Synthesis and Anti - microbial Activity of Novel Isoxazoline Mannich base Derivatives containing Carbazole Ring.
V. Prabhakar1*, K. Sudhakar Babu1, L.K. Ravindranath1, P. V. Ramana Reddy2, J. Latha3
1Department of Chemistry, Sri Krishnadevaraya University, Anantapuramu, (A P) India.
2Department of Chemistry, Loyola Degree College, Sri Yogivemana University, Kadapa , (A P) India.
3Department of Bio-Technology, Sri Krishnadevaraya University College of Engineering and Technology,
S.K. University, Anantapuramu – 515003 (A.P) India
*Corresponding Author E-mail: Virupakshi.prabhakar@gmail.com
ABSTRACT:
A Novel Series of Compounds were synthesized by Condensation reaction of Carbazole aldehyde with Substituted Acetophenone derivatives 4 (a-d) in presence of alcoholic Sodium hydroxide Solution to get Chalcone Intermediates 5 (a-d), which were further treated hydroxyl amine hydrochloride in presence of Pyridine to get Iso Oxazoline Derivatives 6 (a-d). The latter were refluxed with Substituted Primary amines and formaldehyde for 8-10 hrs to get Mannich bases. The Structures of new compounds were confirmed by IR and 1H NMR and 13C NMR Spectral Data. Anti-bacterial and Anti-fungal Activities were evaluated and compared with the standard drugs, some compounds of the series Exhibited Promising Anti-microbial and Anti-fungal Activity Compared to Standard Drugs.
KEYWORDS: Chalcone, Isoxazoline, Carbazoles, Mannich Bases, Anti-bacterial, Antifungal Activity.
Heterocyclic Compounds have so far been Synthesized Mainly due to the wide range of Biological Activities[1. Much attention has paid to the synthesis of heterocyclic compounds bearing nitrogen and oxygen containing ring system, like isoxazoline mainly due to their higher pharmacological activity. Isoxazolines posses medicinal activities such as anti-inflammatory2, antibacterial, anticonvulsant3, antibiotic4, antitubercular5, anti-fungal6 and anxiolytic activity7. The Isoxazole nucleus is a prominent structural moiety found in numerous natural products and synthetic compounds with vital medicinal value8, also possess as anti-Influenza virus activity9. Isoxazoline derivatives controlled botrytis cinera on cucumbers10 has been found to have antiviral properties against herpes type 2 virus11. Penicillin derivatives containing isoxazole ring are found to be antibacterial12. Isoxazole derivatives are used as corrosion inhibitors for fuels and lubricants13.
Its derivatives also show a good potency in animal models of thrombosis14. In addition, isoxazoline derivatives have played a crucial role as intermediates in the organic synthesis of number of heterocyclic pharmacological active compounds.
Fig 1 Structure of Isoxazoline
Chalcones are convenient intermediate compounds for the synthesis of five-, six- and seven – membered heterocycles often exhibiting biological activity. Recently, fluorinated chalcone derivatives have been reported to posse’s anti-inflammatory activity due to their influence on nitric oxide production15.
It is known that introduction of fluorine atom into specific position of organic molecule may cause significant changes in the stability, lipophilicity and biological activities of the resulting molecules16. This has been attributed to the high electro negativity of the halogen, the strong C-F bond and the similar size of the halogen and hydrogen atoms17. For these reason great efforts has been placed on the development and evaluation of biologically active fluorinated materials18, particularly in the field of enzymatic inhibition19. The biological properties of multi fluorine containing compounds have been recently investigated. Owing to their unique properties, such as high thermal stability and lipophilicity, fluoro-organic compounds have been frequently used as biorelated material, medicine and agrochemicals 20,21.
Carbazole is one of Organic Hetero cyclic compound containing a di benzo pyrrole, also known as 9aza -fluorene. Carbazole was synthesized by Borsche-Drechsel Cyclization22. Carbazoles are a large and an interesting group of organic compounds which one can find pharmaceutical activity23, dyestuffs, plastics24-26, and known to possess mutagenic and toxice activities and also to be arecalcitrant molecule27. Carbazole derivatives showed anticancer28, antifungal29, anti malarial30, anti tumor (leuckemia, renal, colon), anti inflammatory, antiallergic antiviral, and anti hypertensive properties31-33. Due to their extensive biological activity carbazole derivatives and their chemistry have been studies at length. Keeping the above facts, we aimed to synthesize new carbazole derivatives to thus obtain new hetero cyclic system which is expected to possess characteristic of biological activites. Structure of Carbazole as shown below.
