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BRINE-SHRIMP LETHALITY BIOASSAY AND ANTIBACTERIAL ACTIVITY OF BIPHENYL ANALOGUES.

Byline: Qamar Ali, Asad Gulzar, Abrar Hussain, Amina Asghar and Ikram Ullah Bajwa

ABSTRACT: A series of biphenyl derivatives were synthesized and evaluated for brine shrimp lethal bioassay and antibacterial activities. The cytotoxic activity was assessed against the reference drug Etoposide. Compound 4a (4, 4'-diiodo-3, 3'-dimethoxybiphenyl) exhibited excellent activity in Brine Shrimp Lethal Bioassay while compound 4b (4-iodo-3, 3'-dimethoxybiphenyl biphenyl) showed moderate activity. Compound 4a also showed low activity against B. subtilis. Other biphenyl derivatives showed moderate brine shrimp lethal bioassay but could not show any antibacterial activity to an appreciable extent.

Keywords: Biphenyl analogues, brine shrimp lethal bioassay, etoposide, cytotoxic

1. INTRODUCTION

Biaryl structures are extensively found in many of the natural products including terpenes, alkaloids, flavonoids, lignan, and tannins etc [1, 2]. They have shown a wide diversity in their applications in biological systems. For example, vancomycin is found to be an integral part of many antibiotics: actinoidin A, complestatin, balhimyicn etc. Gossypol, a natural biaryl pigment found in cottonseed, has shown male antifertility activity. Among liganans, steganacin is a constituent of Steganotaenia araliacea possessing a significant antileukemic activity [3]. In addition, Losatan, an antihypertensive agent, has a biphenyl moiety acting as a spacer within the structure [4].

Heterobiaryls can be employed as ligands used in forming metal complexes that can act as bleaching agents, catalysts, and oxygen-binding molecules [5-7]. Atropisomeric biaryls have shown remarkable applications in agrochemical, pharmaceutical industries, and material sciences. They act as chiral ligands and have been used for development of a variety of catalysts used in transition metal-catalyzed asymmetric transformations [8-15]. Polyaryls such as poly-p-phenylene possess valuable conducting properties and are being used in making rechargeable batteries, solar cells, and a variety of other electrochemical devices [16-18].

In order to find out the cytotoxic and antibacterial activities of synthesized compounds, a variety of biphenyl derivatives having different moieties at 2, 2' and 3, 3' positions were synthesized.

2. MATERIAL AND METHODS

The synthesis of biphenyl derivatives was carried out from two different sources to investigate the difference in activity of synthesized compounds. Two different classes of compounds were synthesized from [1,1'-biphenyl]-2,2'-diol (compound 1) and o-dianisidine bisdiazotated zinc double salt (compound 3).

2.1 Synthesis of Biphenyl Analogues based on [1, 1'-biphenyl] - 2, 2'-diol

The synthesis of biphenyl analogues derived from 1 was achieved through the route outlined in Scheme 1. Compound 1 was dissolved in acetone (HPLC grade) and then K2CO3 was added.The reaction mixture was stirred for 30 minutes and then added tert-butyl bromoacetate. After 3 hours, 1N HCl was added to quech the reaction. The required product was 2a extracted with CH2Cl2 three times and was purified through column chromatography giving 80% yield. Compound 2b was obtained by treating 2a with TFA for 1 hour and after work up with hexane and toluene. Compound 2c was obtained from 1 by refluxing 1 and ethylacetate in the presence of acetone for 36 hours. Compound 2d was obtained by treating dibromomethane (4 equivalents) with 1, while compound 2e was obtained by treating statistical amount of dibromomethane with 1 and K2CO3 in chloroform acting as solvent.

2.2. Synthesis of Biphenyl Analogues based on Fast Blue B salt

The synthesis of biphenyl analogues deriverd from o-dianisidine bisdiazotated zinc double salt (Fast Blue B salt) was achieved through the route outlined in Scheme 2. 28 g (0.17 mmol) of KI was dissolved in 200 mL of water. Then, added 10.00 g (21 mmol) of 3. After stirring for 12 hours at room temperature, 30 mL of dichloromethane was added three times to extract the crude reaction mixture. The resulted crude mixture was purified through column chromatography. Solvent system used for silica gel column was dichloromethane: hexane (1: 4) resulting fine crystals of 4a giving 70 % yield. In addition to 4a, fine crystals of 4b were also obtained giving 25% yield.

