Introduction

The designing of new types of polyheterocyclic compounds along with the refining of procedures for synthesis is an urgent target of the modern heterocyclic chemistry. Amongst the five-membered heterocyclic compounds, much interest has been focused on the pyrazole nucleus which is known to possess a broad spectrum of biological properties such as antipyretic, analgesic and anti-inflammatory (Menozzi et al., 2000; Turan-Zitouni et al., 2001; Palomer et al., 2002; Bekhit et al., 2008), antiparasitic (Rathelot et al., 2002; Kumar et al., 2006), antitubercular (Kaymakci oglu and Rollas 2002; Dixit et al., 2006), anticonvulsant (Kucukguzel et al., 2000), antimicrobial (El-Gaby et al., 2000; Baraldi et al., 2002; Akbas et al., 2005), antiviral (Larsen et al.,1999), anticancer (Poreba et al., 2001; Ishida et al., 2002; Witherington et al., 2003; Rostom et al., 2003; Park et al., 2005), herbicidal (Liu et al., 2007), etc. Likewise, thiazole nucleus present in a number of different types of compounds has been found to be associated with different types of pharmacological properties like anti-inflammatory (Holla et al., 2003; Venugopala and Jayashree, 2003; Pattan et al., 2007; Rostom et al., 2009), antitumor (El-Subbagh et al., 1999; Ramla et al., 2006), antifungal (Chimenti et al., 2011), antihypertensive (William et al., 1992), anti-HIV (Frank et al., 1995), etc. Antimicrobial activities of some substituted thiazoles are well established because it possess (S–C=N) toxophoric unit in the ring. Further, isoxazoles possess analgesic, anti-inflammatory, antimicrobial and other pharmacological activities (Habeeb et al., 2001; Velikorodov and Sukhenkol, 2003; Panda et al., 2009; Madhavi et al., 2010), etc. Numerous biological properties are possessed by the molecules in which both pyrazole and thiazole moieties are present together (Bekhit et al., 2003; Rostom, 2006). Similarly, the compounds containing pyrazole and isoxazole moieties have been shown to exhibit antihyperglycemic, analgesic, anti-inflammatory, antipyretic antibacterial, antiviral, antitumor, antifungal and antidepressant activities (Sugiura et al., 1977; Jungheim, 1989; Xue et al., 1998; Kang et al., 2000). Furthermore, various types of pharmacological properties are exhibited by the molecules containing isoxazole and thiazole pharmacores existing in the same molecule (Rajanarendar et al., 2010). It was, therefore, thought worthwhile to undertake the synthesis of some pyrazole derivatives possessing a thiazole moiety at postion-1 and isoxazole moiety at position-4 to frame potential biological molecules. Keeping in view of growing interest in the reactions of significance available in literature and numerous biological properties possessed by them, we herein report a detailed account of results of our investigations on the multistep synthesis, characterization and antimicrobial activities of 3a–d, 4a–d and the title compounds, 3-methyl-4-(3-methylisoxazol-5-yl)-1-(4-substituted phenylthiazol-2-yl)-1H-pyrazol-5-ol derivatives 5a–d.

Results and discussion

Chemistry

The title compounds 1-(4-(phenyl/4-substituted phenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol 5a–d were synthesized as illustrated in Scheme 1. The thiosemicarbazones 2 were prepared in high yields upon treatment of 2-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one (dehydroacetic acid, DHAA) 1 with thiosemicarbazide in equimolar quantities which were smoothly converted into the corresponding hydrazones 3 upon reaction with different 4-substituted phenacyl bromides in the presence of sodium acetate/ethanol. The hydrazones 3 thus obtained underwent rearrangement in ethanol-acetic acid mixture to afford the key intermediate 1-(5-hydroxy-3-methyl-1-substituted pyrazol-4-yl)-1,3-butanediones 4 which on subsequent treatment with hydroxylamine hydrochloride furnished 1-(4-(phenyl/4-substitutedphenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol 5a–d.

