Indian Journal of Chemistry Vol. 57B, May 2018, pp. 687-699 Synthesis, biological evaluation and docking studies of a new series of tris-heterocycles containing pyrazole, triazole and oxadiazole Gaddam Rajesh Kumar, Kurre Shankar & Cherkupally Sanjeeva Reddy* Department of Chemistry, University College, Kakatiya University, Warangal 506 009, India E-mail: [email protected]Received 16 August 2016; accepted (revised) 8 August 2017 A new series of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]oxadiazoles 7a-j have been synthesized, characterized by spectral means, and evaluated for their antimicrobial and antioxidant properties. Amongst the screened compounds 7a-j, the triazole moiety bearing electron-withdrawing group on the 4 th position of phenyl viz. 4-chlorophenyl 7c, 4-nitrophenyl 7f and 2,4-difluorophenyl 7h exhibit excellent antibacterial activity towards all the tested bacteria viz. Staphylococcus aureus, Bacillus licheniformis, Pseudomonas aeruginosa and Escherichia coli. On the other hand, triazole moiety bearing electron-donating group on the 4 th position of phenyl viz. 4-hydroxyphenyl 7d, 4-methoxy-phenyl 7e and 4-aminophenyl 7g show prominent antifungal activity towards the tested fungi viz. Candida albicans and Fusarium oxysporum. These results are supported by molecular docking studies and through binding interactions as well. Antioxidant studies reveal that the compounds in which the triazole moiety bearing phenyl 7a, 4-methoxyphenyl 7e, 2,4-difluorophenyl 7h and 3-pyridyl 7j is present on the 4 th position, display significant antioxidant properties. In brief, most of the newly synthesized compounds have emerged as potential antimicrobial and antioxidant agents for further development. Keywords: One-pot synthesis, fused triazolo-oxadiazole, pyrazole, antimicrobial activity, antioxidant property, molecular docking studies The fusion of biodynamic heterosystems in a singular molecular framework has attracted considerable interest in heterocyclic chemistry due to their varying pharmacological effects. The prevalence of azoles in several natural products, and as drug candidates such as Itraconazole, Voriconazole, Posaconazole and Fluconazole inspired more research towards azole heterocycles. These are able to bind easily with the enzymes and receptors in organisms through weak interactions such as coordination bonds, hydrogen bonds, ion–dipole, π – π stacking and hydrophobic effects as well as van der Waals forces 1 . Pyrazoles display a broad spectrum of biological activities, among which the prominent are cerebro protectors 2 , antitubercular 3 , antimicrobial 3 , anti- inflammatory 4 , analgesic 4 , antidepressant 5 , anticonvulsant 5 , antiulcer 6 , COX-2 inhibitory 7 , HIV-1 inhibitory 8 , antitumor 9 , antioxidant 10 and anticancer 11 . Hence, pyrazoles have evolved as prominent targets in synthetic organic chemistry. Further, 1,2,4-triazole systems have been widely investigated as biologically potent heterocycles and their N-bridged heterocyclic analogs have shown a wide variety of pharmacological effects including antitumor 12 , antimicrobial 13 , antiviral 13 , anti-inflammatory 14,15 , analgesic 15 , antioxidant 16 and antidepressant 17 . In addition, fused heterocyclic ring system of triazoles possesses a broad spectrum of medicinal applications 18-23 . Similarly, 1,3,4-oxadiazole derivatives are reported to possess significant antioxidant 24,25 , anti-inflammatory 25 , antimicrobial 25,26 and antitubercular 27,28 activities. Inspired by the escalating importance of pyrazole, oxadiazole and triazole in pharmaceutical fields with a broad spectrum of biological profile and in continuation of our research on the synthesis of bioactive heterocycles 