Design, synthesis, and characterization of a fluoro substituted novel pyrazole nucleus clubbed with 1,3,4-oxadiazole scaffolds and their biological applications

Abstract

A novel series of compounds incorporating a fluoro substituted pyrazole nucleus clubbed with 1,3,4-oxadiazole scaffolds (7a–p) was synthesized in good yields (79–89%). The structures of all the compounds were confirmed using elemental analysis, IR, ¹H NMR, and mass spectral data. The newly synthesized compounds were screened for their preliminary in vitro antibacterial activity against a panel of pathogenic strains of bacteria and fungi; for their antituberculosis activity against Mycobacterium tuberculosis H₃₇Rv; and for their and antimalarial activity against Plasmodium falciparum. Compounds 7e, 7o, and 7h were found to possess promising antibacterial potency, while compounds 7c, 7h, and 7j demonstrated better potency against M. tuberculosis H₃₇Rv compared with that of rifampicin. Compounds 7b, 7h, 7i, 7l, and 7o were found to possess excellent activity against a P. falciparum strain compared with quinine (IC₅₀ = 0.826 μM).

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1. Introduction

Malaria is a serious human parasitic infection caused by Plasmodium falciparum and Plasmodium vivax.¹ Currently, a number of vaccine candidates are being clinically tested, which may become significant tools for treatment of malaria. However, the first signs of resistance to artemisinin, the present first-line antimalarial treatment, have appeared in Southeast Asia.^2–4 It is therefore important to develop new antimalarial medications with novel modes of action.⁵

Tuberculosis (TB), a lung infectious disease mostly caused by Mycobacterium tuberculosis (MTB), is a worldwide public health problem, responsible for the death of 2–3 million people annually.^6,7 Moreover, TB is often seen in HIV/AIDS patients who have reduced responses to TB treatment. The emergence and distribution of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of Mycobacterium tuberculosis have become major challenges in treatment with modern anti-TB drugs. Modern anti-TB drugs also suffer from low tolerability or adverse effects.

Over the past two decades, the world population has suffered cruelly with life-threatening infectious disease caused by multidrug-resistant pathogenic bacteria (Gram-positive and Gram-negative bacteria).^8,9 Microbial infections are the second most common disease/condition, after heart attack, causing death in the world, because of their toxicity and resistance towards the available antibiotic drugs.

There is an urgent need for development of novel drugs with fewer side effects and improved efficacy to cure malaria, tuberculosis (TB), and microbial infections. We have designed and synthesized fluoro substituted pyrazole based 1,3,4-oxadiazole scaffolds. Enhancement of hybrid molecules through a combination of diverse pharmacophores in one frame may lead to a pathway to a treatment.

The pyrazole ring is a ubiquitous core in heterocyclic chemistry and represents a key motif in medicinal chemistry because of its potential to exhibit an array of bioactivities such as antimicrobial,¹⁰ anti-inflammatory,¹¹ antipyretic,¹² anticancer,¹³ anti-viral, antitumor,^14,15 analgesic,¹⁶ fungistatic,¹⁷ and anti-hyperglycaemic activities.^18,19 1,3,4-Oxadiazole forms an important class of heterocyclic bioactive compounds which have attracted extensive attention because of their remarkable biological and pharmacological properties, including antibacterial,²⁰ and anti-tubercular,^21,22 anti-inflammatory,²³ antifungal,²⁴ antidepressant,²⁵ anti-proliferative,²⁶ and anti-anxiety²⁷ activities. Moreover, 1,3,4-oxadiazole heterocycles are very good bioisosteres of amides and esters, which contribute substantially to growing pharmacological potency by participating in hydrogen bonding interactions with the receptors. Several biologically active pyrazofurin and pyrazole based 1,3,4-oxadiazole scaffolds have also been reported (Fig. 1, A–C).^21,28,29

Fig. 1 Structures of pyrazofurin and some reported biologically active pyrazole based 1,3,4-oxadiazoles scaffold A, B, C and synthesized compounds 7a–p.

2. Chemistry

The synthetic protocol for a novel series of fluoro substituted pyrazole bearing 1,3,4-oxadiazole scaffolds was performed as outlined in Scheme 1. The starting material 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 2 was prepared by Vilsmeier–Haack reaction of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one.³⁰ 3-Methyl-5-substituted aryloxy-1-phenyl-1H-pyrazole-4-carbaldehydes 4a–d were prepared by refluxing compound 2 and fluoro substituted phenols 3a–d in the presence of anhydrous K₂CO₃ as a basic catalyst in DMF as solvent. Then the derivatives 4a–d were treated with 4-substituted benzohydrazide 5a–d in the presence of a few drops of glacial acetic acid in ethanol. The mixture was refluxed for 1 h to obtain the corresponding hydrazones 6a–p. The obtained hydrazones 6a–p were then subjected to oxidative cyclization using phenyliododiacetate (PhI(OAc)₂) in dichloromethane (MDC) by stirring at room temperature for 20 min to afford the corresponding 1,3,4-oxadiazoles 7a–p.

Scheme 1 Synthesis of fluoro substituted pyrazole bearing 1,3,4-oxadiazole scaffolds.

2.1. Analytical results

The structures of the targeted fluoro substituted pyrazole motifs clubbed with 1,3,4-oxadiazole scaffolds 7a–p were confirmed by mass spectrometry, ¹H NMR, FT-IR, and elemental analysis. The mass spectra of all the compounds showed molecular ion peaks (M⁺) corresponding to their respective molecular weights, which additionally confirmed the molecular framework. The aromatic region resonates in the range of 6.83–7.92 ppm (Ar-H) as a multiplet in ¹H NMR spectra of the compounds. In IR spectra, absorption bands in the range 1621–1638 cm⁻¹ were observed for all the compounds, which may be a result of –C=N stretching. –C=C– stretching appeared at 1589–1598 cm⁻¹. The absorption around 3051–3067 cm⁻¹ is attributed to aromatic C–H stretching. IR spectra of the synthesized scaffolds exhibited characteristic absorption bands in the range 1213–1237 cm⁻¹ resulting from the presence of an ether linkage.

3. Pharmacology

3.1. In vitro antimicrobial activity

Assessment of the antimicrobial activity of the newly synthesized fluoro substituted pyrazole bearing 1,3,4-oxadiazole derivative was carried out by a broth micro dilution method according to National Committee for Clinical Laboratory Standards (NCCLS).³¹ Antibacterial activity was screened against three Gram-positive (Bacillus subtilis MTCC 441, Clostridium tetani MTCC 449, and Streptococcus pneumoniae MTCC 1936) and three Gram-negative (Salmonella typhi MTCC 98, Escherichia coli MTCC 443, and Vibrio cholerae MTCC 3906) bacteria using ampicillin, norfloxacin, chloramphenicol, and ciprofloxacin as the standard antibacterial drugs. Antifungal activity was screened against two fungal species (Aspergillus fumigatus MTCC 3008 and Candida albicans MTCC 227) using nystatin and griseofulvin as the standard antifungal drugs. The strains used in the studies were procured from the Institute of Microbial Technology, Chandigarh (MTCC-Micro Type Culture Collection). Mueller Hinton broth was used as nutrient medium to grow and dilute drug suspensions for the tests. DMSO was used as the diluent to obtain the desired concentrations of compounds to test on the standard bacterial strains. The antimicrobial screening data are shown in Table 1.

