Accepted Manuscript Thiophene and benzodioxole appended thiazolyl-pyrazoline compounds: Microwave assisted synthesis, antimicrobial and molecular docking studies S. Shahavar Sulthana, S. Arul Antony, C. Balachandran, S. Syed Shafi PII: DOI: Reference:

S0960-894X(15)00495-3 http://dx.doi.org/10.1016/j.bmcl.2015.05.033 BMCL 22723

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Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

9 October 2014 29 April 2015 14 May 2015

Please cite this article as: Shahavar Sulthana, S., Arul Antony, S., Balachandran, C., Syed Shafi, S., Thiophene and benzodioxole appended thiazolyl-pyrazoline compounds: Microwave assisted synthesis, antimicrobial and molecular docking studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.05.033

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Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Thiophene and benzodioxole appended thiazolyl-pyrazoline compounds: Microwave assisted synthesis, antimicrobial and molecular docking studies S. Shahavar Sulthanaa , S. Arul Antonya, ∗ , C. Balachandranb and S. Syed Shafic, ∗ a

PG & Research Department of Chemistry, Presidency College, Chennai-600 005, India Division of Microbiology and Cancer Biology, Entomology Research Institute, Loyola College, Chennai-600034, India c Department of Chemistry, Thiruvalluvar University, Serkadu, Vellore-632 115, India b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

A novel series of thiophene and benzodioxole appended thiazolyl- pyrazoline derivatives have been designed, synthesized and evaluated against different bacteria and fungi. The antimicrobial activity of the synthesized compounds were screened using MIC method and were proved synthesized compounds 7o, 7r and 7t to show good antimicrobial activity against bacteria and fungi. In silico molecular docking studies revealed that all the synthesized molecules showed good binding energy towards the target receptor DNA topoisomerase IV, ranging from -10.42 to -11.66 kcal /mol.

Keywords: Thiazole Pyrazoline Antimicrobial Molecular docking

Thiazole moiety plays a vital role in diverse biological activities and is a key feature of some of the most interesting and important classes of compounds. Many thiazole scaffold containing compounds are pharmacologically active, functioning as potent pan-Src kinase inhibitor,1 thymidylate synthase inhibitor, 2 ABCB1 inhibitor,3 metastatic cancer cell migration and invasion inhibitor, 4 tubulin polymerization inhibitor,5 and SIRT1 activator 6. Some novel thiazolones7 and thiazole derivatives especially BILS 179 BS8 were reported to exhibit a high antiviral activity against hepatitis C virus (HCV) and HSV, respectively. Pyrazole derivatives play an important role on designing of new drugs, since they present an interesting pharmacological profile, especially, transforming growth factor β type 1 receptor (ALK5) inhibitors9 and some novel pyrazoles were reported to exhibit a high antiviral activity against hepatitis A virus.10 Interestingly, thiazolyl-pyrazoline derivatives were reported to exhibit variety of significant biological importance such as antimicrobial,11 antiviral,12 anti-inflammatory,13 antiamoebic activity, 14 anticancer,15 β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors,16 selective EP1 receptor antagonists for treatment of overactive bladder,17 EGFR TK inhibitors,18 super oxidase inhibitors and free radical scavengers.19 In addition, thiophene ring containing compounds exhibit different biological activities such as SGLT2 inhibitors,20 antitumor,21 antimicrobial,22 DHODH inhibitors,23 BACE1

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2009 Elsevier Ltd. All rights reserved.

inhibitors,24 antioxidant,25 c-Jun-N-terminal kinase inhibitor,26 5HT2A receptor antagonists,27 anti-inflammatory28 and vitronectin receptor antagonists.29 In addition, 1,3-dioxolane ring containing compounds exhibit different biological activities such as antifungal, plant growth regulator, 30 and VLA-4 antagonists. 31 Our goal in this work was to incorporate these four independently biologically active moieties into one molecule to generate compounds with better biological activities. Thiophene attached thiazolyl-pyrazoline compounds was identified as FabH inhibitors16 and bezodioxole attached thiazolyl-pyrazoline was identified as potential anticancer agents.15b In continuation of our work on synthesis of some pharmacologically important heterocycles, a novel series of thiophene and benzodiozole attached thiazolyl-pyrazolines was designed, synthesized (Scheme 1) and evaluated against different bacteria and fungi. Synthesis of novel thiazolyl-pyrazoline compounds is followed the general pathway outlined in the given Scheme 2.32 Firstly, the chalcone derivatives were obtained by direct condensation by the 2-acetyl thiophene 1 and corresponding benzodioxole aldehyde 2a-b using sodium hydroxide in ethanol. Secondly, cyclization of different chalcone derivatives with thiosemicarbazide 4 under basic condition (KOH) using ethanol as solvent leads to the formation of pyrazole derivatives 5a-b containing thiourea skeleton. Finally, thiazolyl-pyrazoline

