Synthesis, and antitubercular and antimicrobial activity of 1′-(4-chlorophenyl)pyrazole containing 3,5-disubstituted pyrazoline derivatives

Abstract

A new series of 1′-(4-chlorophenyl)-5-(substituted aryl)-3′-(substituted aryl)-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl derivatives (6a–e, 8a–e, 10a–e) have been synthesized, characterized and screened for antimicrobial and antitubercular activity. Among the synthesized compounds, the minimum inhibition concentration of 10e was found to be as low as 1.56 μg ml⁻¹ and that of 10c was 6.25 μg ml⁻¹ as compared to the standard anti-tb drugs pyrazinamide and streptomycin.

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Mycobacterium tuberculosis is one of the most dangerous bacteria, which causes infectious diseases and remains out of control in many developing countries.¹ It is a remarkable pathogenic bacteria, that latently infects inside the body.² The control of tuberculosis is the most challenging in the case of the multi-drug resistance strains of Mycobacterium tuberculosis. The spread of multidrug-resistant TB (MDR-TB) and the appearance of extensively drug-resistant TB (XDR-TB) pose new challenges for the prevention, treatment and control of this deadly disease. Fewer new drugs have been approved to treat tuberculosis with very long and complicated therapies.³ Drug-resistant TB can be treated with multidrug combinations over an average span of six months.²

The azole class of drug derivatives and its research have played an important role in medicinal chemistry. Pyrazoles are well known nitrogen containing heterocyclic compounds. As per the literature, pyrazole and its derivatives represent one of the most desirable classes of compounds with a wide variety of pharmacological activities viz., antitubercular,^4,5 antifungal,⁶ antidepressant,^7,8 antimicrobial,⁹ anti-angiogenic,¹⁰ analgesic,¹¹ anticancer¹² and anticonvulsant.¹³ Moreover, pyrazoles containing pyrazoline derivatives are important drug molecules and exhibit important pharmacophore activities viz. antioxidant,¹⁴ antimicrobial¹⁵ and antidiabetic.¹⁶ Some of the literature reveals that the presence of substituted phenyl, i.e. 4-chlorophenyl or benzene sulfonamide, at the first position of pyrazole causes enhanced biological activities.¹⁷ Based on the above considerations, we hereby report the synthesis of 1-(4-chlorophenyl)pyrazole containing pyrazoline derivatives and their antitubercular activity against Mycobacterial tuberculosis.

The targeted compounds 1′-(4-chlorophenyl)-5-(2,3-dihydrobenzofuran-5-yl)-3′-(substituted aryl)-3,4-dihydro-2H, 1′H-[3,4′]bipyrazolyl (6a–e), 1′-(4-chlorophenyl)-5-(5-methylfuran-2-yl)-3′-(substituted aryl)-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl (8a–e) and 5-biphenyl-4-yl-1′-(4-chlorophenyl)-3′-(substituted aryl)-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl (10a–e) were synthesized according to the steps outlined in Scheme 1.

Scheme 1 Synthetic route for the pyrazole bearing pyrazoline derivatives.

The basic pyrazole skeleton, i.e. 1,3-disubstituted pyrazole-4-carbaldehydes 1, was synthesized by the Vilsmeier–Haack reaction in moderate to good yields.⁹ The other key starting materials 5-acetyl-2,3-dihydrobenzofuran 2, 2-acetyl-5-methylfuran 3 and 4-monoacetylbiphenyl 4 were prepared as per the reported literature¹⁸ and confirmed using IR and NMR. 1,3-disubstituted pyrazole-4-carbaldehyde 1 reacted with 2, 3 and 4 individually to give chalcone derivatives (5a–e, 7a–e and 9a–e, respectively) which on reacting with hydrazine hydrate in ethanol media under reflux temperature gave 6a–e, 8a–e and 10a–e in reasonable yields.

The formation of 6a–e, 8a–e and 10a–e was confirmed by recording their IR, ¹H NMR, ¹³C NMR and mass spectra. The IR analysis of compound 6a showed a peak at 3322 cm⁻¹ which was due to a NH group. The absorption band at 1607 cm⁻¹ was due to a –C=N group and –C=C stretching was observed at 1497 cm⁻¹. The ¹H NMR spectrum of 6a in DMSO-d₆ solvent shows a triplet at δ 2.90–2.96 which was attributed to the H_A proton, and a triplet at δ 3.42–3.49 which was due to the H_B proton, of the pyrazoline ring. The characteristic peak of the –NH proton of pyrazoline was observed as a singlet at δ 8.62. The detailed ¹H NMR resonances are summarized in the experimental section. The mass spectrum of 6a shows a molecular ion peak at m/z = 441.2 (M⁺). This in turn confirmed the formation of a compound with the molecular formula of C₂₆H₂₁ClN₄O. The other compounds 8a–e and 10a–e are explained in the ESI.† The characterization data of the newly synthesized compounds 6a–e, 8a–e and 10a–e are presented in Table 1.

