INTRODUCTION

The heterocyclic compound play vital role in drug chemistry. They exhibit various biological activities against microorganisms. Specially 1,3,4-oxadiazole are very important nucleus in many drug molecules. Presence of fluorine atoms enhances its activity due to the increases the lipophilicity of molecules thus making more superior than non fluorinated compound. Also C–F bond is stronger than C–H which enlarges its bioavibility. Literature suggests that fluorine augments therapeutics efficiency, improves its pharmacological activity of 1,3,4-oxadiazole [1]. In recent days researcher are paying more attention on 1,3,4-oxadiazole due to its various medicinal, pesticidal, and polymer forming properties. 1,3,4-oxdiazole exhibits vast biological activity such anti-microbial [2–4], antitumor [5], antifungal [6, 7], anti-cancer [8–10], antiviral [11–13], anti-inflammatory [14–16], antibacterial [17, 18], anti-diabetic [19], antioxidant [20–22], antallergic [23], anti-tubercular [24], cytotoxic [25], and anti-HIV [26]. The considering this, it is decided to synthesize tetra fluorinated 1,3,4-oxadiazole as importantas pharmacore.

RESULTS AND DISCUSSION

Chemistry

We have synthesized series of phenyl-5-(2,3,5,6-tetrafluoro-phenyl)-[1, 3, 4]-oxadiazole (Va–j) using POCl3 by the oxidative cyclization of 2,3,5,6-Tetrafluoro-benzoic acid hydrazide (3) and various substituted benzoic acid (IVa–j). Compound 2,3,5,6-Tetrafluoro-benzoic acid hydrazide (III) was obtained by the esterification followed by reaction with hydrazine hydrate. The structure of compound (Va–j) were established by spectroscopic method FTIR, 1H NMR and LC-MS. The compound (Va) confirmed by disappearance of the peak at 4.70 for NH2 and 10.30 (1 NH) in 1H NMR spectrum. 1H NMR peaks of aromatic proton appears at δ 7.27–8.91 as multiplet. IR absorption band appears at 1675 cm–1 indicates –C=N group while band at 1172 and 1066 cm–1 C–O–C bending vibration. LC-MS spectra shows peak at m/z 295.

The synthesis was initiated with the reaction between fluorinated benzoic acid hydrazide (III) with 2,3,5,6-tetrafluorobenzoic acid (IVa) suspended in phosphorus oxytrichloride was refluxed for 6 h and obtained yield 75%, while reaction under microwave radiation enhances the yield up to 92% (Table 1). Hence we conclude that the reaction under microwave help to not only increase yield but also reduce the reaction time. Compound (Vb) and (Vc) containing the electron withdrawing group gave highest yield among all synthesized compound. While compound (Vg) containing electron donating group obtained lowest yield. Hence we observed that the effect of product substituent affect on reaction yield of product.

Table 1.   Time and yield data of newly synthesized compounds (Va–j) using conventional and microwave irradiation method
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Table 2.   Antimicrobial activity of compounds (Va–j) in zone of inhibition in mm
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BIOLOGICAL ACTIVITY

Antimicrobial Activity

The synthesized targeted compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes as example of Gram-positive bacteria and Salmonella typhi and Pseudomonas as examples of Gram-negative bacteria. All bacterial strains were maintained on nutrient agar medium at ±37°C and fungi strains were maintained on potato dextrose agar (PDA) at ±27°C. The disc diffusion method was used for the screening of antimicrobial activity. The newly prepared compounds were screened for their antifungal activity against Candida albicans and Aspergillus niger in DMSO by agar dilution method. The lowest concentration (highest dilution) required to arrest the growth of fungus was regarded as minimum inhibitory concentration (MIC). The fungal activity of each compound was compared with fluconazole as standard drug.

Homology Modeling

The Homology modeling technique was employed to build 3D model structure of cytochrome P 450 lanosterol 14α-demethylase of C. albicans with help of V Life MD S 4.3 Promodel molecular modelling tool. The protein sequence was retrieved from Uniprot KB database (Accession no. P 10 613). The homologue template sequencesearch was carried out against the Protein structure database (http://www.rcsb.org/) by using Blast P. The based on default parameters identity and positive criteria appropriate template crystal structure of human lanosterol 14α-demethylase (CYP 51) complexed with Ketocanazole (3 LD 6_B). The secondary structure assignment and sequence realignment was carried out to build final modeled structure of fungal CYP 51. The modeled structure was subjected to various checks like phi-psi, Z score and Cα deviation [27]. The model protein structure and 3D structure of sketched synthesized compound was prepared for molecular docking using Autodock Vina docking tool [28]. The modeled structure was subjected for various checks like phi-psi, Z score and Cα deviation compounds (Va) to (Vj) was carried out using final modeled structure of fungal CYP 51 to understand this mechanism of action of inhibitors molecular interactions were analyzed [29].

