Novel synthetic organic compounds inspired from antifeedant marine alkaloids as potent bacterial biofilm inhibitors

Bioorganic Chemistry 61 (2015) 66–73

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Novel synthetic organic compounds inspired from antifeedant marine alkaloids as potent bacterial biofilm inhibitors Rajesh A. Rane a,1, Rajshekhar Karpoormath a,⇑,1, Shital S. Naphade b,1, Pavankumar Bangalore c,1, Mahamadhanif Shaikh a, Girish Hampannavar a a b c

Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa Government College of Amravati, Maharashtra 444601, India SPP School of Pharmacy and Technology Management, Vile Parle, Mumbai 400056, India

a r t i c l e

i n f o

Article history: Received 23 January 2015 Available online 12 June 2015 Keywords: Marine alkaloids Bromopyrrole 1,3,4-Oxadiazoles Biofilm inhibitors

a b s t r a c t In this paper, we have reported seventeen novel synthetic organic compounds derived from marine bromopyrrole alkaloids, exhibiting potential inhibition of biofilm produced by Gram-positive bacteria. Compound 5f with minimum biofilm inhibitory concentration (MBIC) of 0.39, 0.78 and 3.125 lg/mL against MSSA, MRSA and SE respectively, emerged as promising anti-biofilm lead compounds. In addition, compounds 5b, 5c, 5d, 5e, 5f, 5h, 5i and 5j revealed equal potency as that of the standard drug Vancomycin (MBIC = 3.125 lg/mL) against Streptococcus epidermidis. Notably, most of the synthesized compounds displayed better potency than Vancomycin indicating their potential as inhibitors of bacterial biofilm. The cell viability assay for the most active hybrid confirms its anti-virulence properties which need to be further researched. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction A global trend of indiscriminate and continuous use of antibiotics for the treatment of bacterial infections has led to the development of bacterial resistance for most of the existing antimicrobials [1]. This resistance has led researchers worldwide to search for new molecules active against bacterial pathogens. A promising approach for the development of a new generation of antimicrobial drugs has started from the studies of host–pathogen interactions, prompting a shift of drug targets from bacterial survival to virulence control. For example, receptors involved in bacterial adherence to surfaces as well as signal systems controlling bacterial group behaviour of population, organised in biofilms (such as quorum sensing (QS) molecules) stands as potential non-conventional targets for microbial control [2]. Antimicrobial drugs directed against such non-conventional targets do not risk bacterial survival avoiding the development of resistance [3]. Various organic compounds interfering with bacterial QS were identified as potent QS inhibitors [4–9]. On the other hand, conventional antibiotics eradicates the symptoms of an infection by eliminating ⇑ Corresponding author. 1

E-mail address: [email protected] (R. Karpoormath). These authors contributed equally for this paper.

http://dx.doi.org/10.1016/j.bioorg.2015.06.001 0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

planktonic bacteria arising from adhered populations, but are ineffective towards those bacteria hidden in biofilms [10]. Bromopyrrole alkaloids are a family of marine alkaloids represents a fascinating example of large variety of secondary metabolites formed by marine sponges as a defense towards bacterial biofouling leading to their protection against aquatic feedant. These compounds are known to be involved in defense mechanism of sponges against fishes and other aquatic feedants. They do so by their inhabitant antibiotic as well as antibiofilm action. For instance, Oroidin has been reported to inhibit the growth of P. aeruginosa at 190 lM. Petabromopseudilin derivative (II) inhibited Gram-positive S. aureus growth at 0.005 lg/mL by antibiotic action. Dihydronsventrin and Sventin were reported as biofilm inhibitors at 51 and 74 lM against P. aeruginosa. In addition, Ageliferin, Dibromophakelline, Mauritamide B and Agelanesin C all are reported as QS inhibitors at 11.29, 17.99, 36.76 and 36.30 lM respectively (Fig. 1) [11–12]. These metabolites are identified by their signature 4,5-dibromopyrrole motif with Oroidin as there structural prototype reported as antibiofilm lead compound. Structure–activity relationship on synthetic library inspired form these class of alkaloids indicated that N-methylation of the pyrrole core led to an increased antibiofilm activity towards medically relevant Gram-negative bacterium P. aeruginosa as well as Gram-positive bacterium S. aureus [13,14].

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73

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Fig. 1. Some reported bromopyrrole alkaloids and 1,3,4-oxadiazole with biofilm inhibiting, antibacterial, quorum sensing (QS) inhibiting activity.

Hence continuing with our quest focused on development of novel antibiofilm molecules, we have described in this short communication, the identification of seventeen novel compounds capable of inhibiting biofilm formation for a panel of Gram-positive

Br Br

N CH3

N N Het/Ar

O

R

and Gram-negative pathogenic bacterium. These compounds, consisting of N-methyl-4,5-dibromopyrrole motif integrated with 1,3,4-oxadiazole were chosen for the current study based on their resemblance to known marine antibiofilm agents, QS inhibitors as well as reported antibiotics (Figs. 1 and 2) [13–17]. Based on the potent activity of synthesized adducts towards bacterial biofilm formation, a structure–activity analysis was developed, that can direct future studies involved in the search of novel compounds for biofilm control. Further cell viability data against human VERO cells much above the bacterial MBIC concentrations warrants their suitability for the development of new antibiofilm drugs for therapeutic uses.

