Synthesis, biological evaluation and molecular docking of some substituted pyrazolines and isoxazolines as potential antimicrobial agents

Synthesis, biological evaluation and molecular docking of some substituted pyrazolines and isoxazolines as potential antimicrobial agents

European Journal of Medicinal Chemistry 95 (2015) 96e103 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal h...

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European Journal of Medicinal Chemistry 95 (2015) 96e103

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis, biological evaluation and molecular docking of some substituted pyrazolines and isoxazolines as potential antimicrobial agents Sameena Bano a, Mohammad Sarwar Alam a, *, Kalim Javed a, Mridu Dudeja b, Ayan Kumar Das b, Abhijeet Dhulap c a b c

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India Department of Microbiology, HIMSR, Jamia Hamdard (Hamdard University), New Delhi, India CSIR Unit for Research and Development of Information Products, Pune 411038, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 September 2014 Received in revised form 2 March 2015 Accepted 15 March 2015 Available online 16 March 2015

A series of substituted pyrazolines (2aee, 3aeh and 6aec) and isoxazolines (4aee) were synthesized and their structures were established on the basis of IR, 1H NMR, 13C NMR and mass spectra. All the synthesized compounds were tested against two bacterial and four fungal strains and found to exhibit moderate to potent antifungal activity. Compounds 2b, 4c, 4d and 6aec exhibited significant activity against all tested fungal strains. MIC values of all the active compounds were comparable with standard drug fluconazole. The results of the in silico molecular docking study supported the antifungal activity of the synthesized compounds. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: 2-pyrazolines Isoxazolines antimicrobial Molecular docking 14a-sterol demethylases

1. Introduction Despite significant progress made in the treatment of infectious diseases, caused by bacteria and fungi, it remains a major worldwide health problem due to rapid development of resistance against the existing antimicrobial drugs. Developing novel antimicrobial agents with different mode of action than that of existing drugs is one of the main challenges to overcome the antimicrobial resistance. In view of these facts, it is important to develop more effective antimicrobial agents. Thus, the synthesis and discovery of more efficient antimicrobial agents has been intensively considered during the last decade. Different heterocyclic compounds containing nitrogen, sulphur and oxygen as hetero atoms have been explored for the development of new antimicrobial agents [1e7]. Compounds containing five membered heterocyclic ring systems like pyrazolines and isoxazolines continue to attract considerable interest due to the wide range of biological activities they posses. Pyrazolines have been reported to exhibit a variety of biological activities including anti-tumour [8,9], anti-inflammatory [4,10e15],

* Corresponding author. E-mail address: [email protected] (M.S. Alam). http://dx.doi.org/10.1016/j.ejmech.2015.03.031 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

antiparasitary [16], anticonvulsant [17], antimicrobial [18e22], antinociceptive [23], antimalarial [24], nitric oxide synthase inhibitory, inflammatory arthritis [25], antidepressant [26,27], anticancer [28e30], antibacterial [18], analgesic [31], antioxidant [32], antiamoebic, cytotoxic [33e35], antifungal [36,37], antimycobacterial [38], antihepatotoxic [39] and pesticidal properties [40]. Isoxazoline derivatives have been reported to possess antimicrobial [20,21,41,42], anticonvulsant [43], anti-inflammatory [44], anti-viral [45], analgesic [46] and antitumour activity [47,48]. Penicillin derivatives containing isoxazole ring have been found to possess antibacterial activity [49]. Isoxazoline derivatives also show a good potency in animal models of thrombosis [50]. In addition, isoxazoline derivatives have played a vital role in the theoretical development of heterocyclic chemistry and are also extensively used in organic synthesis [51,52]. Computational biology and bioinformatics play a major role in designing the drug molecules and have the potential of speeding up the drug discovery process. Molecular docking of the drug molecule with the receptor (target) gives important information about drug receptor interactions and is commonly used to find out the binding orientation of drug candidates to their protein targets in order to predict the affinity and activity. Cytochrome P450 14a-sterol

S. Bano et al. / European Journal of Medicinal Chemistry 95 (2015) 96e103

demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. Being a key enzyme of sterol biosynthesis, CYP51 has been found to be a target for antifungal [53] and cholesterollowering [54] drug design. Keeping in view the therapeutic importance of heterocyclic compounds and in continuation of our work on synthesis of biologically active heterocycles [10e12] we hereby report the synthesis, molecular docking and biological studies of pyrazolines and isoxazolines derivatives. The new substituted pyrazolines and isoxazolines in addition to eight previously reported pyrazolines bearing benzenesulfonamide moiety [11] were screened for their antibacterial and antifungal activities. Our group has earlier reported the anti-inflammatory and anti-cancer profile of pyrazolines bearing benzenesulfonamide moiety [11]. The molecular docking study of all the compounds has been done for the better understanding of the drug-receptor interaction. 2. Chemistry The reactions involved in the synthesis of title compounds are given in Scheme 1. The chalcones/flavanones were prepared by reacting acetophenone with appropriate aldehydes in the presence of a base by conventional reactions. Five novel 3,5-diaryl-2pyrazoline (2aee) as well as five 3,5-diaryl-2-isoxazoline (4aee) were synthesized by the condensation of chalcones/flavanones with hydrazine hydrate and hydroxylamine hydrochloride respectively. Three novel 3-aroyl-4-aryl-2-pyrazolines 6aec were synthesized by

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treatment of appropriate chalcones with freshly prepared diazomethane gas dissolved in ether at 0  C. Reaction between synthesized chalcones/flavanones and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol led to synthesis of pyrazolines bearing sulphonamide moiety (3aeh) as reported earlier [11]. All the synthesized compounds were characterized by IR, 1H NMR, 13C NMR, mass and elemental analysis. 3. Results and discussion The IR spectra of all the compounds showed absorption bands in the regions 1517e1614 cm1 corresponding to C¼N stretching because of ring closure. The infrared spectra of (6aec) also revealed CO band at 1642e1600 cm1. In the 1H NMR spectra of all compounds the three hydrogen atoms attached to the C-4 and C-5 carbon atoms of the heterocyclic ring gave an ABX spin system. The pyrazoline/isoxazoline structures were unambigously proved by the measured chemical shifts. In compounds (2aee) and (4aee), H4 (trans) and H-4 (cis) appeared as double doublets and H-5 either appeared as multiplets or double doublets. In 4a and 4e the peak for H-4 (cis) of pyrazolines could not be picked out because of its merger with solvent peak. In compounds (6a-c) H-5 (cis) appeared as multiplets and H-4 appeared either as multiplets or double doublets. The peak for H-5 (trans) could not be identified because of merging with solvent peak. The methyl and aromatic protons were observed at expected ppm. All structures were further supported by 13C NMR spectra which showed the chemical shift values of

Scheme 1. Synthesis of pyrazolines and isoxazolines derivatives.