∗ Corresponding author. Tel.: +91-9444240597; e-mail: [email protected] ∗ Corresponding author. Tel.: +91-9894214051; e-mail: [email protected]

derivatives 7a-t were obtained by microwave irradiation of compound 5a-b with substituted 2-bromoacetophenone 6aj.(Table 1)

Table 1: Syntheis of novel thiazolyl-pyrazoline (7a-t) derivatives S.No

Compound

R1

R2

R3

R4

Yielda

1 2

7a 7b

H H

H CH3

H H

H H

90 92

3 4

7c 7d

H H

NO2 CN

H H

H H

85 87

5 6

7e 7f

H H

H F

CN H

H H

84 86

7

7g

H

H

H

F

88

8 9

7h 7i

H H

Cl Cl

H F

H H

89 82

10 11

7j 7k

H Br

Cl H

H H

F H

84 88

12

7l

Br

CH3

H

H

90

13 14

7m 7n

Br Br

NO2 CN

H H

H H

81 85

15 16

7o 7p

Br Br

H F

CN H

H H

88 84

17

7q

Br

H

H

F

83

18

7r

Br

Cl

H

H

87

19

7s

Br

Cl

F

H

81

20

7t

Br

Cl

H

F

84

a

The structure of novel thiazolyl-pyrazoline derivatives was elucidated with the help of 1H NMR, 13 C NMR and Mass data as illustrated for 7a. In the 1H NMR spectrum of compound 7a, a dd peak at δ: 5.60 ppm with the J value 11.8 and 6.5 Hz for a proton corresponds to C-H proton of pyrazoline ring. The two dd peaks at δ: 3.29 and 3.87 with J value 17.3 : 6.5 Hz and 17.3 :11.9 Hz corresponds to two -CH2 protons of pyrazoline ring respectively. And also two doublets at 5.92 and 5.93 ppm with the J value 1.4 Hz corresponds to -CH2 protons of benzodioxole ring. The peaks in the range of δ: 6.77–7.71 ppm show the 12 aromatic protons. The 13C and DEPT135 NMR of compound 7a revealed that, the peak at δ: 64.5 ppm corresponds to the C-H carbon of pyrazoline ring. The peaks at δ: 44.2 and 101.1 ppm confirmed the presence of two -CH2 carbons in pyrazoline and benzodioxole ring respectively. A distinguishing peak observed at m/z: 432 in the ESI mass spectrum corresponds to [M+H]+ ion of the product 7a. In the course of identifying various novel antimicrobial agents, we are particularly interested in the present work with novel thiazolyl-pyrazoline derivatives. In the present study, the antimicrobial activities of 20 newly synthesized compounds were screened against eight bacteria and two fungi using MIC method (Table 2).33, 34 The results revealed that most of the synthesized compounds exhibited antimicrobial activities against bacteria; Salmonella typhimurium, Klebsiella pneumoniae, Proteus vulgaris, Shigella flexneri, Micrococcus luteus, Enterobacter aerogenes, Staphylococcus aureus and Staphylococcus aureus (MRSA- methicillin resistant); fungi: Candida albicans and Malassesia pachydermatis.