Br N Br

N O CH3

2. Materials and methods

N R

R = SH or S-alkyl or aryl or heteroaromatic ring Fig. 2. Design of novel 2-(1-methyl-4,5-dibromopyrrol-2-yl)-5-substituted-1,3,4 oxadiazoles.

2.1. Synthesis and purification of the compounds In the present work, proposed compounds were synthesized utilizing the reaction sequence as shown in Scheme 1. Trichloroacetylation of 1-methylpyrrole using equimolar quantity of trichloroacetyl chloride gave an excellent yield of 2-trichloroace

68

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73

Br N CH3

CCl3COCl anhydrous ether K2CO3

N CH3

1

COCCl3

Br2/Chloroform

N CH3

Br 3

2

NH2NH2 . H2O

RT, 1 h

Br

Br Br

N H3C

O N N

R-COOH,

R

Br POCl3, reflux

N H3C

CONHNH2

4

5 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m -

COCCl3

R = 2-hydroxyphenylR = benzylR = 4-amino phenylR = 4-chloro phenylR = 4-methoxy phenylR = 2,4- dichlorophenylR = styrylR = 2-chloro-5-fluorophenylR = 4-nitrophenylR = 4,5-dibromo-1H-pyrrol-2-ylR = 2-methoxy-4-vinylphenol R = pyridin-4-ylR = phenyl

CS2/KOH Ethanol,reflux 12 hr

Br Br

O

N H3C

SH

N N 6 Alkyl/Aryl halide,NaOH Ethanol,stir for 10 hr

Br Br

N H3C

O

SR

N N

7 7a - R = methyl7b - R = ethyl7c - R = phenylScheme 1. Synthesis of 2-(1-methyl-4,5-dibromopyrrol-2-yl)-5-substituted-1,3,4-oxadiazoles.

tyl-1-methylpyrrole 2 [18]. Bromination of 2 using two equivalent of bromine in chloroform gave 4,5-dibromo-2-trichloroacetyl-1methylpyrrole 3 in excellent yield [18]. 4,5-Dibromo-2-trichloroacetyl-1-methylpyrrole 3 on stirring with excess of hydrazine hydrate at room temperature gave its acid hydrazide derivative 4 in good yield. Compound 4 on condensation with equimolar quantities different aromatic and hetero-aromatic acids in presence of excess of phosphorus oxychloride at reflux condition gave 2-(4,5-dibromo-1-methylpyrrol-2-yl)-5-aryl-1,3,4-oxadiazoles 5. 5-(4,5-Dibromo-1-methyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 was synthesized by reaction of 4 with carbon disulfide and alcoholic potassium hydroxide at reflux condition. Compound 6 was S-alkylated by reaction with different aryl/alkyl halides in alcoholic sodium hydroxide to give S-alkylated derivatives 7 (Scheme 1). 2.2. Chemistry All reactions were carried out in anhydrous conditions (Molecular sieves, 4 Å 1/1600 pellets) under inert nitrogen atmosphere. Freshly dried and distilled solvents (Ethanol and CAN) over CaCl2 were used. Silica gel of 60–120 mesh and 200–400 mesh was obtained from for column and flash chromatography. Some of the starting materials were obtained from commercially available suppliers and were used without further purification. Analytical Thin layer Chromatography (TLC) was carried out on precoated plates of SiO2 (silica gel 60, F 254, Merck). The IR spectra were recorded on a Bruker Alpha FT-IR spectrometer (Billerica, MA, USA) using

the ATR technique. The 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE 400 (Bruker, Rheinstetten/Karlsruhe, Germany) spectrometer using CDCl3 and/or DMSO-d6. High-resolution mass spectra (HRMS) were recorded on an Autospec mass spectrometer with electrospray ionization (ESI) operating at 70 eV using post-acceleration photomultiplier detector.

2.2.1. General procedure for synthesis of 2,2,2-trichloro-1-(1-methyl1H-pyrrol-2-yl) ethanone 2 In a three-necked round-bottomed flask equipped with a sealed mechanical stirrer, a dropping funnel, and an efficient reflux condenser with a calcium chloride drying tube was charged with 225 g (1.23 mol) of trichloroacetyl chloride and 200 ml of anhydrous diethyl ether. The solution was stirred while 77 g (1.20 mol) of freshly distilled N-methylpyrrole in 640 ml of anhydrous ether was added over 3 h; the heat of reaction causes the mixture to reflux. Following the addition, the mixture was stirred for 1 h before 100 g (0.72 mol) of potassium carbonate in 300 ml of water was slowly added through the dropping funnel. The layers were separated, and the organic phase was dried with magnesium sulphate and filtered. The solvent was removed by distillation on a steam bath, and the residue was dissolved in 225 ml of hexane. The dark solution was cooled on ice to induce crystallization. The tan solid was collected and washed with 100 ml of cold hexane, giving 189–196 g (77–80%) of 1. 2-Pyrrolyltrichloromethyl ketone: m.p. 64–66 °C [18].