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carbon atoms of heterocyclic ring C-3 at (126.46e159.21 ppm), C-4 at (31.02e54.86 ppm) and C-5 at (56.26e76.60 ppm).

Table 2 Minimum inhibitory concentration (mg/mL) of all active compounds against the microorganisms. Compound

4. Pharmacology 4.1. In vitro antimicrobial activity

2a 2b 2c 2d 2e 4a 4b 4c 4d 4e 6a 6b 6c Fluconazole

Antifungal and antibacterial activities were evaluated by using agar well diffusion method [55]. All synthesized compounds were screened for their in-vitro antifungal activity against Candida albicans, Aspergillus fumigatus, Aspergillus versicolor and Aspergillus flavus, and in-vitro antibacterial activity against Gram-negative Escheriochia coli and Gram-positive Staphylococcus aureus. Ciprofloxacin (25 mg/disc) and fluconazole (20 mg/disc) were used as standard references for antibacterial and antifungal activity respectively. After incubation the diameter of the zone of inhibition formed around the cavities and disc of standard drugs was accurately measured in mm. The observed zones of inhibition are presented in Table 1. All the compounds showed significant antifungal activity but weak to moderate antibacterial activity. 3, 5-diaryl-2pyrazoline (2aee), 3, 5-diaryl-2-isoxazoline (4aee) and 3-aroyl-4aryl-2-pyrazolines (6aec) exhibited good antifungal activity comparable with standard drug while pyrazolines bearing sulphonamide moiety (3a-h) showed moderate antifungal activity. Compounds 2b, 4c, 4d and (6aec) exhibited comparable activity against all tested fungal strains. Compounds 2e, 4a, 4b and 4e showed good zone of inhibition against C. albicans and A. fumigatus.

Fungi C. albicans

A. fumigatus

A. versicolor

A. flavus

50 12.5 12.5 12.5 12.5 25 12.5 12.5 12.5 12.5 25 12.5 25 12.5

25 12.5 12. 5 12.5 12.5 12.5 25 6.25 6.25 6.25 6.25 12.5 12.5 12.5

25 6.25 12.5 12.5 12.5 25 25 12.5 12.5 12.5 12.5 12.5 12.5 3.125

6.25 3.125 6.25 25 12.5 6.25 6.25 12.5 12.5 12.5 6.25 6.25 12.5 12.5

activity among all the investigated compounds. 4.3. Molecular docking study Molecular docking provides a powerful tool in understanding different protein functions. Cytochrome P450 belongs to family of hemoproteins and plays an important role in metabolism and detoxification of foreign compounds (xenobiotic molecules) from the human body. Cytochrome P450 14a-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. Thus it provides important target for testing newly designed molecules [53,54]. All 21 molecules were docked against the generated target. Only 12 molecules docked within the generated target grid. The fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) (PDB No: 1EA1) resolved at 2.2 A was the first authentic drug target for cytochrome P450. The active site of CYP51 includes triazole ring which positioned perpendicular to the porphyrin plane with a ring nitrogen atom coordinated to the Hem iron [57].

4.2. MIC of all active compounds against tested fungal strains Minimum inhibitory concentration (MIC) of all active compounds was measured in vitro using two fold serial dilution technique [56]. The results of minimum inhibitory concentration against tested fungi, ranging from 3.12 to 50 mg/mL, are presented in Table 2. MIC values of all the active compounds are comparable with standard drug fluconazole. As revealed from MIC values compounds 2b, 4c, 4d and 6aec exhibited highest antifungal

Table 1 Antimicrobial activity of all synthesized compounds. Compound

Diameter of zone of growth inhibition (mm)a Fungi C. albicans

2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 3f 3g 3h 4a 4b 4c 4d 4e 6a 6b 6c Fluconazole Ciprofloxacin a

18 23 18 20 20 16 17 17 16 16 16 15 15 22 22 22 23 21 19 21 20 21 e

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.2 0.2 0.1

A. fumigatus 18 20 18 21 21 14 13 13 14 16 12 11 10 21 16 20 21 15 20 18 20 22 e

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.1

Values, including diameter of the well (8 mm), are expressed as mean ± SEM.

Bacteria Gram þ ve Gram ve A. versicolor 13 21 13 16 16 13 12 11 13 12 11 12 11 17 15 21 20 14 19 20 19 21 e

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.1 0.1 0.2

A. flavus 12 21 13 17 15 13 11 11 12 12 12 10 12 15 16 20 20 15 21 19 19 21 e

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.2 0.1 0.2 0.1 0.2 0.1 0.1 0.2

S. aureus 11 11 12 11 10 12 13 12 11 12 13 12 13 11 11 12 11 10 10 9 10 e 24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.2

± 0.1

E. coli 8 9 8 8 9 13 14 12 13 14 12 11 12 8 8 7 8 9 8 8 9 e 24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.2

± 0.1

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Fig. 1. 3D crystal structure of fluconazole in binding pocket of CYP51.

The active pocket of CYP450 consists of amino acid residues as Tyr76, Phe78, Leu321, ILE323, Val435, Met433, Leu324, Met79, Ala256, Phe255, Arg96 and Hem 460 as shown in Fig. 1. The binding interactions of fluconazole in binding pocket of CYP450 are as shown in Fig. 2. The generated receptor grid docked by synthesised compounds, exhibited well established bonds with the amino acids of the receptor. The glide docking scores of the synthesised compounds are shown below in Table 3. A set of twelve compounds (2aee, 4aed and 6aec) showed better glide score against the standard drug fluconazole. All the compounds possessed the required binding energy to dock itself with the binding pocket of CYP-51 ranging from 8.37 to 1.73 kcal/mol. Properties like partition coefficient (log P o/w), van der Waals, surface area of polar nitrogen and oxygen atoms (PSA) and aqueous solubility (log S) properties were also within acceptable ranges as shown above in Table 3.