Isolated yield in final step

Scheme 1: Synthetic scheme of novel thiazolyl-pyrazoline derivatives

Significant MIC values were observed against Gram positive and Gram negative bacteria and fungi. Compound 7e, 7o, 7r and 7t showed very good antimicrobial activity when compare with other tested compounds. Among all the compounds tested compound 7o showed very high activity 31.25 µg/mL against tested microbes. Subsequently preliminary SAR studies were performed to deduce how the structure variation and modification could affect the antimicrobial activity. Generally, substitution of functional groups such as F, Cl, Br, CH3 and CN at R1, R2, R3 and R4 positions increases the activity. When compare the MIC values of all the tested compounds Br substitution at R1 position increases the antimicrobial activity. For example, compound 7k shows

MIC of 62.5 µg/mL against P. vulgaris and E. aerogenes whereas compounds 7a shows MIC of 500 and 250 µg/mL respectively. Also, CN substitution at R3 position drastically influence the activity and results. Especially, when compare compounds 7a and 7e, CN substitution at R3 position increases the activity and results in MIC of 31.25 µg/mL against M. luteus and S. aureus. SAR result revealed that Br and CN substitution at R1 and R3 position drastically increase the activity. Among all the synthesised compounds tested for antimicrobial activity, compound 7o shows better antibacterial profile with MIC value of 31.25 µg/mL against S. typhimurium and P. vulgaris, which is comparable with standard drug.

All the synthesised compounds were subjected to molecular docking studies using the AutoDock Tools (ADT)35 version 1.5.6 and AutoDock version 4.2.5.1 docking program to investigate the

potential binding mode of inhibitors. DNA topoisomerase IV receptor is required for maintenance of proper DNA topology during transcription and replication in bacteria. 36

Scheme 2: Mechanism for the formation of novel thiazolyl-pyrazoline derivatives

Table 2: Minimum inhibitory concentration (µg/mL) of synthesized compounds against tested microbes Gram negative bacteria Gram positive bacteria Fungi S. K. P. S. M. E. S. (MRSA) S. C. typhimurium pneumoniae vulgaris flexneri luteus aerogenes aureus aureus albicans 7a 250 500 500 500 250 250 500 500 250 7b 125 125 500 250 500 250 250 125 7c 500 500 500 250 500 500 250 7d 125 250 125 250 125 250 125 250 125 7e 62.5 62.5 250 125 31.25 125 31.25 62.5 125 7f 500 250 500 250 250 125 500 250 125 7g 250 500 500 125 500 250 250 500 7h 250 62.5 125 250 250 250 125 250 500 7i 500 250 500 125 62.5 125 500 250 125 7j 125 125 500 125 250 250 250 125 250 7k 125 250 62.5 250 125 62.5 125 250 125 7l 62.5 62.5 125 125 62.5 125 62.5 250 125 7m 62.5 250 125 250 125 250 250 62.5 125 7n 125 125 62.5 125 62.5 125 62.5 125 125 7o 31.25 62.5 31.25 62.5 31.25 62.5 31.25 62.5 62.5 7p 250 500 250 125 250 250 250 7q 125 250 500 250 125 250 250 250 125 7r 62.5 31.25 250 125 31.25 125 125 62.5 125 7s 125 125 125 125 125 250 125 250 500 7t 31.25 62.5 62.5 62.5 125 125 125 62.5 62.5 C 30 6.25 32a 6.25 6.25 25 6.25 6.25 25 -; no activity; C-Streptomycin, Gentamicina (standard antibacterial agent); C-Ketoconazole, Fluconazoleb (standard antifungal agent) Compound

M. pachydermatis 500 500 500 500 250 250 500 500 500 250 125 250 500 125 125 500 500 125 500 125 24b

Docking of different ligands to protein was performed using AutoDock, following the same protocol used in as that of validation study.37 Docked ligand conformations were analysed in terms of energy, hydrogen bonding, and hydrophobic interaction between ligand and receptor protein DNA topoisomerase IV. Detailed analyses of the ligand-receptor interactions were carried out, and final coordinates of the ligand and receptor were saved. For display of the receptor with the ligand binding site, PyMol software was used. From the docking scores, the free energy of binding (FEB) of all compounds were calculated (Table 3).