Table 1 Characterization data of the compounds 6a–e, 8a–e and 10a–e

Compounds	Ar1	Ar2	Mol. F/mol. wt	M.P. (°C)	Color & nature
6a	Phenyl	5-(2,3-Dihydrobenzofuran)	C26H21ClN4O/440.92	156–157	White solid
6b	4-Methylphenyl	5-(2,3-Dihydrobenzofuran)	C27H23ClN4O/454.95	151–153	Off-white solid
6c	4-Methoxyphenyl	5-(2,3-Dihydrobenzofuran)	C27H23ClN4O/470.95	132–134	Off-white solid
6d	4-Chlorophenyl	5-(2,3-Dihydrobenzofuran)	C26H20Cl2N4O/475.37	171–172	White solid
6e	2-Thiophene	5-(2,3-Dihydrobenzofuran)	C24H19ClN4OS/446.95	140–141	White solid
8a	Phenyl	5-(2-Methylfuran)	C23H19ClN4O/402.88	144–145	White solid
8b	4-Methylphenyl	5-(2-Methylfuran)	C24H21ClN4O/416.90	206–208	Off-white solid
8c	4-Methoxyphenyl	5-(2-Methylfuran)	C24H21ClN4O2/432.9	156–157	Off-white solid
8d	4-Chlorophenyl	5-(2-Methylfuran)	C23H18Cl2N4O/437.32	198–200	Off-white solid
8e	2-Thiophene	5-(2-Methylfuran)	C21H17ClN4OS/408.9	186–187	Off-white solid
10a	Phenyl	4-(Biphenyl)	C30H23ClN4/474.98	164–165	Off-white solid
10b	4-Methylphenyl	4-(Biphenyl)	C31H25ClN4/489.01	157–158	Off-white solid
10c	4-Methoxyphenyl	4-(Biphenyl)	C31H25ClN4O/505.01	160–162	Off-white solid
10d	4-Chlorophenyl	4-(Biphenyl)	C30H22Cl2N4/509.43	184–186	White solid
10e	2-Thiophene	4-(Biphenyl)	C28H21ClN4S/481.01	217–219	Off-white solid

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The antimicrobial activity of all the synthesized compounds was screened against Staphylococcus aureus (Gram positive bacteria), Mycobacterium smegmatis (tubercular variant), and Candida albicans (fungi). The minimum inhibitory concentration (MIC) of all the organisms tested at different concentrations ranged from 500 to 3.9 μg ml⁻¹. Most of the compounds exhibited activity with the MIC ranging from 62.5 to 7.8 μg ml⁻¹. The structure activity relationship of the synthesized compounds was explained based on the MIC. Compound 10e showed the lowest MIC against all the tested organisms due to the presence of a 2-thiophene group at the third position of the pyrazole ring and biphenyl at the third position of the pyrazoline ring. The second lowest MIC was obtained for the following compounds: 6a due to the presence of 2,3-dihydrobenzofuran at the third position of the pyrazoline ring and a phenyl group at the third position of pyrazole, compound 10c due to biphenyl at the third position of pyrazoline and 4-methoxyphenyl at the third position of pyrazole and compound 10d due to biphenyl substitution on pyrazoline at the third position and p-chlorophenyl at the third position of the pyrazole ring. Other compounds 6d, 6e, 8b, 8c and 8d showed moderate activity with the MIC values ranging from 62.5 to 125 μg ml⁻¹ against the tested organisms. The antimicrobial results of the final compounds 6a–e, 8a–e and 10a–e are tabulated in Table 2.

Table 2 The minimum inhibitory concentration (MIC) of 6a–e, 8a–e and 10a–e against various antimicrobial and antitubercular agents

Synthesized compound	MIC in μg ml^−1
	M. smegmatis	S. aureus	C. albicans	M. tuberculosis
ABS; antibacterial standard ciprofloxacin; AFS; antifungal standard fluconazole; PZA; anti-tb standard pyrazinamide; —: no inhibition detected; control; dimethylsulfoxide.
6a	15.6	15.6	31.25	12.5
6b	500	500	500	50
6c	500	500	500	50
6d	62.5	62.5	62.5	25
6e	62.5	125	62.5	25
8a	250	125	125	25
8b	62.5	125	62.5	25
8c	62.5	62.5	62.5	25
8d	62.5	62.5	62.5	25
8e	125	31.25	125	50
10a	125	125	125	50
10b	500	500	500	50
10c	15.6	15.6	31.25	6.25
10d	15.6	62.5	31.25	50
10e	7.8	15.6	31.25	1.56
ABS	<5	<5	—	3.12
AFS	—	—	<10	—
PZA	—	—	—	3.12
Streptomycin	—	—	—	6.25