3. BIOLOGICAL EVALUATION OF SYNTHESIZED BIPHENYL ANALOGUES

3.1 Brine-Shrimp Lethality Bioassay

Cytotoxic activity of synthesized biphenyl analogues was carried out by Artemia salina [19-21]. Test samples of different concentrations (10, 100, 1000 ug/mL) were prepared in DMSO and brine shrimps were hatched in sea water media at 27 C for 2 days. Vials containing test samples, reference drug (Etoposide, LD50= 7.465 ug/mL) were incubated at 27 C for 24 hours, and LD50 values were determined to evaluate the cytotoxic effects of synthesized compounds by using the procedure of Meyer et al [22].

3.2 In Vitro Antibacterial Activity

In vitro antibacterial activity of synthesized biphenyl derivatives was carried out against bacterial strains including Escherichia coli (ATCC 25922), Bacillus subtius (ATCC 6633), Shigella flexenari (clinically isolated), staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhi (ATCC 19430). Antibacterial activity of the synthesized compounds was compared with the reference antibacterial drug (imepinem) by using agar well diffusion method [23].

4. RESULTS AND DISCUSSION

Brine shrimp lethality assay correlates in most of cases with cytotoxic and antitumor properties. The LD50 value of the brine shrimp obtained from 4a were found to be significant as compared to etoposide (LD50= 7.465 ug/mL) used as a reference drug. This significant lethality (0.9 ug/mL) of 4a to brine shrimp is an indicator of the presence of potent cytotoxic components which warrants further investigation. In addition to 4a, 4b (3.17 ug/mL) also showed moderate activity. The lethality associated with 4a and 4b is due to presence of iodine substituent at C-4 of aromatic rings of substituted biphenyl analogues.

In addition to brine shrimp lethal bioassay, in vitro antibacterial activities were also carried out for the synthesized biphenyl analogues. After incubation of plates at 37 C for 14-19 h, the potency was determined by measuring the diameter of zones showing complete inhibition (mm). Most of the compounds showed inactive behavior against all bacterial strains except compound 4a which showed low activity against B. subtilis.

Table-1: Brine shrimp lethality assay of Biphenyl Analogues

Assay###Etoposide###Compounds

###(Reference)###2a###2b###2c###2d###2e###4a###4b

###ug/mL###ug/mL

Brine shrimp###7.465###0###0###0###0###0###0.9###3.17

lethality assay

Table-2: In vitro Anti-bacterial Activity of Biphenyl Analogues Zone of Inhibition of the Tested Compound (mm)

###Tested Compound (mm)

Name of bacteria###Antibacterial Activity Compounds

###2a###2b###2c###2d###2e###4a###4b

E. coli###0###0###0###0###0###0###0

B. subtilis###0###0###0###0###0###15###0

S. flexinari###0###0###0###0###0###0###0

S. aureus###0###0###0###0###0###0###0

P. aeruginosa###0###0###0###0###0###0###0

S. typhi###0###0###0###0###0###0###0

5. CONCLUSION

Synthesis of a series of seven compounds of biphenyl analogues 2(a-e) and 4(a-b) is illustrated in Scheme 1 and Scheme 2. Biphenyl analogues were synthesized by using general synthetic route involving nucleophilic substitution reactions on functional biphenyl analogues. A detailed study on the biological usefulness including brine shrimp lethality assay, and antibacterial activities of some synthetic biphenyl analogues were carried out. Brine shrimp cytotoxicity assay serves as prescreen test for the identification of bioactive compounds to evaluate anticancer potential of any compound. Compound 4a showed significant activity towards Artemia salina which means that this compound has strong potential for the anticancer screening. The biphenyl analogues were also evaluated for their antibacterial activities. Only compound 4a showed low activity against B. subtilis.

6. ACKNOWLEDGEMENT

The authors are indebted to the financial support provided by the HEC (Higher Education Commission, Pakistan) and HEJ-RIC, University of Karachi for providing necessary space and facilities for bioassays.

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Author:Ali, Qamar; Hussain, Abrar; Gulzar, Asad; Asghar, Amina; Ullah, Ikram
Publication:Science International
Article Type:Report
Date:Feb 29, 2016
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