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73

2.2.2. General procedure for synthesis of 2,2,2-trichloro-1-(4,5dibromo-1-methyl-1H-pyrrol-2-yl) ethanone 3 In a three-necked round-bottom flask equipped with a sealed mechanical stirrer, a dropping funnel, and an efficient reflux condenser with a calcium chloride drying tube was charged with 3.0 g (1.0 mol) of 2,2,2-trichloro-1-(1-methyl-1H-pyrrol-2-yl)ethanone 2 in 70 ml of chloroform. The solution was stirred in an ice-bath (0–5 °C) with the help of mechanical stirrer until the solution becomes homogeneous. After that 1.8 ml (2.0 mol) of Bromine (Br2) was added to above solution over a period of 2 h dropwise with the help of dropping funnel. When addition of bromine is completed, the mixture was stirred at room temperature until the HBr gas formed is completely evolved. The reaction mixture was partitioned with 50 ml of water. The organic phase was dried over sodium sulphate and solvent was evaporated in vaccuo. Crystals of brominated pyrrole were collected and further recrystallized from n-hexane, giving 4.5–5.0 g of 2,2,2-trichloro-1-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl) ethanone 3, m.p. 142–144 °C [18]. 2.2.3. General procedure for synthesis of 4,5-dibromo-1-methyl-1Hpyrrole-2-carbohydrazide 4 To a round bottom flask equipped with a sealed mechanical stirrer, solution of 2,2,2-trichloro-1-(1-methyl-1H-pyrrol-2-yl)etha none and 30 ml of ethanol was added while stirring until the solution becomes homogeneous. To the above mixture 2.5 equivalents (3–4 ml) hydrazine hydrate (99% v/v) was added and stirred at room temperature till precipitate starts to appear. The whole reaction mixture was poured over crushed ice. The solid precipitated was filtered under vacuum and dried to obtain 4,5-dibromo-1-me thyl-1H-pyrrole-2-carbohydrazide 4; m.p. 250–252 °C [18]. 2.2.4. General procedure for synthesis of 2-(4,5-dibromo-1-methyl1H-pyrrol-2-yl)-5-aryl-1,3,4-oxadiazole analogues 5 Appropriate aromatic/heteroaromatic acid (1 mmol) and 4 (1 mmol) were stirred in 20 ml phosphorous oxychloride in a round bottom flask. It was then refluxed for 20 h until the 4,5-dibro mo-1-methyl-1H-pyrrole-2-carbohydrazide was consumed (monitored by TLC). The reaction mixture was poured on crushed ice to extract 5 in organic layer. Column purification using flash chromatography (SiO2, 10% MeOH/CHCl3) of the crude product yielded pure 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-aryl-1,3,4-oxa diazole analogues, which was further recrystallized from MeOH. 2-(5-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole2-yl) phenol (5a) Yield: 60%; m.p.: 135–137 °C; IR (KBr) Vmax cm1: 1233(CAOAC of 1,3,4-oxadiazole),1611 (C@N), 3038 (CAH aromatic), 3440 (OH phenol); 1H NMR (300 MHz, DMSO-d6): d 3.59 (s, 3H pyrrole NACH3), 5.3 (s, 1H phenol OH), 6.42 (s, 1H pyrrole 3H), 6.7–7.8 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 167.5, 164.3, 157.8, 130.1, 128.9, 126.6, 121.5, 117.3, 108.6, 107.4, 100.8, 97.4, 27.3; MS m/z: 396.8901 (M+), 398.8910 (M2+), 400.8916 (M4+). 2-Benzyl-5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadia zole (5b) Yield: 70%; m.p.: 158–160 °C; IR (KBr) Vmax cm1: 1232 (CAOAC of 1,3,4-oxadiazole), 1608 (C@N), 2995 (CAH aromatic); 1 H NMR (300 MHz, DMSO-d6): d 3.41 (s, 3H pyrrole NACH3), 4.32 (s, 2H), 6.34 (s, 1H pyrrole 3H), 6.9–7.4 (m, 5H ArH); 13C NMR (75 MHz, DMSO-d6): d 165.2, 164.5, 135.4, 133.3, 128.6, 128.1, 124.7, 108.1, 100.3, 96.8, 27.6; MS m/z: 394.9121 (M+), 396.9125 (M2+), 398.9127 (M4+). 4-(5-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole2-yl) aniline (5c) Yield: 65%; m.p.: 183–185 °C; IR (KBr) Vmax cm1: 1235 (CAOAC of 1,3,4-oxadiazole), 1610 (C@N), 2989 (CAH aromatic), 3320 (NH NH2); 1H NMR (300 MHz, DMSO-d6): d 3.54 (s, 3H pyrrole NACH3), 6.44 (s, 1H pyrrole 3H), 6.2 (s, 2H NH2), 6.9–7.6 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.6,