Fig 2 Structure of Carbazole
Encouraged by the diverse biological activities of isoxazoline compounds, it was decided to prepare a new series of isoxazoline derivatives. These derivatives contains Carbazole nucleus. Literature survey revealed that incorporation of Carbazole ring in isoxazoline Heterocyclic ring enhanced antibacterial and antifungal activity. In the present communication, chalcones (5a-d) were prepared by the action of substituted acetophenone derivatives (4 a-d) with carbazole aldehyde in the presence of aqueous solution of Sodium hydroxide and Ethanol at room temperature by Claisen-Schmidt condensation method. The synthesized chalcones further condensed with hydroxyl amine hydrochloride in presence of pyridine to obtained isoxazoline derivatives (6a-d). The isoxazoline derivatives were reacted with formaldehyde and primary amines to afford Mannich bases 8(a-h).
Scheme 1. The structures of all synthesized compounds were assigned on the basis of IR, Mass, 1H NMR spectral data and Elemental analysis. Further these compounds were subjected for antifungal and antibacterial activity.
MATERIALS AND METHODS:
Laboratory chemicals were provided by Rankem India Ltd. and Ficher Scientific Ltd. Melting points were determined by the open tube capillary method and are not correct. The purity of the compounds was determined by thin layer chromatography (TLC) plates (silica gel G) in the solvent system toluene: ethyl acetate (8:2). The spots were observed by exposure to iodine Vapours or by UV light or P-Anisaldehyde Stain Solution. The IR spectra were received by Perkine Elmer 1720 FT-IR spectrometer (KBr pellets). The 1H NMR and13 C NMR spectra were obtained by Bruker Advance II 400 spectrometer using TMS because the internal standard in CDCl3. Elemental analysis of the new synthesized compounds were obtained by Carlo Erba 1108 analyzer. General Information. Commercial chemicals were treated as follows: DMF, distilled from CaH2 and degassed (freeze and thaw) three times prior to use; THF, ether, hexanes distilled from Na/ benzophenone.
The synthesis of the compounds as per the following Scheme I given below.
The synthetic route was depicted in scheme I
The Title compounds 8(a-h ) were synthesised in Five sequential steps using different reagents and reaction conditions, the 8(a-h) were obtained in moderate yields. The structure were established by spectral (IR, 1H-NMR, 13C-NMR) and analytical data.
SYNTHETIC SCHEME - I
R = - Cl, -Br, -NO2, -OCH3 R1 = -CF3, -F
|
Reagents and Reaction conditions: (a) Methyl Iodide (Me-I), tetra butyl ammomium bromide, potassium hydroxide in acetone, Reflux,1 hr (b) DMF,POCl3,Dry CHCl3, 800C (c) NaOH, Ethanol, RT, 24–36 h. (d) Hydroxyl amine hydrochloride, dry ethanol, Pyridine (e) formaldehyde,Methanol, Reflux |
|
Compound |
8a |
8b |
8c |
8d |
8e |
8f |
8g |
8h |
|
R |
-Cl |
-Cl |
-Br |
-Br |
-NO2 |
-NO2 |
-OCH3 |
-OCH3 |
|
R1 |
-CF3 |
-F |
-CF3 |
-F |
-CF3 |
-F |
-CF3 |
-F |
POSSIBLE MECHANISM FOR FORMATION OF ISO OXAZOLINE HETEROCYCLIC RING 6(A-D) FORMATION
EXPERIMENTAL SECTION:
All reactions were carried out under argon in oven-dried glassware with magnetic stirring. Unless otherwise noted, all materials were obtained from commercial suppliers and were used without further purification. All solvents were reagent grade. THF was distilled from sodium benzophenone ketyl and degassed thoroughly with dry argon directly before use. Unless otherwise noted, organic extracts were dried with anhydrous Na2SO4, filtered through a fitted glass funnel, and concentrated with a rotary evaporator (20–30 Torr). Flash chromatography was performed with silica gel (200–300 mesh) by using the mobile phase indicated. The NMR spectra were measured with a 400 MHz Bruker Avance spectrometer at 400.1 and 100.6 MHz for 1H for 13C, respectively, in CDCl3 solution with tetra methyl silane as internal standard. Chemical shifts are given in ppm (δ) and are referenced to the residual proton resonances of the solvents. Proton and carbon magnetic resonance spectra (1H NMR and 13C NMR) were recorded using tetramethylsilane (TMS) in the solvent of CDCl3-d or DMSO-d6 as the internal standard (1H NMR: TMS at 0.00 ppm, CDCl3 at 7.26 ppm, DMSO at 2.50 ppm; 13C NMR: CDCl3 at 77.16 ppm, DMSO at 40.00 ppm).