molecular docking studies revealed all the synthesized molecules showed good binding energy toward the target receptor DNA topoisomerase IV, ranging from -10.42 to -11.66 kcal /mol. Generally, substitution of functional groups such as F, Cl, Br, CH3 and CN at R1, R2, R3 and R4 positions increases the free energy of binding. Interestingly, Br substituted compounds show higher binding energy values compare with unsubstituted compounds, for example, Br substitution at R1 position increases binding energy from 10.46 to 10.88 kcal /mol. Also, CN substitution at R3 position drastically change the binding energy from 10.46 to 11.26 kcal /mol. This result revealed that Br and CN substitution at R1 and R3 position increase the activity. Hence, among all the compounds docked, compound 7o, which fits exactly in the active site and forms one hydrogen bonds (Bond length=2.9Å) with ASP78 amino acid of the 4EMV protein, exhibits very high binding energy value -11.66 kcal /mol. (Figure 1). Table 3: Free energy of binding (FEB) Binding energy (kcal/mol)a

Compound

(a) Method validation using crystallised and docked ligands of 4EMV

(b) Interaction of most active compound 7o with active site residues of 4EMV

(c) Docking pose of most active compound 7o with 4EMV Figure 1: Molecular docking studies with 4EMV receptor

The docking of synthesised compounds with receptor DNA topoisomerase IV exhibited well established bonds with one or more amino acids in the receptor active pocket. The active pocket consisted of 16 amino acid residues as ASN51, ALA52, ASP54, GLU55, ASP78, GLY80, ARG81, GLY82, MET83, PRO84, THR94, ILE98, PHE99, HIS120, SER124, and THR172. In silico

7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s 7t a Calculated by Autodock

DNA Topoisomerase IV 10.46 10.69 10.42 10.92 11.26 10.53 10.50 10.84 10.84 10.87 10.88 11.24 10.92 11.26 11.66 10.67 10.95 11.31 11.23 11.30

In summary, we have reported the synthesis of novel thiophene and benzodioxole appended thiazolyl-pyrazoline compounds. These novel compounds were evaluated for their antimicrobial activity against eight bacteria and two fungi. The results indicated that the synthesised thiazolyl-pyrazoline compounds showed significant antimicrobial activities. In silico molecular docking simulation was performed to position all the synthesised compounds into the DNA topoisomerase IV receptor structure active site to determine the probable binding model. This studies revealed that all the synthesized molecules showed good binding energy towards the target receptor DNA topoisomerase IV, ranging from -10.42 to -11.66 kcal /mol. Among all the synthesised compounds tested for antimicrobial activity, compound 7o showed better activity profile against tested microbes. These thiophene and benzodioxole appended thiazolyl-pyrazoline compounds can be lead compounds to get better antimicrobial activity. References and notes 1.