29-33 , it is of our interest to incorporate these three active pharmacophores in a single molecular frame work, to obtain a new class of fused / linked heterocyclics with potential biological activity. We here in report the synthesis, antimicrobial and antioxidant studies of a new series of 3- aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)- [1,2,4] triazolo[3,4-b][1,3,4]oxadia-zoles. Results and Discussion The starting material, (E)-2-acetyl-3-(dimethylamino)- 2-propenoate 2, required for the synthesis of title compounds was obtained on the condensation of N,N-
Transcript
Indian Journal of Chemistry Vol. 57B, May 2018, pp. 687-699
Synthesis, biological evaluation and docking studies of a new series of tris-heterocycles containing pyrazole, triazole and oxadiazole
Received 16 August 2016; accepted (revised) 8 August 2017
A new series of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]oxadiazoles 7a-j have been synthesized, characterized by spectral means, and evaluated for their antimicrobial and antioxidant properties. Amongst the screened compounds 7a-j, the triazole moiety bearing electron-withdrawing group on the 4th position of phenyl viz. 4-chlorophenyl 7c, 4-nitrophenyl 7f and 2,4-difluorophenyl 7h exhibit excellent antibacterial activity towards all the tested bacteria viz. Staphylococcus aureus, Bacillus licheniformis, Pseudomonas aeruginosa and Escherichia coli. On the other hand, triazole moiety bearing electron-donating group on the 4th position of phenyl viz. 4-hydroxyphenyl 7d, 4-methoxy-phenyl 7e and 4-aminophenyl 7g show prominent antifungal activity towards the tested fungi viz. Candida
albicans and Fusarium oxysporum. These results are supported by molecular docking studies and through binding interactions as well. Antioxidant studies reveal that the compounds in which the triazole moiety bearing phenyl 7a, 4-methoxyphenyl 7e, 2,4-difluorophenyl 7h and 3-pyridyl 7j is present on the 4th position, display significant antioxidant properties. In brief, most of the newly synthesized compounds have emerged as potential antimicrobial and antioxidant agents for further development.
The fusion of biodynamic heterosystems in a singular molecular framework has attracted considerable interest in heterocyclic chemistry due to their varying pharmacological effects. The prevalence of azoles in several natural products, and as drug candidates such as Itraconazole, Voriconazole, Posaconazole and Fluconazole inspired more research towards azole heterocycles. These are able to bind easily with the enzymes and receptors in organisms through weak interactions such as coordination bonds, hydrogen bonds, ion–dipole, π – π stacking and hydrophobic effects as well as van der Waals forces1.
Pyrazoles display a broad spectrum of biological activities, among which the prominent are cerebro protectors2, antitubercular3, antimicrobial3, anti-inflammatory4, analgesic4, antidepressant5, anticonvulsant5, antiulcer6, COX-2 inhibitory7, HIV-1 inhibitory8, antitumor9, antioxidant10 and anticancer11. Hence, pyrazoles have evolved as prominent targets in synthetic organic chemistry. Further, 1,2,4-triazole systems have been widely investigated as biologically potent heterocycles and their N-bridged heterocyclic analogs have shown a wide variety of pharmacological effects including antitumor12, antimicrobial13, antiviral13,
anti-inflammatory14,15, analgesic15, antioxidant16 and antidepressant17. In addition, fused heterocyclic ring system of triazoles possesses a broad spectrum of medicinal applications18-23. Similarly, 1,3,4-oxadiazole derivatives are reported to possess significant antioxidant24,25, anti-inflammatory25, antimicrobial25,26 and antitubercular27,28 activities.