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144.9, 135.6, 127.3, 116.8, 115.9, 107.6, 105.3, 97.5, 27.5; MS m/z: 395.9075 (M+), 397.9095 (M2+), 399.9105 (M4+). 2-(4-Chlorophenyl)-5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)1,3,4-oxadiazole (5d) Yield: 70%; m.p.: 172–174 °C; IR (KBr) Vmax cm1: 1239 (CAOAC of 1,3,4-oxadiazole), 1603 (C@N), 3074 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.51 (s, 3H pyrrole NACH3), 6.54 (s, 1H pyrrole 3H), 7.5–8.0 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.7, 132.8, 130.9, 129.3, 128.7, 125.2, 106.6, 103.4, 97.9, 27.3; MS m/z: 414.8655 (M+), 416.8658 (M2+), 418.8661 (M4+). 2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-(4-methoxyphenyl)1,3,4-oxadiazole (5e) Yield: 60%; m.p.: 203–205 °C; IR (KBr) Vmax cm1: 1238 (CAOAC of 1,3,4-oxadiazole), 1615 (C@N), 2909 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.47 (s, 3H pyrrole NACH3), 4.19 (s, 3H OCH3), 6.36 (s, 1H pyrrole 3H), 7.1–8.0 (m, 4H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.8, 163.5, 134.2, 128.4, 115.9, 115.6, 108, 105.1, 96.2, 55.4, 27.9; MS m/z: 410.9208 (M+), 412.9022 (M2+), 414.9028 (M4+). 2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-(2,4-dichlorophenyl)1,3,4-oxadiazole (5f) Yield: 60%; m.p.: 197–199 °C; IR (KBr) Vmax cm1: 1236 (CAOAC of 1,3,4-oxadiazole), 1605 (C@N), 3083 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H pyrrole NACH3), 6.74 (s, 1H pyrrole 3H), 7.1–8.1(m, 3H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.6, 135.7, 135.3, 134.5, 133.4, 131.4, 129.8, 127.6, 108.3, 104.2, 97.6, 27.2; MS m/z: 448.8105 (M+), 450.8115 (M2+), 452.8121 (M4+). 2-(4,5-Dibromo-1-methyl-1H-pyrrol-2-yl)-5-styryl-1,3,4-oxadia zole (5g) Yield: 80%; m.p.: 200–202 °C; IR (KBr) Vmax cm1: 1229 (CAOAC of 1,3,4-oxadiazole), 1620 (C@N), 2999 (CAH aromatic); 1 H NMR (300 MHz, DMSO-d6): d 3.49 (s, 3H pyrrole NACH3), 6.33 (s, 1H pyrrole 3H), 6.67 (d, 1H CH@CH), 6.72 (d, 1H CH@CH), 7.0–7.7 (m, 5H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.6, 163.6, 137.2, 133.7, 133.1, 128.9, 128.6, 127.7, 124.5, 108.3, 100.5, 96.3, 27.5; MS m/z: 406.9100 (M+), 408.9102 (M2+), 410.9108 (M4+). 2-(2-chloro-5-fluorophenyl)-5-(4,5-dibromo-1-methyl-1H-pyrrol2-yl)-1,3,4-oxadiazole (5h) Yield: 70%; m.p.: 178–180 °C; IR (KBr): Vmax cm1: 1237 (CAOAC of 1,3,4-oxadiazole), 1623 (C@N), 3015 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.56 (s, 3H pyrrole NACH3), 6.22 (s, 1H pyrrole 3H), 7.1–8.0 (m, 3H ArH); 13 C NMR (75 MHz, DMSO-d6): d 164.4, 160.5, 138.8, 134.2, 127.4, 127.1, 116.3, 115.8, 105.3, 101.6, 98.1, 27.7; MS m/z: 432.8266 (M+), 434.8269 (M2+), 436.8272 (M4+). 2-(4,5-Dibromo-1-methyl-1H-pyrrol-2yl)-5-(4-nitrophenyl)1,3,4-oxadiazole (5i) Yield: 60%; m.p.: 287–289 °C; IR (KBr) Vmax cm1: 1240 (CAOAC of 1,3,4-oxadiazole), 1619 (C@N), 2935 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.61 (s, 3H pyrrole NACH3), 6.34 (s, 1H pyrrole 3H), 7.1–8.4 (m, 4H ArH); 13 C NMR (75 MHz, DMSO-d6): d 164.3,147.9, 135.3, 132.1, 131.6, 128.4,103.4, 100.5, 95.2, 27.6; MS m/z: 425.9799 (M+), 427.9800 (M2+), 429.9801 (M4+). 2,5-bis(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole (5j) Yield: 50%; m.p.: 210–212 °C; IR (KBr) Vmax cm1: 1245 (CAOAC of 1,3,4-oxadiazole), 1628 (C@N), 2940 (CAH aromatic); 1 H NMR (300 MHz, DMSO-d6): d 3.52 (s, 6H pyrrole NACH3), 6.46 (s, 2H pyrrole 3H); 13C NMR (75 MHz, DMSO-d6): d 164.5, 136.2, 106.5, 103.6, 96.8, 27.6; MS m/z: 525.8100 (M+), 527.7080 (M2+), 529.7085 (M4+). 4-(2-(5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazol2-yl)vinyl)-2-methoxyphenol (5k) Yield: 75%; m.p.: 160–162 °C; IR (KBr) Vmax cm1: 1231 (CAOAC of 1,3,4-oxadiazole), 1627 (C@N), 2950 (CAH Aromatic), 3441 (OH phenol); 1H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H pyrrole NACH3), 3.89 (s, 3H OCH3), 5.2 (s, 1H OH), 6.54 (s, 1H pyrrole 3H), 6.70 (d, 1H CH@CH), 6.73(d, 1H CH@CH), 7.0–7.38 (m, 3H ArH); 13C NMR (75 MHz, DMSO-d6): d 164.5, 160.6, 149.4, 147.2, 133.8, 130.8, 128.9,