Scheme 1
scheme 1

Synthesis of 1-(4-(4-substituted-phenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ols 5

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The structures of all the products were established on the basis of elemental analyses, IR, NMR (1H and 13C) and mass spectral data. The hydrazones 3 exhibited medium intensity NH, OH broad stretching bands in the region 3,600–3,100 cm−1 and strong C=O stretching bands in the region 1,699–1,684 cm−1 in their IR spectra. The 1H NMR (400 MHz) spectra of these hydrazones displayed singlet in the region at δ 5.89–6.05 due to pyrone ring-H and singlet in the region at δ 6.62–6.85 due to thiazole ring-H which are in agreement with the results as reported in the literature [Singh et al., 1993]. The IR spectra of 4 exhibited absorption bands in the regions 3,600–3,100 and 1,718–1,706 and 1,646–1,633 cm−1 assigned to OH and CO functionalities, respectively. The 1H NMR (400 MHz) spectra of 4 displayed four signals due to each of the CH3 groups present in pyrazolyl moiety in enol and keto forms, in the regions at δ 2.04–2.07 and 2.54–2.66 and δ 2.20–2.29 and δ 2.50–2.62, respectively, and one signal in each case due to CH2 and oleifinic-H in the region at δ 4.04–4.06 and 6.67–6.69 (exchangeable with D2O), respectively. The singlet observed in the region at δ 7.29–7.62 was assigned to thiazole ring-H. The ratio of aromatic to aliphatic protons was found satisfactory. In 13C NMR spectra of 4 displayed peaks in the regions at δ 188.48–188.87, 186.94–187.44 and 183.02–183.24 due to carbonyl carbons. All these characteristic features corroborate existence of keto-enol tautomerism in 4 (Fig. 1). Further, the % ratio of keto/enol forms of 4 (4A:4B) were calculated from 1H NMR (400 MHz) spectral data, and were found as 4a (35:65), 4b (25:75), 4c (50:50) and 4d (37:63). A careful analysis of spectral data shows an interesting trend that 4a, 4b and 4d exist predominantly in enolic form but in 4c keto-enol forms are in equal amounts.

Fig. 1
figure 1

Keto-enol tautomeric forms of 1-(1-(4-(4-substituted-phenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)butane-1,3-diones 4

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The title compounds 1-(4-(phenyl/4-substituted phenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol 5a–d exhibited the characteristics broad absorption bands of medium intensity in the region 3,432–3,204 cm−1 due to OH of pyrazole moiety in their IR spectra. The 1H NMR (400 MHz) spectra of 5a–d showed a singlet in the region δ 6.51–6.55 due to isoxazole-H and another singlet in the region δ 7.25–7.71 assigned to thiazole-H. The mass spectral studies and elemental analysis results were found satisfactory. Physical and chemical data of the compounds 3a–d, 4a–d and 5a–d are detailed in Table 1.

Table 1 Physical and analytical data of compounds 3a–d, 4a–d and 5a–d
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Antimicrobial activity

All the newly synthesized compounds has been assayed for their in vitro antibacterial activity against Gram-positive Bacillus subtilis, Staphylococcus aureus and Gram-negative Escherichia coli and in vitro antifungal activity against Candida albicans and Aspergillus niger. The bacterial and fungal strains selected for this study are most common and easily available. B. subtilis may contaminate food and produces the proteolytic enzyme subtilisin and is responsible for causing ropiness. S. aureus is a common cause of boils, sties and skin infections and may cause serious infections in debilitated persons. E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. The fungus C. albicans is often a benign member of the mucosal flora; however, it commonly causes mucosal disease with substantial morbidity and in vulnerable patients it causes life-threatening bloodstream infections. A. niger is not only a xerophilic fungi, but is also a thermo-tolerant organism. Double strength nutrient broth and Sabouraud dextrose broth were employed for bacterial and fungal growth, respectively. The pMICs (negative logarithm of minimum inhibitory concentrations) were recorded as the minimum concentration of a compound that inhibits the growth of tested microorganisms. pMICs were determined by means of standard serial dilution method. The pMIC values are presented in Table 2. Most of the synthesized compounds showed good antibacterial activity with pMICs ranging from 1.435 to 1.778. The new title compounds isoxazolyl thiazolyl pyrazoles 5a–d and their precursors 3a–d and 4a–d were successfully synthesized. The synthesized compounds were found to exhibit moderate antibacterial activity, e.g. 5a–d against B. subtilis; 3a, 4b, 4d, 5a–c against S. aureus and 4b, 5a, 5b, 5d against E. coli, whereas the compounds 5b, 5c were found to be active fungicidal agent against A. niger. However, amongst the newly synthesized title compounds, 1-(4-(4-chlorophenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol 5d was found to exhibit the promising antibacterial activity against S. aureus (pMIC = 2.077, in comparison to the standard reference norfloxacin, pMIC = 2.698). Almost all the compounds showed moderate level of antifungal activity with pMICs ranging from 1.435 to 1.478 in comparison to the standard reference fluconazole (pMIC = 3.000). However, 5b (pMIC = 1.750) and 5c (pMIC = 1.771) exhibited good fungicidal activity. The results of antimicrobial activity illustrate that the presence of electron donating groups, CH3 and OCH3 on the phenyl ring, in compounds 3–5 increases the antibacterial as well as antifungal activities. Further, the presence of electron withdrawing group, Cl on the phenyl ring, in compounds 5d enhances the antibacterial activity to a greater extent. The graphical data of antimicrobial activities of all the synthesized compounds is depicted in Fig. 2.

