INTRODUCTION:

Organic chemistry is the art of building up of organic compounds from smaller entities. The area of organic chemistry that involves the study of carbon and hydrogen related compounds. Hydrocarbons led to synthesize seemingly an ‘n’ number of compounds. This science has found application in the production of organic compounds of commercial interest in the construction of new, potentially bioactive molecules derived from rational design. Naphthoquinone is a class of organic compound derived from naphthalene (Y.B Rokade et al, 2009 ). Molecules with the quinonoid structure constitute one of the most interesting classes of compounds in organic chemistry. Quinone and naphthoquinone moieties are prevalent motifs in various natural products and are associated with diverse biological activities (Deepak et al, 2009).

The naphthoquinone isomers (mainly of 1,4-naphthoquinone, juglone, lawsone and plumbagin) are the most widely spread phytochemicals containing 1,4-naphthoquinone skeleton as their parent moiety (Mandala et al, 2009). Naphthalene-1, 4-dione derivatives have been known to possess a wide spectrum biological activities such as antibacterial, antifungal, antiinflammatory, anticancer, antidiabetic and antimalarial. Interestingly, Plumbagin and juglone have strong sterilizing activity against mycobacterium strains. Keeping this context in mind an attempt has been made to synthesis and to study the in-vitro antimicrobial efficacy of some biologically active lawsones (BA1-BA5). This possibly led to the development of compounds with probable antimicrobial activity especially in antibacterial study to overcome the strains those are resistant with earlier 1,4- naphthoquinone derivatives by developing structural modifications.

MATERIALS AND METHODS:

The chemicals used in the present studies are of synthetic grade, sigma-aldrich company ltd. The products were characterized by FT-IR, ¹H NMR and DART-MS spectra. The melting points were determined by open capillary methods and are uncorrected. The FT-IR spectra were recorded on Model IR- affinity-1 (SHIMADZU) FTIR instrument by using KBr pellet technique. ¹H NMR spectra were recorded in dimethylsulphoxide (DMSO) on Bruker, Advance-II 400, NMR Spectrophotometer using tetra methyl silane as an internal standard. The purity of the compounds were checked by by thin layer chromatography. The crude products were crystallised from 10 % ethanol. All the synthesized test compounds were tested for their antimicrobial activity against various bacterial strains like gram positive bacteria’s; Bacillus subtilis (ATCC-6633), gram negative bacteria’s; Escherichia coli (NCIM-2118)

Typical procedure for the preparation of 2-hydroxynaphthalene-1,4- diones

A mixture of 2-hydroxynaphthalene-1,4-dione (1), 1 mmol, substituted amines (2), 1 mmol, substituted aldehydes (3), 1 mmol and ZnCl₃ was refluxed in water (5 mL) for appropriate time (0-6 h). After completion of the reaction confirmed by TLC (Eluent: Toluene: Ethyl acetate: Methanol; 7.7: 1.7: 0.4), the reaction mixture was filtered and the precipitate was washed with water (10 ml) and rectified spirit (10 ml) to afford the pure product (4) .

SCHEME:

Table 1: Different substitution and yield of each substitution

PRODUCT	X	Y	TIME (h)	Yield (%)
BA1	H	H	6	71.42
BA2	H	2-NO₂	6	42.00
BA3	H	4-OCH₃	6	68.00
BA4	H	4-Br	6	54.36
BA5	H	4-F	6	51.62

Characterization of Compounds (Ba1-Ba5)

BA1: 2-hydroxy-3[phenyl(phenylamino)methyl] naphthalene-1,4-dione. Dark brown powder (yield 1.42%); mp 145-148°C. FT-IR (KBr) (ν_max): 3342.64 (Ar-OH), 1292.64 (C–N of 2° N-H), 1624.06 (C=O), 1489 (Ring C=C stretch) cm^-1. ¹H-NMR (400 MHz, DMSO-d₆): δ_H (ppm) 7.280 (1H, s, Ar-OH), 6.226 (1, s, NH), 7.233–8.108, (14H, m, H-Ar). DART-MS, m/z: 354.76 (M⁺).

