INTRODUCTION:

Benzoxazine derivatives are an important class of heterocyclic rings which have gained great significance due to their wide range of biological activities but still remains little studied. Benzoxazine are heterocyclic systems with benzene ring fused to a six membered ring with one oxygen and one nitrogen atom. A wide spectrum of benzoxazines from herbicides, fungicides to therapeutically usable drugs has been studied in the recent past. In view of this we have planned to synthesize different substituted benzoxazine derivatives.

In the present study (8E) -8- benzylidene -6- Methoxy -4- phenyl -1, 4, 5, 6, 7, 8-hexahydro-2H-3, 1-benzoxazin-2-imine derivatives 4(a-f) (Scheme-1) have been synthesized from 2, 6-diarylidene-4-methoxy cyclohexanone 3(a-f). 4-methoxy cyclohexanone has been reacted with different aromatic aldehydes which is an example for Claisen-Schmidt condensation. According to literature survey benzoxazine derivatives are in the stage of development and can be used as potential new drugs in future. These compounds have been reported to posses antiplatelet^1-2, anti-microbial^3-5, antitubercular⁶, anti-inflammatory⁷ and antidiabetic⁸. Hence the present study was aimed to synthesize some new benzoxazine derivatives and explore their plausible antimicrobial properties.The compounds were characterized by IR, ¹H NMR and mass spectroscopy.

MATERIALS AND METHODS:

Melting points of the newly prepared compounds were determined by using DBK. Instruments melting point apparatus and are uncorrected. The Infra Red spectra of the newly prepared compounds were recorded on BRUKER Attenuated Total Reflectance Infra Red (ATR-IR) spectrophotometer. The ¹H NMR spectral analysis was done using CDCl₃ as solvent on INOVA 400 MHz NMR spectrometer. Electron Impact mass spectra (EIMS) were recorded on VG Autospec MS. Purity of the compounds and reaction completion was checked by using TLC. Ethylacetate and chloroform in the ratio 8:2 was used as mobile phase for elution and the spots were detected in iodine chamber. Bacterial and fungal strains were purchased from IMTECH, Chandigarh, India. Ampicillin and Amphotericin B were procured from Sigma Aldrich, Bangalore,

EXPERIMENTAL:

Synthesis Procedure:

Step 1: General method of synthesis of 2,6-diarylidene-4-methoxycyclohexanones, 3(a-f).

To a mixture of 30ml of 10% of sodium hydroxide and 4-methoxy cyclohexanone (0.01ml) in 50ml of ethyl alcohol, aromatic aldehyde (0.02ml) was added and stirred at 20-25⁰C for 2 h. Later, reaction mixture was kept in an ice chest over night. The product obtained was filtered, washed with ice cold water followed by ice cold ethanol, dried and recrystallized from dimethylformamide⁹.

Step 2: General procedure for the preparation of (8E)-8-benzylidene-6-methoxy-4-phenyl-1,4,5,6,7,8-hexahydro-2H-3,1-benzoxazin-2-imine, 4(a-f).

A mixture of 2,6-darylidene-4-methoxycyclohexanones (0.01mol), urea (0.015mol) and potassium hydroxide (0.01mol) dissolved in 10ml of water and refluxed in isopropyl alcohol for 16 h. Later, solvent was removed by distillation and residue obtained was treated with ice cold water, filtered, dried and recrystallized from ethanol.

Antimicrobial Screening:

All synthesized compounds were evaluated for in vitro antibacterial activity by using two Gram-positive organism viz., Staphylococcus aureus (MTTC 3160) and Bacillus subtilis (MTCC 441), two Gram-negative organisms, viz., Escherichia coli (MTCC 443) and pseudomonas aeruginosa (MTCC 424) and antifungal activity by using two fungal organism viz., Aspergillus niger (MTCC 282) and Candida albicans (MTCC 227) by cup plate method at 100µg/0.1ml. Ampicillin and Amphoterician B were used as standard reference for comparing the antibacterial and antifungal activity respectively.