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124.1, 122.8, 116.2, 109.5, 107.3, 100.3, 96.4, 55.9, 27.1; MS m/z: 452.9320 (M+), 454.9325 (M2+), 456.9328 (M4+). 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(pyridin-4-yl)1,3,4-oxadiazole (5l) Yield: 70%; m.p.: 240–242 °C; IR (KBr) Vmax cm1: 1245 (CAOAC of 1,3,4-oxadiazole), 1613 (C@N), 2899 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.7 (s, 3H pyrrole NACH3), 6.41 (s, 1H pyrrole 3H), 7–8.27 (m, 4H pyridine); 13C NMR (75 MHz, DMSO-d6): d 164.2, 149.9, 143.9, 128.4, 121.8, 107.1, 100.5, 97.2, 27.3; MS m/z: 381.9001 (M+), 383.9006 (M2+), 385.9009 (M4+). 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-phenyl-1,3,4-oxadiazole (5m) Yield: 80%; m.p.: 270–272 °C; IR (KBr) Vmax cm1: 1239 (CAOAC of 1,3,4-oxadiazole), 1614 (C@N), 1710 (C@O), 2931 (CAH aromatic); 1H NMR (300 MHz, DMSO-d6): d 3.55 (s, 3H pyrrole NACH3), 6.38 (s, 1H pyrrole 3H), 7.44–7.62 (m, 5H ArH); 13 C NMR (75 MHz, DMSO-d6): d 177.9, 164.9, 160.3, 157.8, 149.3, 135.2, 132.7, 128.7, 128.4, 127.2, 123.8, 123.2, 117.8, 115.3, 107.2, 100.9, 97.6, 27.6; MS m/z: 474.9022 (M+), 476.9026 (M2+), 478.9033 (M4+). 2.2.5. General procedure for synthesis of 5-(4, 5-dibromo-1-methyl1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 In a two necked round bottom flask equipped with sealed mechanical stirrer and an efficient reflux condenser, 4,5-dibro mo-1-methyl-1H-pyrrole-2-carbohydrazide 4 (0.003 mol) in ethanol (20 ml) was added. To this solution potassium hydroxide (0.006 mol) and carbon disulfide (4 ml) was added with stirring. The reaction mixture was refluxed for 12 h. The solvent was removed under reduced pressure; the residue was treated with water and then filtered. The filtrate was cooled and neutralized to pH 6 using dilute hydrochloric acid. The precipitated product was filtered, washed with water and dried to obtain 5-(4,5-dibro mo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6. 5-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole2-thiol (6) Yield: 80%; m.p.: 190–192 °C; IR (KBr) Vmax cm1: 1242 (CAOAC of 1,3,4-oxadiazole), 1625 (C@N), 2751 (SH group). 1 H NMR (300 MHz, DMSO-d6): d 3.41 (s, 3H pyrrole NACH3), 3. 2(s, 1H SH), 6.61(s, 1H pyrrole 3H); 13C NMR (75 MHz, DMSO-d6): d 164.4, 160.5, 127.9, 107.5, 100.2, 96.8, 27.5; MS m/z: 336.8369 (M+), 338.8376 (M2+), 340.8379 (M4+). 2.2.6. General procedure for synthesis of 2-(4,5-dibromo-1-methyl1H-pyrrol-2-yl)-5-(alkylnarylthio)-1,3,4-oxadiazole 7 In a two necked round bottom flask 5-(4,5-dibromo-1-me thyl-1H-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 (1 mmol), alkyl/aryl halide (1 mmol) and sodium hydroxide (5 mmol) in ethanol were stirred for 10 h at room temperature. The solvent was removed under reduced pressure. The residue obtained was dried to give 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(alkyln arylthio)-1,3,4-oxadiazole 7. 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(methylthio)-1,3,4-ox adiazole (7a)Yield: 80%; m.p.: 186–188 °C; IR (KBr) Vmax cm1: 1247 (CAOAC of 1,3,4-oxadiazole), 1629 (C@N), 2749 (SH); 1H NMR (300 MHz, DMSO-d6): d 3.41 (s, 3H pyrrole NACH3), 6.57(s, 1H pyrrole 3H), 2.36(s, 3H); 13C NMR (75 MHz, DMSO-d6): d 164.8, 160.2, 126.2, 107.8, 100.4, 96.9, 27.3, 16.9; MS m/z: 350.8567 (M+), 352.8571 (M2+), 354.8575 (M4+). 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(ethylthio)-1,3,4-oxa diazole (7b) Yield: 55%; m.p.: 150–152 °C; IR (KBr) Vmax cm1: 1229 (CAOAC of 1,3,4-oxadiazole), 1632 (C@N), 2751 (SH); 1H NMR (300 MHz, DMSO-d6): d 1.3 (t, 3H), 3.1(m, 2H), 3.62 (s, 3H pyrrole NACH3), 6.62(s, 1H pyrrole 3H); 13C NMR (75 MHz, DMSO-d6): d 165.3, 160.1, 127.3, 106.5, 100.3, 97.1, 27.9, 27.3, 16.8; MS m/z: 364.8676 (M+), 366.8682 (M2+), 368.8688 (M4+). 2-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-5-(phenylthio)-1,3,4-ox adiazole (7c) Yield: 65%; m.p.: 170–172 °C; IR (KBr) Vmax cm1:

1222 (CAOAC of 1,3,4-oxadiazole), 1616 (C@N), 2699 (SH group); 1 H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H pyrrole NACH3), 6.43 (s, 1H pyrrole 3H), 7.14–7.47 (m, 5H ArH); 13C NMR (75 MHz, DMSO-d6): d 166.4, 160.8, 136.2, 128.4, 127.4, 126.7, 123.3, 105.5, 100.9, 97.8, 28.1; MS m/z: 412.8632 (M+), 414.8654 (M2+), 416.8658 (M4+).