Inspired by the escalating importance of pyrazole, oxadiazole and triazole in pharmaceutical fields with a broad spectrum of biological profile and in continuation of our research on the synthesis of bioactive heterocycles29-33, it is of our interest to incorporate these three active pharmacophores in a single molecular frame work, to obtain a new class of fused / linked heterocyclics with potential biological activity. We here in report the synthesis, antimicrobial and antioxidant studies of a new series of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[3,4-b][1,3,4]oxadia-zoles. Results and Discussion
The starting material, (E)-2-acetyl-3-(dimethylamino)-2-propenoate 2, required for the synthesis of title compounds was obtained on the condensation of N,N-
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dimethyldimethoxymethanamine with ethyl acetoacetate 1
34, which on cyclocondensation with phenyl hydrazine gave 5-methyl-1-phenyl-1H-4-pyrazolecarboxylate 3
34. Compound 3 on hydrazinolysis with hydrazine hydrate resulted 5-methyl-1-phenyl-1H-4-pyrazolecarbohydrazide 4 in 78% yield. This 4 on reaction with carbon disulfide and potassium hydroxide, in ethanol, followed by acidification afforded 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-1,3,4-oxadiazol-2-yl-hydrosulfide 5 in 74% yield, which on reflux with hydrazine hydrate, in the presence of potassium hydroxide, in ethanol for 4 h, afforded 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-1,3,4-oxadiazol-2-yl-hydrazine 6 in 71% yield. One-pot cycloconden-sation of compound 6 with different aroyl/heteroyl chlorides in the presence of POCl3, in pyridine at reflux temperature, resulted the new series
of title compounds, 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]-oxadiazoles 7a-j (Scheme I).
The plausible mechanism for the formation of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]oxadiazoles 7a-j involves two steps. The initial step involves Schotten–Baumann condensation reaction between 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-1,3,4-oxadiazol-2-yl-hydrazine 6 and aroyl/heteroyl chloride in pyridine. Addition of pyridine is required to neutralize the resultant acid (Scheme II).
In the subsequent step, the intermediate undergoes Bischler–Napieralski cyclo-condensation type reaction in presence of POCl3 (as shown in Scheme III) to give the title compounds 7a-j via dichlorophosphoryl imine-ester intermediate.
Scheme I — Schematic route for the synthesis of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]oxadiazoles 7a-j.
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Structures of all the newly synthesized compounds have been satisfactorily established on the basis of elemental analyses, Electron Ionization (EI) mass, IR, 1H- and 13C NMR spectral data. In IR spectra of compounds 7a-j, disappearance of the broad band in the range of 3300-3200 cm−1 (NH2) shows the evidence for ring closure involving –NH2 group. Similarly, the absence of 1H NMR signals for the –NH2 and –NH protons at δ 5.32 and 8.10 respectively, well support the structures. Appearance of prominent 13C NMR signals at about δ 156.9 and 159.4 are further proof of evidence of their structures. In summary, all the newly synthesized compounds
exhibited satisfactory spectral data consistent with their chemical structures. Biological Activity
Antibacterial activity
The newly synthesized compounds 7a-j were screened for their in-vitro antibacterial activity against two representative Gram-positive bacteria viz.
Staphylococcus aureus and Bacillus licheniformis and two Gram-negative bacteria viz. Pseudomonas
aeruginosa and Escherichia coli by using the agar diffusion method35,36. Ciprofloxacin was used as a standard, and the results are depicted in Table I. The
Scheme II
Scheme III
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structure–activity relationship reveal that, the compounds in which the triazole moiety bearing phenyl 7a, 4-methoxyphenyl 7e and 3-pyridyl 7j showed least antibacterial activity; 3-nitrophenyl 7b,
4-hydroxyphenyl 7d and 4-aminophenyl 7g, exhibited moderate antibacterial activity, while 4-chlorophenyl 7c, 4-nitrophenyl 7f and 2,4-difluorophenyl 7h
displayed excellent antibacterial activity against all the tested bacteria and is comparable to the standard drug (Table I). The minimum inhibitory concentration (MIC) values were determined for the selected compounds with significant growth inhibition zones using two-fold serial dilution method37,38 (Table I).
Antifungal activity All the newly synthesized compounds 7a-j were
also screened for their in vitro antifungal activity against Candida albicans and Fusarium oxysporum
by Agar-diffusion method35,36. The structure–activity relationship for the tested compounds on comparison with the standard drug Itrazole, reveal that the compounds in which triazole moiety bearing 4-hydroxyphenyl 7d, 4-methoxyphenyl 7e and
4-methoxyphenyl 7g exhibited prominent antifungal activity, nearly equal to the Itrazole standard against
all the tested fungi (Table I). The remaining compounds showed moderate to good antifungal activity. The MIC values were determined for the selected compounds with significant growth inhibition zones using two-fold serial dilution method37,38 (Table I). Antioxidant properties
Antioxidant activity of the newly synthesized compounds 7a-j was assessed on the basis of radical scavenging effect of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical39, which is mostly used to characterize antioxidants.