Binding poses of highly ranked docked molecules are presented in the Fig. 3. Compound 2b showed H-bond formation with residue ARG-90 and MET - 433. Furthermore there was pep stacking with PHE-78 and TYR-76 and p-cation stacking was observed with HEM - 460. Compound 6a did not show any H-bond formation but it showed pep stacking observed with TYR e 76. Compound 6b showed no H-bond formation but p-cation stacking was observed with ARG - 96. Compound 2d showed H-bond formation with residue ARG e 96, pep stacking with PHE-78, TYR-76 and p-cation stacking with HEM - 460. The docking results of the compounds fully supported the determined activity of these compounds as antifungal agents. 5. Conclusion In the present study sixteen pyrazolines and five isoxazolines have been synthesized. The structures proposed for all new compounds were confirmed by spectroscopic data and elemental analysis. All the synthesized compounds were evaluated for their antifungal and antibacterial properties. All the compounds showed Table 3 Docking scores of reference ligand and 12 new ligands with Cytochrome P450 target (PDB No: 1EA1).

Fig. 2. Binding interaction of fluconazole in binding pocket of CYP51.

Ligand

GScore

Glide energy

Mol Wt

logP o/w

Log S

PSA

2b 6a 6b 2d 2c 2a 6c 4a 4c 4d 4b 2e Fluconazole

8.37 7.56 7.37 7.63 7.43 7.32 6.89 6.71 6.61 5.99 5.96 5.76 5.63

42.8 21.59 43.72 40.52 33.71 37.3 46.04 23.36 18.49 21.31 10.05 24.76 42.58

316.786 342.824 372.85 360.839 343.855 330.813 387.822 301.772 336.217 336.217 331.798 390.866 306.274

2.947 4.417 4.312 3.966 4.211 3.848 3.414 3.528 3.912 3.936 3.586 4.676 0.538

4.735 4.995 5.149 5.482 6.136 5.325 4.863 4.583 5.251 4.862 4.752 6.846 2.18

70.715 54.398 64.069 61.098 54.741 55.392 100.547 46.622 44.843 41.207 51.513 67.162 78.63

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S. Bano et al. / European Journal of Medicinal Chemistry 95 (2015) 96e103

Fig. 3. Binding poses of highly ranked conformers for compounds number 2b, 6a, 6b and 2d inside the binding pocket of CYP450.

significant antifungal activity but weak to moderate antibacterial activity. The molecular docking study of the compounds was carried out for the better understanding of the drug-receptor interaction. Compounds 2b, 4c, 4d and 6aec exhibited comparable activity against all tested fungal strains. MIC values of all the active compounds are comparable with standard drug fluconazole. A set of twelve compounds (2aee, 4aed and 6aec) showed better glide score against the standard drug fluconazole. All the compounds possessed the required binding energy to dock itself with the binding pocket of CYP51 ranging from 8.37 to 1.73 kcal/mol. The antifungal activity of these compounds was fully supported by the in silico molecular docking study. 6. Experimental protocols 6.1. Chemistry Melting points were uncorrected and measured using Electrothermal IA 9100 apparatus. All the Fourier Transform Infra Red (FTIR) spectra were measured on a Brukers Vector 22 spectrophotometer in film; nmax values are given in cm1. 1H NMR spectra were recorded on a Bruker Spectrospin DPX 300-MHz spectrometer using DMSO-d6 as a solvent and tetramethyl silane (TMS) as an internal standard. Chemical shifts are given in d (ppm) scale and coupling constants (J values) are expressed in Hz. Mass spectra (MS) were scanned by using a FAB ionization JEOL-JMS-DX 303 system equipped with a direct inlet probe system. The m/z values of the more intense peaks are mentioned. 13C NMR spectra was recorded on a Bruker spectrospin DPX at 75 MHz using deuterated DMSO as a solvent and tetramthyl silane (TMS) as internal standard. Purity of the compounds was checked on TLC plates (silica gel G) which were visualized by exposing to iodine vapours. Elemental analysis was carried out on CHNS Elementar (Vario EL III). 6.2. General procedure for the synthesis of 3, 5-diaryl)-2pyrazolines 2a-e Appropriate chalcones or flavanone (0.001 mol) and hydrazine