Cup plate method:

The diameter of zone of inhibition of all synthesized compounds were determined by cup plate method¹⁰ .A test tube containing sterile liquefied soft agar (2% in distilled water, 6ml) was maintained to 50⁰C and inoculated with 0.2ml suspension of the test culture, mixed well and transferred in the pre-sterilized Petri-dishes having sterile nutrient agar medium and allowed to solidify for 5.0 min. The cup borer was sterilized by dipping into absolute ethanol and flaming it and allowed to cool it down. The cup borer was used to prepare cups of 8mm diameter in the agar media. Standards and test solutions of synthesized compounds were prepared at a concentration of 100µg/0.1ml for antibacterial and antifungal assay with DMF. A 0.1 ml of test as well as standard solution was added to the cups with a micropipette. Then the test sample and standard were allowed to diffuse for 1h in refrigerator at 4-5⁰C. The plates were incubated in upright position at 37±1⁰C/24h and 25⁰C/48 h for bacteria and fungi respectively.DMF was used as blank. The diameter of zone of inhibition surrounding each cup was measured in mm and the data was shown in table 2.

Minimum Inhibitory Concentration Method:

The minimum inhibitory concentrations of all synthesized compounds were determined by serial dilution method¹¹. The lowest concentration of a compound preventing the appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). All the synthesized compounds were dissolved separately to prepare stock solution containing 1000µg/ml in DMF. The test compounds (4a-f), were dissolved in 2ml of the DMF and 1ml of this solution was sterile nutrient broth media, thus 1ml of the resulted solution gives 1000µg/ml. 1ml of the above solution was transferred to 1ml of DMF to give half concentration of first. Thus sequential concentration like 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90 and 1.95µg/ml were prepared in a similar manner up to eight dilutions. From 8^th dilution; 1ml of the solution is removed and discarded. The tubes were mixed well after each addition. The experimental MIC values are presented in table 3.

Scheme 1 : Synthesis of benzoxazine derivatives (4a-f)

RESULTS AND DISCUSSION:

The structures of newly synthesized benzoxazines (4a-f) were characterized by IR, proton NMR and Mass spectroscopy. Physical data of the compounds 4(a-f) was shown in the table 1. The compounds substituted with electron withdrawing groups enhanced the activity namely 4b and 4c found to be more active against the selected bacteria. The presence of lipophilic group greatly enhanced the antifungal activity of compound 4b against A.niger. The formation of final products 4a, 4b, 4c, 4d, 4e and 4f were confirmed by IR, ¹H-NMR and mass spectra. The interpreted data is mentioned below.

4a: ATR-IR (v_max cm⁻¹): 3228 –imine NH stretching; 3138 cyclic NH stretching; 1082 -C-N stretching; 1538,1486 -C=C stretching; 1593 -C=N –stretching; 2920, 2850 -C-H stretching; ¹HNMR – δ3.2(s, (OCH₃), 3H), δ2.0 (d, CH₂, 2H), δ2.4 (d, CH₂, 2H), δ2.9 (m, CH, 1H) δ8.4 (s, imine NH, 1H), δ8.0 (s, cyclic NH, 1H), δ6.6-7.2 (m, ArH, 8H), -H, δ6.3 (s, benzylic1H),δ 3.8(s, (OCH₃)₂, 6H), δ4.9 (s, (CH-O)_,1H) MS-m/z 407(M+).

4b: ATR-IR (v_max cm⁻¹): 3299 –imine NH stretching; 3163 cyclic NH stretching; 1073 -C-N stretching; 1514,1414 -C=C stretching; 1583 -C=N –stretching; 2963, 2866 -C-H stretching; ¹HNMR – δ3.8(s, (OCH₃), 3H), δ2.0 (d, CH₂, 2H), δ2.2 (d, CH₂, 2H), δ 8.3 (s, imine, 1H), δ7.9 (s, cyclic NH, 1H), δ6.8-7.4 (m, ArH, 8H), δ6.4 (s, benzylic-H, 1H),δ 4.8(s, (CH-O), 1H), MS-m/z 415(M+).

4c ATR-IR (v_max cm⁻¹): 3230 –imine NH stretching; 3162 cyclic NH stretching; 1090 -C-N stretching; 1514,1411 -C=C stretching; 1583 -C=N –stretching; 2938, 2867 -C-H stretching; ¹HNMR – δ3.4(s, (OCH₃), 3H), δ2.2 (d, CH₂, 4H), δ2.4 (d, CH₂, 1H), δ 7.6 (s, imine, 1H), δ7.2-7.4 (m, ArH, 8H), δ6.6 (s, benzylic-H, 1H),δ 4.9(s, (CH-O), 1H), MS-m/z 436(M+).