Table 2 In vitro antimicrobial activity (pMIC) of compounds 3a–d, 4a–d and 5a–d
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Fig. 2
figure 2

In vitro antimicrobial activity (pMIC) of compounds 3a–d, 4a–d and 5a–d

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Conclusion

The synthesized compounds were found to exhibit moderate antibacterial activity, e.g. 5a–d against B. subtilis; 3a, 4b, 4d, 5a–c against S. aureus and 4b, 5a, 5b, 5d against E. coli, whereas the compounds 5b, 5c were found to be active fungicidal agent against A. niger. However, amongst the newly synthesized title compounds, 1-(4-(4-chlorophenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol 5d was found to exhibit the promising antibacterial activity against S. aureus.

Experimental

Melting points were determined in open capillaries and are uncorrected. The IR absorption spectra were scanned on Perkin Elmer Spectrum, BX II FTIR spectrometer using potassium bromide (KBr) pellets and the wave numbers were given in cm−1. The 1H NMR and 13C NMR spectra were recorded on Bruker Avance II 400 spectrometer at 400 and 100 MHz, respectively, in deuteriochloroform or deuteriochloroform + dimethyl sulfoxide-d6 or dimethyl sulfoxide-d6. The chemical shifts are reported in parts per million (δ ppm) using tetramethylsilane (TMS) as an internal standard. Coupling constants J are valued in Hertz (Hz). Mass spectra were recorded on Waters Micromass Q-Tof Micro (ESI) spectrometer. Elemental analysis was carried out using Vario Micro Cube Elementar CHNS analyser. Analytical results for C, H and N were within ±0.4% of the theoretical values. The purities of the compounds were checked by thin layer chromatography (TLC) using readymade silica gel (SIL G/UV254, ALUGRAM) plates. The spots were visualized under ultraviolet (UV) lamp. Solvents were dried using standard literature procedures. The thiosemicarbazones 2 were prepared by the condensation of equimolar quantities of 2-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one and thiosemicarbazide in absolute alcohol (Singh et al., 1993).

General procedure for the synthesis of 4-hydroxy-6-methyl-3-(1-(2-(4-substituted-phenylthiazol-2-yl)hydrazono)ethyl)-2H-pyran-2-ones (3a–3d)

To a solution of 2 (2.41 g, 0.01 mol) and anhydrous sodium acetate (0.82 g, 0.01 mol) in absolute ethanol, the appropriate p-substituted phenacyl bromide (0.01 mol) was added slowly with stirring. The reaction mixture was refluxed gently for 25–35 min. The yellowish crystalline solid thus obtained was filtered, washed with cold ethanol, and recrystallized from dimethyl formamide and water. Physical and analytical data are recorded in Table 1.

4-Hydroxy-6-methyl-3-(1-(2-(4-phenylthiazol-2-yl)hydrazono)ethyl)-2H-pyran-2-one (3a)

Yield 76%; mp 188–190°C (Lit. mp 190–191°C; Singh et al., 1993); IR (KBr): 3,400–3,100 (NH, OH), 1,698 cm−1(CO); MS: (TOF MS ES+) m/z 342.2 [M + H]+.

4-Hydroxy-6-methyl-3-(1-(2-(4-tolylthiazol-2-yl)hydrazono)ethyl)-2H-pyran-2-one (3b)

Yield 71%; mp 204–206°C; IR (KBr) 3,550–3,200 (NH, OH), 1,699 cm−1(CO); 1H NMR (DMSO-d6) δ 2.16 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.70 (s, 3H, CH3), 5.89 (s, 1H, pyrone-H), 6.62 (s, 1H, thiazole-H), 7.23 (d, 2H, J = 8.0 Hz, H-3″,5″), 7.61 (d, 2H, J = 8.08 Hz, H-2″,6″); MS: (TOF MS ES+) m/z 356.1 [M + H]+; C18H17N3O3S; Calc. C 60.83, H 4.82, N 11.82; Found C 60.34, H 4.71, N 12.08.