hydrate (98%, 0.001 mol) were dissolved in ethanol (20e30 ml) and refluxed for 24 h. After completion of the reaction the reaction mixture was concentrated to 10e15 ml. It was left at room temperature to give crystalline compound. It was filtered and crystallized with ethanol to give pure compound. 6.2.1. 3-(50 -Chloro-20 -hydroxy-40 ,60 -dimethylphenyl)-5-(4methoxyphenyl)-D2-pyrazoline (2a) As light brown crystals in 34% yield, m.p. 134e135  C, Rf ¼ 0.46, [petroleum ether (60e80 ): acetone; 8: 2]. IR ymax (KBr): 3319 (OH), 2839 (NH), 1611 (C¼N), 1028 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.34 [3H, s, CH3-40 ], 2.46 [3H, s, CH3-60 ], 3.19 [1H, dd, J ¼ 6 Hz, 15 Hz, H-4 trans (pyrazoline)], 3.60 [1H, dd, J ¼ 6 Hz, 15 Hz, H-4, cis (pyrazoline)], 3.80 [3H, s, OCH3-4], 4.83 [1H, m, H-5 (pyrazoline)], 5.91[1H, brs, NH], 6.79 [1H, s, H-30 ], 6.89 [2H, d, J ¼ 9 Hz, H-3, H-5], 7.29 [2H, d, J ¼ 9 Hz, H-2, H-6], 11.34 [1H, s, OH (chelated)]. 13C NMR (DMSO, d): 20.67 (-CH3), 20.92 (-CH3), 34.54 (C-4, pyrazoline), 76.60 (C-5, pyrazoline), 111.99 (C-200 and C-600 ), 117.54 (C-30 ), 120.29 (C-10 ), 126.46 (C-2 and C-6), 127.38 (C-3 and C5), 128.37 (C-50 ), 128.40 (C-4), 128.56 (C-300 and C-500 ), 133.96 (C-40 ), 134.08(C-400 ), 136.63C-60 ), 139.63C-1), 140.05100 ), 148.28C-3 (pyrazoline), 155.56 (C-20 ). FAB-MS (m/z): 330 [Mþ], 331 [Mþ1, base peak]. Elemental Analysis: Calculated for the molecular formula C18H19CI N2O2; Calculated; C ¼ 65.35, H ¼ 5.74, N ¼ 8.47, Found: C ¼ 65.61, H ¼ 5.14, N ¼ 8.29. 6.2.2. 3-(50 -Chloro-20 -hydroxy-40 ,6'-dimethylphenyl)-5-(3hydroxyphenyl)-D2-pyrazoline (2b) As brown crystals in 45% yield, m.p. 164e165  C, Rf ¼ 0.26, [petroleum ether (60e80 ): acetone; 8: 2].IR ymax (KBr): 3323 (OH), 2937 (NH), 1599 (C]N), 1455 cm1.1H NMR (300 MHz, DMSO, d): 2.34 [3H, s, CH3-40 ], 2.45 [3H, s, CH3-60 ], 3.20 [1H, dd, J ¼ 8.9 Hz, 16.2 Hz, H-4 trans (pyrazoline)], 3.59 [1H, dd, J ¼ 10 Hz, 16.2 Hz, H-4, cis (pyrazoline)], 4.83 [1H, m, H-5 (pyrazoline)], 5.42 [1H, brs, NH], 5.96 [1H, s, H-30 ], 6.77e7.25 [5H, m, AreH], 11.30 [1H, s, OH (chelated)]. 13C NMR (DMSO, d):18.12 (-CH3), 20.75 (-CH3), 44.72 (C4, pyrazoline), 63.42 (C-5, pyrazoline), 113.56 (C-4) 114.07 (C-2), 115.44 (C-30 ), 117.47 (C-10 ), 120.15 (C-6), 124.31 (C-50 ), 129.37 (C-5),

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135.12 (C-40 ), 136.18 (C-1), 144.63 (C-60 ), 149.00 (C-3, pyrazoline), 154.46 (C-3), 157.44 (C-20 ). FAB-MS (m/z): 316 [Mþ,], 317 [Mþ1, base peak]. Elemental Analysis: Calculated for the molecular formula C17H17CI N2O2; Calculated; C ¼ 64.46, H ¼ 5.41, N ¼ 8.84, Found: C ¼ 64.40, H ¼ 5.52, N ¼ 9.0. 6.2.3. 3-(50 -Chloro-20 -hydroxy-40 ,6'-dimethylphenyl)-5-(4-(N,Ndimethyl)-phenyl)-D2-pyrazoline (2c) As brown crystals in 38% yield, m.p. 120e121  C, Rf ¼ 0.51, [petroleum ether (60e80 ): acetone; 8: 2]. IR ymax (KBr): 3316 (OH), 2837(NH), 1614 (C]N), 1570, 1521, 1444 cm1.1H NMR (300 MHz, DMSO, d): 2.34 [3H, s, CH3-40 ], 2.47 [3H, s, CH3-60 ], 2.94 (6H, s, N(CH3)2), 3.21 [1H, dd, J ¼ 8.7 Hz, 16.2 Hz, H-4 trans (pyrazoline)], 3.57 [1H, dd, J ¼ 9.8 Hz, 16.2 Hz, H-4, cis (pyrazoline)], 4.79 [1H, m, H-5 (pyrazoline)], 5.86 [1H, brs, NH], 6.69 [2H, d, J ¼ 8.6 Hz, H-3, H5], 6.79 [1H, s, H-30 ], 7.21 [2H, d, J ¼ 9 Hz, H-2, H-6], 11.38 [1H, s, OH(chelated)]. 13C NMR (DMSO, d):18.23 (-CH3), 20.82 (-CH3), 40.35 (-N (CH3)2), 44.64 (C-4, pyrazoline), 63.20 (C-5,pyrazoline), 112.53 (C-3 and C-5) 115.51 (C-30 ), 120.28 (C-10 ), 124.39(C-50 ), 127.50 (C-2 and C-6), 130.43 (C-40 ), 135.14 (C-1), 136.23(C-60 ), 149.30(C-4), 149.95 (C-3, pyrazoline), 154.59 (C-20 ). FAB-MS (m/z): 343 [Mþ], 344 [Mþ1, base peak]. Elemental Analysis: Calculated for the molecular formula C19H22Cl N3O; Calculated: C ¼ 66.37, H ¼ 6.40, N ¼ 12.22, Found: C ¼ 66.97, H ¼ 6.53, N ¼ 11.99. 6.2.4. 3-(50 -Chloro-20 -hydroxy-40 ,6'-dimethylphenyl)-5-(3,4dimethoxyphenyl)-D2-pyrazoline (2d) As yellow crystals in 38% yield, m.p. 147e148  C, Rf ¼ 0.34, [petroleum ether (60e80 ): acetone; 8: 2]. IR ymax (KBr): 3333 (OH), 2997(NH), 1600 (C¼N), 1023 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.35 [3H, s, CH3-40 ], 2.47 [3H, s, CH3-60 ], 3.19 [1H, dd, J ¼ 15 Hz, 18 Hz, H-4 trans (pyrazoline)], 3.60 [1H, dd, J ¼ 9 Hz, 15 Hz, H-4, cis (pyrazoline)], 3.80 [6H, two closely packed singlets, OCH3-3, 4], 4.83 [1H, m, H-5 (pyrazoline)], 5.94 [1H, brs, NH], 6.80 [1H, s, H-30 ], 6.82 [1H, s, H-2], 6.91 [2H, m, H-5, H-6], 11.47 [1H, s, chelated OH]. 13C NMR (DMSO, d):18.06(-CH3), 20.69 (-CH3), 44.54 (C-4, pyrazoline), 55.42 (OCH3 at C-3), 55.56 (OCH3 at C-4), 63.04 (C-5, pyrazoline), 110.32 (C-2), 111.60 (C-5), 115.36 (C-30 ), 118.67 (C10 ), 120.17 (C-50 ), 124.23 (C-6), 133.06 (C-1), 135.63 (C-40 ), 136.10 (C60 ), 147.95 (C-4), 148.73 (C-30, 148.91 (C-3, pyrazoline), 154.46 (C20 ). FAB-MS (m/z): 360 [Mþ], 361 [Mþ1, base peak]. Elemental Analysis: Calculated for the molecular formula C19H21CI N2O3; Calculated: C ¼ 63.24, H ¼ 5.82, N ¼ 7.76, Found: C ¼ 62.98, H ¼ 5.54, N ¼ 7.99. 6.2.5. 3-(50 -Chloro-20 -hydroxy-40 ,60 -dimethylphenyl)-5-(3,4,5trimethoxyphenyl)-D2-pyrazoline (2e) As colourless crystals in 50% yield, m.p. 145e146  C, Rf ¼ 0.31, [petroleum ether (60e80 ): acetone; 8: 2]. IR ymax (KBr): 3241 (OH), 2940 (NH), 2835,1593 (C]N), 1504, 1124 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.32 [3H, s, CH3-40 ], 2.44 [3H, s, CH3-60 ], 3.14 [1H, dd, J ¼ 10 Hz, 16.2 Hz, H-4 trans (pyrazoline)], 3.55 [1H, dd, J ¼ 10 Hz, 16.2 Hz, H-4, cis (pyrazoline)], 3.81[3H, s, OCH3], 3.86[6H, s, 2xOCH3], 4.83 [1H, m, H-5 (pyrazoline)], 6.4[1H, brs, NH], 6.66 [2H, s, H-2, 6], 6.74 [1H, s, H-30 ], 10.76 [1H, s, OH (chelated)]. 13C NMR (DMSO, d):18.01(-CH3), 20.64 (-CH3), 44.48 (C-4, pyrazoline), 55.18 (OCH3 at C-3, C-5), 59.96 (OCH3 at C-4), 63.04 (C-5, pyrazoline), 103.79 (C-2 andC-6), 115.35 (C-30 ), 120.12 (C-10 ), 124.24 (C-50 ), 135.07 (C-1), 136.11 (C-40 ), 136.48 (C-4), 139.01 (C-60 ), 148.84 (C-3, pyrazoline), 152.18 (C-3 and C-5), 154.45 (C-20 ). FAB-MS (m/z): 390 [Mþ], 391 [Mþ1, base peak], 392 [Mþ2]. Elemental Analysis: Calculated for the molecular formula C20H23 CI N2O4; Calculated: C ¼ 61.46, H ¼ 5.89, N ¼ 7.17, Found: C ¼ 61.57, H ¼ 5.44, N ¼ 7.44.