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28. Isloor, A. M.; Kalluraya, B.; Sridhar Pai, K. Eur. J. Med. Chem. 2010, 45, 825. 29. Bubenik, M.; Meerovitch, K.; Bergeron, F.; Attardo, G.; Chan, L. Bioorg. Med. Chem. Lett. 2003, 13, 503. 30. Xu, L. Z.; Zhang, S. S.; Niu, S. Y.; Qin, Y. Q.; Li, X. M.; Jiao, K. Molecules 2004, 9, 913. 31. Rehman, A.; Soni, A.; Naik, K.; Nair, S.; Palle, V. P.; Dastidar, S.; Ray, A.; Alam, M. S.; Salman, M.; Cliffe, I. A.; Sattigeri, V. Bioorg. Med. Chem. Lett. 2010, 20, 5514. 32. General procedure for the preparation of compounds (3a-b): A mixture of 2-acetylthiophene (1.0 mmol) (1), piperonal (1.0 mmol) (2a-b), and sodium hydroxide (2.5 mmol) in abs.ethanol (4 mL) was stirred at room temperature. Reaction was monitored by TLC, with eluent 8:2 pet ether : ethyl acetate. After the reaction completion, evidenced by TLC the resulting solid obtained was filtered through Buckner funnel and washed with water. The solid was dried and recrystallized from abs.ethanol afforded pure chalcone derivative (3a-b). 3-(Benzo[d][1,3]dioxol-5-yl)-1(thiophen-2-yl)prop-2-en-1-one (3a): Pale yellow solid; Yield 93%; 1 H NMR (400 MHz, CDCl 3) δ 7.85 (dd, J = 3.8, 0.9 Hz, 1H), 7.77 (d, J = 15.5 Hz, 1H), 7.67 (dd, J = 4.9, 1.0 Hz, 1H), 7.25 (d, J = 15.5 Hz, 1H), 7.20 – 7.15 (m, 2H), 7.12 (dd, J = 8.1, 1.5 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.02 (s, 2H); 13 C NMR (100 MHz, CDCl3 ) δ 181.98, 149.99, 148.42, 145.69, 143.94, 133.67, 131.59, 129.18, 128.23, 125.34, 119.66, 108.71, 106.69, 101.67. ESI-MS m/z = 259 [M+H]+. Anal. Calcd for C14H10 O3S: C, 65.10; H, 3.90; Found: C, 64.97; H, 3.93. 3-(6bromobenzo[d][1,3]dioxol-5-yl)-1-(thiophen-2-yl)prop-2-en-11 one (3b): Yellow solid; Yield 95%; H NMR (400 MHz, CDCl 3) δ 8.13 (d, J = 15.5 Hz, 1H), 7.85 (dd, J = 3.8, 1.0 Hz, 1H), 7.68 (dd, J = 4.9, 1.0 Hz, 1H), 7.25 – 7.15 (m, 3H), 7.08 (s, 1H), 6.05 (s, 2H); 13C NMR (100 MHz, CDCl3 ) δ 181.84, 150.30, 147.94, 145.35, 142.34, 133.95, 131.88, 128.28, 128.05, 122.60, 118.83, 113.36, 106.50, 102.35. ESI-MS m/z = 337 [M+H]+. Anal. Calcd for C14H9BrO3S: C, 49.87; H, 2.69; Found: C, 49.92; H, 2.71. General procedure for the preparation of compounds (5a-b): To a suspension of chalcone derivative (1.0 mmol) (3a-b) and potassium hydroxide (2.5 mmol) in abs.ethanol (4 mL), thiosemicarbazide (1.2 mmol) (4) was added. The mixture was stirred and refluxed. The reaction was monitored by TLC. After the completion of reaction, evidenced by TLC, the resulting solid was poured in crushed ice and the solid mass which separated out was filtered, dried and recrystallized from ethanol afforded pure product (5a-b). 5-(benzo[d][1,3]dioxol-5-yl)-3-(thiophen-2-yl)4,5-dihydro-1H-pyrazole-1-carbothioamide (5a): Colourless solid; Yield 87%; 1H NMR (400 MHz, CDCl3) δ 7.48 (dd, J = 5.1, 1.1 Hz, 1H), 7.26 – 7.23 (m, 1H), 7.08 (dd, J = 5.1, 3.7 Hz, 1H), 7.00 (s, 1H), 6.79 – 6.65 (m, 3H), 6.08 (s, 1H), 5.99 – 5.87 (m, 3H), 3.82 (dd, J = 17.5, 11.3 Hz, 1H), 3.16 (dd, J = 17.5, 3.5 Hz, 1H); 13C NMR (100 MHz, CDCl3 ) δ 176.41, 151.41, 148.13, 147.09, 135.56, 134.05, 130.06, 129.83, 127.91, 119.05, 108.58, 105.92, 101.17, 63.37, 43.81. ESI-MS m/z = 332 [M+H]+. Anal. Calcd for C15H13 N3 O2S2 : C, 54.36; H, 3.95; N, 12.68; Found: C, 54.38; H, 3.91; N, 12.62. 5-(6-bromobenzo[d][1,3]dioxol-5-yl)-3(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (5b): Pale yellow solid; Yield 90%; 1H NMR (400 MHz, DMSO) δ 8.15 (s, 1H), 7.78 (d, J = 4.8 Hz, 1H), 7.68 (s, 1H), 7.50 (d, J = 3.1 Hz, 1H), 7.24 (s, 1H), 7.19 – 7.09 (m, 1H), 6.33 (s, 1H), 6.16 – 5.94 (m, 3H), 4.00 (dd, J = 17.8, 11.4 Hz, 1H), 3.03 (dd, J = 17.8, 3.2 Hz, 1H); 13C NMR (100 MHz, DMSO) δ 176.13, 151.64, 147.94, 147.78, 135.12, 134.05, 131.79, 130.97, 128.62, 113.15, 111.33, 105.80, 102.50, 63.49, 42.47. ESI-MS m/z = 410 [M+H]+. Anal. Calcd for C15 H12BrN3O2S2: C, 43.91; H, 2.95; N, 10.24; Found: C, 44.01; H, 2.91; N, 10.18;. General procedure for the preparation of compounds (7a-t): To a suspension of compound (1.0 mmol) (5a-b) in ethanol (3 mL) and phenacyl bromide (1.0 mmol) (6a-j) were placed in a pyrex vial and irradiated with 200 W. Reaction was monitored by TLC, after completion of reaction, the reaction mixture was cooled to room temperature and concentrated. The solid obtained was washed with little amount of hexane, filtered and dried under reduced pressure to give the desired thiazolyl-pyrazoline compound (7a-t). 2-(5(benzo[d][1,3]dioxol-5-yl)-3-(thiophen-2-yl)-4,5-dihydro-1Hpyrazol-1-yl)-4-phenylthiazole (7a): Yellow solid; Yield 90%; 1H NMR (400 MHz, CDCl3 ) δ 7.74 – 7.67 (m, 2H), 7.41 (dd, J = 5.1, 1.0 Hz, 1H), 7.37 – 7.29 (m, 2H), 7.25 – 7.21 (m, 1H), 7.20 (dd, J = 3.6, 1.0 Hz, 1H), 7.06 (dd, J = 5.0, 3.7 Hz, 1H), 6.94 (dd, J = 8.0, 1.7 Hz, 1H), 6.87 (d, J = 1.7 Hz, 1H), 6.82 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 5.93 (d, J = 1.4 Hz, 1H), 5.92 (d, J = 1.4 Hz, 1H),