The structure–activity relationship among the tested compounds 7a-j, reveal that the triazole moiety bearing phenyl (7a), 4-methoxyphenyl (7e), 2,4-difluoro-phenyl (7h) and 3-pyridyl (7j) showed significant antioxidant activity (Table I). Other compounds were found to be moderate to good. Molecular Docking Studies
These studies open the door to unfold the biological processes within a shorter computational timeframe, without compromising the accuracy, provide new insights to evaluate biological properties
Table I — Antimicrobial and antioxidant activity of the newly synthesized compounds 7a-j
Compd Antibacterial activity
(Inhibition zone diameter, mm) Antifungal activity
(Inhibition zone diameter, mm) Antioxidant activity
(% Inhibition at 100 µM)
P. aeruginosa E. coli S. aureus B. licheniformis F. oxysporum C. albicans 7a 8 12 9 11 11 8 70.2 7b 14 16 12 12 17 14 50.2 7c 25
(12.50)a 20
(25.00) 15
(25.00) 17
(12.50) 13 11 42.6
7d 20 18 14 13 24 (12.50)
18 (6.25)
60.3
7e 12 14 8 10 20 (6.25)
14 (12.50)
70.3
7f 29 (3.12)
26 (6.25)
19 (6.25)
21 (6.25)
9 9 58.9
7g 17 15 12 14 28 (3.12)
22 (3.12)
48.6
7h 22 (6.25)
24 (12.50)
16 (12.50)
18 (12.50)
14 11 70.6
7i 10 10 10 9 6 6 65.7 7j 12 13 14 12 16 12 69.7
Ciprofloxacin 20 (25.00)
25 (12.50)
18 (12.5)
19 (25.00)
― ― ―
Itrazole ― ― ― ― 25 (12.50)
19 (6.25)
―
aValues in parentheses indicate Minimum inhibitory concentration (MIC) in µg/mL of selected compounds. 1% DMSO was used as a control
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of the molecule in further stages of analysis at the atomic level.
InhA, the enoyl acyl carrier protein reductase (ENR), is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target40, and Candida albicans dihydrofolate reductase (CaDHFR)41 is a drug target that is essential for DNA and protein synthesis. These two proteins viz., ENR
and CaDHFR were selected as targets to find out selective antimicrobial agents from the newly synthesized compounds. Antibacterial – docking: Protein ID: 4TZK vs.
newly synthesized compounds 7a-j Analysis of the binding patterns and their
corresponding docking score between each newly synthesized compounds 7a-j against target protein
Table II — Antibacterial docking: Protein ID: 4TZK vs. newly synthesized compounds 7a-j
Compd Hydrophobic Interactions Binding energy (K Cal mol−1)
(ENR) with protein ID: 4TZK (Table II) reveal that, compounds bearing electron-withdrawing group on either 2nd or 4th positions showed best fitness than other compounds.
Visual inspection of docked structures of newly synthesized compounds 7a-j with receptor showed antagonist hydrophobic interaction with Leu63 residue is the key binding interaction responsible for varied binding affinities in receptor protein, in addition to H-bonding between Nitrogen of oxadiazole and -NH2 of Gly96 (Table II). On the other hand, we observed that electron-donating group at 4th position of phenyl on triazole moiety and π-π interaction between residue Phe149 and phenyl are agonistic in nature. Thus, compounds 7c, 7f (Figure 1) and 7h containing 4-chlorophenyl, 4-nitrophenyl and 2,4-difluorophenyl substituents respectively evolved as potential antibacterial agents. Antifungal – docking: Protein ID: 3QLS vs. newly
synthesized compounds 7a-j The theoretical outcome of binding patterns and
their corresponding binding free energies of each newly synthesized compounds 7a-j in the target protein CaDHFR with protein ID: 3QLS (Table III) depict that, triazole moiety bearing electron-donating group on 4th position of phenyl viz. 4-hdroxyphenyl 7d, 4-methoxyphenyl 7e and 4-aminophenyl 7g showed higher binding affinities than other compounds.