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6.3. General procedure for the synthesis of 3, 5-diaryl-1-(psulfamylphenyl)-2-pyrazolines 3aeh 3, 5-diaryl-1-(p-sulfamylphenyl)-2-pyrazolines were prepared as per the previously reported method by us [8]. 6.4. General procedure for the synthesis of 3, 5-diaryl-2isoxazolines 4aee Appropriate chalcone or flavanone (0.001 mol) and hydroxylamine hydrochloride (0.002 mol) and KOH (0.002 mol) were dissolved in ethanol (25 ml) and refluxed for 6e8 h. After completion of the reaction monitored by TLC the reaction mixture was acidified with acetic acid. The resulting solid was washed with water and crystallized with alcohol to give pure compound. 6.4.1. 3-(40 -Chloro-20 -hydroxy-30 ,5'-dimethylphenyl)-5-phenyl-D2isoxazoline(4a) As light brown crystals in 62% yield, m.p. 185e186  C, Rf ¼ 0.61, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3306 (OH), 1595 (C]N), 1546, 1449, 1304, 1187 cm1. 1H NMR (300 MHz, DMSO, d): 2.31 [3H, s, CH3-40 ], 2.64 [3H, s, CH3-60 ], 2.84 [1H, dd, J ¼ 12.5 Hz, 16.9 Hz, H-4 trans (isoxazoline)], 5.12 [1H, m, H-5 (isoxazoline)], 6.91 [1H, s, H-30 ], 7.35e7.49 [5H, m, AreH], 11.49 [1H, s, OH-20 -Chelated]. 13C NMR (DMSO, d): 20.22 (-CH3), 20.83 (-CH3), 31.25 (C-4, isoxazoline), 76.28 (C-5, isoxazoline), 117.56 (C-30 ), 117.89 (C-10 ), 126.21 (C-3and C-5), 127.97 (C-1), 128.16 (C-50 ), 128.47 (C-2and C-6), 134.37(C-4), 137.46 (C-40 ), 139.88 (C-60 ), 149.65 (C-20 ), 155.12 (C-3, isoxazoline). FAB-MS (m/z): 301 [Mþ,], 302 [Mþ1, base peak], 303 [Mþ2], 284. Elemental Analysis: Calculated for the molecular formula C17H16CI NO2; Calculated; C ¼ 67.66, H ¼ 5.30, N ¼ 4.64, Found: C ¼ 67.61, H ¼ 5.24, N ¼ 4.69. 6.4.2. 3-(40 -Chloro-20 -hydroxy-30 ,50 -dimethylphenyl)-5-(4methoxyphenyl)-D2-isoxazoline (4b) As off white crystals in 45% yield, m.p. 161e162  C, Rf ¼ 0.76, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3303 (OH), 1605 (C]N), 1033 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.23 [3H, s, CH3-40 ], 2.48 [3H, s, CH3-60 ], 2.73 [1H, dd, J ¼ 12 Hz, 16 Hz, H-4 trans (isoxazoline)], 3.36 [1H, dd, J ¼ 4 Hz, 16 Hz, H-4, cis (isoxazoline)], 3.71 [3H, s, OCH3-4], 4.85 [1H, dd, J ¼ 4 Hz, 12 Hz, H-5 (isoxazoline)], 6.78 [1H, s, H-30 ], 6.82 [2H, d, J ¼ 8 Hz, H-3, H-5], 7.27 [2H, d, J ¼ 8 Hz, H-2, H-6], 10.67 [1H, s, OH-20 ]. 13C NMR (DMSO, d): 17.49 (-CH3), 20.61 (-CH3), 40.32 (C-4, isoxazoline), 55.11 (OCH3 at C-4), 76.03 (C-5, isoxazoline), 114.11 (C-3 and C-5), 115.39 (C-30 ), 117.69 (C-10 ), 119.82 (C-1), 123.55 (C-50 ), 128.08 (C-2 and C-6), 131.94 (C-40 ), 134.33 (C-60 ), 152.21 (C-4), 154.85 (C-20 ), 159.21 (C-3, isoxazoline). FAB-MS (m/z): 331 [Mþ], 332 [Mþ1, base peak]. Elemental Analysis: Calculated for the molecular formula C18H18CI NO3; Calculated; C ¼ 65.16, H ¼ 5.41, N ¼ 4.22, Found: C ¼ 64.99, H¼ 6.4.3. 3-(40 -Chloro-20 -hydroxy-30 , 50 -dimethylphenyl)-5-(4chlorophenyl)-D2-isoxazoline (4c) As off white crystals in 30% yield, m.p. 159e160  C, Rf ¼ 0.59, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3304 (OH), 1595 (C¼N), 1494, 1190 cm1. 1H NMR (300 MHz, DMSO, d): 2.31 [3H, s, CH3-40 ], 2.64 [3H, s, CH3-60 ], 2.84 [1H, dd, J ¼ 11.3 Hz, 16.8 Hz, H-4 trans (isoxazoline)], 3.16 [1H, d, J ¼ 5 Hz, H-4, cis (isoxazoline)], 5.16 [1H, dd, J ¼ 3.2 Hz, 11 Hz, H-5 (isoxazoline)], 6.91 [1H, s, H-30 ], 7.46 [2H, d, J ¼ 8.7 Hz, H-3, H-5], 7.50 [2H, d, J ¼ 8.7 Hz, H-2, H-6], 11.51 [1H, s, OH-20 -chelated]. 13C NMR (DMSO, d): 20.17 (-CH3), 20.80 (-CH3), 31.02 (C-4, isoxazoline), 75.58 (C-5,isoxazoline), 117.53 (C-30 ), 117.55 (C-1), 128.10 (C-3 and C-5), 128.46 (C-2 and C-6), 132.68 (C-10 ), 134.37 (C-50 ), 137.51 (C-4), 138.87 (C-40 ), 149.34 (C-60 ), 154.86 (C-20 ), 159.18 (C-3, isoxazoline). FAB-MS (m/z):