Comparative Modeling

The sequence identity and atomic resolution are two key parameters during the selection of template structure. It was observed to be 44% and 2.8 Å which satisfy the basic criteria for comparative modeling. The final model was subjected for structure validations tool like Procheck, ProSA and SPDBV were 99.7% of residue followed in allowed region. Overall quality of model was evaluated using Prosa were Z-score = –3.2 and Cα deviation = 0.45 Å respectively. The Validation study of model suggested that it was perfect for further computation study.

The synthesized compounds (Va–j) docked in active site of modeled CACYP 51 using Autodock Vina docking tool. Results of docking are shown in Table 3. Analysis of docking interaction has been done and it was found that aryl substituted 1,3,4 oxadiazole are mainly responsible for interaction. The aryl substituted 1,3,4-oxadiazole derivatives (Vb), (Ve) and (Vf) are most active agents. It also reproduced similar result as that of in vitro activity data. All active compounds efficiently bind in the active site residues like PRO104, PHE108, VAL130, SER414, MET413, CYS439, HIS504 and CYS50.

Table 3.   In-vitro antibacterial, antifungal activity and molecular docking of synthesized compounds in the study
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The Nitro phenyl substituted compound (Vb) has highest docking score with free energy of binding –6.04 kcal/mol. The positive charged polar Amino acid residue ARG417 forms conventional hydrogen bond interactions with oxygen atoms of Nitro group with the distance of 2.4 and 2.01Å respectively. Aliphatic amino acid VAL 130 form carbon hydrogen bond with one of the meta substituted fluorine atom with distance of 2.42 Å. The Non polar hydrophobic amino acid residues PRO 104 and PHE108 forms halogen fluoride bond interactions with another meta substituted fluorine atom with the distance of 3.15 and 2.25 Å respectively. The Non polar hydrophilic and hydrophobic amino acid residues CYS439, MET413 and SER414 forms Pi-sulfur, Pi-lone pair, Amide Pi stacked and Pi-alkyl interactions with 1,3,4-oxadizoline substituted phenyl and 1,3,4-oxadizoline with various distance shown in Fig. 1. The second most active derivative (Ve) have very good docking score with free energy of binding of –5.17 kcal/mol. The aliphatic Amino acid residue GLY500 forms carbon hydrogen bond interactions with ortho substituted fluorine atoms with the distance of 2.51 Å. The Non Polar and Polar amino acid LEU 412, MET413, PRO 442, SER 441 and SER 414 forms fluoride bond interaction with ortho and meta substituted fluorine atom with various distance. Non charged and charged amino acid residues HI S504 and CYS506 forms Pi bond interaction with Pi electron cloud of phenyl ring such as Pi-line pair and Pi-alkyl interactions of 2.72 and 5.83Å respectively shown in Fig. 2.

Fig. 1.
figure 1

Binding Pose and molecular interactions of (Vb) in the active site of cytochrome P450 lanosterol 14α-demethylase.

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Fig. 2.
figure 2

Binding Pose and molecular interactions of (Ve) in the active site of cytochrome P 450 lanosterol 14α-demethylase of (Ve) in the active site of cytochrome P450 lanosterol 14α-demethylase.

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EXPERIMENTAL

Materials and Methods

All the solvents and reagents used were of AR grade from Aldrich and used without further purification. The NMR spectra were recorded on a Bruker DPX 400 (1H at 400.13 MHz and (13C at 100.63 MHz) spectrometer, 5 mm sample tubs, 298 K, digital resolution of ±0.01 ppm, 0.5 M in CDCl3, containing TMS as internal standard. Mass spectra were achieved using an HP 6890 GC connected to HP 5973 MSD and interfaced by a Pentium PC.