BA2: 2-hydroxy-3{[(2-nitrophenyl)amino][phenyl} napthalene1,4-dione. Orange Powder (yield 42%); mp 185-190 °C. FT-IR (KBr) (ν_max): 3346.64 (Ar-OH), 1282.57 (C–N of 2° N-H), 1610.00 (C=O), 1348.33 (NO2) 1478.22 (Ring C=C stretch) cm^-1. ¹H-NMR (400 MHz, DMSO-d₆): δ_H (ppm) 7.232 (1H, s, Ar-OH), 6.252 (1, s, NH), 7.246–8.111, (13H, m, H-Ar). DART-MS, m/z: 401.05 (M⁺).

BA3: 2-Hydroxy-3 {[4-methoxy phenyl) amino] (phenyl) methyl} naphthalene-1,4-dione. Yellow Powder (yield 86%); mp 192-95 °C. FT-IR (KBr) (ν_max): 3340.02(Ar-OH), 1280.52 (C–N of 2° N-H), 1614.03 (C=O), 1453 (Ring C=C stretch), 1048.33 (C–O–C of OCH₃), ¹H-NMR (400 MHz, DMSO-d₆): δ_H (ppm) 7.187 (1H, s, Ar-OH), 6.935 (1, s, NH), 7.210–8.107, (13H, m, H-Ar), 3.693 (3H, s, O–CH₃). DART-MS, m/z: 367.06 (M⁺).

BA4: 2-Hydroxy3-{[(4-Bromophenyl) amino] (phenyl) methyl]} naphthalene-1,4-Dione .Brown colour Powder (yield 54.36%); mp 170-175 °C. FT-IR (KBr) (ν_max): 3350.64 (Ar-OH), 1290.66 (C–N of 2° N-H), 1612.08 (C=O), 1479 (Ring C=C stretch), 698.02 (C–Br) cm^-1. ¹H-NMR (400 MHz, DMSO-d₆): δ_H (ppm) 7.292 (1H, s, Ar-OH), 6.876 (1, s, NH), 7.293–8.121, (13H, m, H-Ar). DART-MS, m/z: 428.08 (M⁺).

BA5: 2-Hydroxy-3-{[(4-flurophenyl) amino (phenyl) methyl]} naphthalene-1,4-Dione. Red Powder (yield 86%); mp 145-148 °C. FT-IR (KBr) (ν_max): 3346.60 (Ar-OH), 1287.12 (C–N of 2° N-H), 1611.01 (C=O), 1450 (Ring C=C stretch) 1150.02 (C–F) cm^-1cm^-1.¹H-NMR (400 MHz, DMSO-d₆): δ_H (ppm) 7.254 (1H, s, Ar-OH), 6.298 (1, s, NH), 7.248–8.081, (13H, m, H-Ar). DART-MS, m/z: 372.23 (M⁺).

Table 2: Physicochemical characteristic of substituted naphthoquinone derivatives


Comp.	X	Y	M. F	M.W	%YIELD	M.P (°C)
BA1	H	NH₂	C₂₃H₁₇NO₃	358.38	42.00	142-145
BA2	H	2-NO₂	C₂₃H₁₆N₂O₅	400.38	68.00	190-192
BA3	H	4-OCH₃	C₂₄H₁₉NO₄	385.41	71.42	160-165
BA4	H	4-Br	C₂₃H₁₆BrNO₃	434.28	54.36	185-188
BA5	H	4-F	C₂₃H₁₆FNO₃	373.37	51.62%	190-192

ANTIMICROBIAL ACTIVITY:

Antibacterial Activity

The test compounds (BA1-BA5) were tested for their Minimum Inhibitory Concentration (MIC) against gram positive bacteria; Bacillus subtilis and gram negative bacteria; Escherichia coli by broth dilution technique. The present investigations were undertaken to test to identify the antimicrobial activity with that of MIC of these compounds against some gram positive and gram-negative bacteria by using agar diffusion or disc diffusion technique against the gram positive and gram-negative strains and the results so obtained were compared with the standard antibiotic

