Synthesis of Novel Substituted 3,4-Dihydroquinoxaline Derivatives as Antimicrobial Agents

 

Charoo S. Garg*, Jimit S. Patel and Dhrubo Jyoti Sen

Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Arvind Baug, Mehsana-384001, Gujarat

*Corresponding Author E-mail: charoo23pharma@gmail.com

ABSTRACT:

As a part of research project on the synthesis of number of substituted quinoxaline derivatives with electron donating as well as electron withdrawing groups has been done and evaluated them for antibacterial and antifungal activity. First of all different acetanilides were prepared by taking chloro acetyl chloride and substituted anilines. Then they were condensed with benzene diamine to form novel quinoxaline derivatives. Further quinoxalines were benzoylated by using 4-methoxy benzoyl chloride and 4-chloro benzoyl chloride. The purity of all compounds have been checked by the TLC monitoring and the confirmation of the structure was checked by different spectral analysis like UV, IR, Mass and NMR and evaluated for antibacterial agent using ciprofloxacin as a standard drug and for antifungal activity using clotrimazole as a standard drug

.

 

INTRODUCTION:

The heterocycles are those cyclic organic compounds in which N, O or S elements have been replaced by one or more of the ring carbon atoms. The sulphur and nitrogen atoms are important components of functional materials since heteroatoms present in their rings stabilize ion radical species and extended π-conjugation facilitate in decreasing columbic repulsion. Quinoxaline is one of the benzo-fused six-membered heterocycles shown in figure.¹

 

Quinoxaline

 

Quinoxaline is an important part of antibiotic echinomycin.² Quinoxaline holds up as a core unit in number of biologically active compounds includes anticancer, antibacterial, antiviral and anti-inflammatory, anti HIV activity.³

 

 

Quinoxaline displays a wide spectrum of potential pharmacological activities for examples, imidazo-quinoxalines ribonucleosides as antiviral.⁴ Pyrazolo-quinoxaline showed a relatively high antibacterial activity and also quinoxaline-1,4-di-N-oxides are used for the treatment of bacterial disease.^5,6 Also some 6(7)-substituted-3-methyl-2-phenylthio-quinoxaline-1,4-di-N-oxides display good antitubercular activity.⁷ Quinoxaline derivatives such as pyrazolo-quinoxaline is used as a antifungal agents.⁸ The importance of quinoxalines as pharmaceutical agents was recognized by the Brimonidins. The drug acts through reducing the intraocular pressure, thus alleviates the symptoms of glaucoma.⁹ Recently, a new series of 2-alkylcarbonyl and 2-benzoyl-3-trifluromethylquinoxaline-1,4-di-N-oxide derivatives have been recognized and evaluated for in-vitro antitumor activity against a 3-cell line panel (breast, lung, CNS). For the above reasons novel series of quinoxaline derivatives were planned to synthesize.

 

EXPERIMENTAL:

The entire chemicals were supplied by S.D. Fine Chem. (Mumbai), Finar Chem. Ltd (Ahmadabad) and Loba Chemie Pvt. Ltd. (Mumbai). Melting points were determined by open tube capillary method and were uncorrected. Purity of compounds was checked by thin layer chromatography (TLC) on silica gel G in solvent system hexane-ethyl acetate (3:2), the spots were located under iodine vapours or UV light. IR spectra of all compounds were recorded on FT-IR 8400S Shimadzu spectrophotometer using KBr. Mass spectra were obtained using 2010EV LCMS Shimadzu instrument.

 

General procedure for the synthesis of 2-chloro-N-(4-substituted phenyl)-acetamide:

(0.01mole) of p-substituted aniline was dissolved in acetone and kept under stirring in cold conditions. Now (0.01mole) chloro acetyl chloride was added drop wise. Mixture was allowed to stir for half an hour. White precipitates appeared. Cold water was added; the precipitates were filtered and recrystallized with methanol.¹⁰

Compounds: 3 (R=Cl, NO₂, CH₃)

 

General procedure for the synthesis of N-(4-substitutedphenyl)-3,4-dihydroquinoxalin-2-amine:

To a solution of benzene-1,2-diamine (0.01mol) in glacial acetic acid (10ml GAA in 30ml water), solution of acetamide in rectified spirit was added and refluxed for about 18-20 hrs. On adding water precipitates were obtained. They were washed with water, dried and hexane wash were given and recrystallized in ethanol.¹¹

Compounds: 5-a, 5-b, 5-c

 

General procedure for the synthesis of {3-[(4-substitutedphenyl)-amino]-quinoxalin-1(2H)-yl}(4-     substituted phenyl)-methanone:

(0.1 mole) of quinoxaline was taken in a clean and dry beaker. Now 0.1 mole of 4-substituted benzoyl chloride was added. The mixture was triturated for about 10-15 mins with the help of glass rod. Now crushed ice was added and trituration was continued. Solid product was obtained. After filtration recrystallization was done in methanol.¹²

Compounds: 5-d, 5-e, 5-f and 5-g, 5-h, 5-i

Following compounds were synthesized:

 

                                       

5-a: N-(4-chlorophenyl)-3,4-dihydroquinoxalin-2-amine

5-b: {3-[(4-chlorophenyl)-amino]-quinoxalin-1(2H)-yl}(4-methoxyphenyl)-methanone

                                       

5-c: (4-chlorophenyl){3-[(4-chlorophenyl)amino]-quinoxalin-1(2H)-yl}methanone

5-d: N-(4-nitrophenyl)-3,4-dihydroquinoxalin-2-amine

                                       

5-e: (4-methoxyphenyl){3-[(4-nitrophenyl)amino]-quinoxalin-1(2H)-yl}methanone

5-f: (4-chlorophenyl){3-[(4-nitrophenyl)amino] quinoxalin-1(2H)-yl}methanone                                     

             

5-g: N-(4-methylphenyl)-3,4-dihydroquinoxalin-2-amine

                              

5-h: (4-methoxyphenyl){3-[(4-methylphenyl)amino]-quinoxalin-1(2H)-yl}methanone

5-i: (4-chlorophenyl){3-[(4-methylphenyl)amino]-quinoxalin-1(2H)-yl}methanone

 

Scheme:

 

 

Table: 1-Physical Characteristics of synthesized compounds

Compound Code	Molecular Formula	Molecular Weight (g/mol)	Melting Point (oC)	% Yield (%w/w )	Rf
5-a	C14H12ClN3	257.72	150-152	44	0.48
5-b	C22H18ClN3O2	390.86	170-172	82	0.6
5-c	C21H15Cl2N3O	396.27	155-160	79	0.35
5-d	C14H12N4O2	268.27	142-144	23	0.67
5-e	C22H18N4O4	402.4	150-155	71	0.57
5-f	C21H15ClN4O3	406.8	160-165	54	0.48
5-g	C15H15N3	237.3	140-143	51	0.52
5-h	C23H21N3O2	371.43	146-150	88	0.5
5-i	C22H18ClN3O	375.85	142-145	81	0.32

 

Table: 2-Spectral data of synthesized compounds

Compound Code	UV (λmax, nm)	IR ( cm-1 )	Mass (m/z)	NMR (δ, ppm)
5-a	535	3182 (-NH), 1647 (-C=N), 1176 (-C-N), 775(Cl)	257 [M+] 259 [M+2]	____
5-b	627	3170 (-NH), 1685 (-N-C=O), 1427 (-OCH3 ), 771 (p-Cl)	391.2[M+] 393.2[M+2]	____
5-c	536	3190.04 (-NH), 1689.96 (-N-C=O), 744.47(p-Cl), 1627.81 (-C=N)	---	7.23-8.5(m,12H,ArH), 6.23(s,1H,NH), 3.35(s,2H,CH2)
5-d	405	3483(-NH), 1670.23 (-C=N), 1589.23 (-NO2), 1334.5,(C-N)	269 [M+]	____
5-e	569	3166.90(-NH),1674.10(-N-C=O),1604.66(C=N), 1299.93(-OCH3) 1508.23(NO2)	402.1[M+]	____
5-f	554	3166.90(-NH),1677.95(-N-C=O),1616.24(C=N), 1500.23(NO2), 752.19 (p-Cl)	---	7.22-9.07(m,12H,ArH), 6.2(s,1H,NH),3.349(s,2H,CH2)
5-g	509	3179.63 (-NH), 1601.76 (-C=N)	237.1[M+]	____
5-h	620	3124.47(-NH), 1685.67 (-N-C=O), 1604.66(-C=N), 1172.64 (-C-N)	371.1[M+]	____
5-i	535	3150(-NH), 1600(-C=N), 1176.50 (-C-N), 1685.67(-N-C=O)	375.1[M+] 377.1[M+2]	6.8-8.27(m,12H,ArH), 6.26(s,1H,NH), 3.349(s,2H,CH2), 2.27(s,3H,CH3)