4-Hydroxy-3-(1-(2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)ethyl-6-methyl-2H-pyran-2-one (3c)

Yield 73%; mp 189–190°C; IR (KBr): 3,600–3,220 (NH, OH), 1,687 cm−1 (CO); 1H NMR (DMSO-d6): δ 2.08 (s, 3H, CH3), 2.69 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 6.05 (s, 1H, pyrone-H), 6.69 (s, 1H, thiazole-H), 6.94 (d, 2H, J = 8.72 Hz, H-3″,5″), 7.88 (d, 2H, J = 8.72 Hz, H-2″,6″); MS: (TOF MS ES+) m/z 372.2 [M + H]+; C18H17N3O4S; Calc. C 58.21, H 4.61, N 11.31; Found C 57.94, H 4.96, N 11.59.

3-(1-(2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazono)ethy)-4-hydroxy-6-methyl-2H-pyran-2-one (3d)

Yield 67%; mp 178–180°C; IR (cm−1): 3,500–3,200 (NH, OH), 1,684 cm−1 (CO); 1H NMR (DMSO-d6) δ 2.20 (s, 3H, CH3), 2.63 (s, 3H, CH3), 5.89 (s, 1H, pyrone-H), 6.85 (s, 1H, thiazole-H), 7.38–7.76 (m, 4H, Ar–H) MS: (TOF MS ES+) m/z 376.2 [M + H]+; C17H14ClN3O3S; Calc. C 54.33, H 3.75, N 11.18; Found C 54.72, H 3.83, N 11.26.

General procedure for the synthesis of 1-(1-(4-(4-substituted-phenyl)thiazol-2-yl)-5-hydroxy -3-methyl-1H-pyrazol-4-yl)butane-1,3-diones (4a–4d)

The hydrazone 3 (0.01 mol) was added in one lot to a hot solution of acetic acid and refluxed for approximately 2 h. The solid thus obtained, on cooling the reaction mixture overnight, was filtered, washed with little cold ethanol and crystallised from acetonitrile. Physical and analytical data are recorded in Table 1.

1-(5-Hydroxy-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-4-yl)butane-1,3-dione (4a)

Yield 57%; mp 142–144°C (Lit. mp 145°C; Singh et al., 1993); IR (KBr) 3,550–3,200 (OH), 1,706, 1,633 cm−1(CO); 1H NMR (DMSO-d6) δ 2.04, 2.27 (two s, 3H, CH3), 2.59, 2.62 (two s, 3H, CH3), 4.04 (s, 2H, CH2), 6.68 (s, 1H, olefinic-H), 7.29–7.84 (m, 6H, ArH and thiazole-H); 13C NMR (DMSO-d6) (enol + keto forms) δ 13.49, 13.71, 24.17, 30.97, 56.01, 97.76, 100.22, 103.84, 108.77, 126.41, 128.31, 128.68, 133.96, 134.05, 149.80, 149.84, 152.19, 152.70, 153.52, 159.05, 160.08, 183.13, 187.28, 188.5; MS: (TOF MS ES+) m/z 342.2 [M + H]+.

Hydroxy-3-methyl-1-(4-tolylthiazol-2-yl)-1H-pyrazol-4-yl)butane-1,3-dione (4b)

Yield 59%; mp 110–112°C; IR (KBr) 3,600–3,200 (OH), 1,707, 1,646 cm−1(CO); 1H NMR (DMSO-d6) δ 2.06, 2.27 (two s, 3H, CH3), 2.37, (s, 3H, CH3), 2.60, 2.64 (two s, 3H, CH3), 4.04 (s, 2H, CH2), 6.69 (s, 1H, olefinic-H), 7.21 (d, 2H, J = 7.9 Hz, H-3″, 5″), 7.39 (s, 1H, thiazole-H), 7.84 (d, 2H, J = 7.9 Hz, H-2″,6″); 13C NMR (DMSO-d6) (enol + keto forms) δ 13.49, 13.79, 21.34, 24.15, 30.95, 56.00, 97.73, 100.19, 103.81, 108.01, 126.33, 129.22, 129.36, 131.60, 137.49, 149.84, 149.88, 152.12, 152.71, 153.52, 159.08, 160.12, 183.24, 187.17, 188.48; MS: (TOF MS ES+) m/z 356.1 [M + H] +; C18H17N3O3S; Calc. C 60.83, H 4.82, N 11.82; Found C 61.14, H 5.08, N 12.23.