2.3. Antibiofilm study 2.3.1. Detection of biofilm formation by tissue culture plate (TCP) assay Detection of biofilm formation was confirmed by Tissue culture plate (TCP) assay as described in literature [13]. Tissue culture plate (TCP) assay as described in literature is most widely used and was considered as standard test for detection of biofilm formation [11]. In our present study, we have screened Methicillin-sensitive Staphylococcus aureus (MSSA; ATCC 35556), Methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43866), Streptococcus epidermidis (SE; ATCC 35984) and Pseudomonas aeruginosa (PA) bacterium for their ability to form biofilm by TCP method. Isolates from fresh agar plates were inoculated in respective media and incubated for 18 h at 37 °C in stationary condition and diluted 1 in 100 with tryptic soy broth (TSB). Individual wells of sterile, polystyrene, 96-well flat-bottom tissue culture plates were filled with 0.2 mL aliquots of the diluted cultures and only broth served as control to check sterility and nonspecific binding of media. The tissue culture plates were incubated for 18 h and 24 h at 37 °C. After incubation, content of each well was gently removed by tapping the plates. The wells were washed four times with 0.2 mL of phosphate buffer saline (PBS, pH 7.2) to remove free floating ‘‘planktonic’’ bacteria. Biofilms formed by adherent ‘‘ses sile’’ organisms in the plate were fixed with sodium acetate (2%) and stained with crystal violet (0.1% w/v). Excess stain was rinsed off by thorough washing with de-ionized water and plates were kept for drying. Adherent staphylococcal cells usually formed biofilm on all side wells and were uniformly stained with crystal violet. Optical densities (ODs) of stained adherent bacteria were determined with a micro ELISA auto reader (California) at wavelength of 570 nm (OD 570 nm). These OD values were considered as an index of bacteria adhering to surface and forming biofilms. Experiment was performed in triplicate and repeated three times, the data was then averaged and standard deviation was calculated. To compensate for background absorbance, OD readings from sterile medium, fixative and dye were averaged and subtracted from all test values. The mean OD value obtained from media control well was deducted from all the test OD values as given in Table 2.

2.3.2. Antibiofilm assay For antibiofilm concentration experiment, overnight cultures of bacterium Methicillin-sensitive Staphylococcus aureus (MSSA; ATCC 35556), Methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43866), Streptococcus epidermidis (SE; ATCC 35984) and Pseudomonas aeruginosa (PA; ATCC 27853) were diluted (1:10) in TSB (tryptic soy broth) (OD 600 = 0.6–0.8) and were then diluted to 1:200 in Mueller–Hinton. The bacterial suspension was inoculated into sterile 96-well polystyrene microtiter plates (Falcon) incubated at 37 °C for 6 h. The plates with young biofilm were washed gently four times with sterile PBS before adding fresh TSB containing various concentrations of compounds and incubated at 37 °C for 24 h. The initial dilution was 200 lg/mL and minimum biofilm inhibitory concentration (MBIC) values were defined as the lowest concentration at which no visible growth was observed. Positive control was performed for all the evaluated bacteria. A high level of biofilm growth was observed in case of positive control. For all assays, Vancomycin was used as reference standard [13].

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73

2.3.3. Cell viability test Bacterial growth after treatment was measured by quantifying cell viability after incubation with 5f. MSSA, MRSA and SE cells were incubated with 5f at various concentrations as shown in Fig. 3 in isotonic saline solutions at 37 °C under 200 rpm shaking speed for 4 h. After the treatment, 100 lL of reaction mixture was made up to 1 mL, and then from 1 mL, 50 lL was taken for plating. Loss of viability was evaluated by the colony-counting method. Briefly, a series of 20-fold cell dilutions (100 lL each) were spread onto LB plates and left to grow overnight at 37 °C. Colonies were counted and compared with those on control plates to calculate changes in cell-growth inhibition. For control, we used isotonic saline solution without 5f. All treatments were prepared in triplicate and repeated at least in three independent experiments.

71

P

Q

2.4. Cytotoxicity of compounds (selectivity assay) The synthesized analogs were further studied for their mammalian toxicity (IC50) using VERO cell lines. Viability was assessed, after 72 h, by the conversion of MTT into formazan product using Promega Cell Titre 96 non-radioactive cell proliferation assay. All the hybrids were found to be non-toxic and the data is displayed in Table 1. 3. Results and discussion

R

3.1. Synthesis of the compounds The Spectral data (IR, 1H NMR, 13C NMR and MS) of all synthesized compounds were in agreement with the proposed structures. The MS of all the compounds exhibited the [M+.] as molecular ion peak, confirming the molecular weight. Bromination of pyrrole ring was confirmed by the presence of [M2+] and [M4+] peaks in mass spectra. 1H NMR spectra of these hybrids showed singlet peak of pyrrole-NACH3 proton in the range of d 3.7–3.4. Thiol group of 6 exhibited NMR peak at d 3.2, while proton peaks of aryl substitutions attached to oxadiazole ring resonated between 8.2 and 6.1 ppm. A proton signal at third position of pyrrole ring was observed to resonate between d 6.7–6.3. 5-(4,5-dibromo-1-me thyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-thiol 6 displayed distinct IR peaks at 2751 cm1 for ASH group, while for the alkylated product [5-(4,5-dibromo-1-methyl-pyrrol-2-yl)-1,3,4-oxadiazole-2-methyl thiol] the ASH signal was absent. 3.2. Quantification of biofilm inhibition activities The respective bacteria were evaluated for their ability to form biofilm by TCP method and the results are presented in Table 2. Here ‘‘high’’ biofilm forming isolate were selected for future antibiofilm study and the selected isolates reproducibly formed biofilm in the assay performed. Table 1 shows the results of biofilm inhibition for each assayed compound as well as the concentrations at which these inhibition levels were observed. All experiments were performed in triplicate and the mean values were taken as minimum biofilm inhibitory concentration (MBIC). Most of the synthesized compounds disclosed significant biofilm inhibition against MSSA and MRSA bacterial strains. In the case of MSSA strain, compound 5f (MBIC = 0.39 lg/mL) having electron withdrawing substituents (2,4-dichlo-) on phenyl ring exhibited maximum biofilm inhibition with four-fold greater activity than the standard drug (MBIC = 3.125 lg/mL). Further, five compounds (5c, 5d, 5e, 5i and 5j) exhibited three-fold superior biofilm inhibition (MBIC = 0.78 lg/mL) than standard drug. Generally, substituting phenyl ring at para-position was beneficial for inhibition of biofilm produced by MSSA. Comparison of anti-biofilm activity of 5m