4d: ATR-IR (v_max cm⁻¹): 3274 –imine NH stretching; 3124 cyclic NH stretching; 1061 -C-N stretching; 1517,1426 -C=C stretching; 1593 -C=N –stretching; 2980, 2889 -C-H stretching;.¹HNMR – δ3.1 (s, (OCH₃), 3H), δ1.8 (d, CH₂, 2H), δ2.2(d, CH₂, 2H), δ3.4 (m, CH, 1H) δ 9.8 (s, imine, 1H), δ7.8 (s, cyclic NH, 1H), δ6.7-7.4 (m, ArH, 6H), δ6.6 (s, benzylic-H, 1H), δ 3.8(s, (OCH₃)₂, 6H), δ4.2 (s,(OCH₃)_2,6H), δ 5.0(s, (CH-O), 1H), MS-m/z 467(M+).

4e: ATR-IR (v_max cm⁻¹): 3284 –imine NH stretching; 3164 cyclic NH stretching; 1013 -C-N stretching; 1469,1416 -C=C stretching; 1556 -C=N –stretching; 2914, 2868 -C-H stretching; ¹HNMR – δ2.6 (s, (OCH₃), 3H), δ2.1 (d, CH₂, 2H), δ2.4 (d, CH₂, 2H), δ3.2 (m, CH, 1H) δ 7.6 (s, imine, 1H), δ7.4 (s, cyclic NH, 1H), δ6.6 (s, 2H ArH), δ6.5 (s, 2H ArH, ),δ 3.0(s, (OCH₃)₂, 12H), δ3.7 (s,(OCH₃)_2,6H), δ 4.8(s, (CH-O), 1H), MS-m/z 527(M+).

4f: ATR-IR (v_max cm⁻¹): 3209 –imine NH stretching; 3128 cyclic NH stretching; 1072 -C-N stretching; 1471,1426 -C=C stretching; 1533 -C=N –stretching; 2957, 2847 -C-H stretching;¹HNMR – δ2.8 (s, (OCH₃), 3H), δ1.3 (m, (CH₂)₂, 4H), δ3.0 (m, CH, 12H) δ 9.7 (s, imine, 1H), δ7.0-8.0 (m, ArH, 8H), δ6.2 (s, benzylic-H, 1H), δ 4.8(s, (CH-O), 1H), MS-m/z 434(M+).

Table 1: Physical data of the compounds 4(a-f)

Compound Name	R	Molecular formula	Molecular Weight	M.P (⁰C)	% Yield	Rf value
4a	-4-OCH₃	C₂₄H₂₆N₂O₄	407	120-121⁰C	86	0.82
4b	-4-CL	C₂₂H₂₀Cl₂N₂O₂	415	109-110⁰C	92	0.72
4c	-3-NO₂	C₂₂H₂₀N₄O₆	436	217-218⁰C	73	0.65
4d	-3,4(OCH₃)₂	C₂₆H₃₀N₂O₆	467	99-100⁰C	81	0.78
4e	3,4,5(OCH₃)₃	C₂₈H₃₄N₂O₈	527	199-200⁰C	85	0.85
4f	4-N-(CH₃)₂	C₂₆H₃₂N₄O₂	434	201-202⁰C	78	0.64

Table-2: In vitro antimicrobial activity of the newly synthesised compounds (4a-f)

Compound. no.	Zone of Inhibition in mm
	Gram-positive Bacteria		Gram-negative Bacteria		Fungi
	*S.aureus*	*B.subtilis*	*E.coli*	*P.aeruginosa*	*A.niger*	*C.albicans*
4 a	24	25	18	17	13	14
4 b	28	26	25	24	16	13
4 c	20	22	24	25	13	12
4 d	18	16	09	10	10	11
4 e	19	15	15	13	11	10
4f	14	17	18	16	12	13
Ampicillin	37	31	32	30	--	--
Amphotericin-B	--	--	--	--	18	17

Table-3: MIC of the synthesised compounds (4a-f)

Compound. no.	Minimum Inhibitory Concentration (MIC) in µg/ml
	Gram-positive Bacteria		Gram-negative Bacteria		Fungi
	*S.aureus*	*B.subtilis*	*E.coli*	*P.aeruginosa*	*A.niger*	*C.albicans*
4 a	62.5	31.25	250	62.5	125	125
4 b	7.81	7.81	7.81	31.25	125	31.25
4 c	31.25	31.25	500	125	250	62.5
4 d	62.5	62.5	62.5	62.5	62.5	500
4 e	62.5	62.5	125	250	500	62.5
4f	31.25	62.5	31.25	125	250	125
Ampicillin	1.95	1.95	7.81	15.62	--	--
Amphotericin-B	--	--	--	--	3.90	7.81