General procedure for the preparation of 9-methyl-9H-carbazole (2)34:
Carbazole (10 m.mol), tetra butyl ammonium bromide (1 m.mol) and potassium hydroxide (40 m.mol) dissolved in acetone (20 ml) were added to a 100 ml three-necked flask, the mixture solution was stirred for 40 minutes. Methyl Iodide (12 m.mol) was added drop wise to the solution with constant stirring. The mixture was refluxed for 1 h. The reaction mixture was then poured into ice water (100 ml) with vigorous stirring to obtain a great deal of deposit. The mixture was Extracted with DCM, washed with water and the Purified by Column Chromatography to give N-Methyl carbazole (2).
Yield :68%.
1H-NMR (400 M.HZ, DMSO-d6): δ 7.3-8.4(8H,m,Ar-H), 3.9(3H,S, N-CH3)
IR(KBr,cm-1): 3110 cm-1 (Ar C-H stret), 1550 cm-1 (C=C Stret), 2900 cm-1 (SP3 C-H Stretch),1240 cm-1 (C-N Stretch) Wave numbers respectively.
General procedure for the preparation of 9-Methyl-9H-carbazole-3-carbaldehyde (3)35:
9-Methyl carbazole (2) (10 m.mol) was dissolved in dry DMF and dry CHCl3 (20 mL. 3:7) under anhydrous condition. It was cooled to 00 C, POCl3 (1.87 mL) was added drop wise for 30 min and stirring continued for 4 h at 800 C. After completion of reaction (TLC), the reaction mass was poured over crushed ice (50 g), basified with NaOH, extracted with chloroform and dried over anhydrous Na2SO4. Organic layer was concentrated and purified through silica gel column using chloroform as eluting solvent to yield product 3.
Yield: 55%; off white solid; mp: 76–78°C;
1H-NMR (400 M.HZ, CDCl3-d1): δ 7.3-8.4(7H, m, Ar-H), 3.9(3H,S, N-CH3), 9.9 (1H,S, H-C=O)
IR(KBr,cm-1): 3110 cm-1 (Ar C-H stret), 1550 cm-1 (C=C Stret), 2900 (SP3 C-H Stretch), 1725 cm-1 (C=O Stretch) Wave numbers respectively.
General procedure for the preparation of (E)-3-(9-methyl-9H-carbazol-2-yl)-1-(4-chloro/Bromo/Nitro phenyl)prop-2-en-1-one 5(a-d) [36] :
A mixture of Acetophenone Derivatives 4(a-c) (0. 01m. mol), Carbazole aldehyde (3) (0.1 m.mol) in ethanol (4 mL) and aqueous Sodium hydroxide (70%, 10 ml) was stirred and kept at room temperature for 12 h. The mixtures were poured on crushed ice and acidified with dil 2N.HCl. The precipitate obtained after acidification were filtered and washed thoroughly with distilled water till it is free from acid and dried. The dry residue was re crystallized from a suitable solvent. The physical and spectral data of the compounds are the following.
Table 1 Yields and Melting Points of Corresponding Compounds
(5 a-d) :
|
S.NO |
Yield (%) |
Melting Point (°C) |
|
5a |
80 |
122-124 |
|
5b |
78 |
153-154 |
|
5c |
75 |
186-187 |
|
5d |
70 |
126-127 |
Table: 2 IR(KBr,cm-1) data of Compounds 5 (a-d):
|
Compound |
vmax, cm-1 |
|
5a |
3110 cm-1 (Ar C-H stret), 2940 cm-1 (SP3 C-H Stretching), 1550 cm-1 (C=C Stret), 770 cm-1 (C-Cl Stretch) Wave numbers respectively. |
|
5b |
3110 cm-1 (Ar C-H stret), 2920 cm-1 (SP3 C-H Stretching), 1580 cm-1 (C=C Stret), 568 cm-1 (C-Br Stretch) Wave numbers respectively. |
|
5c |
3110 cm-1 (Ar C-H stret), 1610 cm-1 (C=C Stret), 2900 (SP3 C-H Stretch), 1525 and 1350 cm-1 ( two bands,N-O Stretch in –NO2 Group) Wave numbers respectively. |
|
5d |
3110 cm-1 (Ar C-H stret), 1610 cm-1 (C=C Stret), 2900 (SP3 C-H Stretch), 1150 cm-1 (C-O Stretch in Methoxy Group) Wave numbers respectively. |
Table: 3 1H –NMR data of Synthesised compounds 5(a-c):
|
Compound |
1H-NMR (CDCl3-d1) (δ ppm) |
|
5a |
δ 3.9(3H,S, N-CH3), 7.4-8.4(7H,m,Ar-H), 8(1H,d,J=14HZ,Beta alkene Proton from carbonyl group), 7.6(1H, d, J=14Hz,alpha alkene proton from carbonyl group), 7.9(2H,d,J=8HZ, meta to chloro group), 7.7(2H,d,J=8HZ,ortho to Chloro group ) |
|
5b |