Table: 3-Log P value of Synthesized Compounds

Compound	Log P
5-a	2.44
5-b	3.61
5-c	4.21
5-d	1.90
5-e	3.08
5-f	3.68
5-g	1.91
5-h	3.08
5-i	3.68
Ciprofloxacin	-0.81
Clotrimazole	3.5

 

Figure: 1 Histogram of Log P

 

Biological Evaluation:

Biological evaluation holds a great significance in screening the new chemical entities. The newly synthesized compounds were screened for antifungal and antimicrobial activity.

 

ANTIMICROBIAL ACTIVITY^13-17::

In our current study, evaluation of antimicrobial activity was carried out by using the methods mentioned below. Here responses of microorganisms to the synthesized compounds were measured with that of the standard reference drug. The standard reference drug used was ciprofloxacin.

 

Antibacterial Activity:

The microbiological assay is based upon a comparison of inhibition of growth of micro-organisms by measured concentrations of test compounds with that produced by known concentration of a standard antibiotic. Two methods generally employed are turbidometric (tube-dilution) method and cylinder plate (cup-plate) method. In the turbidometric method inhibition of growth of microbial culture in a uniform dilution of antibiotic in a fluid medium is measured. It is compared with the synthesized compounds. Here the presence or absence of growth is measured. The cylinder plate method depends upon diffusion of antibiotic from a vertical cylinder through a solidified agar layer in a petri dish or plate to an extent such that growth of added micro-organisms is prevented entirely in a zone around the cylinder containing solution of the antibiotics. The cup-plate method is simple and measurement of inhibition of microorganisms is also easy. Here this method has been used for antibacterial screening of the test compounds.

 

Name of Microorganisms:

Ø  Gram +Ve microorganisms

Staphylococcus aureus (MTCC  No. 96)

Bacillus subtilis (MTCC No. 121).

Ø  Gram -Ve microorganisms

Escherichia coli (MTCC No. 521).

 

Preparation of medium:-

Ø  Nutrient agar 2%

Ø  Peptone 1%

Ø  Beef extract 1%

Ø  Sodium chloride 0.5%

Ø  Distilled water up to 100ml

 

All the ingredients were weighed and added to water. This solution was heated on water bath for about one and half-hour till it became clear. This nutrient media was sterilized by autoclave.

 

Apparatus:-

All the apparatus like petri dishes, pipettes, glass rods, test-tubes were properly wrapped with papers and sterilized in hot air oven.

 

Agar plate disc diffusion method:

Ø  The antibacterial activity was assayed by agar plate disc diffusion method at the concentration of 50 μg per disk.

Ø  All the synthesized compounds were tested in-vitro for their antibacterial activity against microorganisms such as Staphylococcus aureus, Bacillus subtilis (gram-positive), Escherichia coli (gram negative) strains.

Ø  Each test compounds were dissolved in dimethylsulphoxide (DMSO) to get a concentration of 10 mg/mL.

Ø  The disc (6 mm in diameter) was impregnated with 5 μL of each test solution to get 50 μg/disc, air dried and placed on the agar medium, previously seeded with 0.2 mL of broth culture of each organism for 18 hours. 

Ø  The plates were incubated at 37°C for 24 hours and the inhibition zones measured in mm.

Ø  Discs impregnated with DMSO were used as a control and ciprofloxacin discs as antibacterial reference standard.