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336 [Mþ, base peak], 335 [M1], 337 [Mþ1], 338 [Mþ2]. Elemental Analysis: Calculated for the molecular formula C17H15CI2 NO2; Calculated; C ¼ 60.62, H ¼ 4. 45, N ¼ 4.16, Found: C ¼ 60.13, H ¼ 4. 75, N ¼ 4.27. 0

0

0

0

6.4.4. 3-(4 -Chloro-2 -hydroxy-3 , 5 -dimethylphenyl)-5-(2chlorophenyl)-D2-isoxazoline (4d) As off white crystals in 50% yield, m.p. 165e166  C, Rf ¼ 0.62, (toluene: ethyl acetate: formic acid, 5: 4: 1).IR ymax (KBr): 3273 (OH), 1594 (C¼N), 1550, 1477 cm1. 1H NMR (300 MHz, DMSO, d): 2.32 [3H, s, CH3-40 ], 2.65 [3H, s, CH3-60 ], 2.76 [1H, dd, J ¼ 11.8 Hz, 16.8 Hz, H-4 trans (isoxazoline)], 3.49 [1H, dd, J ¼ 3.1 Hz, 16.9 Hz, H4, cis (isoxazoline)], 5.33 [1H, dd, J ¼ 3 Hz, 11.7 Hz, H-5 (isoxazoline)], 6.92 [1H, s, H-30 ], 7.43 [2H, m, H-4, H-5], 7.51 [1H, d, J ¼ 7 Hz, H-3], 7.64 [1H, d, J ¼ 7 Hz, H-6], 11.55 [1H, s, OH-20 -chelated]. 13C NMR (DMSO, d): 20.17 (-CH3), 20.80 (-CH3), 29.82 (C-4, isoxazoline), 73.58 (C-5, isoxazoline), 117.4 3(C-30 ), 117.79 (C-10 ), 127.63 (C-5), 128.25 (C-50 ), 129.51 (C-3), 129.93 (C-4), 131.17 (C-6), 134.47 (C-2), 136.94 (C-1), 137.60 (C-40 ), 149.12 (C-60 ), 154.93 (C-20 ), 159.18 (C-3, isoxazoline). FAB-MS (m/z): 336 [Mþ, base peak], 335 [M1], 337 [Mþ1], 338 [Mþ2], 300. Elemental Analysis: Calculated for the molecular formula C17H15CI2 NO2; Calculated; C ¼ 60.62, H ¼ 4. 45, N ¼ 4.16, Found: C ¼ 60.43, H ¼ 4. 65, N ¼ 4.37. 6.4.5. 3-(40 -Chloro-20 -hydroxy-30 , 50 -dimethylphenyl)-5-(3, 4dimethoxyphenyl)-D2-isoxazoline (4e) As off white crystals in 46% yield, m.p. 123e124  C, Rf ¼ 0.82, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3274 (OH), 1595 (C¼N), 1024 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.30 [3H, s, CH3-40 ], 2.50 [3H, s, CH3-60 ], 2.88 [1H, dd, J ¼ 9 Hz, 15 Hz, H-4 trans (isoxazoline)], 3.75 [3H, s, OCH3-4], 3.76 [3H, s, OCH3-3], 5.03 [1H, dd, J ¼ 3 Hz, 15 Hz, H-5 (isoxazoline)], 6.89 [1H, s, H-30 ], 6.98 [2H, m, H-5, H-6], 7.08 [1H, s, H-2], 11.48 [1H, s, OH-20 chelated]. 13C NMR (DMSO, d): 17.47 (-CH3), 20.60 (-CH3), 40.32 (C4, isoxazoline), 55.46 (OCH3 at C-3, C-4), 76.03 (C-5, isoxazoline), 109.56 (C-5), 111.60 (C-2), 115.39 (C-30 ), 119.86 (C-10 ), 123.75 (C-6), 129.56 (C-5), 132.37 (C-1), 134.00 (C-40 ), 135.79 (C-60 ), 148.87 (C-4), 152.20 (C-3), 154.89 (C-20 ), 159.21(C-3, isoxazoline).FAB-MS (m/z): 361 [Mþ], 362 [Mþ1, base peak], 360 [M1]. Elemental Analysis: Calculated for the molecular formula C19H20CI NO4; Calculated; C ¼ 63.07, H ¼ 5.53, N ¼ 3.87, Found: C ¼ 63.47, H ¼ 5.14, N ¼ 3.99. 6.5. General procedure for the synthesis of 3-aroyl-4-aryl-2pyrazolines 6a-c Appropriate chalcone (50 mg) was treated with freshly prepared diazomethane gas dissolved in ether (20 ml) at 0  C and reaction mixture was kept overnight at room temperature to give crystalline compound. It was recrystallized with acetone to give pure compound. Diazomethane gas was prepared through reported method [55]. 6.5.1. 3-(40 -Chloro-20 -methoxy-30 , 5'-dimethylbenzoyl)-4-phenylD2-pyrazoline (6a) As off white crystals in 60% yield, m.p. 207e208  C, Rf ¼ 0.77, (toluene: ethyl acetate: formic acid, 5: 4: 1). IRymax(KBr):3314 (NH), 2943,1614 (CO),1517 (C¼N), 1435, 1320, 1098 cm1(OCH3). 1H NMR (300 MHz, DMSO, d): 1.97 [3H, s, CH3-40 ], 2.34 [3H, s, CH3-60 ], 3.67 [3H, s, OCH3], 4.08 [1H, m, H-5 cis (pyrazoline)], 4.46 [1H, m, H-4 (pyrazoline)], 6.92 [1H, s, H-30 ], 7.19e7.35 [5H, m, AreH], 8.9 [1H, s, NH]. 13C NMR (DMSO, d):17.04 (-CH3), 20.91 (-CH3), 45.93 (C-4, pyrazoline), 55.85(C-5, pyrazoline), 58.61(OCH3 at C-20 ), 111.75(C30 ), 125.36(C-10 ),126.78 (C-4), 127.28 (C-2 and C-6), 128.61(C-3 and C-5), 129.59 (C-50 ), 132.53 (C-1), 136.61 (C-60 ), 142.29 (C-40 ), 149.90 (C-3, pyrazoline),154.26 (C-20 ), 188.88 (C¼O). FAB-MS (m/z): 342