Fig. 3. Cell viability of MSSA (P), MRSA (Q) and SE (R) on treatment with 5f at different concentrations. These experiments were performed in triplicate (C = Control).

(MBIC = 12.5 lg/mL) with other compounds in the series revealed that the presence of substituents like chloro-, fluro-, bromo-, methoxy-, nitro- and amino- on phenyl ring was beneficial than the un-substituted phenyl ring. In addition, six compounds (5a, 5b, 5g, 5h, 5k and 5l) presented two-fold higher anti-biofilm activity (MBIC = 1.56 lg/mL) than the standard drug Vancomycin against MSSA. Compounds with substitution at ortho- or metaposition of aromatic rings displayed lower antibiofilm activity relative to para- substituted molecules. Also, hetero-aromatic substitution at R position decreased the anti-biofilm activity for this class of compounds. For the MRSA, most active compound was found to be 5f with four-time superior antibiofilm activity (MBIC = 0.78 lg/mL) than standard Vancomycin (MBIC = 6.25 lg/mL). Compounds 5c, 5d, 5e, and 5j showed three-time more potency (MBIC = 1.56 lg/ml), whereas 5h, 5i and 5l exhibited two-time potency (MBIC = 3.125 lg/mL) towards biofilm produced by MRSA as compared to the standard drug. Compounds 5a, 5b, 5g, 5m, 6 and 7a were found to be equipotent to that of standard drug Vancomycin (MBIC = 6.25 lg/mL). Consequently, substituting phenyl ring at para-position with halogen-, methoxy-, nitro- and amino- groups was beneficial for the biofilm inhibition. On the other hand, substituting 1,3,4-oxadiazole ring with thio-phenyl and thio-ethyl at second position decreased the anti-biofilm activity of compounds 7b and 7c (MBIC = 25 lg/mL). This could be due to the presence of

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Table 1 Antibiofilm results: (MBIC in lg/mL).a,b

Br Br

N H3C

O R N N

S. no.

R

MSSA

MRSA

S. epidermidis

P. aeruginosa

Cytotoxicity (lM) VERO cells

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 6 7a 7b 7c Standardb

2-HydroxyphenylBenzyl4-Aminophenyl4-Chlorophenyl4-Methoxyphenyl2,4-DichlorophenylStyryl2-Chloro-5-fluorophenyl4-Nitrophenyl4,5-Dibromo-1H-pyrrol-2-yl2-Methoxy-4-vinylphenolPyridin-4-ylPhenylThiolThiomethylThioethylThiophenyl-

1.56 1.56 0.78 0.78 0.78 0.39 1.56 1.56 0.78 0.78 1.56 1.56 12.5 6.25 12.5 12.5 12.5 3.125

6.25 6.25 1.56 1.56 1.56 0.78 6.25 3.125 3.125 1.56 25 3.125 6.25 6.25 6.25 25 25 6.25

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

25 12.5 25 25 6.25 12.5 50 6.25 6.25 6.25 50 6.25 50 100 50 50 50 3.125

NDc ND 69.0 137.9 248.1 297.0 ND ND 78.2 206.5 ND ND ND ND ND ND ND ND

Italic values indicates the compounds tested for antibiofilm activity and bold value denotes the most active compounds of the series. a The initiate dilution was 200 lg/ml. b For all assay standard used was Vancomycin. c ND – not determined.

Table 2 Screening of various bacterial strains for biofilm formation by TCP method in tryticase soy broth with 1% glucose at 18 and 24 h of incubation. Biofilm formation (OD

570nm)

High (>0.240 ± 0.020) Moderate (0.120–0.240 ± 0.024) Weak/non (<0.12 ± 0.014)