In order to define the key binding interactions, where specific roles of each individual pharmacophore in the active pocket of target protein were explored and many conserved hydrophobic
interactions were found, viz., Ile16, Ile95, Leu63, Val65, Phe41, Ile122, Phe97, Met147, Phe149, Ala191, Pro193, Ile194 and Ile21 (Figure 2). These binding interactions in the active pocket of targeted protein reveal that the molecular interactions with residue Ile122 are significant in determining the antagonist properties of molecules, while, π-π interactions between N-phenyl-Phe36 residue are agonist in nature (Table III) and are the key binding sites to regulate the action of protein. Thus, compounds 7d, 7e and 7g are promising candidates which have emerged as potential antifungal agents.
Correlation of in vitro and in silico
It was interesting to observe varied degree of interactions in the in silico molecular docking studies and MIC values in the in vitro antimicrobial studies, though the core structure of all the compounds was similar. It is pertinent to note that correlation studies of both, in vitro and in silico results, were more effectual in demonstrating the antimicrobial activity.
The in vitro antibacterial MIC values correlated well with binding energies obtained through molecular docking with enoyl acyl carrier protein reductase (ENR), protein ID: 4TZK. From the comparative analysis, the minimum bacterial inhibition potencies of 7c, 7f and 7h (Table I) showed correlation with minimum binding energies −9.688, −9.988 and −9.855 K Cal mol−1, respectively with targeted protein (Table II). These studies reveal that, antagonist hydrophobic interaction with Leu63 residue and H-bonding between Nitrogen of oxadiazole and -NH2 of Gly96 (Table II) are crucial in
Figure 1 — Possible 3D (left) and 2D (right) orientations of compound 7f vs. 4TZK showing hydrophobic interactions with Ile16, Ile95, Leu63, Val65, Phe41, Ile122, Phe97, Met147, Phe149, Ala191, Pro193, Ile194, Ile21 and H-bonds between N of oxadiazole and -NH2 of Gly96 (2.32 Å); -NO2 of 3-NO2-phenyl and -NH2of Val65 (2.76 Å).
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designing potent heterocycles that can function as excellent antibacterial agents.
Similarly, from the comparative analysis of in vitro antifingal MIC values and molecular binding energies
Table III — Antifungal docking: Protein ID: 3QLS vs. newly synthesized compounds 7a-j
Compd Hydrophobic Interactions Binding energy (K Cal mol−1)
−6.253 -NH2 of Lys24 -OH of Furan H-donor 2.46 Imidazole – Arg79
8
-NH2 of Thr58 O− of PO4− H-acceptor 2.16
-NH2 of Ala11 -C=O of Amide H-acceptor 1.80
-C=O of Ala11 -NH2 of Amide H-donor 2.16
-C=O of Ile19 -NH2 of Amide H-donor 2.09
-NH2 of Arg56 O of Furan H-acceptor 2.70
-NH2 of Arg56 O− of PO4− H-acceptor 2.01
-NH of Arg56 -P=O of PO4− H-acceptor 1.70
=NH2+ of
Arg79 O− of PO4
− H-acceptor 2.04
-COO− of Glu120
-NH2 of Amine H-donor 2.17
-NH2 of Lys57 -O- of PO4− H-acceptor 2.70
-NH2 of Ile117 -P=O of PO4− H-acceptor 2.39
-NH2 of Gly114
-P=O of PO4− H-acceptor 2.05
-NH2 of Glu116
O− of PO4− H-donor 1.88
-NH2 of Ala115
-O- of PO4− H-acceptor 2.23