δ 3.9(3H,S, N-CH3), 7.4-8.3(7H,m,Ar-H), 8(1H,d,J=14HZ,Beta alkene Proton from carbonyl group), ), 7.5(1H, d, J=14Hz,alpha alkene proton from carbonyl group), 7.9(2H,d,J=8HZ, meta to Bromo group), 7.8(2H,d,J=8HZ,ortho to Bromo group ) |
|
5c |
δ 3.9(3H,S, N-CH3), 7.4-8.4(7H,m,Ar-H), 8(1H,d,J=14HZ,Beta alkene Proton from carbonyl group), 7.5(1H, d, J=14Hz,alpha alkene proton from carbonyl group), 8.1(2H,d,J=8HZ, meta to nitro group), 8.5(2H,d,J=8HZ, ortho to nitro group). |
|
5d |
δ 3.9(3H,S, N-CH3), 7.4-8.4(7H,m,Ar-H), 8(1H,d,J=14HZ,Beta alkene Proton from carbonyl group), 7.6(1H, d, J=14Hz,alpha alkene proton from carbonyl group), 8.1(2H,d,J=8HZ, meta to Methoxy group), 7.2(2H,d,J=8HZ, ortho to Methoxy group). |
General procedure for the preparation of 3-(4-chloro/Bromo/Nitro/Methoxy phenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazole (6 a-d)37:
A mixture of chalcones (0.01 m.mole) and hydroxylamine hydrochloride (0.1 m. mole) in 10 mL pyridine was refluxed for 2hrs. On cooling the reaction mixture was poured over crushed ice and conc. HCl. The solid obtained was filtered, washed with water and crystallized from Ethanol Physical and analytical data of compounds are given in Table 4.
Table 4. Physical data of isoxazolines (6 a-d).
|
Compound code |
*M.P.oC |
**Rf |
% Yield |
|
6a |
185-188 |
0.5 |
72 |
|
6b |
135-138 |
0.52 |
75 |
|
6c |
142-145 |
0.42 |
65 |
|
6d |
156-158 |
0.45 |
76 |
*All melting points are uncorrected.
**Mobile phase- Chloroform: Methanol (8:2).
Recrystallization solvent: Ethanol.
Table 5. Spectral data of isoxazolines (6 a-d)
|
Compound code |
IR (KBr, cm-1) |
1H NMR (δ ppm) |
|
6a |
3119.60 (Ar -H), 1653.05(C=N), 1506.46 (C=C), 1352.14(C-O-N), 844.85 (C-Cl) |
7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –Cl group), 8 (2H, d, Ar-H, meta to –Cl group), 5.8(1H, dd, Hc), 3.85 (1H, dd, Ha), 3.6(1H, dd, Hb), 3.9(3H,S, N-CH3). |
|
6b |
3109.98 (Ar -H), 1610.22 (C=N), 1513.12(C=C), 1339.61 (C-O-N), 568 cm-1 (C-Br) |
7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –-Br group ), 7.7 (2H, d, Ar-H, meta to –Br group 5.95(1H, dd, Hc), 3.85 (1H, dd, Ha), 3.6(1H, dd, Hb), 3.8(3H,S, N-CH3). |
|
6c |
3088.95 (Ar -H), 1523.82 (C=N), 1344.43 (C-O-N), 1535 and 1360 cm-1 ( two bands,N-O Stretch in –NO2 Group) |
7.4-8.2(7H, m, Ar-H), 8.2(2H, d, Ar-H, Ortho to –NO2 group), 8.05 (2H, d, Ar-H, meta to – NO2 group), 5.90(1H, dd, Hc), 3.85 (1H, dd, Ha), 3.6(1H, dd, Hb), 3.8(3H,S, N-CH3). |
|
6d |
3108.95 (Ar -H), 1523.82 (C=N), 1344.43 (C-O-N), 1165 cm-1 (C-O Stretch in –Methoxy Group) |
7.4-8.2(7H, m, Ar-H), 7.2(2H, d, Ar-H, Ortho to –OCH3 group ), 8.05 (2H, d, Ar-H, meta to – OCH3 group), 5.90(1H, dd, Hc), 3.85 (1H, dd, Ha), 3.6(1H, dd, Hb), 3.9(3H,S,-OCH3) 3.8(3H,S, N-CH3). |
General procedure for the preparation of N-((3-(4-chlorophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8a), N-((3-(4-chlorophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-fluoroaniline (8b), N-((3-(4-bromophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8c), N-((3-(4-bromophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-fluoroaniline (8d), N-((5-(9-methyl-9H-carbazol-3-yl)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8e), 4-fluoro-N-((5-(9-methyl-9H-carbazol-3-yl)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-4-yl)methyl)aniline (8f), N-((3-(4-methoxyphenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8g), 4-fluoro-N-((3-(4-methoxyphenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)aniline (8h) [38] :
To a Solution of Compounds 6 (a-d) (0.01 m.mol) in methanol (10 mL), formaldehyde (0.02 m.mol) and corresponding Substituted Anilines 7(a-b) (0.02 m.mol) were added. The reaction mixture was refluxed for 7-10 hrs. The Solvent was distilled off and the residue was poured into ice water. The Precipitate Solid was filtered off, dried and re crystallised from Ethanol.