5.60 (dd, J = 11.9, 6.5 Hz, 1H), 3.87 (dd, J = 17.3, 11.9 Hz, 1H), 3.29 (dd, J = 17.3, 6.5 Hz, 1H); 13 C NMR (100 MHz, CDCl 3) δ 164.71, 151.47, 148.04, 147.31, 147.17, 135.52, 135.11, 134.96, 128.43, 128.13, 127.71, 127.56, 127.46, 125.88, 120.37, 108.25, 106.84, 103.55, 101.12, 64.59, 44.22; ESI-MS m/z = 432 [M+H]+. Anal. Calcd for C23 H17N3O2S2: C, 64.02; H, 3.97; N, 9.74; Found: C, 63.94; H, 4.01; N, 9.78. 2-(5-(6-bromobenzo[d][1,3]dioxol-5yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4phenylthiazole (7k): Pale yellow solid; Yield 93%; 1 H NMR (400 MHz, CDCl3 ) δ 7.77 – 7.68 (m, 2H), 7.40 (dd, J = 5.0, 1.0 Hz, 1H), 7.35 – 7.28 (m, 2H), 7.25 – 7.20 (m, 1H), 7.18 (dd, J = 3.6, 1.0 Hz, 1H), 7.10 – 7.01 (m, 2H), 6.86 (s, 1H), 6.76 (s, 1H), 6.01 – 5.93 (m, 1H), 5.92 (d, J = 1.3 Hz, 1H), 5.90 (d, J = 1.3 Hz, 1H), 3.96 (dd, J = 17.4, 12.0 Hz, 1H), 3.12 (dd, J = 17.4, 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl3) δ 164.40, 151.56, 147.99, 147.82, 147.47, 134.94, 134.83, 133.97, 128.43, 128.24, 127.91, 127.57, 127.49, 125.92, 112.73, 112.42, 107.01, 103.73, 101.85, 64.22, 43.37; ESI-MS m/z = 510 [M+H]+. Anal. Calcd for C23 H16BrN3O2S2: C, 54.12; H, 3.16; N, 8.23; Found: C, 54.23; H, 3.19; N, 8.20. 33. Balachandran, C.; Duraipandiyan, V.; Al-Dhabi, N.A.; Balakrishna, K.; Kalia, N.P.; Rajput, V.S.; Khan, I.A.; Ignacimuthu, S. Indian .J. Microbiol. 2012, 52, 676. 34. Minimum inhibitory concentration (MIC): The following bacteria and fungi were used for the experiment. Bacteria: Salmonella typhimurium MTCC 1251, Klebsiella pneumoniae MTCC 109, Proteus vulgaris MTCC 1771, Shigella flexneri MTCC 1457, Micrococcus luteus MTCC 106, Enterobacter aerogenes MTCC 111, Staphylococcus aureus MTCC 96 and Staphylococcus aureus (MRSA- methicillin resistant); The reference cultures were obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India-160 036. Fungi: Candida albicans MTCC 227 and Malassesia pachydermatis; All the cultures were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India. Minimum inhibitory concentration studies of the synthesised compounds were performed according to the standard reference methods for bacteria, for filamentous fungi and yeasts.33 The required concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.62 and 7.81µg/mL) of the compound were dissolved in DMSO (2%), and diluted to give serial two-fold dilutions that were added to each medium in 96 well plates. An inoculum of 100 from each well was inoculated. The antifungal agents Ketoconazole for fungi and Streptomycin for bacteria were included in the assays as positive controls. For fungi, the plates were incubated for 48 to 72 hours at 28°C and for bacteria the plates were incubated for 24 h at 37 °C. The MIC for fungi was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 µl of tested broth was placed on the sterile MHA plates for bacteria and incubated at respective temperature. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate.