 

ANTIFUNGAL ACTIVITY^13-17::

Principle:

The antifungal activity of all newly synthesized quinoxaline derivatives were examined against Candida albicans. Antifungal screening of all the derivatives was done by using filter disc method. Clotrimazole was used as a standard drug. Activity of the compounds was recorded by measuring the zone of inhibition in mm and compared with the standard zone of inhibition produced by clotrimazole. This determination indicates whether the organism was sensitive or resistant to the compound.

 

Materials used

·         Test organisms: Candida albicans was used for the determination of the activity.

·         Growth Media: The activity was conducted on the Sabouraud dextrose agar media.

 

Composition

Ø  Enzymatic digest of Casein 5g

Ø  Enzymatic digest of Animal Tissue 5g

Ø  Dextrose 20g

Ø  Final pH 5.6 ±0.2 at 25 °C

Ø  Purified water 1000ml

 

Apparatus:

Ø  Petri plate: Glass plate, which was previously sterilized by Dry Heat Sterilization was used.

Ø  Pipette: Micropipette was used for adding the required concentration of the analogues to the plates.

Ø  Glass wares: 500ml conical flask and test tubes were used.

Ø  Compounds screened: All the synthesized quinoxaline derivatives.

Ø  Solvent used: Dimethyl sulfoxide.

Ø  Standard used: Clotrimazole

 

Preparation of standard solution:

The standard drug clotrimazole was dissolved in appropriate quantity of ethanol to obtain the concentration range of 500, 750 and 1000μg/ml and the zone of inhibition was checked.

 

Preparation of test solution: Specified quantity (100mg) of the compound was accurately weighed and dissolved in 100ml of DMSO to get the 1000μg/ml stock solution. Further dilution was made to obtain the concentration in the range 500μg/ml, 750μg/ml and 1000μg/ml.

 

Procedure

30g of the medium was suspended in 1000ml of purified water. The mixture was allowed to boil till it forms a homogeneous solution. The medium was autoclaved at 121°C for 15 minutes at 15psi.

 

Media was cooled to the temperature of approximately 40°C temperature and microorganisms were inoculated to the media. 150ml was transferred to a petri plates aseptically. Two such plates were prepared for each organism and plates were allowed to cool for 20 minutes.

 

Compounds were dissolved in DMSO and diluted in same to get concentration of 500μg/ml, 750μg/ml and 1000μg/ml.

 

Both high and low strength discs were applied for each compound to be tested and the organism is reported as being sensitive if clear zone appears around both discs.

 

 

 

Figure: 2 Histogram of antibacterial activity

 

Figure: 3 Histogram of antifungal activity

 

 

 

Table: 4-Screening of antibacterial activity of synthesized compounds

 

Table: 5-Screening of antifungal activity of synthesized compounds

 

RESULT AND DISCUSSION:

The physicochemical parameters of the synthesized compounds were shown in Table: 1 and spectral datas in Table: 2. Antimicrobial and antifungal screening datas were shown in Table: 4 and Table: 5 respectively. All the synthesized compounds were screened for their anti-microbial activities against S.aureus, B.subtilis and E.coli and for anti-fungal activities against Candida albicans. Compounds 5-a, 5-b, 5-d, 5-e and 5-f showed anti-fungal activity but less potent as compared to standard reference drug clotrimazole. Compounds 5-d, 5-e and 5-f showed good anti-bacterial activity but less potent as compared to standard reference drug ciprofloxacin. Compounds 5-c, 5-g, 5-h and 5-i were found to be very least potent towards anti-fungal activity. Log P of all the compounds were displayed in Table: 3 and histogram in Figure: 1. Rest all the synthesized compounds were found to be very less potent towards anti-bacterial activity as compared to standard reference drug ciprofloxacin. Figure: 2 represents the histogram of antibacterial activity of the synthesized compounds for S.aureus, B.subtilis and E.coli and Figure: 3 represents the histogram of antifungal activity of the synthesized compounds for Candida albicans.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Quality Assurance Department of Shri Sarvajanik Pharmacy College, Mehsana for UV and FTIR spectras, Oxygen Heathcare, Ahmedabad for Mass spectras and Punjab University, Chandigarh for NMR spectras.

 

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Received on 15.05.2012        Modified on 08.06.2012

Accepted on 20.06.2012        © AJRC All right reserved