Table 6 Physical data of target compounds (8 a-h):
|
Comp-ound code |
Molecular formula |
Mole-cular weight |
Melting Point ( °C) |
Rf |
Yield (%) |
|
8a |
C30H23ClF3N3O |
534 |
85-87 |
0.64 |
65 |
|
8b |
C29H23ClFN3O |
484 |
55-57 |
0.62 |
70 |
|
8c |
C30H23BrF3N3O |
578 |
75-78 |
0.69 |
72 |
|
8d |
C29H23BrFN3O |
528 |
60-63 |
0.63 |
71 |
|
8e |
C30H23F3N4O3 |
545 |
45-47 |
0.65 |
80 |
|
8f |
C29H23FN4O3 |
495 |
58-60 |
0.63 |
76 |
|
8g |
C31H26F3N3O2 |
530 |
77-80 |
0.67 |
82 |
|
8h |
C30H26FN3O2 |
480 |
52-55 |
0.65 |
67 |
|
*All melting points are uncorrected. **Mobile phase- Benzene: Methanol (7:3). Recrystallization solvent: Ethanol . |
|||||
N-((3-(4-chlorophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8a)
IR (KBr, cm-1): N-H (3336.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1255), C-O-N (1181.44), C-Cl (827.49).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –Cl group ), 8 (2H, d, Ar-H, meta to –Cl group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.4 (2H, d, Ortho to –CF3 Group), 6.6 (2H, d, meta to –CF3 Group), 3.9(3H,S, N-CH3).
N-((3-(4-chlorophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-fluoroaniline (8b)
IR (KBr, cm-1): N-H (3302.29), Ar Stretch C-H (3006.29), Aliphatic C-H (2903.83), C=N (1526.15), C=C (1318.38), C-O-N (1178.98), C-Cl (821.21), C-F (1270).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –Cl group ), 8 (2H, d, Ar-H, meta to –Cl group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.38(2H,d,HN-CH2-CHISOXAZOLINE RING), 7 (2H, d, Ortho to fluoro Group), 6.8 (2H, d, meta to fluoro Group), 3.9(3H,S, N-CH3).
N-((3-(4-bromophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8c)
IR (KBr, cm-1): N-H (3345.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1255), C-O-N (1181.44), 588 cm-1 (C-Br) .
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –Br group ), 7.8 (2H, d, Ar-H, meta to –Br group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.4 (2H, d, Ortho to –CF3 Group), 6.55 (2H, d, meta to –CF3 Group), 3.9(3H,S, N-CH3).
N-((3-(4-bromophenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-fluoroaniline (8d)
IR (KBr, cm-1): N-H (3336.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1265), C-O-N (1181.44), 570 cm-1 (C-Br) .
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.6(2H, d, Ar-H, Ortho to –Br group ), 7.8 (2H, d, Ar-H, meta to –Br group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.05 (2H, d, Ortho to fluoro Group), 6.92 (2H, d, meta to fluoro Group), 3.9(3H,S, N-CH3).
N-((5-(9-methyl-9H-carbazol-3-yl)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8e)
IR (KBr, cm-1): N-H (3346.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1285), C-O-N (1181.44), 1535 and 1360 cm-1 ( two bands,N-O Stretch in –NO2 Group).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 8.2(2H, d, Ar-H, Ortho to –NO2 group ), 8.05 (2H, d, Ar-H, meta to – NO2 group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.4 (2H, d, Ortho to –CF3 Group), 6.55 (2H, d, meta to –CF3 Group), 3.9(3H,S, N-CH3).