1-(5-Hydroxy -1-(4-(4-methoxyphenyl)thiazol-2-yl) -3-methyl -1H-pyrazol-4-yl)butane-1,3-dione (4c)

Yield 64%; mp 158–160°C; IR (KBr) 3,600–3,300 (OH), 1,718, 1,646 cm−1(CO); 1H NMR (DMSO-d6) δ 2.05, 2.20 (two s, 3H, CH3), 2.50, 2.54 (two s, 3H, CH3), 3.81 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 6.67 (s, 1H, olefinic-H), 7.02 (d, J = 8.7 Hz, H-2″, 6″), 7.62 (s, 1H, thiazole-H), 7.93 (d, J = 8.7 Hz, H-3″,5″); 13C NMR (DMSO-d6) (enol + keto forms) δ 13.39, 13.76, 24.14, 30.91, 55.40, 56.41, 98.61, 100.10, 103.78, 108.88, 114.28, 123.90, 126.89, 136.71, 149.81, 149.93, 152.17, 152.87, 153.18, 159.47, 159.70, 161.51, 183.22, 186.94, 188.87; MS: (TOF MS ES+) m/z 372.04 [M + H] +; C18H17N3O4S; Calc. C 58.21, H 4.61, N 11.31; Found C 58.53, H 4.93, N 10.96.

1-(1-(4-(4-Chlorophenyl)thiazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)butane-1,3-dione (4d)

Yield 61%; mp 120–122°C; IR (KBr) 3,550–3,100 (OH), 1,717, 1,636 cm−1(CO); 1H NMR (DMSO-d6) δ 2.07, 2.29 (two s, 3H, CH3), 2.62, 2.66 (two s, 3H, CH3), 4.06 (s, 2H, CH2), 6.68 (s, 1H, olefinic-H), 7.40 (d, 2H, J = 8.4 Hz, H-2″,6″), 7.45 (s, 1H, thiazole-H), 7.933 (d, J = 8.4 Hz, H-3″,5″); 13C NMR (DMSO-d6) (enol + keto forms) δ 13.49, 13.69, 24.17, 30.94, 55.94, 97.75, 100.25, 103.86, 109.28, 127.80, 128.76, 132.64, 132.70, 133.58, 148.66, 152.32, 152.79, 153.64, 159.13, 160.15, 183.02, 187.44, 188.51; MS: (TOF MS ES+) m/z 376 [M + H] +; C17H14ClN3O3S; Calc. C 54.33, H 3.75, N 11.18; Found C 54.56, H 3.61, N 11.47.

General procedure for the synthesis of 1-(4-(4-substituted-phenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ols (5a–d)

The solution of 4 (0.01 mol) and hydroxylamine hydrochloride (0.69 g, 0.01 mol) in 50 ml of ethanol and 50 ml of acetic acid was heated at reflux for approximately 2 h. The reaction mixture was cooled and then allowed to stand overnight. The solid thus obtained, was collected and recrystallised from acetonitrile. Physical and analytical data are recorded in Table 1.

3-Methyl-4-(3-methylisoxazol-5-yl)-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5-ol (5a)

Yield 68%; mp 208–210°C; IR (KBr) 3,204 cm−1 (OH); 1H NMR (DMSO-d6) δ 2.27 (s, 3H, CH3), 2.61 (s, 3H, CH3), 6.51 (s, 1H, isoxazole-H), 7.33–7.46 (m, 3H, Ar–H)), 7.71 (s, 1H, thiazole-H), 8.01 (d, 2H, J = 7.36 Hz, H-2″,6″); 13C NMR (DMSO-d6) δ 11.37, 12.92, 93.91, 99.32, 109.4, 126.45, 128.44, 128.96, 134.18, 148.09, 149.63, 152.51, 158.07, 159.42, 163.49; MS: (TOF MS ES+) m/z 339.3 [M + H]+; C17H14N4O2S; Calc. C 60.34, H 4.17, N 16.56; Found C 60.28, H 4.37, N 16.58.