35. http://autodock.scripps.edu/resources/references 36. Manchester, J. I.; Dussault, D. D.; Rose, J. A.; Boriack-Sjodin, P. A.; Uria-Nickelsen, M.; Ioannidis, G.; Bist, S.; Fleming, P.; Hull, K. G. Bioorg. Med. Chem. Lett. 2012, 22, 5150. 37. Molecular docking studies: The DNA topoisomerase IV structure was obtained from the Protein Data Bank (PDB ID: 4EMV). The co-crystallized ligand in the DNA topoisomerase IV structure was removed. Then the polar hydrogen atoms were added, lower occupancy residue structures were deleted, and any incomplete side chains were replaced using the ADT. Further ADT was used to remove crystal water, Gasteiger charges were added to each atom, and merged the non-polar hydrogen atoms to the protein structure. The distance between donor and acceptor atoms that form a hydrogen bond was defined as 1.9 Å with a tolerance of 0.5 Å, and the acceptor– hydrogen–donor angle was not less than 120˚. The structures were then saved in PDBQT file format, for further studies in ADT. Ligand 2D structures were drawn using ChemDraw Ultra 7.0 (ChemOffice 2002). Chem3D Ultra 7.0 was used to convert 2D structure into 3D and the energy minimized using semi-empirical AM1 method. Minimize energy to minimum RMS gradient of 0.100 was set in each iteration. All structures were saved as .pdb file format for input to ADT. All the ligand structures were then saved in PDBQT file format, to carry out docking in ADT. A grid box with dimension of 40 × 40 × 40 Å3 with 0.375 Å spacing and centered on 14.860, 29.555, 6.941 was created around the binding site of ligand on DNA topoisomerase IV using ADT. The center of the box was set at ligand center and grid energy calculations were carried out. For the AutoDock docking calculation, default parameters were used and 20 docked conformations were generated for each compound. The energy calculations were done using genetic algorithms. All dockings were taken into 2.5 million energy evaluations were performed for each of the test molecules. In order to verify the reproducibility of the docking calculations, the bound ligand was extracted from the complex and submitted for one-ligand run calculation. This reproduced top scoring 20 conformations falling within root-mean-square deviation (rmsd) value of 0.47 Å to 1.92 Å from bound X-ray conformation for DNA topoisomerase IV, suggesting this method is valid enough to be used for docking studies of other compounds.

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Thiophene and benzodioxole appended thiazolylpyrazoline compounds: Microwave assisted synthesis, antimicrobial and molecular docking studies

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S. Shahavar Sulthanaa , S. Arul Antonya, *, C. Balachandranb , S. Syed Shafic, ∗

Thiophene and benzodioxole appended thiazolyl-pyrazoline compounds: Microwave assisted synthesis, antimicrobial and molecular docking studies.

A novel series of thiophene and benzodioxole appended thiazolyl-pyrazoline derivatives have been designed, synthesized and evaluated against different...
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