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and interactions with target protein CaDHFR, protein ID: 3QLS depict that, the minimum fungal inhibition potencies of compounds 7d, 7e and 7g (Table I) followed the order of the best fitness score −6.487, −6.463, −6.681 K Cal mol−1, respectively with targeted protein (Table III). These studies reveal that, π-π interactions between N-phenyl-Phe36 residues are agonist in nature, while, molecular interactions with residue Ile122 are significant in determining the antagonist properties of molecules (Table III) to regulate the action of protein. Experimental Section
All the solvents and chemicals were of commercial grade and used as received. Melting points weredetermined on a Fisher-Johns melting point apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on Merckpre-coated silica gel F254 plates and the compounds are visualized either by exposure to UV light or dipping in 1% aqueous potassium permanganate solution. Column chromatography was performed over silica gel 60 (0.043–0.060 mm). Infra red spectra were recorded on a Perkin-Elmer FTIR 5000 spectrometer, using KBr pellets. The 1H and 13C NMR spectra were recorded in DMSO-d6 and CDCl3 on a Varian Gemini 300 MHz and 75 MHz spectrometer respectively. Chemical shifts are reported in δ units down field using TMS as an internal standard, and coupling constants (J) are reported in Hz. Mass spectra were obtained on a VG micro mass 7070H spectrometer. Elemental analyses
(C, H, N) were determined by a Perkin-Elmer 240 CHN elemental analyzer. Shimadzu UV-2450 spectrophotometer was used to monitor the antioxidant properties. Synthesis of 5-methyl-1-phenyl-1H-4
pyrazolecarbohydrazide, 4 Hydrazine hydrate (5 mmol) was added to a
stirring solution of compound 3 (5 mmol) in ethanol (15 mL) and refluxed for 4 h, cooled to RT and the separated product was filtered, dried and purified by recrystallization from methanol to give white crystalline compound 4. Yield 78%. m.p. 164-66°C. IR (KBr): 3269 (broad, NH2), 3062 (C–H, aromatic), 2942 (C–H, aliphatic), 1662 (C=O), 1506 (C=C) cm−1; 1H NMR (300 MHz, CDCl3): δ 2.61 (s, 3H, CH3), 5.61 (s, 2H, NH2), 7.15-7.30 (m, 5H, Ar-H), 8.26 (s, 1H, Ar-H), 8.96 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): δ 15.6 (CH3, C-1′′′), 112.7 (C, C-4′′), 124.9 (CH, C-2′, C-6′), 127.7 (CH, C-4′), 128.3 (CH, C-3′, C-5′), 138.5 (CH, C-1′), 139.6 (C, C-5′′), 143.0 (CH, C-3′′), 165.1 (C=O); ESI-MS: m/z 216 (M+). Anal. Calcd for C11H12N4O: C, 61.10; H, 5.59; N, 25.91. Found: C, 61.06; H, 5.55; N, 25.85%. Synthesis of 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-
1,3,4-oxadiazol-2-ylhydrosulfide, 5
A mixture of compound 4 (5 mmol), potassium
hydroxide (5 mmol) and carbon disulfide (7.5 mmol), in ethanol (100 mL) was heated under reflux with stirring for 12 h and the solvent was distilled off in vacuo. The separated product was poured over crushed ice and acidified with 10% hydrochloric acid.
Figure 2 — Possible 3D (left) and 2D (right) orientations of compound 7g vs. 3QLS, showing many conserved hydrophobic interactions viz., Ile16, Ile95, Leu63, Val65, Phe41, Ile122, Phe97, Met147, Phe149, Ala191, Pro193, Ile194, Ile21 and π-π interactions with N-Phenyl-Phe36 residue.
