exhibited remarkable antifungal properties. Notably, compound 3f demonstrated the highest antifungal activity, registering zones of inhibition at 25 µg/ml (10mm), 50 µg/ml (14mm), 75 µg/ml (15mm), and 100 µg/ml (18 mm) against C. albicans. Following closely, compound 3b displayed inhibition zones of 9mm, 12mm, and 18mm at concentrations of 50 µg/ml, 75 µg/ml, and 100 µg/ml, respectively. Compound 3c exhibited a 18 mm inhibition zone at 100 µg/ml. while compound 3a displayed zones of 11mm and 17mm at 75 µg/ml and 100 µg/ml. Compound 3g demonstrated inhibition zones of 11mm, 14mm, and 15mm at concentrations of 50 µg/ml, 75 µg/ml, and 100 µg/ml. compound 3d shows a zones of 8mm and 15mm at concentrations of 75,100 µg/ml. also compound 3h showed a zone of 10mm at 50 µg/ml and 16mm at 100µg/ml respectively, against C. albicans, as illustrated in Fig. 2 and summarized in Table 2.

Table 2: The antifungal activity on Candida albicans fungal strain with the compounds 3 and (3 a-h) using Fluconazole as reference standard

S. No.	Compound	Concentration (µg) / Zone of Inhibition (mm)
S. No.	Compound	25	50	75	100
1	3	0	0	13	15
2	3a	0	0	11	17
3	3b	0	9	12	18
4	3c	0	0	0	18
5	3d	0	0	8	15
6	3e	0	0	0	17
7	3f	10	14	15	18
8	3g	0	11	14	15
9	3h	0	10	0	16
10	Fluconazole	0	0	8	15

3.2 Molecular Docking study:

Molecular docking has been extensively utilized in pharmaceutical research for the development of structure-based drugs. In this work, the interaction behaviour of the 3 and 3(a-h) derivatives with UDP-N-acetylglucosamine-enol pyruvate reductase Mtb MurB (PDB ID: 5JZX). It is common knowledge that the, Mur proteins family (Mur A-F, Y and G) may serve as a catalyst for more than ten biosynthetic transformations in the formation of the peptidoglycan layer on bacterial cell walls ^[44-45]. UDP- N-acetylglucosamine-nolpyruvate reductase (MurB) additionally has a significant impact in the binding of the EP-UDPGIcNAc or NADPH in E. coil MurB, Because of this reason receptor for our docking study. The most minimal energy docked posture of the protein-ligand complex portray the hydrogen bonds that structure between the named ligands and the designated proteins because it is an efficient strategy for determining how a small ligand interacts with a target protein (shown in Fig. 3 and Table 3). The docking studies of titled derivatives well accords with experimental. All compounds demonstrated favourable docking scores within the range of -5.91 to -7.56 kcal/mol, as outlined in Table 3. Notably, among the compounds, compound 3h stood out with the highest docking score of -7.56. Specifically, compound 3f exhibited a score of -7.46 kcal/mol, while compounds 3d and 3g showed scores of -7.34 kcal/mol, indicating their robust binding affinity.

Fig. 2. Zone of inhibitions, for the anti-fungal activity of the newly synthesized compounds 3, 3(a-h) using various concentrations Candida albicans as fungal strain.

Table 3: Binding Energies of compounds of 3 and 3(a-h), against receptor UDP-N-acetyl glucosamine-enol pyruvate reductase MurB

S. No.	Compound	Binding Energies (Kcal mol^-1)
		Mtb MurB (PDB ID: 5JZX)
		Binding energy	No. of H Bonds	Residues involved in bonding
1	3	-5.91	6	SER130, GLY69, ALA67(2), VAL 65(2)
2	3a	-6.98	6	GLY69(2), ALA67, GLY68(3)
3	3b	-7.13	5	GLY69(2), SER70(2), GLY68
4	3c	-6.89	5	SER 130, SER70(2), GLY69, ALA67
5	3d	-7.34	5	SER70(2), GLY69(2), ALA67
6	3e	-7.07	4	SER130(2), SER 70, ALA67
7	3f	-7.46	6	SER130, SER70(2), GLY69(2), ALA 67
8	3g	-7.34	5	SER70(2), GLY68, SER130, ALA67
9	3h	-7.56	5	SER70(2), GLY69(2), GLY68

Fig 3: Showing the binding poses and interactions of compound 3 and 3(a-h) to binding sites of target protein: against receptor UDP-N-acetylglucosamine-enol pyruvate reductase Mtb MurB(PDB ID: 5JZX).

CONCLUSION:

In conclusion, we have successfully devised a straightforward and effective approach for synthesizing a series of novel compounds, namely 5-(Pyrazine-2-yl)1,3,4-oxadiazol-2-amine (3) and its derivatives 3(a-h), as confirmed by comprehensive spectral analyses. The synthesized compounds underwent evaluation for their antibacterial and antifungal properties. Notably, compounds 3(f), 3(b), 3(c), 3(a), 3(g), and 3(h) exhibited significant antifungal activities against the Candida albicans fungal strain. Molecular docking studies further demonstrated that compounds 3h, 3f, 3d, and 3g interacted more efficiently with the target protein. This research thus highlights the promising antimicrobial potential of the newly synthesized compounds, emphasizing their potential application in combating fungal infections.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regards to examination.

ACKNOWLEDGMENTS:

The authors are indebted to Honourable Vice-Chancellor, Mahatma Gandhi University, Nalgonda and chairman B V Raju Institute of technology, for supplying the research facilities and constant support to authors. Exceptional thanks to Pharma Laboratory, Hyderabad for providing support in the spectral analysis

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Received on 23.12.2023 Modified on 09.03.2024

Research J. Pharm. and Tech 2024; 17(10):5023-5030.

DOI: 10.52711/0974-360X.2024.00772