[Mþ], 343 [Mþ1, base peak], 173. Elemental Analysis: Calculated for the molecular formula C19H19 CI N2O2; Calculated; C ¼ 66.57, H ¼ 5.54, N ¼ 8.17, Found: C ¼ 66.97, H ¼ 5.59, N ¼ 8.44. 6.5.2. 3-(40 -Chloro-20 -methoxy-30 , 5'-dimethylbenzoyl)-4-(2methoxyphenyl) -D2-pyrazoline (6b) As light yellow crystals in 40% yield, m.p. 189e190  C, Rf ¼ 0.74, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3353 (NH), 2838, 1642 (CO), 1597 (C¼N), 1495, 1323, 1096 cm1 (OCH3). 1 H NMR (300 MHz, DMSO, d): 1.85 [3H, s, CH3-40 ], 2.34 [3H, s, CH360 ], 3.65 [3H, s, OCH3-20 ], 3.83 [3H, s, OCH3-2], 3.95 [1H, m, H-5 cis (pyrazoline)], 4.68 [1H, m, H-4 (pyrazoline)], 6.74e7.25 [6H, m, AreH], 9.6 [1H, s, NH]. 13C NMR (DMSO, d): 16.03 (-CH3), 20.07 (-CH3), 54.86 (C-4, pyrazoline), 56.26 (C-5, pyrazoline), 75.57 (OCH3 at C-20 ), 110.22 (C-3), 110.74 (C-10, 118.16 (C-30 ), 119.64 (C-5), 124.50 (C-4), 127.02 (C-10 ), 127.32 (C-50 ), 127.45 (C-6), 131.75 (C-60 ), 135.84 (C-40 ), 146.72 (C-3, pyrazoline), 153.41 (C-2), 155.37 C-20 ), 188.09 (C]O). FAB-MS (m/z): 372 [Mþ], 373 [Mþ1]. Elemental Analysis: Calculated for the molecular formula C20H21CI N2O3; Calculated; C ¼ 64.43, H ¼ 5.63, N ¼ 7.51, Found: C ¼ 64.97, H ¼ 5.64, N ¼ 7.44. 6.5.3. 3-(40 -Chloro-20 -methoxy-30 , 5'-dimethylbenzoyl)-4-(2nitrophenyl)-D2-pyrazoline (6c) As light yellow crystals in 42% yield, m.p. 219e220  C, Rf ¼ 0.80, (toluene: ethyl acetate: formic acid, 5: 4: 1). IR ymax (KBr): 3352(NH), 2837, 1600(CO), 1522 (C¼N), 1452, 1353,1033 cm1 (OCH3). 1H NMR (300 MHz, DMSO, d): 2.35 [3H, s, CH3-40 ], 2.50 [3H, s, CH3-60 ], 3.72 [3H, s, OCH3-20 ], 4.24 [1H, m, H-5 cis (pyrazoline)], 4.84 [1H, dd, J ¼ 5.7 Hz, 13 Hz, H-4 (pyrazoline)], 6.74 [1H, s, H-30 ], 7.30 [1H, d, J ¼ 7.5 Hz, H-6], 7.53 [1H, m, H-4], 7.59 [1H, m, H-5], 7.98 [1H, d, J ¼ 7.5 Hz, H-3], 9.18 [1H, s, NH]. 13C NMR (DMSO, d): 17.14 (-CH3), 20.90 (-CH3), 41.34 (C-4, pyrazoline), 55.94 (C-5, pyrazoline), 58.12 (OCH3 at C-20 ), 111.70 (C-30 ), 124.53 (C-3), 125.45 (C-1), 128.43 (C-10 ), 129.19 (C-4), 132.64 (C-5), 133.88 (C-50 ), 133.90 (C-6), 135.66 (C-60 ), 136.85 (C-40 ), 147.61 (C-3, pyrazoline), 148.43 (C-2), 154.29 (C-20 ), 188.64 (C¼O). FAB-MS (m/z): 387 [Mþ], 388 [Mþ1], 197. Elemental Analysis: Calculated for the molecular formula C19H18 CI N3O4; Calculated; C ¼ 58.84, H ¼ 4.64, N ¼ 10.83, Found: C ¼ 58.97, H ¼ 4.44, N ¼ 10.84. 6.6. Pharmacology 6.6.1. In vitro antimicrobial activity Antifungal and antibacterial activities were evaluated by using agar well diffusion method [55]. The sabouraud dextrose agar (Hi Media, Mumbai, India) and nutrient agar medium (peptone, beef extract, NaCl and agareagar) were used for antifungal and antibacterial screening respectively. The inoculums of the different fungi and bacteria were spread over agar medium. After the media had cooled, wells of bore size (6 mm) were made in solid medium by using a sterile metallic borer and 100 mL test drug (2.0 mg/ml in DMSO) was poured in each cavity of different plates. Standard drug, Ciprofloxacin (25 mg/disc) was used against bacteria S. aureus (ATCC25923) and E. coli (ATCC25922) and, fluconazole (20 mg/disc) against fungi C. Albicans (ATCC12031), A. Fumigatus (ATCC1022), A. Versicola (ATCC10072) and A. Flavus (ATCC 9643) were placed aseptically in a separate petri dish. The plates were kept at room temperature for one hour to diffuse the drug in surrounding medium and then incubated at 37  C for 24 h. The diameter of the zone of inhibition formed around the cavities and disc of standard drug after incubation was accurately measured in mm, which is presented in Table 1. 6.6.2. MIC of all active compounds MIC measurements of all active compounds were carried out