Table 1 In vitro antimicrobial activity (MIC, μM) of compounds 7a–p^a

Compound	Gram positive bacteria			Gram negative bacteria			Fungi
	S.P.	C.T.	B.S.	S.T.	V.C.	E.C.	C.A.	A.F.
	MTCC	MTCC	MTCC	MTCC	MTCC	MTCC	MTCC	MTCC
	1936	449	441	98	3906	443	227	3008
a S.P.: Streptococcus pneumoniae, B.S.: Bacillus subtilis, C.T.: Clostridium tetani, E.C.: Escherichia coli S.T.: Salmonella typhi, V.C.: Vibrio cholerae, C.A.: Candida albicans, A.F.: Aspergillus fumigatus, MTCC: Microbial Type Culture Collection. A: ampicillin, B: chloramphenicol, C: ciprofloxacin, D: norfloxacin, E: nystatin, F: griseofulvin.b n. t.: not tested.
7a	469	586	134	586	469	469	1173	104
7b	1017	508	203	203	508	203	1017	508
7c	468	468	234	586	234	146	2344	1172
7d	524	524	209	209	131	209	2098	>2098
7e	139	559	279	139	447	447	>2237	>2237
7f	230	184	115	184	369	461	>1847	159
7g	452	282	452	452	565	452	1130	>2260
7h	293	152	508	508	223	293	>2035	>2035
7i	282	565	452	252	452	226	1130	1130
7j	468	586	586	586	283	234	>2344	172
7k	254	407	407	407	508	508	>2035	135
7l	223	279	559	123	447	559	>2237	2237
7m	447	447	447	223	279	1118	2237	>2237
7n	226	282	226	282	565	265	1130	130
7o	102	503	201	503	1006	402	2012	2012
7p	507	507	1015	406	1015	126	1015	>2203
A	286	715	715	286	286	286	n. t.^b	n. t.
B	154	154	154	154	154	154	n. t.	n. t.
C	150	301	150	75	75	75	n. t.	n. t.
D	31	313	310	31	31	31	n. t.	n. t.
E	n. t.	n. t.	n. t.	n. t.	n. t.	n. t.	107	107
F	n. t.	n. t.	n. t.	n. t.	n. t.	n. t.	1147	283

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3.2. In vitro antituberculosis activity

The primary in vitro antituberculosis activity of the newly synthesized fluoro substituted pyrazole bearing 1,3,4-oxadiazole derivatives was assessed at 250 μg mL⁻¹ against Mycobacterium tuberculosis H₃₇Rv strain using Lowenstein–Jensen medium as described by Rattan.³² The obtained results are presented in Table 2 in the form of % inhibition. Rifampicin and isoniazid were used as the standard drugs.

Table 2 In vitro antituberculosis activity (% inhibition) of pyrazole based 1,3,4-oxadiazole derivatives against M. tuberculosis H₃₇Rv (at concentration 250 μg mL⁻¹)

Comp.	% inhibition	Comp.	% inhibition
7a	54	7j	93
7b	23	7k	85
7c	95	7l	65
7d	62	7m	35
7e	88	7n	88
7f	91	7o	87
7g	20	7p	65
7h	94	Rifampicin	98
7i	32	Isoniazid	99

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3.3. In vitro antimalarial activity

In vitro antimalarial activity of the newly synthesized fluoro substituted pyrazole bearing 1,3,4-oxadiazole derivatives against P. falciparum strain was assessed using chloroquine and quinine as the reference compounds. Results of the antimalarial screening are expressed as the drug concentration resulting in 50% inhibition (IC₅₀) of parasite growth and are listed in Table 3.

Table 3 In vitro antimalarial activity of pyrazole based 1,3,4-oxadiazole scaffolds

Compound	IC50 (μM)	Compound	IC50 (μM)
7a	2.956	7j	2.884
7b	0.709	7k	2.361
7c	4.385	7l	0.797
7d	2.329	7m	2.081
7e	2.304	7n	2.712
7f	1.570	7o	0.610
7g	2.825	7p	2.396
7h	0.506	Chloroquine	0.062
7i	0.536	Quinine	0.826

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4. Biological section

4.1. In vitro antibacterial activity

Evaluation of antibacterial data (Table 1) revealed that most of the tested compounds exhibited moderate to excellent antibacterial activity and good to moderate antifungal activity against all the tested microbial strains.

Among them, compounds 7e (139 μM) and 7o (102 μM) exhibited excellent potency against S. pneumoniae compared with ciprofloxacin (150 μM), chloramphenicol (154 μM) and ampicillin (286 μM), while compounds 7f (230 μM), 7i (282 μM), 7k (254 μM), 7l (223 μM), and 7n (226 μM) displayed activities comparable with that of ampicillin (286 μM).

Compound 7h (152 μM) illustrated superior potency against C. tetani compared with all the standard drugs. Compounds 7a (134 μM) and 7f (115 μM) exhibited greater activity against C. tetani compared with all the standard drugs. Most of the compounds displayed excellent activity towards Gram-positive bacteria, i.e. B. subtilis and C. tetani, compared with ampicillin as well as norfloxacin.

In the case of Gram-negative bacteria against S. typhi, compounds 7e (139 μM) and 7l (123 μM) demonstrated excellent potency compared with that of chloramphenicol (154 μM) as well as ampicillin (286 μM), while compounds 7b (203 μM), 7d (209 μM), 7f (184 μM), 7i (252 μM), 7m (223 μM), and 7n (282 μM) exhibited comparable potency with that of ampicillin (286 μM).

Against V. cholerae, compound 7d (131 μM) showed brilliant activity compared with that of chloramphenicol (154 μM) as well as ampicillin (286 μM), while compounds 7c (234 μM), 7h (223 μM), 7j (283 μM), and 7m (279 μM) demonstrated reduced potency compared with that of chloramphenicol (154 μM) but showed comparable potency with ampicillin (286 μM).

Compounds 7c (146 μM) and 7p (126 μM) showed the highest activity at inhibiting Gram-negative bacteria E. coli compared with chloramphenicol (154 μM) as well as ampicillin (286 μM), while compounds 7b (203 μM), 7d (209 μM), 7i (226 μM), 7j (234 μM), and 7c (265 μM) illustrated good potency approaching that of ampicillin (286 μM).

4.2. In vitro antifungal activity

Evaluation of antifungal activity (Table 1) showed that all the compounds had moderate activity against C. albicans as compared with the standard drugs nystatin and griseofulvin. Against C. albicans, compounds 7b (1017 μM), 7g (1130 μM), 7i (1130 μM), 7n (1130 μM), and 7p (1015 μM) illustrated good potency compared with that of griseofulvin (1147 μM), but they were found to be less active compared with nystatin (107 μM). Compound 7a (104 μM) showed comparable potency against A. fumigatus with that of nystatin (107 μM) but superior to that of griseofulvin (283 μM). Compounds 7f (159 μM), 7j (172 μM), 7k (135 μM), and 7n (130 μM) also exhibited better activity against A. fumigatus compared with that of griseofulvin (283 μM).

4.3. In vitro antituberculosis activity

Antituberculosis screening of the synthesized fluoro substituted pyrazole nuclei clubbed with 1,3,4-oxadiazole scaffolds were conducted at 250 μg mL⁻¹ concentration against the M. tuberculosis H₃₇Rv strain.

Compounds 7c, 7f, 7h, 7j, 7n, and 7o demonstrated excellent activities of 95%, 91%, 94%, 93%, 88%, and 87%, respectively, at 250 μg mL⁻¹ against M. tuberculosis H₃₇Rv (Table 2) compared with that of rifampicin, 98%. The remaining compounds had poor inhibition against M. tuberculosis growth. From the above results, it can be concluded that compounds 7c, 7h, and 7j could be new antituberculosis agents in this series.

4.4. In vitro antimalarial activity

All the synthesized fluoro substituted pyrazole nuclei clubbed with 1,3,4-oxadiazole scaffolds were evaluated for their antimalarial activity against a chloroquine- and quinine-sensitive strain of P. falciparum. All experiments were performed in duplicate, and the results were used to calculate mean IC₅₀ values, as listed in Table 3.

Compounds 7b, 7h, 7i, 7l, and 7o had IC₅₀ values in the range of 0.506 μM to 0.797 μM against the P. falciparum strain. As compared with quinine (IC₅₀ = 0.826 μM), these compounds displayed promising activity against the P. falciparum strain. The remaining compounds were less active against the chloroquine-sensitive strain of P. falciparum.

5. Conclusion

Novel fluoro substituted pyrazole bearing 1,3,4-oxadiazole derivatives (7a–p) were synthesized in good yields via a four-step protocol from accessible 3-methyl-1-phenyl-1-H-pyrazol-5-(4H)-one, with the final step using phenyliododiacetate (PhI(OAc)₂) in dichloromethane at room temperature. The derivatives were evaluated for in vitro antimicrobial, antituberculosis, and antimalarial activities. From the series, compounds 7e, 7o, and 7h showed promise against two Gram-positive bacteria, S. pneumoniae and C. tetani. Compounds 7e and 7l displayed superior potency against Gram-negative bacteria, S. typhi. In terms of antifungal activity, all the compounds showed moderate activity against C. albicans. Compounds 7c, 7h, and 7j demonstrated better potency against the M. tuberculosis H₃₇Rv strain. Compounds 7b, 7h, 7i, 7l, and 7o showed excellent activity compared with quinine (IC₅₀ = 0.826 μM) against a P. falciparum strain. Compound 7h was identified as the most biologically active member of the series, with admirable antimicrobial, antituberculosis, and antimalarial activities as compared with standard drugs.