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The MIC against the pathogenic bacteria Mycobacterium tuberculosis H₃₇Rv at different concentrations ranging from 100 to 0.8 μg ml⁻¹ is also represented in Table 2. Most of the compounds exhibited activity with the MIC ranging from 50 μg ml⁻¹ to 1.56 μg ml⁻¹. Compound 10e showed the lowest MIC (1.56 μg ml⁻¹) among all other compounds and it was more active than the standard first-line anti-tb drug pyrazinamide (MIC value of 3.12 μg ml⁻¹). The second lowest MIC against the tested microorganisms (6.25 μg ml⁻¹) was obtained for compound 10c and it was similarly active to the standard anti-tb drug streptomycin (MIC value of 6.25 μg ml⁻¹). Compound 6a showed the third lowest MIC (12.5 μg ml⁻¹). Other compounds 6d, 6e, 8a, 8b, 8c and 8d showed moderate activity with an MIC value of 25 μg ml⁻¹ against tested Mycobacterium tuberculosis. This indicates that most of the pyrazole containing pyrazoline compounds with 5-methylfuran substitution at the third position are able to show moderate activity against M. Tuberculosis. Compounds 6b, 6c, 8e, 10a, 10b and 10d show less activity against the tested organisms with an MIC value of 50 μg ml⁻¹.

An in vitro cytotoxicity study was carried out using HeLa cells at Stellixir Biotech Pvt. Ltd, Bangalore. The five compounds 6a, 6d, 8b, 10c and 10e which had the highest to moderate antimicrobial and antituberculosis activity were tested for cytotoxicity with HeLa cells, represented in Fig. 1. The IC₅₀ values of the synthetic compounds were found to be moderately effective for the HeLa cells. The control cells which are not treated with any compound show 100% viability. One of the synthetic compounds, 8b, shows the highest cytotoxicity (12.83 μg ml⁻¹) among the tested compounds. The compounds which have cytotoxicity below a 50 μg ml⁻¹ IC₅₀ value are usually considered as toxic compounds.

Fig. 1 Cytotoxicity of 6a, 6d, 8b, 10c and 10e with a HeLa cell line.

In conclusion, compounds 6a–e, 8a–e and 10a–e were synthesized, characterized and investigated for their in vitro antimicrobial activity by the Resazurin reduction method. These compounds were used for determining the MIC in 96 well microplates,¹⁹ and antitubercular activity using a microplate alamar blue assay (MABA) method,²⁰ and proved to be very good antimicrobial and antitubercular agents. The results are consistent with specific substitution utilised in tuberculosis chemotherapy and antimicrobial agents. Compounds 10e and 10c showed the best screening results among all the synthesized compounds. This indicates that the newly synthesized pyrazole containing pyrazoline compounds might emerge as antituberculosis drugs.

Experimental

All the chemicals were purchased from Sigma Aldrich and Spectrochem-India. Melting points were determined using the open capillary method and are uncorrected. The IR spectra (in KBr pellet) were recorded using a Perkin-Elmer FTIR-4000–400 cm⁻¹ spectrophotometer. The NMR spectra were obtained using a Bruker Avance-400 spectrometer (400 MHz) for ¹H NMR and ¹³C NMR using tetramethylsilane (TMS) as the internal standard. The mass spectrum was recorded using a LC-MS Applied biosystems MDS SCIEX-API 4000 spectrometer. Elemental analysis was performed using a Flash EA 1112 series CHNS-O analyzer. The completion of the reaction was checked using thin layer chromatography (TLC) with ready-made aluminium sheets (Merck F₂₅₄). The names of the structures were mentioned as per chemdraw.

A mixture of 1,3-disubstituted pyrazole-4-carbaldehyde (1a–e) (0.01 mol) and an acetyl derivative (2, 3 and 4) (0.91 g, 0.01 mol) in methanol (10 ml) was stirred in the presence of 10% sodium hydroxide solution (2 ml) at ambient temperature for 5 h. The resultant yellow color reaction mass was filtered and washed with 5 ml of ethanol to get a chalcone intermediate (5a–e, 7a–e and 9a–e) in reasonably good yields (80–95%). The compound (5a–e, 7a–e and 9a–e) (0.005 mol) was taken in ethanol (10 ml) and an excess amount of hydrazine hydrate (1.3 g, 0.015 mol) was added. The reaction mixture was heated at reflux temperature for 2 h and the reaction was monitored using TLC [hexane : ethylacetate (4 : 1)]. The reaction mass was cooled to room temperature and stirred for 0.5 h. The solid product was filtered and washed with ethanol to get the final compound (6a–e, 8a–e and 10a–e).