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The precipitated crude product was filtered, washed with water, dried and purified by recrystallization from ethanol to give pure compound 5 as orange yellow solid. Yield 74%. m.p. 147-49°C. IR (KBr): 3162 (S–H), 2914 (C–H), 1612 (C=N), 1343 (C-S), 1152 (C-O) cm−1; 1H NMR (300 MHz, CDCl3): δ 2.52 (s, 3H, CH3), 7.20-7.45 (m, 5H, Ar-H), 8.20 (s, 1H, Ar-H). 11.61 (s, 1H, NH/SH); 13C NMR (75 MHz, DMSO-d6): δ 13.7 (CH3, C-1′′′), 123.5 (C, C-4′′), 126.5 (CH, C-2′, C-6′), 127.5 (CH, C-4′), 129.8 (CH, C-3′, C-5′), 136.7 (C, C-5′′), 138.5 (CH, C-3′′), 139.7 (C, C-1′), 158.8 (O-C=N), 171.0 (N=C-SH); ESI-MS: m/z 258 (M+). Anal. Calcd for C12H10N4OS: C, 55.80; H, 3.90; N, 21.69. Found: C, 55.76; H, 3.82; N, 21.64%. Synthesis of 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-
1,3,4-oxadiazol-2-yl-hydrazine, 6 To a stirred solution of compound 5 (5 mmol) and
potassium hydroxide (5 mmol) in ethanol (30 mL), 80% hydrazine hydrate (0.03 mol) was added drop-wise and then refluxed for 6 h. The solvent was distilled off in vacuo, cooled and the separated crystals were filtered, washed with cold ethanol and purified by recrystallization from methanol to give the pure compound 6 as yellow solid. Yield 71%, m.p.132-34°C. IR (KBr): 3300-3200 (NH2), 3145 (Ar-H), 2915 (C-H), 1614 (C=N), 1540 (C=C), 1268(C-O) cm−1; 1H NMR (300 MHz, CDCl3): δ 2.55 (s, 3H, CH3), 5.32 (s, 2H, NH2), 7.18-7.26 (m, 5H, Ar-H), 8.19 (s, 1H, Ar-H), 8.10 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): δ 13.9 (CH3, C-1′′′), 123.8 (C, C-4′′), 125.9 (CH, C-2′, C-6′), 127.7 (CH, C-4′), 129.6 (CH, C-3′, C-5′), 136.8 (C, C-5′′), 138.2 (CH, C-3′′), 139.8 (C, C-1′), 158.8 (O-C=N), 169.3 (N=C-NH-NH2); ESI-MS: m/z 256 (M+). Anal. Calcd for C12H12N6O: C, 56.24; H, 4.72; N, 32.79. Found: C, 56.22; H, 4.69; N, 32.75%. General procedure for the synthesis of 3-aryl /
hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-
[1,2,4] triazolo[3,4-b][1,3,4]oxadiazole, 7a-j To a solution of compound 6 (5 mmol) in POCl3
(10 mL), a solution of corresponding acid chlorides (5 mmol) in dry pyridine (20 mL) was added in drops and the mixture was stirred at RT for about 1 h. Then the reaction mixture was heated under reflux for about 2 h on a steam bath. After completion of reaction (TLC), the solution was poured onto crushed ice and the excess POCl3 was neutralized with 10% sodium bicarbonate solution. The separated solid was filtered,
dried and purified by column chromatography to afford pure compounds 7a-j.
Standard sterilized Whatmann filter paper disks (5 mm diameter) impregnated with a solution of the test compound in DMSO (1 mg/mL) was placed on an agar plate seeded with the appropriate test organism in triplicate. Ciprofloxacin and Itrazole were used as standard antibacterial and antifungal agents respectively. DMSO was used as control and all the solutions were made in DMSO. The plates were incubated at 37°C for 1–3 days. Antimicrobial activity was determined by measuring the diameter of the zone of inhibition surrounding microbial growth35,36. Compounds that showed significant growth inhibition zones were further evaluated for their MICs. Minimum inhibitory concentration (MIC)
measurement
The microorganism’s susceptibility tests in nutrient and potato dextrose broths were used for the determination of MIC. Stock solutions of the tested compounds, ciprofloxacin and itrazole were prepared in DMSO at concentration of 100 µg/mL followed by dilutions at concentrations of 100-3.12 µg/mL. The microorganism suspensions were inoculated into different concentrations of corresponding compounds
and control experiments. These were incubated at 37°C for 1–3 days for MIC determination37,38. In vitro antioxidant assay
Reversible reduction property of DPPH and the odd electron in the DPPH free radical, which is purple color, gives a strong absorption maximum at 517 nm (λmax), makes it suitable for spectrophotometric studies. Purple color decolorizes when a radical scavenging antioxidant reacts with the stable free radical DPPH and converts it into 1,1-diphenyl-2-picrylhydrazine, which is stoichiometric with respect to the number of electrons captured and resulting change in the absorbance produced in this reaction has been used to measure the antioxidant property.