4-fluoro-N-((5-(9-methyl-9H-carbazol-3-yl)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-4-yl)methyl)aniline (8f)
IR (KBr, cm-1): N-H (3336.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1265), C-O-N (1181.44), 1565 and 1340 cm-1 ( two bands,N-O Stretch in –NO2 Group).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 8.2(2H, d, Ar-H, Ortho to –NO2 group ), 8.05 (2H, d, Ar-H, meta to – NO2 group), 5.283(1H, s, HN-Ar, 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.4 (2H, d, Ortho to –CF3 Group), 6.55 (2H, d, meta to –CF3 Group), 3.9(3H,S, N-CH3).
N-((3-(4-methoxyphenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)-4-(trifluoromethyl)aniline (8g)
IR (KBr, cm-1): N-H (3336.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1265), C-O-N (1181.44), 2910 cm-1 (C-H Stretch in –OCH3 Group).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.1(2H, d, Ar-H, Ortho to – OCH3 group ), 7.9 (2H, d, Ar-H, meta to – OCH3 group), 8.1(1H, s, HN-Ar), 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7.4 (2H, d, Ortho to –CF3 Group), 6.50 (2H, d, meta to –CF3 Group), 3.73 (3H, S, -OCH3), 3.9(3H,S, N-CH3).
4-fluoro-N-((3-(4-methoxyphenyl)-5-(9-methyl-9H-carbazol-3-yl)-4,5-dihydroisoxazol-4-yl)methyl)aniline (8h)
IR (KBr, cm-1): N-H (3336.00), Ar Stretch C-H (3130.34), Aliphatic C-H (2834.23), C=N (1608.69), C=C (1344.43), C-F (1265), C-O-N (1181.44), 2930 cm-1 (C-H Stretch in –OCH3 Group).
1H NMR (δ ppm, 400 MHZ, CDCl3): 7.4-8.2(7H, m, Ar-H), 7.1(2H, d, Ar-H, Ortho to – OCH3 group), 7.9 (2H, d, Ar-H, meta to – OCH3 group), 8.1(1H, s, HN-Ar), 2.4(1H,dt, HC-CH2-NH), 4.5(1H,d, -CH), 3.4(2H,d,HN-CH2-CHISOXAZOLINE RING), 7 (2H, d, Ortho to Fluoro atom ), 6.8 (2H, d, meta to – Fluoro atom), 3.73 (3H, S, -OCH3), 3.9(3H,S, N-CH3).
Table: 7 13C –NMR data of of Novel Synthesised Isoxazoline mannich base derivatives 8(a-h):
|
Structure of the compound |
13CNMR (100 M.HZ, CDCl3-d1, δ ppm) |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In isoxazoline ring), 30-40(3 Aliphatic Carbons),126 (-CF3 carbon) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In Isoxazoline ring), 30-40(3 Aliphatic Carbons) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 158.4 (N=C In isoxazoline ring), 30-42(3 Aliphatic Carbons), 125 (-CF3 carbon) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In Isoxazoline ring), 30-40(3 Aliphatic Carbons) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C -In isoxazoline ring), 30-40(3 Aliphatic Carbons), 128(-CF3 carbon) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In Isoxazoline ring), 30-40(3 Aliphatic Carbons) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In isoxazoline ring), 30-40(3 Aliphatic Carbons),124 (-CF3 carbon) respectively. |
|
|
105-155 (20 Aromatic carbons), 87(O-CH), 159 (N=C In Isoxazoline ring), 30-40(3 Aliphatic Carbons) respectively. |
ANTI-MICROBIAL ACTIVITY
Media and chemicals
Nutrient Broth, Nutrient agar and 5 mm diameter antibiotic assay were obtained from Hi-Media Laboratories Limited, India. Barium chloride dehydrate GR, concentrated sulphuric acid GR, Dimethyl sulphoxide GR, Sodium chloride AR and Potassium dichromate were obtained from Ranbaxy Laboratories Ltd, Chemical Division, India. The standard bacterial and fungal strains were procured from National Centre from Cell Science (NCCS), Pune, India. The bacterial included two Gram positive bacterial isolates Staphylococcus aureus NCCS 2079 and Bacillus cereus NCCS 2106 and two Gram negative bacterial isolates Escherichia coli NCCS2065 and Pseudomonas aeruginosa NCCS 2200. The fungicidal organisms included were Aspergillus nigeri NCCS 1196 (AN) and Candida albicans NCCS 3471(CA). The bacteria were grown and maintained on nutrient agar (Hi-Media, Mumbai) and were subculture when needed.