6. Experimental section

All the reagents and solvents used were of commercial grade and were employed without any further purification. The progress of the reactions as well as the purity of the compounds were checked by thin-layer chromatography on aluminium plates coated with silica gel 60 F₂₅₄, 0.25 mm thickness (Merck), and the developed chromatograms were visualized under UV light and iodine vapours. Melting points were determined in open capillaries using μThermoCal10 melting point apparatus (Analab Scientific Pvt. Ltd, India) and were not corrected. IR spectra were recorded on a Shimadzu FTIR 8401 spectrophotometer using potassium bromide pellets in the range 4000–400 cm⁻¹ and the frequencies of only the characteristic peaks were expressed in cm⁻¹. Mass spectra were recorded on a Shimadzu LCMS 2010 spectrometer. ¹H NMR spectra were recorded on a Bruker Avance 400F (MHz) spectrometer (Bruker Scientific Corporation Ltd., Switzerland) using CDCl₃ as the solvent and tetramethylsilane (TMS) as the internal standard. Elemental analyses were performed on a PerkinElmer 2400 series-II elemental analyzer (PerkinElmer, USA). All compounds were found within ±0.4% of their theoretical values.

6.1. General procedure for the synthesis of 3-methyl-5-substituted aryloxy-1-phenyl-1H-pyrazole-4-carbaldehyde (4a–d)

5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 2 (1 mmol), substituted phenols 3a–d (1 mmol), and anhydrous potassium carbonate (2 mmol) in dimethylformamide (10 mL) were charged in a 100 mL round bottom flask equipped with a mechanical stirrer and a condenser. The reaction mixture was heated at 90 °C for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction as confirmed by TLC, the reaction mixture was poured into 100 mL of ice-water and filtered, washed thoroughly with water, dried and recrystallized from hot ethanol to obtain a white solid.

6.1.1 5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (4a). Yield 85%, mp 225–227 °C; IR (KBr, ν_max, cm⁻¹): 1215 (C–O–C); 1720 (–C=O str.), 3053 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.56 (s, 3H, pyrazole–CH₃), 6.99–7.11 (m, 2H, Ar-H), 7.12–720 (m, 2H, Ar-H), 7.34–7.37 (m, 1H, Ar-H), 7.36–7.45 (m, 2H, Ar-H), 7.47–7.69 (m, 2H, Ar-H), 9.61 (s, 1H, –CHO); ¹³C APT (400 MHz, CDCl₃) δ 14.5, 108.5, 117.6, 118.3, 123.4, 125.8, 128.1, 128.9, 136.8, 144.2, 150.8, 151.0, 152.2, 153.4, 182.6; ESI-MS (m/z): 297.2 (M⁺); anal.% calculated for C₁₇H₁₃FN₂O₂: C, 68.91; H, 4.42; N, 9.45; found: C, 68.69; H, 4.20; N, 9.22.

6.1.2 5-(3-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (4b). Yield 78%, mp 210–212 °C; IR (KBr, ν_max, cm⁻¹): 1218 (C–O–C); 1715 (–C=O str.); 3051 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.58 (s, 3H, pyrazole–CH₃), 675–6.87 (m, 3H, Ar-H), 7.25–7.33 (m, 2H, Ar-H), 7.37–7.45 (m, 2H, Ar-H), 7.61–7.63 (m, 2H, Ar-H), 9.70 (s, 1H, –CHO); ¹³C APT (400 MHz, CDCl₃) δ 14.3, 104.4, 109.1, 117.7, 122.8, 128.2, 129.3, 131.1, 136.7, 150.9, 151.3, 157.7, 162.2, 164.6, 182.7; ESI-MS (m/z): 297.3 (M⁺); anal.% calculated for C₁₇H₁₃FN₂O₂: C, 68.91; H, 4.42; N, 9.45; found: C, 68.69; H, 4.21; N, 9.22.

6.1.3 3-Methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazole-4-carbaldehyde (4c). Yield 81%, mp 226–228 °C; IR (KBr, ν_max, cm⁻¹): 1219 (C–O–C); 1719 (–C=O str.); 3056 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.59 (s, 3H, pyrazole–CH₃), 7.03–7.06 (m, 4H, Ar-H), 7.29–7.39 (m, 1H, Ar-H), 7.42–7.46 (m, 2H, Ar-H), 7.63–7.65 (m, 2H, Ar-H), 9.66 (s, 1H, –CHO); ¹³C APT (400 MHz, CDCl₃) δ 14.4, 101.9, 116.9, 116.9, 118.5, 124.8, 128.7, 129.2, 137.8, 151.9, 152.3, 152.9, 158.2, 160.5, 182.9; ESI-MS (m/z): 347.2 (M⁺); anal.% calculated for C₁₈H₁₃F₃N₂O₂: C, 62.42; H, 3.78; N, 8.09; found: C, 62.19; H, 3.54; N, 7.87.

6.1.4 5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (4d). Yield 82%, mp 245–247 °C; IR (KBr, ν_max, cm⁻¹): 1218 (C–O–C); 1717 (–C=O str.); 3055 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.57 (s, 3H, pyrazole–CH₃), 7.00–7.05 (m, 4H, Ar-H), 7.28–7.37 (m, 1H, Ar-H), 7.42–7.46 (m, 2H, Ar-H), 7.63–7.65 (m, 2H, Ar-H), 9.65 (s, 1H, –CHO); ¹³C APT (400 MHz, CDCl₃) δ 14.4, 101.8, 116.7, 116.9, 117.5, 122.8, 128.1, 129.2, 136.8, 150.9, 152.3, 152.8, 158.0, 160.4, 182.8; ESI-MS (m/z): 297.1 (M⁺); anal.% calculated for C₁₇H₁₃FN₂O₂: C, 68.91; H, 4.42; N, 9.45; found: C, 68.66; H, 4.21; N, 9.74.

6.2. Synthesis of (E)-N′-((5-(substituted-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-substituted benzohydrazide (6a–p)

A mixture of 3-methyl-5-substituted aryloxy-1-phenyl-1H-pyrazole-4-carbaldehydes 4a–d (10 mmol), 4-substituted benzohydrazide 5a–d (10 mmol), and a catalytic amount of glacial acetic acid in ethanol (50 mL) was refluxed for 1 h. After completion of the reaction, the reaction mixture was stirred magnetically for further 10 min. After cooling, the separated solid mass was collected by filtration, washed well with ethanol (10 mL), dried, and crystallized from hot ethanol (10 mL) to afford compounds (6a–p).

6.2.1 (E)-N′-((5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methylbenzohydrazide (6a). Yield 77%, mp 166–168 °C; IR (KBr, ν_max, cm⁻¹): 3441 (–NH str.); 1722 (C=O); 1630 (C=N); 1230 (C–O–C); 3028 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.35 (s, 3H, Ar-CH₃), 2.61 (s, 3H, pyrazole–CH₃), 7.04 (m, 1H, Ar-H), 7.19–7.22 (m, 3H, Ar-H), 7.27–7.31 (m, 2H, Ar-H), 7.34 (m, 3H, Ar-H), 7.36–7.41 (m, 2H, Ar-H), 7.56–7.70 (m, 2H, Ar-H); 8.15 (s, 1H, =CH–), 9.40 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 21.4, 104.5, 112.5, 118.7, 120.5, 121.9, 122.4, 124.8, 126.8, 126.9, 127.7, 129.6, 130.6, 132.5, 137.4, 139.6, 143.6, 149.9, 154.5, 156.6, 164.6; ESI-MS (m/z): 429.2 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₂: C, 70.08; H, 4.94; N, 13.08; found: C, 69.87; H, 4.69; N, 12.87.