1′-(4-Chlorophenyl)-5-(2,3-dihydrobenzofuran-5-yl)-3′-phenyl-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl (6a)

IR (KBr ν_max cm⁻¹): 3322 (N–H str), 3064 (Ar–H str), 2919 (C–H aliphatic str), 1607 (C=N str), 1497 (C=C str), 824 (C–Cl str); ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 2.90–2.96 (t, 1H, H_A, J = 12.8 Hz), 3.19 (t, 2H, –CH₂), 3.42–3.49 (t, 1H, H_B, J = 13.6 Hz), 4.56 (t, 2H, –CH₂), 4.89–4.92 (t, 1H, H_X, J = 10.4 Hz), 6.77 (m, 1H, Ar–H), 7.33–7.36 (m, 2H, Ar–H), 7.43–7.56 (m, 6H, Ar–H), 7.77–7.95 (m, 4H, Ar–H), 8.62 (s, 1H, –NH); ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 160.5, 151.2, 138.8, 133.2, 130.7, 130.1, 129.9, 129.7, 129.1, 128.6, 128.4, 128.2, 128.0, 126.3, 122.9, 121.0, 120.2, 109.2, 71.7, 55.5, 29.3; MS: m/z = 441.2 (M⁺), anal. calcd for C₂₆H₂₁ClN₄O; calcd: C, 70.82; H, 4.80; N, 12.71; found: C, 70.83; H, 4.80; N, 12.72.

1′-(4-Chlorophenyl)-5-(5-methylfuran-2-yl)-3′-phenyl-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl (8a)

IR (KBr ν_max cm⁻¹): 3316 (N–H str), 3117 (Ar–H str), 2923 (C–H aliphatic str), 1594 (C=N str), 1499 (C=C str), 829 (C–Cl str); ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 2.31 (s, 3H, –CH₃), 2.84–2.90 (t, 1H, H_A, J = 13.0 Hz), 3.31 (m, 1H, H_B), 4.89–4.95 (t, 1H, H_X, J = 10.6 Hz), 6.17 (s, 1H, Ar–H), 6.50 (s, 1H, Ar–H), 7.44–7.76 (m, 8H, Ar–H), 7.94–7.95 (d, 2H, Ar–H, J = 6.0 Hz), 8.59 (s, 1H, –NH); ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 152.9, 151.1, 147.3, 142.1, 138.7, 133.2, 130.7, 129.9, 129.1, 128.6, 128.4, 128.0, 123.7, 120.2, 110.9, 108.3, 55.2, 13.9; MS: m/z = 403.2 (M⁺), anal. calcd for C₂₃H₁₉ClN₄O; calcd: C, 68.57; H, 4.75; N, 13.91; found: C, 68.60; H, 4.76; N, 13.92.

5-Biphenyl-4-yl-1′-(4-chlorophenyl)-3′-phenyl-3,4-dihydro-2H,1′H-[3,4′]bipyrazolyl (10a)

IR (KBr ν_max cm⁻¹): 3312 (N–H str), 3073 (Ar–H str), 2921 (C–H aliphatic str), 1593 (C=N str), 1497 (C=C str), 830 (C–Cl str); ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 2.97–3.03 (dd, 1H, H_A, J = 10.9 Hz), 3.49–3.56 (dd, 1H, H_B, J = 10.7 Hz), 4.97–5.03 (dt, 1H, Hx), 7.30–7.38 (m, 2H, Ar–H), 7.45–7.55 (m, 6H, Ar–H), 7.64–7.65 (d, 1H, Ar–H, J = 3.3 Hz), 7.68 (s, 1H, pyrazole-5H), 7.70–7.71 (dd, 5H, Ar–H, J = 1.4 Hz), 7.80–7.82 (d, 2H, Ar–H, J = 8.5 Hz), 7.89–7.91 (d, 2H, Ar–H, J = 7.7 Hz), 8.61 (s, 1H, –NH); MS: m/z = 475.1 (M⁺), anal. calcd for C₃₀H₂₃ClN₄; calcd: C, 75.86; H, 4.88; N, 11.80; found: C, 75.90; H, 4.90; N, 11.90.

Acknowledgements

AMI thanks the Director of the National Institute of Technology Karnataka, India for providing the research facilities. The authors extend their appreciation to The Deanship of Scientific Research at King Saud University for funding the work through research group project no. RGP-VPP-207.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nj02237a

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