Glass wares and Apparatus
Glass petridish, Glass tubes, Beakers, Erlenmeyer flasks, Bacterial loop and measuring cylinder. All the glass wares were of Borosilicate grade. Digital electronics balance (Shankar Scientific supplies, India), Yorco Horizontal Laminar air flow bench (Yorco sales Pvt. Ltd, New Delhi, India), Ausco incubator, Zone reader (Cintex industrial Corporation, India), hot air oven, autoclave and UV/Visible spectrophotometer (Shimadzu corporation, Japan).
Antibacterial activity
The antibacterial activity of synthesized compounds was studied by the disc diffusion method against the following pathogenic organisms. The gram-positive bacteria screened were Staphylococcus aureus NCCS 2079 (SA) and Bacillus cereus NCCS 2106 (BC). The gram negative bacterial screened were Escherichia coli NCCS 2065 (EC) and Pseudomonas aeruginosa NCCS 2200 (PA). The synthesized compounds were used at the concentration of 250 µg/ml and 500 µg/ml using DMSO as a solvent. The amoxicillin 10 µg/disc and Streptomycin 30 µg/disc were used as a standard (Himedia laboratories limited, Mumbai).
Disc Diffusion Method
A suspension of Staphylococcus aureus (SA) was added to sterile nutrient agar at 45ºC. The mixture was transferred to sterile petridishes to give a depth of 3 to 4 mm and allowed to solidify. Precautions were observed to reduce uniform layer of medium on the plate. Sterile discs 5mm in diameter (made from Whatman Filter paper) were immersed in the solutions of synthesized compounds (250µg/ml) and maintain an untreated control sample for comparison. Leave the plates to stand for 1hour at room temperature as a period of preincubation diffusion to minimize the effects of variations in different time. Then the plates were incubated at 37ºC for 24 hours and observed for antibacterial activity. The diameter of the zone of inhibition was measured for each plate in which the zone of inhibition was observed. The average zone of inhibition was calculated and compared with that of standard. A similar procedure was adopted for studying the antibacterial activity against the other organisms.
Antifungal activity
The antifungal activity3 of synthesized compounds were studied by disc diffusion method against the organisms of Aspergillus nigeri NCCS 1196 (AN) and Candida albicans NCCS 3471(CA). Compounds were treated at the concentrations of 250 µg/ml using DMSO as a solvent. The standard used was Ketaconazole 50 µg/ml and Griseofulvin 50 µg/ml against both the organisms.
Disc Diffusion Method
A suspension of Aspergills nigeri NCCS 1196 (AN) was added to a sterile sabouraud dextrose agar at 45ºC. The mixture was transferred to sterile petridishes and allowed to solidify. Sterile discs 5 mm in diameter (made from Whatmann Filter paper) immersed in the solutions of synthesized compounds and control were placed on the surface of agar medium with forceps and pressed gently to ensure even contact. Leave the plates to stand for 1 hour at room temperature as a period of preincubation diffusion to minimize the effects of variation at 37ºC for 13 hours and observed for antibacterial activity. The diameters of the zone of inhibition were measured for the plates in which the zone of inhibition was observed. The average zone of inhibition was calculated with that of standard. The Carbazole-Isoxazoline Mannich base derivates containing Chloro with –CF3 (8a) and Nitro with –CF3 (8e) showed more activity than other substituent’s
The order of activity was 8a>8e>8c>8g>8f>8b>8d>8h.
Anti-Microbial Evaluation of Novel compounds 8 (a-h):
Table 8. Antimicrobial activity and antifungal activity of synthesized compounds 8(a-h):
|
Compound No |
Zone of inhibition in mm |
|||||
|
Antibacterial activity |
Antifungal activity |
|||||
|
S.aureus |
E.coli |
P.aeruginosa |
C. albicans |
A. flavus |
A.fumigatus |
|
|
8a |
24 |
22 |
23 |
12 |
10 |
11 |
|
8b |
19 |
17 |
17 |
11 |
10 |
11 |
|
8c |
22 |
20 |
21 |
10 |
9 |
10 |
|
8d |
18 |
15 |
16 |
9 |
9 |
10 |
|
8e |
23 |
21 |
22 |
11 |
9 |
10 |
|
8f |
20 |
17 |
18 |
10 |
9 |
10 |
|
8g |
21 |
18 |
19 |
10 |
9 |
10 |
|
8h |
17 |
14 |
15 |
9 |
9 |
10 |
|
Ampicillin |
20 |
21 |
22 |
21 |
- |
- |
|
Flucanazole |
22 |
20 |
23 |
22 |
- |
-- |
RESULTS AND DISCUSSIONS:
The Title compounds 8a-8h were synthesized in good yields (scheme-I). All these compounds were tested for Anti-bacterial and Anti-fungal activity showed considerable activity when compared to the standard drug Amoxicillin. It is interesting to note that the compound 8a, 8e possessed the maximum activity. It clearly indicates the favourable effect of Electron with drawing substituent’s on the anti-bacterial and anti-fungal activity of the Isoxazoline Mannich bases.