6.2.2 (E)-4-Bromo-N′-((5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6b). Yield 80%, mp 208–210 °C; IR (KBr, ν_max, cm⁻¹): 3446 (–NH str.); 1720 (C=O); 1629 (C=N); 685 (C–Br); 1232 (C–O–C); 3026 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.62 (s, 3H, pyrazole–CH₃), 6.65 (m, 2H, Ar-H), 6.72–6.77 (m, 1H, Ar-H), 7.21–7.25 (m, 1H, Ar-H), 7.29–7.32 (m, 1H, Ar-H), 7.38–7.42 (m, 4H, Ar-H), 7.57 (m, 2H, Ar-H), 7.74–7.80 (m, 2H, Ar-H), 8.10 (s, 1H, =CH–), 9.35 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 103.6, 103.8, 110.8, 110.9, 111.5, 118.9, 122.6, 127.5, 128.7, 129.4, 130.8, 130.9, 137.4, 143.8, 149.6, 154.5, 156.4, 162.3, 164.7; ESI-MS (m/z): 494.3 (M⁺); anal.% calculated for C₂₄H₁₈BrFN₄O₂: C, 58.43; H, 3.68; N, 11.36; found: C, 58.19; H, 3.43; N, 11.13.

6.2.3 (E)-N′-((5-(3-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methylbenzohydrazide (6c). Yield 75%, mp 172–174 °C; IR (KBr, ν_max, cm⁻¹): 3447 (–NH str.); 1715 (C=O); 16 276 (C=N); 1235 (C–O–C); 3025 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, Ar-CH₃), 2.64 (s, 3H, pyrazole–CH₃), 6.69 (m, 2H, Ar-H), 6.75–6.79 (m, 1H, Ar-H), 7.18–7.23 (m, 3H, Ar-H), 7.37–7.41 (m, 2H, Ar-H), 7.58–7.60 (m, 2H, Ar-H), 7.70 (m, 2H, Ar-H), 8.11 (s, 1H, =CH–), 9.17 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 15.1, 21.5, 103.7, 103.9, 110.8, 111.1, 122.2, 126.8, 127.4, 129.2, 129.8, 130.5, 130.9, 137.2, 142.8, 143.5, 149.4, 154.5, 156.9, 162.2, 164.6; ESI-MS (m/z): 429.3 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₂: C, 70.08; H, 4.94; N, 13.08; found: C, 69.86; H, 4.71; N, 12.86.

6.2.4 (E)-4-Methyl-N′-((3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)benzohydrazide (6d). Yield 79%, mp 177–179 °C; IR (KBr, ν_max, cm⁻¹): 3413 (–NH str.); 1721 (C=O); 1627 (C=N); 1232 (C–O–C); 3029 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.39 (s, 3H, Ar-CH₃), 2.62 (s, 3H, pyrazole–CH₃), 7.06 (m, 1H, Ar-H), 7.18–7.20 (m, 3H, Ar-H), 7.26–7.30 (m, 2H, Ar-H), 7.33 (m, 3H, Ar-H), 7.36–7.40 (m, 2H, Ar-H), 7.56–7.69 (m, 2H, Ar-H); 8.16 (s, 1H, =CH–), 9.41 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 21.4, 104.4, 112.9, 118.5, 120.5, 121.9, 122.3, 124.7, 126.8, 126.9, 127.6, 129.5, 130.6, 132.6, 137.1, 139.6, 143.5, 149.8, 154.5, 156.6, 164.6; ESI-MS (m/z): 479.2 (M⁺); anal.% calculated for C₂₆H₂₁F₃N₄O₂: C, 65.27; H, 4.42; N, 11.71; found: C, 65.05; H, 4.21; N, 11.48.

6.2.5 (E)-4-Chloro-N′-((5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6e). Yield 81%, mp 212–214 °C; IR (KBr, ν_max, cm⁻¹) 3435 (–NH str.); 1713 (C=O); 1630 (C=N); 750 (C–Cl); 1231 (C–O–C); 3030 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.62 (s, 3H, pyrazole–CH₃), 6.84–6.97 (m, 4H, Ar-H), 7.28–7.31 (m, 1H, Ar-H), 7.37–7.41 (m, 4H, Ar-H), 7.59 (m, 2H, Ar-H), 7.75–7.82 (m, 2H, Ar-H), 8.10 (s, 1H, =CH–), 9.27 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 116.4, 116.7, 116.9, 122.2, 124.8, 124.9, 126.9, 129.2, 129.8, 130.5, 130.7, 130.9, 137.2, 143.5, 149.8, 150.9, 157.6, 158.2, 160.1; ESI-MS (m/z): 449.4 (M⁺); anal.% calculated for C₂₄H₁₈ClFN₄O₂: C, 64.22; H, 4.04; N, 12.48; found: 63.99; H, 3.82; N, 12.24.

6.2.6 (E)-4-Bromo-N′-((3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)benzohydrazide (6f). Yield 72%, mp 190–192 °C; IR (KBr, ν_max, cm⁻¹): 3436 (–NH str.); 1720 (C=O); 1631 (C=N); 684 (C–Br); 1230 (C–O–C); 3024 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.63 (s, 3H, pyrazole–CH₃), 7.00–7.11 (m, 2H, Ar-H), 7.22 (m, 5H, Ar-H), 7.29–7.33 (m, 4H, Ar-H), 7.37–7.72 (m, 2H, Ar-H), 8.15 (s, 1H, =CH–), 9.29 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 118.5, 120.6, 122.4, 122.6, 126.8, 126.9, 129.4, 129.8, 130.2, 130.8, 131.5, 131.9, 132.4, 135.6, 137.2, 143.5, 149.8, 154.6, 155.7, 163.6; ESI-MS (m/z): 544.2 (M⁺); anal.% calculated for C₂₅H₁₈BrF₃N₄O₂: C, 55.26; H, 3.34; N, 10.31; found: C, 55.05; H, 3.09; N, 10.09.

6.2.7 (E)-N′-((5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methoxybenzohydrazide (6g). Yield 79%, mp 210–212 °C; IR (KBr, ν_max, cm⁻¹): 3443 (–NH str.); 1718 (C=O); 1625 (C=N); 1235 (C–O–C); 3025 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.53 (s, 3H, pyrazole–CH₃), 3.83 (s, 3H, –OCH₃), 6.85 (m, 6H, Ar-H), 7.25–7.29 (m, 1H, Ar-H), 7.35–7.39 (m, 2H, Ar-H), 7.59–7.62 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), 8.09 (s, 1H, =CH–), 9.71 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 15.2, 55.3, 104.2, 113.6, 116.3, 116.7, 116.9, 122.2, 124.5, 125.8, 128.9, 128.9, 129.2, 137.5, 138.8, 143.5, 147.8, 152.7, 157.2, 159.8, 162.3; ESI-MS (m/z): 445.3 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₃: C, 67.56; H, 4.76; N, 12.61; found: 67.32; H, 4.51; N, 12.38.

6.2.8 (E)-4-Bromo-N′-((5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6h). Yield 77%, mp 195–197 °C; IR (KBr, ν_max, cm⁻¹): 3437 (–NH str.); 1723 (C=O); 1638 (C=N); 686 (C–Br); 1239 (C–O–C); 3027 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.61 (s, 3H, pyrazole–CH₃), 6.85–6.99 (m, 4H, Ar-H), 7.25–7.35 (m, 1H, Ar-H), 7.38–7.43 (m, 4H, Ar-H), 7.57 (m, 2H, Ar-H), 7.75–7.85 (m, 2H, Ar-H), 8.09 (s, 1H, =CH–), 9.29 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 116.3, 116.7, 116.8, 122.2, 124.7, 124.9, 126.9, 129.5, 129.8, 130.6, 130.7, 130.9, 137.4, 143.4, 149.7, 150.9, 157.6, 158.3, 160.2; ESI-MS (m/z): 494.2 (M⁺); anal.% calculated for C₂₄H₁₈BrFN₄O₂: C, 58.43; H, 3.68; N, 11.36; found: C, 58.21; H, 3.45; N, 11.12.