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using the two fold serial dilution technique [56]. Twofold serial dilutions of the selected compounds were prepared using proper nutrient broth. Compounds were prepared in the concentration range of 100, 50, 25, 12.5, 6.25 and 3.125 mg/mL. The microorganism suspensions (106 CFU/mL) were used to inoculate the test compounds in their suitable broth. The plates were incubated at 37  C for 24 and 48 h for bacteria and fungi, respectively. At the end of experiment the growth of microorganisms was observed by turbidity measurements. The lowest concentrations showing no growth was taken as the minimum inhibitory concentration (MIC). 6.6.3. Molecular docking study As the synthesised compounds showed promising antifungal activity in in-vitro studies, they were virtually docked into the binding pocket of Cytochrome P450 protein. The docking was carried out by using Schrodinger Software (V 9.4). The target protein (PDB No: 1EA1) was obtained from Protein Data Bank and further optimized and minimized to obtain a low energy and structural correct target protein. Target grid was generated using fluconazole as the reference ligand. The synthesised compounds were virtually prepared by Ligprep Module, wherein the lowest energy conformers were docked against the prepared target grid with extra precision mode and “write XP descriptor” information. This provides lowest energy ligands, docked into the target grid with best possible pose. The compounds are quantified using the glide score and scoring function were used to predict the binding affinity and ranking of the ligands [58]. Acknowledgements The authors thank Vice Chancellor, Dr. G. N. Qazi for providing necessary facilities to the Department of Chemistry. Thanks are also due to Central Drug Research Institute (CDRI), Lucknow for providing mass spectra. Appendix A. Supplementary data Supplementary data related to this chapter can be found at http://dx.doi.org/10.1016/j.ejmech.2015.03.031. References [1] A.M.S. Marwa, Ei-Sharief, S.Y. Abbas, A.M. Khairy, El-Bayouki, Eur. J. Med. Chem. 67 (2013) 263e268. [2] P.C. Shyma, B. Kaiiuraya, S.K. Peethamber, S. Telker, T. Arulmoli, Eur. J. Med. Chem. 68 (2013) 394e404. [3] T. Nasr, S. Bondock, S. Eid, Eur. J. Med. Chem. 84 (2014) 491e504. [4] P.K. Sharma, S. Kumar, P. Kumar, P. Kaushik, D. Kaushik, Y. Dhingra, K.R. Aneja, Eur. J. Med. Chem. 45 (2010) 2650e2655. [5] Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang, Y. Liu, J. Yao, Z. Miao, W. Zhang, C. Sheng, Eur. J. Med. Chem. 82 (2014) 490e497. [6] Y. Zou, S. Yu, R. Li, Q. Zhao, X. Li, M. Wu, T. Huang, X. Chai, H. Hu, Q. Wu, Eur. J. Med. Chem. 74 (2014) 366e374. _ ulları, T. Iça, [7] S. Mert, R. Kasımog F. Çolak, A. Altun, S. Ok, Eur. J. Med. Chem. 78 (2014) 86e96. [8] M. Johnson, B. Younglove, L. Lee, R. LeBlanc, H. Holt, P. Hills, H. Mackay, T. Brown, L.S. Mooberry, M. Lee, Bioorg. Med. Chem. Lett. 17 (2007) 5897e5901. [9] Z. Ratkovic, Z.D. Juranic, T. Stanojkovic, D. Manojlovic, R.D. Vukicevic, N. Radulovic, M.D. Joksovi c, Bioorg. Med. Chem. 38 (2010) 26e32. [10] R. Bashir, S. Ovais, S. Yaseen, H. Hamid, M.S. Alam, M. Samim, S. Singh, K. Javed, Bioorg. Med. Chem. Lett. 21 (2011) 4301e4305. [11] S. Bano, K. Javed, S. Ahmed, I.G. Rathish, S. Singh, M.S. Alam, Eur. J. Med. Chem. 46 (2011) 5763e5768. [12] I.G. Rathish, K. Javed, S. Ahmed, S. Bano, M.S. Alam, S.K.K. Pillai, B. Singh, Vivek, Eur. J. Med. Chem. 19 (2009) 255e258. [13] R.S. Joshi, P.G. Mandhane, S.D. Diwakar, S.K. Dabhade, C.H. Gill, Bioorg. Med. Chem. Lett. 20 (2010) 3721e3725. [14] M. Amir, H. Kumar, S.A. Khan, Bioorg. Med. Chem. Lett. 18 (2008) 918e922.

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