Screening for Antimicrobial activity

The antimicrobial activity of all the synthesized compounds (BA1-BA5) was determined by well plate or agar diffusion method [13]. The medium used were double strength nutrient broth (Hi-Media) for antibacterial activity. The in-vitro antibacterial activity was carried out against 24 h old cultures of bacterial strain. The different strains of bacteria (gram positive bacteria; Bacillus subtilis and gram negative bacteria; Escherichia coli). Pure cultures of the test microorganisms were procured from Institut of Microbial Technology, Chandigarh and National Chemical Laboratory, Pune. The compounds were tested at the concentrations of 10 and 50 µg/ml and solutions were prepared by dissolving in dimethyl sulfoxide (DMSO). The petridishes used for antibacterial screening were incubated at 37±1ºC for 24 h. Nutrient agar plates were prepared aseptically by pouring agar in to petridish to get a thickness of 5-6 mm. The plates were allowed to solidify (plates were inverted to prevent the condensate falling on the agar surface) on a perfectly horizontal surface so that the agar occupies a constant depth throughout the petry dish. The plates were dried at 37 °C before inoculation. The test organisms were poured in to the surface of the medium and remove excess of the fluid. With the petry dish lid in plate, allow 3-5 mins for the surface of the agar to dry. After that, bore has been made using a sterile cork borer about 10 mm in diameter.

TABLE 3: Evaluation of In-vitro antibacterial activity of 2-OH-1,4-naphthoquinone derivatives

COMPOUND CODE	ANTIBACTERIAL ACTIVITY (Zone of Inhibition in mm)
	GRAM POSITIVE		GRAM NEGATIVE
	*Bacillus substilis*		*Escherichia coli*
	Concentration (µg/ml)
	10	50	10		50
BA1	11	13	10		12
BA2	15	17	14		16
BA3	12	15	11		14
BA4	16	17	15		16
BA5	15	17	13		16
STANDARD (Gentamycin; 10 µg/ml)	20		17.5
CONTROL	-	-	-	-

(-) indicates no activity

The agar disc is placed by a vaccum device or a splayed-out pen nib. The drug solutions of 10 µg/ml and 50 µg/ml (test, standard, control) were applied in to the disc and incubate it aerobically at 37 °c for 18-24 h. After overnight incubation, observations were made for zone of inhibition of the test compounds with control by measuring the diameter of each zone of inhibition in mm.The end point of inhibition is where growth starts. The results were compared with the standard drug Gentamycin at 10µg/ml by measuring zone of inhibition in mm. The antibacterial results were presented in Table 3.

Fig.1: Effect of test compounds (10 and 50μg/ml) on in Vitro antifungal activity

RESULT AND DISCUSSION:

The results of antimicrobial activity indicated that all the compounds BA1-BA5 were found to be very good activity against all bacterial strains used in this in-vitro bioassay at the concentration range of 10 and 50 µg/ml. BA2, BA4 and BA5 shows significant activity when compared to standard drug Gentamycin at 10 µg/ml. BA1 and BA3 shows moderate activity against gram positive Bacillus subtilis (ATCC-6633) at 10 and 50 µg/ml concentrations. On the other hand against Escherichia coli, compounds BA2, BA4 and BA5 shows significant activity when compared to standard drug Gentamycin at 10 µg/ml. BA1 and BA3 shows comparatively moderate activity with that of standard drug Gentamycin at 10 µg/ml. The electron withdrawing groups present (NO₂, Br, F) at BA2, BA4 and BA5 were the potential activators for the antibacterial efficacy than that of the other (BA1 and BA3) molecules.

CONCLUSION:

In the present attempt, all the newly synthesized substituted naphthoquinones BA1-BA5 are nothing but Lawsones. Therefore, the probable mechanism of antibacterial activity is to inhibit the growth of other microorganisms. The results of in-vitro antimicrobial susceptibility testing are valuable for selecting chemotherapeutic agents active against the infecting organism. Extensive work has been performed in an effort to standardize the testing methods and improve the clinical predictive values of the results. Thus, the beta-lactum antibiotic such as gentamycin (aminoglycoside) generally act as bactericidal agents. These antibiotics exert their effort by passing through the bacterial outer membrane (in gram negative bacteria), cell wall and cytoplasmic membrane to the cytoplasam, Where they inhabit bacterial protein synthesis by irreversibly binding to the 30S ribosomal proteins. This attachment to the ribosome have two effects: production of abberent proteins as the result of misreading of the messenger RNA (mRNA), and interruption of proteins synthesis by causing the premature release of the ribosome from mRNA.

ACKNOWLEDGMENT:

Author would like to express their deepest gratitude to Grace College of Pharmacy, for providing all facilities required for my paper work.

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Received on 13.12.2013 Modified on 10.01.2014

Asian J. Research Chem. 7(3): March 2014; Page 281-284