6.2.9 (E)-N′-((5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methoxybenzohydrazide (6i). Yield 76%, mp 189–191 °C; IR (KBr, ν_max, cm⁻¹): 3438 (–NH str.); 1720 (C=O); 1634 (C=N); 1234 (C–O–C); 3022 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.55 (s, 3H, pyrazole–CH₃), 3.82 (s, 3H, –OCH₃), 6.83 (m, 6H, Ar-H), 7.24–7.28 (m, 1H, Ar-H), 7.35–7.38 (m, 2H, Ar-H), 7.57–7.59 (m, 2H, Ar-H), 7.80 (m, 2H, Ar-H), 8.10 (s, 1H, =CH–), 9.73 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 15.1, 55.3, 104.2, 113.7, 116.3, 116.6, 116.8, 122.2, 124.3, 125.4, 128.8, 128.9, 129.2, 137.3, 138.3, 143.5, 147.8, 152.8, 157.5, 159.9, 162.3; ESI-MS (m/z): 445.3 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₃: C, 67.56; H, 4.76; N, 12.61; found: C, 67.35; H, 4.52; N, 12.36.

6.2.10 (E)-N′-((5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methylbenzohydrazide (6j). Yield 78%, mp 210–212 °C; IR (KBr, ν_max, cm⁻¹): 3435 (–NH str.); 1722 (C=O); 1608 (C=N); 1230 (C–O–C); 3021 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.40 (s, 3H, Ar-CH₃), 2.61 (s, 3H, pyrazole–CH₃), 9.60–6.94 (m, 4H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.36–7.40 (m, 1H, Ar-H), 7.59–7.60 (m, 2H, Ar-H), 7.70 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), 8.09 (s, 1H, =CH–), 9.36 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 15.1, 21.5, 116.4, 116.6, 116.8, 122.2, 122.8, 125.4, 126.8, 127.3, 129.1, 129.9, 131.8, 137.3, 142.5, 143.9, 145.2, 147.9, 157.5, 158.5, 160.1; ESI-MS (m/z): 429.3 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₂: C, 70.08; H, 4.94; N, 13.08; found: C, 69.85; H, 4.71; N, 12.85.

6.2.11 (E)-4-Bromo-N′-((5-(2-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6k). Yield 74%, mp 180–182 °C; IR (KBr, ν_max, cm⁻¹): 3413 (–NH str.); 1723 (C=O); 1609 (C=N); 685 (C–Br); 1232 (C–O–C); 3024 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.63 (s, 3H, pyrazole–CH₃), 6.90–6.99 (m, 1H, Ar-H), 7.04 (m, 2H, Ar-H), 7.08–7.14 (m, 1H, Ar-H), 7.29–7.33 (m, 1H, Ar-H), 7.39–7.44 (m, 4H, Ar-H), 7.64–7.67 (m, 2H, Ar-H), 7.75–7.85 (m, 2H, Ar-H), 8.10 (s, 1H, =CH–), 9.35 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 116.7, 117.5, 117.8, 122.4, 124.6, 127.6, 129.5, 129.7, 130.8, 130.9, 131.5, 137.5, 137.9, 141.7, 143.8, 149.8, 154.4, 159.6, 163.4; ESI-MS (m/z): 494.1 (M⁺); anal.% calculated for C₂₄H₁₈BrFN₄O₂: C, 58.43; H, 3.68; N, 11.36; found: C, 58.22; H, 3.45; N, 11.12.

6.2.12 (E)-4-Chloro-N′-((5-(2-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6l). Yield 75%, mp 198–200 °C; IR (KBr, ν_max, cm⁻¹): 3435 (–NH str.); 1711 (C=O); 1599 (C=N); 751 (C–Cl); 1233 (C–O–C); 3025 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.62 (s, 3H, pyrazole–CH₃), 6.92–6.99 (m, 1H, Ar-H), 7.00 (m, 2H, Ar-H), 7.08–7.12 (m, 1H, Ar-H), 7.26–7.30 (m, 1H, Ar-H), 7.37–7.41 (m, 4H, Ar-H), 7.62–7.64 (m, 2H, Ar-H), 7.75–7.83 (m, 2H, Ar-H), 8.11 (s, 1H, =CH–), 9.37 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 116.6, 117.2, 117.8, 122.2, 124.6, 127.5, 129.2, 129.3, 130.5, 130.8, 131.5, 137.2, 137.9, 141.5, 143.8, 149.5, 154.2, 159.2, 163.8; ESI-MS (m/z): 449.5 (M⁺); anal.% calculated for C₂₄H₁₈ClFN₄O₂: C, 64.22; H, 4.04; N, 12.48; found: C, 63.98; H, 3.81; N, 12.25.

6.2.13 (E)-4-Chloro-N′-((5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)benzohydrazide (6m). Yield 73%, mp 192–194 °C; IR (KBr, ν_max, cm⁻¹): 3412 (–NH str.); 1722 (C=O); 1605 (C=N); 752 (C–Cl); 1236 (C–O–C); 3029 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.63 (s, 3H, pyrazole–CH₃), 6.68 (m, 2H, Ar-H), 6.75–6.79 (m, 1H, Ar-H), 7.20–7.24 (m, 1H, Ar-H), 7.28–7.31 (m, 1H, Ar-H), 7.37–7.41 (m, 4H, Ar-H), 7.58 (m, 2H, Ar-H), 7.75–7.81 (m, 2H, Ar-H), 8.11 (s, 1H, =CH–), 9.39 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 103.6, 103.9, 110.8, 110.9, 111.0, 118.9, 122.5, 127.5, 128.7, 129.2, 130.8, 130.9, 137.2, 143.8, 149.5, 154.5, 156.2, 162.4, 164.8; ESI-MS (m/z): 449.3 (M⁺); anal.% calculated for C₂₄H₁₈ClFN₄O₂: C, 64.22; H, 4.04; N, 12.48; found: C, 63.99; H, 3.81; N, 12.24.

6.2.14 (E)-N′-((5-(3-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-4-methoxybenzohydrazide (6n). Yield 73%, mp 192–194 °C; IR (KBr, ν_max, cm⁻¹): 3431 (–NH str.); 1720 (C=O); 1633 (C=N); 1239 (C–O–C); 3028 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.61 (s, 3H, pyrazole–CH₃), 3.86 (s, 3H, –OCH₃), 6.65–6.73 (m, 2H, Ar-H), 6.75–6.77 (m, 1H, Ar-H), 6.89–6.91 (m, 2H, Ar-H), 7.17–7.26 (m, 1H, Ar-H), 7.30–7.36 (m, 1H, Ar-H), 7.40 (m, 2H, Ar-H), 7.57–7.59 (m, 2H, Ar-H), 7.79 (m, 2H, Ar-H), 8.09 (s, 1H, =CH–), 9.34 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 15.8, 55.3, 103.7, 103.9, 110.7, 110.9, 122.2, 127.4, 129.2, 130.8, 130.9, 132.2, 135.7, 137.2, 143.4, 149.8, 150.2, 154.9, 158.2, 162.1, 164.6; ESI-MS (m/z): 445.3 (M⁺); anal.% calculated for C₂₅H₂₁FN₄O₃: C, 67.56; H, 4.76; N, 12.61; found: C, 67.33; H, 4.53; N, 12.38.

6.2.15 (E)-4-Chloro-N′-((3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)benzohydrazide (6o). Yield 70%, mp 158–160 °C; IR (KBr, ν_max, cm⁻¹): 3445 (–NH str.); 1728 (C=O); 1621 (C=N); 750 (C–Cl); 1235 (C–O–C); 3022 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.59 (s, 3H, pyrazole–CH₃), 6.91–6.98 (m, 1H, Ar-H), 7.10–7.18 (m, 1H, Ar-H), 7.20–7.25 (m, 3H, Ar-H), 7.30–7.34 (m, 2H, Ar-H), 7.36–7.38 (m, 2H, Ar-H), 7.55 (m, 2H, Ar-H), 7.76 (m, 3H, Ar-H⁺=CH–), 9.88 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.8, 104.1, 112.8, 116.4, 118.5, 120.6, 121.8, 122.3, 124.6, 125.4, 126.5, 126.8, 128.9, 132.6, 137.1, 144.2, 150.1, 154.4, 155.8, 163.4, 164.1; ESI-MS (m/z): 499.3 (M⁺); anal.% calculated for C₂₅H₁₈ClF₃N₄O₂: C, 60.19; H, 3.64; N, 11.23; found: C, 59.96; H, 3.40; N, 10.99.