Synthesis of methyl 2,3,5,6-tetrafluorobenzoate (II). The Fluorinated benzoic acid (0.01 mol) was dissolved in 20 mL absolute methanol followed by the addition of 3.0 mL concentrated sulphuric acid in a 100 mL round bottom (RB) flask. The mixture was refluxed for 3h. Reaction progress was monitored on TLC (ethyl acetate : n-hexane, 30 : 70), after conformation product formation the reaction mixture was poured into a 500 mL separating funnel. Then 100 mL distilled water and 10% sodium bicarbonate solution was added that to remove unreacted carboxylic acid. 100 mL diethyl ether was added and ether layer containing compound isolated. The solvent was distilled off to afford the transparent esters, with yields 74%.

Synthesis of 2,3,5,6-tetra fluoro benzo hydrazide (III). Fluorinated methyl ester of benzoic acid (II) (0.01 mol) was added to 25 mL methanol in 100 mL RB flask followed by 4.0 mL of 80% hydrazine hydrate. The mixture was refluxed consequently for 5h till the completion of reaction, supervised by TLC (ethyl acetate : n-hexane, 30 : 70). The precipitates of solid products was generated after addition of excess of distilled water which was filtered and washed with distilled water to afford (III) with yields 73%.

Conventional synthesis of 1,3,4-oxadiazoles from Fluorinated benzoic acid hydrazide (Va–j). A mixture of fluorinated benzoic acid hydrazide (III) (0.01 mol) with substituted benzoic acid (IV) (0.01 mol) suspended in phosphorus oxytrichloride (10 mL) was refluxed. The progress of reaction was monitored by TLC. After completion of reaction the reaction mass was cooled and poured on to crushed ice with continuous stirring. The separated solid mass was neutralized with Sodium bicarbonate. The mixture was left overnight for Room temperature. The resulting solid (Va–j) thus obtained was collected by filtration, washed well with cold water, dried and recrystallized from absolute ethanol.

Microwave assisted synthesis of 1,3,4-oxadiazoles from fluorinated benzoic acid hydrazide (Va–j). A mixture of fluorinated benzoic acid hydrazide (3) (0.01 mol) with substituted benzoic acid (IV) (0.01 mol) suspended in phosphorus oxytrichloride (10 mL) was irradiated at 110°C for 15 min. The completion of the reaction was monitored by TLC. After completion of the reaction the RBF was removed from the oven. The reaction mixture was poured on to crushed ice with continuous stirring. The separate was left overnight at Room temperature. The resulting solid (Va–j) thus obtained was collected by filtration, washed well with cold water, dried and recrystallized from absolute ethanol.

Phenyl-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Va). Yield 91%, mp 95°C; IR (KBr, cm–1): 1675 (C=N stretching), 1172 (C–O–C stretching), 1549 (C=C stretching, aromatic ring), 1066 (C=N stretching). 1H NMR (400 MHz, CDCl3) δ: 7.33 (s, 1H), 7.53 (dd, J = 8.6 , 6.0 Hz, 1H), 7.58 (dd, J = 6.0, 8.6 Hz, 2H), 8.14 (d, J = 8.24 Hz, 2H), ESI-MS: m/z 295 (M + 1).

2-(4-Nitro-phenyl)-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Vb). Yield 92%, mp 208°C; IR (KBr, cm–1): 1606 (C=N stretching), 1176 (C–O–C stretching), 1507 (C=C stretching, aromatic ring), 1440 (N–O stretching); 1H NMR (400 MHz, CDCl3) δ: 7.3 (s, 1H), 8.35 (d, J = 8.88 Hz, 2H) 8.44 (d, J = 8.88 Hz, 2H). ESI-MS: m/z 340 (M + 1).

2-(3-Nitro-phenyl)-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Vc). Yield 92%, mp 156°C; IR (KBr, cm–1): 1588 (C=N stretching), 1174 (C–O–C stretching), 1506 (C=C stretching aromatic ring), 1421 (N–O stretching); 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H), 7.78 (dd, J = 8.08 Hz), 8.45 (d, J = 6.8 Hz, 1H), 8.51 (d, J = 7.8 Hz, 1H), 8.98 (s, 1H), ESI-MS: m/z 340 (M + 1).

2-(4-Methoxy-phenyl)-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Vd). Yield 84%, mp 191°C; IR (KBr, cm–1): 1609 (C=N stretching), 1015 (C–O–C stretching), 1551 (C=C stretching, aromatic ring), 2896 (C–OCH3 stretching); 1H NMR (400 MHz, CDCl3) δ: 3.9 (s, 3H, OCH3), 7.054 (s, 1H, Ar–H), 7.055 (d, J = 8.4 Hz, 2H, Ar–H), 8.081 (d, J = 8.4 Hz, 2H, Ar–H). ESI-MS: m/z 325 (M + 1).