Solutions of the synthesized compounds (100 µM) were added to DPPH (100 µM) in methanol, kept in dark for 30 min and the absorbance were measured at 517 nm (λmax), using a spectrophotometer. Control experiment was conducted with equal amount of solvent in an identical manner. The experiments were carried out in triplicate. Difference between the control and the test experiment was expressed as the percent scavenging of the DPPH radical. Molecular modelling studies
The molecular docking studies of the ligands to protein active sites were performed by molecular docking software program, Schrodinger suite Maestro-9.3 version. The X-ray crystallographic structures of selected proteins, InhA, the enoyl acyl carrier protein reductase (Protein ID: 4TZK)40 and Candida albicans dihydrofolate reductase (Protein ID: 3QLS)41 were obtained from Protein Data Bank (RCSB PDB) and are used for docking studies by using Maestro. It was further optimized with prepared Wizard and the refined protein structure minimized with Macro Model Minimization40, which is a method to get low energy conformations of the protein and to release any unwanted strain in protein crystal structure as well in homology model, and the refined protein used for grid generation with glide module from Schrödinger41-45. Ligands preparation
All the molecules were drawn with Maestro interface, further minimized with conformational search40 to get global minima of the structure for the individual molecule. The low energy conformer used for docking41 and docking score was recorded
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(Table II and Table III). The docking method is XP (Extra Precision). The molecular modeling software installed on high end workstations (Dell Precision T5500, with 500GB hard disk and 16GB RAM with 4GB graphic card capacity).
To find strongest potential molecular targets, microscopic binding interactions between each protein and the newly synthesized compounds were studied, to sort out the key interactions between receptor protein and ligand. Conclusions
A new series of 3-aryl/hetaryl-6-(5-methyl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4] oxadiazoles 7a–j have been synthesized by one pot cyclocondensation of 5-(5-methyl-1-phenyl-1H-4-pyrazolyl)-1,3,4-oxadiazol-2-yl-hydrazine (6) with different aroyl/heteroyl chlorides and screened for their antimicrobial and antioxidant properties. Among the screened compounds 7a-j, the triazole moiety bearing electron-withdrawing group on the 4th position of phenyl viz. 4-chlorophenyl 7c, 4-nitrophenyl 7f and 2,4-difluorophenyl 7h exhibited significant anti-bacterial activity towards the tested bacteria. Triazole moiety bearing electron-donating group on the 4th position of phenyl viz. 4-hdroxy-phenyl 7d, 4-methoxyphenyl 7e and 4-aminophenyl 7g showed prominent activity towards the tested fungi. The results were further supported by molecular docking studies and their binding interactions as well. Antioxidant activity revealed that, compounds in which triazole moiety bearing phenyl 7a, 4-methoxyphenyl 7e, 2,4-difluorophenyl 7h and 3-pyridyl 7j displayed considerable antioxidant property. Most of the newly synthesized compounds showed marked inhibitory activity against the tested microorganisms and considerable antioxidant activity, and emerged as potential molecules for further development. Acknowledgements
Authors are thankful to the Director, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad; Secretary, Viswabhara Educational Society, for providing facilities to carry out the biological screening; Dr. T. Balraj, Indian Institute of Science Education and Research, Kolkata, for his support in molecular docking analysis. Financial assistance from the UGC, New Delhi, India, is gratefully acknowledged.
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