6.2.16 (E)-4-Methoxy-N′-((3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)methylene)benzohydrazide (6p). Yield 68%, mp 172–174 °C; IR (KBr, ν_max, cm⁻¹): 3433 (–NH str.); 1726 (C=O); 1632 (C=N); 1231 (C–O–C); 3025 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.80 (s, 3H, pyrazole–CH₃), 3.86 (s, 3H, –OCH₃), 6.89–6.91 (m, 2H, Ar-H), 7.04–7.06 (m, 1H, Ar-H), 7.12–7.14 (m, 1H, Ar-H), 7.19 (m, 1H, Ar-H), 7.26–7.32 (m, 3H, Ar-H), 7.35–7.40 (m, 3H, Ar-H), 7.57–7.88 (m, 2H, Ar-H), 8.12 (s, 1H, =CH–), 9.20 (s, 1H, –NH); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 55.3, 104.3, 112.9, 118.5, 119.1, 120.5, 122.3, 125.2, 126.3, 127.6, 128.5, 130.6, 132.6, 135.2, 137.1, 143.5, 149.4, 154.5, 156.6, 163.4, 164.2; ESI-MS (m/z): 495.2 (M⁺); anal.% calculated for C₂₆H₂₁F₃N₄O₃: C, 63.16; H, 4.28; N, 11.33; found: C, 62.93; H, 4.05; N, 11.10.

6.3. Synthesis of 2-(5-(substituted-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(p-substituted)-1,3,4-oxadiazole (7a–p)

A mixture of compounds 6a–p (10 mmol) was dissolved in DCM (20 mL) and stirred. To this solution, PhI(OAc)₂ (10 mmol) was added and the mixture was stirred for 15–20 min at room temperature. After completion of the reaction as monitored by TLC (ethyl acetate : hexane: 3 : 7), the solvent was evaporated and the residue was washed with diethyl ether, filtered (5 mL), dried, and then crystallized from acetone to afford target compounds (7a–p).

6.3.1 2-(5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(p-tolyl)-1,3,4-oxadiazole (7a). Yield 79%, mp 176–178 °C; IR (KBr, ν_max, cm⁻¹): 1215 (C–O–C); 1622 and 1594 (C=N and C=C); 3054 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, Ar-CH₃), 2.74 (s, 3H, pyrazole–CH₃), 6.83–6.94 (m, 3H, Ar-H), 6.96–6.99 (m, 1H, Ar-H), 7.00–7.02 (m, 2H, Ar-H), 7.12 (m, 1H, Ar-H), 7.15–7.48 (m, 2H, Ar-H), 7.70–7.92 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 21.5, 95.0, 116.4, 117.0, 117.2, 120.9, 122.6, 124.5, 124.6, 126.6, 127.9, 129.3, 129.6, 137.1, 141.9, 144.1, 144.2, 146.9, 149.3, 150.6, 150.7, 153.1, 158.0, 163.7; ESI-MS (m/z): 427.1 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₂: C, 70.41; H, 4.49; N, 13.14; found: C, 70.17; H, 4.26; N, 12.93.

6.3.2 2-(4-Bromophenyl)-5-(5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7b). Yield 81%, mp 154–156 °C; IR (KBr, ν_max, cm⁻¹): 1215 (C–O–C); 1621 and 1592 (C=N and C=C); 3051 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.75 (s, 3H, pyrazole–CH₃), 6.80–6.82 (m, 3H, Ar-H), 7.28 (m, 1H, Ar-H), 7.36–7.38 (m, 1H, Ar-H), 7.43–7.7.47 (m, 2H, Ar-H), 7.57–7.65 (m, 4H, Ar-H), 7.67–7.68 (m, 2H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 95.3, 103.6, 103.8, 110.8, 110.9, 111.1, 122.5, 122.6, 126.1, 127.9, 128.0, 129.3, 130.9, 131.0, 132.3, 137.1, 149.3, 150.1, 152.6, 158.5, 162.3, 162.7, 164.7; ESI-MS (m/z): 492.2 (M⁺); anal.% calculated for C₂₄H₁₆BrFN₄O₂: C, 58.67; H, 3.28; N, 11.40; found: C, 58.44; H, 3.03; N, 11.14.

6.3.3 2-(5-(3-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(p-tolyl)-1,3,4-oxadiazole (7c). Yield 80%, mp 181–183 °C; IR (KBr, ν_max, cm⁻¹): 1213 (C–O–C); 1622 and 1589 (C=N and C=C); 3051 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, Ar-CH₃), 2.76 (s, 3H, pyrazole–CH₃), 6.79–6.84 (m, 3H, Ar-H), 7.23–7.29 (m, 3H, Ar-H), 7.34–7.38 (m, 1H, Ar-H), 7.43–7.64 (m, 2H, Ar-H), 7.66–7.68 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 21.5, 95.6, 103.7, 110.8, 11.09, 111.0, 120.1, 122.6, 126.6, 127.9, 129.3, 129.6, 130.8, 130.9, 137.0, 142.0, 146.4, 149.3, 157.6, 157.7, 158.0, 162.3, 163.2, 164.7; ESI-MS (m/z): 427.4 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₂: C, 70.41; H, 4.49; N, 13.14; found: C, 70.18; H, 4.25; N, 12.87.

6.3.4 2-(3-Methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)-5-(p-tolyl)-1,3,4-oxadiazole (7d). Yield 82%, mp 172–174 °C; IR (KBr, ν_max, cm⁻¹): 1216 (C–O–C); 1617 and 1598 (C=N and C=C); 3052 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.41 (s, 3H, Ar-CH₃), 2.76 (s, 3H, pyrazole–CH₃), 7.10–7.12 (m, 1H, Ar-H), 7.21–7.23 (m, 2H, Ar-H), 7.35–7.40 (m, 3H, Ar-H), 7.43–7.48 (m, 3H, Ar-H), 7.57–7.59 (m, 2H, Ar-H), 7.65–7.67 (m, 2H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 21.5, 95.4, 113.2, 113.3, 117.9, 120.6, 120.6, 120.7, 122.7, 126.5, 127.1, 128.1, 129.4, 129.6, 130.7, 132.8, 135.2, 137.1, 142.1, 145.7, 149.4, 156.7, 157.9, 163.6; ESI-MS (m/z): 477.4 (M⁺); anal.% calculated for C₂₆H₁₉F₃N₄O₂: C, 65.54; H, 4.02; N, 11.76; found: C, 65.30; H, 3.81; N, 11.54.

6.3.5 2-(4-Chlorophenyl)-5-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7e). Yield 81%, mp 168–170 °C; IR (KBr, ν_max, cm⁻¹): 1218 (C–O–C); 1625 and 1595 (C=N and C=C); 3053 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.73 (s, 3H, pyrazole–CH₃), 6.99–7.00 (m, 4H, Ar-H), 7.34–7.37 (m, 1H, Ar-H), 7.41–7.46 (m, 4H, Ar-H), 7.66–7.71 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 95.1, 116.4, 116.5, 116.6, 116.7, 121.7, 122.1, 122.6, 125.7, 127.6, 128.0, 129.3, 137.0, 137.7, 147.3, 149.3, 152.5, 152.6, 157.6, 158.5, 160.0, 161.4, 162.5; ESI-MS (m/z): 447.7 (M⁺); anal.% calculated for C₂₄H₁₆ClFN₄O₂: C, 64.51; H, 3.61; N, 12.54; found: C, 64.29; H, 3.38; N, 12.28.

6.3.6 2-(4-Bromophenyl)-5-(3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7f). Yield 86%, mp 158–160 °C; IR (KBr, ν_max, cm⁻¹): 1225 (C–O–C); 1625 and 1590 (C=N and C=C); 3057 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.76 (s, 3H, pyrazole–CH₃), 7.10–7.12 (d, 1H, Ar-H), 7.33 (m, 2H, Ar-H), 7.38–7.53 (m, 4H, Ar-H), 7.58–7.61 (m, 4H, Ar-H), 7.65–7.67 (d, 2H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 15.0, 113.2, 113.2, 117.9, 120.1, 120.7, 122.4, 122.7, 126.2, 127.2, 128.2, 129.4, 130.8, 135.4, 142.3, 149.5, 150.3, 152.6, 153.4, 154.8, 155.2, 156.7, 156.9, 163.7; ESI-MS (m/z): 542.2 (M⁺); anal.% calculated for C₂₅H₁₆BrF₃N₄O₂: C, 55.47; H, 2.98; N, 10.35; found: C, 55.24; H, 2.77; N, 10.09.