No. of isolates MRSA, ATCC 43866 Incubation

SE, MSSA, ATCC ATCC 35984 35556 time in hours

PA ATCC 27853

18

24

18

24

18

24

18

24

1 6 54

4 8 62

12 21 92

17 28 92

5 11 64

7 18 70

5 11 61

12 20 79

that most the synthesized molecules were antibacterial at more than three-fold MBIC concentration against respective bacterium. Further most active compound 5f was subjected to the cell viability assay at respective MBIC as shown in Fig. 3. It was observed that there are no effects on the viability of the bacteria at the concentrations at which compound 5f showed antibiofilm activities against respective bacterium indicating that antibiofilm effects are not simply due to a decreased number of bacteria but because of the antivirulence properties of this class of compounds that needs to be investigated further and is the ongoing work at our laboratory. 4. Conclusion

sulphur bridge/linker between 1,3,4-oxadiazole ring and R (alkyl/aromatic) group. In case of S. epidermidis (SE) bacterium, compounds with para-substitution on the benzene ring exhibited equipotent biofilm inhibition activity (MBIC = 3.125 lg/mL) compared to the standard drug Vancomycin (MBIC = 3.125 lg/ml). Moreover, substitution of phenyl ring with chloro-, fluro-, bromo-, nitro-, amino- and methoxy- group (5b, 5c, 5d, 5e, 5f, 5h, 5i and 5j) improved the anti-biofilm activity against SE. No significant biofilm inhibition activity was observed for compounds with substitutions at orthoand meta- position on the phenyl ring. In addition to this, these synthesized compounds were also screened against P. aeruginosa. All compounds displayed less activity (MBIC = 6.25 to 100 lg/mL) towards P. aeruginosa (compared to the standard drug Vancomycin; MBIC = 3.125 lg/mL), indicating that this structural motif was less potent against biofilm produced by Gram-negative bacterium as compared to Gram-positive. Further in order to confirm the antivirulence properties of these compounds they were evaluated against test microbes for bactericidal activity (minimum inhibitory concentration). It was observed

In summary, this study identified compound 5f with MBIC of 0.39, 0.78 and 3.125 lg/mL as promising lead against biofilm produced by MSSA, MRSA and SE respectively. Additionally, eight compounds displayed equal potency with the standard drug Vancomycin against S. epidermidis. An important observation was that the compounds which displayed promising biofilm inhibition activity also exhibited low toxicity towards human VERO cells. The cell viability assay for the most active hybrid confirms its anti-virulence properties which need to be further studied. This could be advantageous for their use in therapeutics and functionalization of materials such as biofouling control in closed systems. Currently, evaluation of such potential along with studies on the mechanism of biofilm inhibition for these compounds is underway. Furthermore, we are also working towards consolidation of the potential use of these compounds on a range of bacterial clinical isolates chosen in accordance with their foreseen applications. Conflict of interest Authors in this manuscript declare no conflict of interest.

R.A. Rane et al. / Bioorganic Chemistry 61 (2015) 66–73

Acknowledgments The authors sincerely thank College of Health Science, University of KwaZulu-Natal, Durban, and NRF South Africa for funding and scholarships.

References [1] R. Laxminarayan, A. Duse, C. Wattal, A.K. Zaidi, H.F. Wertheim, N. Sumpradit, et al., Lancet. Infect. Dis 13 (2013) 1057–1098. [2] M. Safari, R. Amache, E. Esmaeilishirazifard, T. Keshavarz, Appl. Microbiol. Biotechnol. 98 (2014) 3401–3412. [3] J.L. Martinez, F. Baquero, Clin. Microbiol. Rev. 15 (2002) 647–679. [4] K.R. Hardie, K. Heurlier, Nat. Rev. Microbiol. 6 (2008) 635–643. [5] K.M. Smith, Y. Bu, H. Suga, Chem. Biol. 10 (2003) 563–571. [6] G.D. Geske, R.J. Wezeman, A.P. Siegel, H.E. Blackwell, J. Am. Chem. Soc. 127 (2005) 12762–12763. [7] G. Brackman, U. Hillaert, S. Van Calenbergh, H.J. Nelis, T. Coenye, Res. Microbiol. 160 (2009) 144–151.

73

[8] C. Lu, C.K. Maurer, B. Kirsch, A. Steinbachand, R.W. Hartmann, Angew. Chem. 53 (2014) 1109–1112. [9] M.P. Storz, C.K. Maurer, C. Zimmer, N. Wagner, C. Brengel, J.C. de Jong, S. Lucas, M. Müsken, S. Häussler, A. Steinbach, R.W. Hartmann, J. Am. Chem. Soc. 134 (2012) 16143–16146. [10] R.M. Donlan, J.W. Costerton, Clin. Microbiol. Rev. 15 (2002) 167–193. [11] R.A. Rane, N.U. Sahu, C.P. Shah, R. Karpoormath, Curr. Top. Med. Chem. 14 (2014) 253–273. [12] S. Dobretsov, M. Teplitski, M. Bayer, S. Gunasekera, P. Proksch, V.J. Paul, Biofouling 27 (2011) 893–905. [13] R.A. Rane, N.U. Sahu, C.P. Shah, N.K. Shah, J. Enzyme Inhibit. Med. Chem. 29 (2014) 401–407. [14] J.J. Richards, T.E. Ballard, R.W. Huigens, C. Melander, Chem. Bio. Chem. 9 (2008) 1267–1279. [15] B. Chandrakantha, P. Shetty, V. Nambiyar, N. Isloor, A.M. Isloor, Eur. J. Med. Chem. 45 (2010) 1206–1210. [16] Y. Li, Y. Luo, Y. Hu, D.D. Zhu, S. Zhang, Z.J. Liu, et al., Bioorg. Med. Chem. 20 (2012) 4316–4322. [17] G.C. Ramaprasad, B. Kalluraya, B.S. Kumar, R.K. Hunnur, Eur. J. Med. Chem. 45 (2010) 4587–4593. [18] R.A. Rane, S.D. Gutte, N.U. Sahu, Bioorg. Med. Chem. Lett. 22 (2012) 6429– 6432.