CONCLUSION:

A new series of 3,1-benzoxazines were synthesized and characterized by IR, NMR and Mass spectroscopy, evaluated for antibacterial and antifungal activity. Benzoxazine nucleus was found to exhibit variable pharmacological activities. Modifications on benzoxazine nucleus have to be further continued to develop potentially new therapeutic agents. Thus the research to explore benzoxazine nucleus needs to be everlasting. The compounds 4b and 4c found to be more active against the tested bacteria. The compounds 4a, 4d, 4e and 4f were found to be moderate to weakly active against the tested bacteria and fungi.

ACKNOWLEDGEMENT:

The authors are grateful to, Indian Institute of Chemical Technology for providing the Spectras and Dr.A.Venkateshwar Reddy, Principal, Anwarul Uloom College of Pharmacy, Hyderabad, for providing lab facilities to carry out this work.

REFERENCES:

1. Saleh Ihmaid, Jasim Al-Rawi and Christopher B. Synthesis, structural elucidation and DNA-dependent protein kinase and antiplatelet studies of 2-amino-[5,6,7,8-mono and 7,8-di-substituted]-1,3-benzoxazines. European Journal of Medicinal Chemistry.2010; 45(11): 4934-4946.

2. Saleh K Ihmaid et al. Synthesis, DNA-PK inhibition, anti-platelet activity studies of 2-(N-substituted-3-aminopyridine)-substituted-1,3-benzoxazines and DNA-PK and PI3K inhibition, homology modelling studies of 2-morpholino-(7,8-di and 8- substituted)-1,3- benzoxazines. European Journal of Medicinal Chemistry. 2012; 57(0): 85-101.

3. Thierry Bessonand Charles WR. Antimicrobial evaluation of 3,1-benzoxazin-4-ones,3,1-benzothiazin-4-ones, 4-alkoxyquinazolin-2-carbonitriles and N-arylimino-1,2,3-dithiazoles. Bioorganic and Medicinal Chemistry Letters. 1996; 6(19): 2343-2348.

4. Sayaji SD and Piste BP. Novel one-pot synthesis and anti-microbial activity of 6-chloro-2,4-diphenyl 3,4-dihydro-2H-1,3-benzoxazines derivatives. Internet J chem. Tech Research, 2013; 5(5): 2199-2203.

5. Waisser K et al. In vitro antifungal activity of 3-phenyl-2H-benzoxazine-2,4(3H)-diones. Folia Microbiologica. 2002; 47 (5):488-492.

6. Waisser K et al. Influence of the replacement of the oxo function with the thioxo group on the antimycobacterial activity of 3-aryl-6,8-dichloro-2H-1,3-benzoxazine-2,4(3H)-dinoes and 3-arylquinazoline-2,4(1H,3H)-diones. Farmaco.2001; 56(10):803-807

7. Mymoona Akhter et al. Synthesis of some new 3,4-dihydro-2H-1,3-benzoxazines under microwave irradiation in solvent-free conditions and their biological activity. Medicinal Chemistry Research. 2011; 20(8):1147-1153.

8. Madhavan GR et al. Dual PPAR-alpha and gamma activators derived from novel benzoxazinone containing thiazolidinediones having antidiabetic and hypolipidemic potential. Bioorganic Medicinal Chemistry Letters. 2006; 14(2);584-591.

9. Bharath Rathna Kumar P, Srinivasa Murthy M and Jayaveera KN. Synthesis, characterization and antimicrobial evaluation of 4-aryl-8-arylidene-5,6-dihydro-2-imino-6-methoxy-4H,7H-[3,1]benzothiazines. Indo American Journal of Pharmaceutical Research. 2014; 4(8):14-19.

10. Black JG. Microbiology principles and exploration. 4^th Ed, prentice-hall. New delhi, 1991; pp.163.

11. Goto S, Jo K, Kawakita T, Mitsuhashi S, Nishino T, Ohsawa N, et al. Determination method of minimum inhibitory concentrations. Chemother., 1981; pp.76-79

Received on 08.07.2019 Modified on 05.09.2019

Research J. Pharm. and Tech 2020; 13(3):1167-1170.

DOI: 10.5958/0974-360X.2020.00214.0