6.3.7 2-(5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (7g). Yield 80%, mp 172–174 °C; IR (KBr, ν_max, cm⁻¹): 1229 (C–O–C); 1621 and 1598 (C=N and C=C); 3056 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.73 (s, 3H, pyrazole–CH₃), 3.87 (s, 3H, –OCH₃), 6.82–6.84 (m, 1H, Ar-H), 6.85–7.04 (m, 4H, Ar-H), 7.15–7.20 (m, 1H, Ar-H), 7.33–7.44 (m, 1H, Ar-H), 7.46–7.75 (m, 2H, Ar-H), 7.76–7.78 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 55.4, 95.1, 114.3, 116.2, 116.4, 117.2, 122.6, 124.5, 124.6, 124.6, 124.7, 127.9, 128.4, 129.3, 137.1, 144.1, 144.2, 146.8, 149.2, 150.6, 153.1, 157.8, 162.1, 163.5; ESI-MS (m/z): 443.4 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₃: C, 67.87; H, 4.33; N, 12.66; found: C, 67.64; H, 4.11; N, 12.39.

6.3.8 2-(4-Bromophenyl)-5-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7h). Yield 80%, mp 177–179 °C; IR (KBr, ν_max, cm⁻¹): 1237 (C–O–C); 1636 and 1594 (C=N and C=C); 3053 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.75 (s, 3H, pyrazole–CH₃), 6.80–6.88 (m, 3H, Ar-H), 7.29 (m, 1H, Ar-H), 7.36–7.40 (m, 1H, Ar-H), 7.48–7.50 (m, 2H, Ar-H), 7.57–7.67 (m, 4H, Ar-H), 7.68–7.69 (m, 2H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 95.4, 103.6, 103.8, 110.7, 110.9, 111.5, 122.4, 122.6, 126.3, 127.9, 128.0, 129.5, 130.9, 131.3, 132.2, 137.4, 149.5, 150.4, 152.8, 158.7, 162.5, 162.7, 164.7; ESI-MS (m/z): 492.3 (M⁺); anal.% calculated for C₂₄H₁₆BrFN₄O₂: C, 58.67; H, 3.28; N, 11.40; found: C, 58.39; H, 3.07; N, 11.15.

6.3.9 2-(5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (7i). Yield 85%, mp 157–159 °C; IR (KBr, ν_max, cm⁻¹): 1227 (C–O–C); 1623 and 1593 (C=N and C=C); 3058 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.73 (s, 3H, pyrazole–CH₃), 3.87 (s, 3H, –OCH₃), 6.81–6.89 (m, 1H, Ar-H), 6.87–7.08 (m, 4H, Ar-H), 7.19–7.25 (m, 1H, Ar-H), 7.33–7.48 (m, 1H, Ar-H), 7.46–7.79 (m, 2H, Ar-H), 7.79–7.81 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 55.3, 95.1, 114.2, 116.4, 116.8, 117.3, 122.5, 124.6, 124.7, 124.8, 124.9, 127.0, 128.4, 129.5, 137.4, 144.3, 144.2, 146.7, 149.3, 150.7, 153.3, 157.8, 162.3, 163.5; ESI-MS (m/z): 443.4 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₃: C, 67.87; H, 4.33; N, 12.66; found: C, 67.63; H, 4.07; N, 12.44.

6.3.10 2-(5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(p-tolyl)-1,3,4-oxadiazole (7j). Yield 86%, mp 178–180 °C; IR (KBr, ν_max, cm⁻¹): 1229 (C–O–C); 1625 and 1595 (C=N and C=C); 3054 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.42 (s, 3H, Ar-CH₃), 2.74 (s, 3H, pyrazole–CH₃), 6.99–7.04 (m, 4H, Ar-H), 7.24–7.26 (m, 2H, Ar-H), 7.33–7.37 (m, 1H, Ar-H), 7.43–7.47 (m, 2H, Ar-H), 7.65–7.68 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 21.5, 95.3, 116.4, 116.4, 116.6, 116.7, 120.9, 122.6, 123.4, 126.5, 127.9, 128.2, 129.3, 129.6, 137.1, 142.0, 147.2, 149.2, 150.3, 152.6, 157.6, 158.1, 160.0, 163.6; ESI-MS (m/z): 425.4 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₂: C, 70.41; H, 4.49; N, 13.14; found: C, 70.18; H, 4.26; N, 12.88.

6.3.11 2-(4-Bromophenyl)-5-(5-(2-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7k). Yield 86%, mp 184–186 °C; IR (KBr, ν_max, cm⁻¹): 1227 (C–O–C); 1638 and 1595 (C=N and C=C); 3060 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.74 (s, 3H, pyrazole–CH₃), 6.82–6.84 (m, 1H, Ar-H), 6.82–6.84 (m, 1H, Ar-H), 6.86–6.99 (m, 2H, Ar-H), 7.00–7.04 (m, 1H, Ar-H), 7.16–7.21 (m, 1H, Ar-H), 7.34–7.56 (m, 2H, Ar-H), 7.59–7.73 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 116.3, 117.0, 117.2, 122.6, 122.6, 124.6, 127.7, 124.8, 125.6, 126.1, 128.0, 129.3, 132.2, 135.7, 137.0, 144.1, 145.7, 149.3, 150.5, 153.0, 154.8, 158.5, 162.8; ESI-MS (m/z): 492.3 (M⁺); anal.% calculated for C₂₄H₁₆BrFN₄O₂: C, 58.67; H, 3.28; N, 11.40; found: C, 58.41; H, 3.07; N, 11.13.

6.3.12 2-(4-Chlorophenyl)-5-(5-(2-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7l). Yield 79%, mp 190–192 °C; IR (KBr, ν_max, cm⁻¹): 1226 (C–O–C); 1638 and 1598 (C=N and C=C); 3061 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.74 (s, 3H, pyrazole–CH₃), 6.82–6.84 (m, 1H, Ar-H), 6.86–6.93 (m, 1H, Ar-H), 6.95–7.01 (m, 1H, Ar-H), 7.02–7.04 (m, 2H, Ar-H), 7.16–7.34 (m, 4H, Ar-H), 7.36–7.74 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 94.8, 116.4, 117.0, 117.2, 122.1, 122.6, 124.6, 124.7, 124.7, 127.8, 128.0, 129.3, 137.0, 137.7, 144.1, 144.2, 147.0, 149.3, 150.6, 153.0, 154.9, 158.4, 162.7; ESI-MS (m/z): 447.7 (M⁺); anal.% calculated for C₂₄H₁₆ClFN₄O₂: C, 64.51; H, 3.61; N, 12.54; found: C, 64.28; H, 3.36; N, 12.27.

6.3.13 2-(4-Chlorophenyl)-5-(5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7m). Yield 87%, mp 177–179 °C; IR (KBr, ν_max, cm⁻¹): 1227 (C–O–C); 1622 and 1597 (C=N and C=C); 3056 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.75 (s, 3H, pyrazole–CH₃), 6.80–6.83 (m, 3H, Ar-H), 7.24–7.30 (m, 1H, Ar-H), 7.34–7.36 (m, 1H, Ar-H), 7.38–7.65 (m, 4H, Ar-H), 7.65–7.68 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 95.3, 103.6, 103.9, 110.8, 110.9, 111.1, 133.6, 127.8, 128.0, 129.3, 129.3, 130.9, 131.0, 137.0, 137.7, 14.02, 149.3, 157.7, 157.6, 158.4, 162.3, 162.6, 164.7; ESI-MS (m/z): 447.8 (M⁺); anal.% calculated for C₂₄H₁₆ClFN₄O₂: C, 64.51; H, 3.61; N, 12.54; found: C, 64.26; H, 3.37; N, 12.28.