Chemistry
The Target compounds were synthesized as shown in Scheme 1. Carbazole (1) on methylation with methyl iodide gave 9-methyl carbazole (2), which on Vilsmeier–Haack formylation gave 3-formyl-9-methylcarbazole (3). Chalcones was synthesized by the reaction of 4-substituted acetophenone derivatives and 3-formyl-9-methylcarbazole (3) in the presence of NaOH i.e. compounds (5 a-d). Compounds (5a–5d) on cyclization with Hydroxyl amine hydrochloride in the presence of pyridine yielded Isoxazline Heterocyclic compounds (6a–6d) respectively. Compounds 6a-6d further undergoes Mannich reaction with different substituted anilines and Formaldehyde to afford compounds 8a-8h.
All the synthesized compounds (8a-8h) were characterized by IR, 1H NMR, 13C NMR.
Characterization:
The IR spectrum of the title Compounds 8(a-h) has given stretching vibration at 3100cm-1, due to the stretching vibration corresponding to Ar-H Stretching vibrations. The absorption peak at 2935 cm-1 is due to The stretching vibration corresponding to the SP3 C-H ( methyl gp).The strong Intensity absorption at 1350 and 1530 cm-1 is due to The stretching vibration of -N-O Stretching in Nitro group, 1360 cm-1 is due to The stretching vibration of C-F bond. 760 cm-1 is due to the stretching vibration of C-Cl bond. 560 cm-1 is due to the stretching vibration of C-Br bond. The weak Intensity absorption at 1620 cm-1 corresponds to a C=N Stretching vibration.1150cm-1 corresponding to C-O-C Stretching.
It has been observed from chemical structure of compound 9(a-f) that different pair of protons. The protons of Methyl group which is attached to benzene ring appeared as a singlet at δ =2.3 ppm, The protons of Methyl group appeared as a Singlet at δ =3.8 ppm, . The protons attached benzene ring appeared between δ =7.2-8.4 ppm respectively.
The chemical shifts of the final compound carbon vary from δ = 165 to 23 ppm. The carbon nucleus under the influence of a strong electronegative environment appeared down field, the carbon chemical shift of the methyl group at δ= 23 ppm. The carbon chemical shift of the Tri fluoro methyl carbon group at δ= 124 ppm.
Readily available starting materials and simple synthesizing procedures make this method very attractive and convenient for the synthesis of Isoxazoline Mannich base derivatives. Formation of products was confirmed by recording their 1H NMR, 13C, FT-IR.
Biological Activity screening:
The results of biological studies of newly synthesized compounds reveal that the compounds possess significant anti-bacterial and anti-fungal activities. The results of these studies are given in Table 8. From Anti-bacterial and Anti-fungal activity screening results, it has been observed that compounds 8a, 8e possess good activity.
CONCLUSION:
The approach of the present study was to synthesize various mannich bases and evaluate the anti-bacterial and anti-fungal activities. From result generated it can be concluded that test compounds 8a, 8e, 8c, 8b were found to possess moderate antibacterial activity against gram positive bacteria and gram negative bacteria compared with Amoxicillin. The observed antimicrobial and antifungal activities are attributed to the substitution of –CF3 group at 4 position of benzene amine ring and -Cl Group in Isoxazoline ring of synthesized compounds. These results suggest that the Mannich bases of appropriately substituted isoxazolines have good potential for further development as antimicrobial agents. The data reported in this article may be helpful guide for the medicinal chemist as well as Synthetic Chemist who is working in this area.
ACKNOWLEDGMENTS:
Authors are thankful to our Research Supervisor Dr. K. Sudhakar Babu for providing us required facilities and motivation for completion of the Research work. We also extend our gratitude towards Department of Chemistry, Sri Krishnadevaraya University for providing us facilities of IR Spectra, 1H NMR for characterization of Novel Synthesized compounds.
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Received on 31.01.2016 Modified on 17.02.2016
Accepted on 05.03.2016 © AJRC All right reserved
Asian J. Research Chem. 9(4): April, 2016; Page 159-169
DOI: 10.5958/0974-4150.2016.00026.2