2-(4-Chloro-phenyl)-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Ve). Yield 90%, mp 154°C; IR (KBr, cm–1): 1638 (C=N stretching), 1151 (C–O–C stretching), 1499 (C=C C stretching, aromatic ring); 1H NMR (400 MHz, DMSO-d6) δ: 7.56 (s, 1H, Ar–H), 7.8 (d, J = 6.12 Hz, 2H, Ar–H), 8.8 (d, J = 6.12 Hz, 2H, Ar–H), ESI-MS: m/z 329 (M + 1).

4-[5-(2,3,5,6-Tetrafluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-phenylamine (Vf). Yield 85%, mp 97°C IR (KBr, cm–1): 1605 (C=N stretching), 1171 (C–O–C stretching), 1492 (C=C stretching); 3415 (NH2 stretching): 1H NMR (400 MHz, CDCl3) δ: 3.73 (s, 2H, Ar–NH 2), 6.78 (s, H, Ar–H), 7.32 (d, J = 7.8 Hz, 2H, Ar–H), 7.87 (d, J = 9 Hz, 2H, Ar–H). ESI-MS: m/z 310 (M + 1).

2-(2,3,5,6-Tetrafluoro-phenyl)-5-p-tolyl-[1,3,4]oxadiazole (Vg). Yield 80%, mp 123°C, IR (KBr, cm–1): 1601 (C=N stretching), 1173 (C–O–C stretching), 1492 (CH stretching, CH3 group), 1H NMR (400 MHz, CDCl3) δ: 2.44 (s, 3H, Ar–CH3), 7.26 (s, H, Ar–H), 7.34 (d, J = 8.4 Hz, 2H, Ar–H), 8.01 (d, J = 6 Hz, 2H, Ar–H). ESI-MS: m/z 309 (M + 1).

2-(2-Chloro-phenyl)-5-(2,3,5,6-tetrafluoro-phenyl)-[1,3,4]oxadiazole (Vh). Yield 87%, mp 130°C; IR (KBr, cm–1): 1600 (C=N), 1171 (C–O–C), 1497 (C=C); 1H NMR (400 MHz, CDCl3) δ: 7.36 (s, H, Ar–H), 7.44 (dd, J = 6.04 Hz, H, Ar–H), 7.52 (dd J = 7.3 Hz, 1H, Ar–H), 7.61 (d, J = 8.04 Hz, 1H, Ar–H), 8.1 (d, J = 6 Hz, 1H, Ar–H), ), ESI-MS: m/z 329 (M + 1).

3-3-[5-(2,3,5,6-Tetrafluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-phenol (Vi). Yield 85%, mp 294°C; IR (KBr) cm–1: 1608 (C=N), 1171 (C–O–C), 1467 (C=C); (O–H stretching aromatic): 1H NMR (400 MHz, CDCl3) δ: 7.04 (s, 1H, Ar–OH), 7.27 (s, 1H, Ar–H), 7.8 (d, J = 9.0 Hz, 2H, Ar–H),7.1 (d, J = 9.0 Hz, 2H, Ar–H). ESI-MS: m/z 311 (M + 1).

3-3-[5-(2,3,5,6-Tetrafluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-phenol (Vj). Yield 87%, mp 253°C; IR (KBr) cm–1: 1605 (C=N), 1159 (C–O–C), 1430 (C=C); 3260 (O–H stretching aromatic), 1H NMR (400 MHz, DMSO, dppm): δ: 7.05 (s, 1H, Ar–H), 7.3 (s, 1H, Ar–H), 7.31 (d, J = 7.84, 1H), 7.37 (d, J = 7.84 Hz, 1H, Ar–H) 7.48 (dd, J = 7.84, 6.68 Hz, 1H, Ar–H), 7.70 (d, J = 6.8 Hz, 1 H, Ar–H). ESI-MS: m/z 311 (M + 1).

CONCLUSION

A series of phenyl-5-(2,3,5,6-tetrafluoro-phenyl)- [1,3,4]-oxadiazole derivatives was synthesized by using conventional as well as microwave-assisted technique and evaluated for the antimicrobial activity. In vitro antimicrobial screening revealed that (Vb), and (Ve) can serve as important lead moiety as they replicating in vitro activity in the inhibition assay and in silico molecular docking study. They can be used in scaffold hoping for the design and development of new lead as antibacterial and antifungal agents.

Scheme 1.