6.3.14 2-(5-(3-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (7n). Yield 82%, mp 169–171 °C; IR (KBr, ν_max, cm⁻¹): 1231 (C–O–C); 1631 and 1593 (C=N and C=C); 3061 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.57 (s, 3H, pyrazole–CH₃), 3.69 (s, 3H, –OCH₃), 6.62–6.65 (m, 3H, Ar-H), 6.75–6.77 (m, 2H, Ar-H), 7.05–7.11 (m, 1H, Ar-H), 7.16–7.19 (m, 1H, Ar-H), 7.25–7.29 (m, 2H, Ar-H), 7.48–7.53 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 10.2, 50.6, 99.2, 106.1, 106.2, 111.4, 117.8, 123.2, 126.6, 124.5, 126.1, 126.2, 132.3, 141.6, 144.5, 145.8, 152.8, 152.9, 153.0, 155.4, 157.4, 158.6, 160.2, 162.4, 163.7; ESI-MS (m/z): 443.4 (M⁺); anal.% calculated for C₂₅H₁₉FN₄O₃: C, 67.87; H, 4.33; N, 12.66; found: C, 67.66; H, 4.07; N, 12.38.

6.3.15 2-(4-Chlorophenyl)-5-(3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7o). Yield 88%, mp 194–196 °C; IR (KBr, ν_max, cm⁻¹): 1234 (C–O–C); 1634 and 1594 (C=N and C=C); 3062 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.77 (s, 3H, pyrazole–CH₃), 7.10–7.12 (d, 1H, Ar-H), 7.33 (s, 3H, Ar-H), 7.36–7.38 (m, 3H, Ar-H), 7.42–7.54 (m, 2H, Ar-H), 7.61–7.67 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 15.0, 113.2, 113.2, 117.9, 120.1, 120.7, 122.4, 122.7, 126.2, 127.1, 128.2, 129.3, 130.8, 135.5, 142.4, 149.7, 150.2, 152.8, 153.6, 154.7, 155.3, 156.8, 156.9, 163.8; ESI-MS (m/z): 497.8 (M⁺); anal.% calculated for C₂₅H₁₆ClF₃N₄O₂: C, 60.43; H, 3.25; N, 11.28; found: C, 60.15; H, 3.04; N, 11.05.

6.3.16 2-(4-Methoxyphenyl)-5-(3-methyl-1-phenyl-5-(3-(trifluoromethyl)phenoxy)-1H-pyrazol-4-yl)-1,3,4-oxadiazole (7p). Yield 84%, mp 188–190 °C; IR (KBr, ν_max, cm⁻¹): 1237 (C–O–C); 1637 and 1595 (C=N and C=C); 3067 (Ar, –CH str.); ¹H NMR (400 MHz, CDCl₃) δ 2.76 (s, 3H, pyrazole–CH₃), 3.86 (s, 3H, –OCH₃), 6.90–6.93 (d, 2H, Ar-H), 7.10–7.12 (d, 1H, Ar-H), 7.36–7.40 (m, 2H, Ar-H), 7.44–7.47 (m, 4H, Ar-H), 7.62–7.67 (m, 4H, Ar-H); ¹³C APT (400 MHz, CDCl₃) δ 14.9, 55.4, 95.4, 113.3, 113.3, 114.3, 116.0, 117.9, 130.6, 120.6, 122.7, 128.0, 128.2, 129.4, 130.7, 132.4, 132.8, 137.0, 146.1, 149.4, 156.7, 157.7, 160.4, 162.2, 163.4; ESI-MS (m/z): 493.3 (M⁺); anal.% calculated for C₂₆H₁₉F₃N₄O₃: C, 63.41; H, 3.89; N, 11.38; found: C, 63.18; H, 3.61; N, 11.14.

7. Biological evaluation

7.1. In vitro antimicrobial assay

Assay of antimicrobial activity of fluoro substituted pyrazole containing 1,3,4-oxadiazole scaffolds was carried out by a broth micro dilution method. DMSO was used as the diluent to get the desired concentration of compounds to test on standard bacterial strains. Mueller–Hinton broth was used as a nutrient medium to grow and dilute the compound suspension for the test bacteria. Sabouraud dextrose broth was used for fungal nutrition. Inoculum size for the test strain was adjusted to 10⁸ CFU mL⁻¹ by comparing the turbidity. Serial dilutions were prepared for primary and secondary screening. Each synthesized compound and the standard drugs were diluted, to obtain 2000 μg mL⁻¹ concentration as the stock solution. The compounds found to be active in the primary screening (i.e. 500, 250, and 200 μg mL⁻¹ concentrations) were further screened in a second set of dilutions at 100, 50, 25 and 12.5 μg mL⁻¹ concentration against all microorganisms. Suspensions (10 μL) were further inoculated on appropriate media and growth was noted after 24 and 48 h. The control tube containing no antibiotic was instantaneously subcultured (before inoculation) by evenly spreading a loopful of medium over an area of plate suitable for growth of the test organism. The tubes were then incubated overnight at 37 °C. The highest dilution preventing appearance of turbidity after spot subculture was considered to be the minimal inhibitory concentration (MIC, mM), as listed in Table 1. All tubes showing no visible growth (the same as the control tube) were subcultured and incubated overnight at 37 °C, and compared with the amount of growth in the control tube before incubation. In this study ampicillin, norfloxacin, chloramphenicol, and ciprofloxacin were used as the standard antibacterial drugs. Nystatin and griseofulvin were used as the standard antifungal drugs. Results are summarized in Table 1.

7.2. In vitro antituberculosis assay

All fluoro substituted pyrazole containing 1,3,4-oxadiazole derivatives were screened for their antitubercular activity against Mycobacterium tuberculosis H₃₇Rv using a Lowenstein–Jensen method with a minor modification in which 250 μg mL⁻¹ dilution of each compound was added to Lowenstein–Jensen medium and then media was uncontaminated by an inspissation method. A culture of Mycobacterium tuberculosis H₃₇Rv grown on Lowenstein–Jensen medium was harvested in 0.85% saline in a bijou bottle. Stock solutions of title compounds (100 μg mL⁻¹) were prepared in DMSO. These tubes were then incubated at 37 °C for 24 h followed by streaking of Mycobacterium tuberculosis H₃₇Rv (5 × 10⁴ bacilli per tube). Growth of bacilli was observed after 2 weeks, 3 weeks, and finally after 4 weeks of incubation. The tubes containing the compounds were compared with control tubes in which the medium alone was incubated with Mycobacterium tuberculosis H₃₇Rv. The concentration at which complete inhibition of colonies occurred was taken to be the active concentration of the tested compound. The standard strain Mycobacterium tuberculosis H₃₇Rv was also tested with the known drugs rifampicin and isoniazid for comparison. Results are summarized in Table 2.

7.3. In vitro antimalarial assay

In vitro antimalarial activity of the fluoro substituted pyrazole based 1,3,4-oxadiazole derivatives was screened against a P. falciparum strain. The P. falciparum strain was acquired from Shree R. B. Shah Mahavir Super-speciality hospital, Surat, Gujarat, India and was used in in vitro tests. The strain was cultivated by a modified method described by Trager and Jensen.³³ Compounds were dissolved in DMSO. The final concentration of DMSO used was not toxic and did not interfere with the assay. The antiparasitic effect of the compounds was measured by growth inhibition percentage as described by Carvalho and Krettli.³⁴ For experimental purposes, the cultures were synchronized with 5% D-sorbitol when the parasites were in the ring stage.³⁵ The parasite suspension, consisting of predominately the ring stage, was adjusted to 1–2% parasitaemia and 2.5% haematocrit in hypoxanthine-free RPMI-1640 culture medium with 10% human plasma, then exposed to seven concentrations of each compound for a single cycle of parasitic growth for 48 h at 37 °C. A positive control with reference to antimalarial drugs in standard concentrations was used for each experiment. The stock solutions were additionally diluted in whole medium (RPMI 1640 plus 10% human serum) to each of the used concentrations. The concentration that inhibited 50% of parasite growth (IC₅₀ value) was determined by an interpolation method using Microcal Origin software. The standard drugs chloroquine and quinine were used as the reference antimalarial drugs, blood smears were read blind, and each duplicate experiment was repeated three times. Results are summarized in Table 3.

Acknowledgements

The authors are thankful to Head, Department of Chemistry, Sardar Patel University for providing necessary research facilities and constant encouragement. SCK and VBP gratefully acknowledge the University Grants Commission, New Delhi, India for meritorious fellowships awards 2013–2015.

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Footnote

† Electronic supplementary information (ESI) available: The spectral data of synthesized compound are shown. See DOI: 10.1039/c6ra01349j

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