3-Methyl-4-(3-methylisoxazol-5-yl)-1-(4-p-tolylthiazol-2-yl)-1H-pyrazol-5-ol (5b)

Yield 63%; mp 220–222°C; IR (KBr) 3,431 cm−1 (OH); 1H NMR (DMSO-d6) δ 2.23 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.62 (s, 3H, CH3), 6.52 (s, 1H, isoxazole-H), 6.91–7.84 (m, 5H, Ar–H, thiazole-H); 13C NMR (DMSO-d6) δ 12.84, 11.41, 12.84, 21.36, 94.26, 99.31, 108.79, 126.30, 129.25, 129.39, 137.90, 147.72, 150.0, 151.37, 158.11, 159.41, 163.49; MS (TOF MS ES+) m/z 353.4 [M + H]+; C18H16N4O2S; Calc. C 61.35, H 4.58, N 15.90; Found C 61.72, H 4.81, N 15.59.

1-(4-(4-Methoxyphenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5ol (5c)

Yield 74%; mp 232–234°C; IR (cm−1) 3,432 cm−1 (OH); 1H NMR (DMSO-d6) δ 2.30 (s, 3H, CH3), 2.64 (s, 3H, CH3), 3.84 (s, 3H, CH3), 6.54 (s, 1H, isoxazole-H), 6.93 (d, 2H, J = 2.84 Hz, H-3″,5″), 7.25 (s, 1H, thiazole-H), 7.86 (d, 2H, J = 2.84 Hz, H-2″,6″); 13C NMR (DMSO-d6) δ 11.39, 12.83, 55.35, 94.37, 99.32, 106.71, 114.04, 126.94, 127.22, 127.62, 127.70, 147.69, 149.87, 159.46, 159.63, 163.44; MS (TOF MS ES+) m/z 369.2 [M + H]+; C18H16N4O3S; Calc.C 58.68, H 4.38, N 15.21; Found C 59.01, H 4.10, N 15.07.

1-(4-(4-Chlorophenyl)thiazol-2-yl)-3-methyl-4-(3-methylisoxazol-5-yl)-1H-pyrazol-5-ol (5d)

Yield 69%; mp 258–260°C; IR (KBr) 3,431 cm−1 (OH); 1H NMR (DMSO-d6) δ 2.31 (s, 3H, CH3), 2.66 (s, 3H, CH3), 6.55 (s, 1H, isoxazole-H), 7.41 (d, 2H, J = 8.4 Hz, H-2″,6″)), 7.44 (s, 1H, thiazole-H), 7.94 (d, 2H, J = 8.4 Hz, H-3″, 5″); 13C NMR (DMSO-d6) δ 11.38, 12.84, 94.43, 99.40, 109.1, 127.76, 128.76, 132.75, 133.63, 138.9, 147.92, 148.81, 155.4, 159.51, 163.34; MS (TOF MS ES+) m/z 373.3 [M + H]+; C17H13ClN4O2S; Calc. C 54.77, H 3.51, N 15.03; Found C 55.13, H 3.16, N 15.16.

Antimicrobial activity

All the twelve newly synthesized compounds 3a–d, 4a–d and 5a–d were screened for their in vitro antimicrobial activity against five microorganisms, two Gram-positive bacteria B. subtilis (MTCC 441) and S. aureus (MTCC 7443) and one Gram-negative bacteria E. coli (MTCC 42) and two fungi C. albicans (MTCC 183) and A. niger (MTCC 282) by serial tube dilution technique using two solid media Double strength nutrient broth and Sabouraud dextrose broth for bacterial and fungal growth, respectively. The stock solutions (100 μg/ml) of all the test compounds were prepared by dissolving 1 mg of the test compound in 10 ml of dimethylsulphoxide. Norfloxacin and fluconazole were used as reference against bacteria and fungi, respectively. The fresh cultures were obtained by inoculation of respective microorganism in suitable medium (Double strength nutrient broth in case of bacteria and Sabouraud dextrose broth in case of fungi) followed by incubation at 37 ± 1°C. The stock solutions of the test compounds were serially diluted in test tubes containing 1 ml of sterile medium to get the concentration of 50–3.12 μg/ml and then inoculated with 100 μl of suspension of respective microorganism in sterile saline. The inoculated test tubes were incubated at 37 ± 1°C for 24 h in case of B. subtilis, S. aureus and E. coli, at 37 ± 1°C for 48 h in case of Candida albicans and at 37 ± 1°C for 120 h in case of A. niger and their pMICs(-log of Minimum inhibitory concentrations) were determined. Minimum inhibitory concentration (MIC), in microbiology, is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after incubation.

The reference compounds Norfloxacin and fluconazole were also tested under similar conditions to compare the results of tested compounds. The data for the antibacterial and antifungal studies are listed in Table 2 and expressed in Fig. 2.