Introduction
Formerly heterocyclic compounds have been investigated for their biological activities. 2-amino benzothiazole, a heterocyclic compound containing N and S atoms serve as an eccentric and a versatile scaffold for experimental drug design1 and its hybrids have numerous biological activities like anti-inflammatory, antitumor,2 anthelmintic,3 anti-tubercular,4 anticonvulsant,5 antimicrobial6 and anti-corrosion activity.7 Quinazoline nucleus is the cardinal skeleton of umpteen compounds which can be synthesized from anthranilic acid. Quinazolines CBR417 and CBR490 acts as antifilarial by eliminating bacterial endo symbiont Wolbachia which is essential for worm viability and reproduction.8 Quinazoline hybrids have an enormous number of biological activities like anti-hepatitis A,9 histone deacetylase inhibition,10 phosphoinositide-3-kinase inhibition, antiphospholipases, antiproteases, antidiabetic.11, 12 In the present work, a novel series of diethyl amino Quinazolinone derivatives were synthesized from benzothiazoles which were screened for antimicrobial activity.
Materials and Methods
Purification of the synthesized compounds was done by recrystallization. The melting points were determined by open capillary method and were uncorrected. The IR and NMR spectra were recorded on ABB BOMEM FTIR Spectrometer using a KBr disc and 400MHz NMR Spectrometer in DMSO using TMS as an internal standard respectively. Chemical shift (δ) was given in ppm. The absorbance of the solutions during in vitro studies were measured using UV-Visible Spectrophotometer SL-159, Elico India Ltd.
Synthesis of 2-amino-6-fluoro-7-chloro-1, 3-benzothiazole13, 14: 8gm (0.08Mol) of potassium thiocyanate and 1.45g (0.01Mol) of 4-Fluoro-3-chloro aniline (Compound 1) was added to glacial acetic acid (20ml) cooled below room temperature. The above mixture was placed in an ice water bath on a magnetic stirrer. 1.6ml of bromine in 6ml of glacial acetic acid was added slowly in a dropwise manner into the mixture in such a way that the temperature never rise beyond room temperature. The stirring of solution for 2 hours below room temperature continued after complete addition of bromine. Then the mixture was further stirred a room temperature for 10 hours and allowed to stand overnight. The orange precipitate produced during overnight standing was heated with 10ml glacial acetic acid, heated to 850 C and filtered hot. The filtrates obtained were to 850C after addition of 6ml of water and filtered hot. Then again the orange residue was treated combined, cooled and neutralized with ammonia solution to a pH 6.0. The dark yellow precipitate collected was treated with activated charcoal, recrystallized with benzene and ethanol (1:1) mixture to obtain yellow crystals of 2-amino-6-fluoro-7-chloro-1, 3-benzothiazole (compound 2).
Synthesis of 3-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one15, 16: 2-amino-7-chloro-6-fluoro benzothiazole (0.01 Mol) in glacial acetic acid was added to the mixture obtained from 4 hours reflux of anthranilic acid (0.01 Mol), acetic anhydride and refluxed for 4hrs. The reaction mixture was poured onto the crushed ice and kept overnight. The solid obtained was filtered, washed with cold distilled water, dried and re-crystallized from ethanol (95%).
Synthesis of 1-((diethylamino)methyl)-3-(7-chloro-6-fluorobenzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one17: A mixture of Compound 2 (0.01mol), formaldehyde (40%, 1.5ml) and diethyl amine (0.01mol) were stirred for 4hrs in presence of methanol and left overnight at room temperature. The solid collected by filtration was washed with ethanol, dried and re-crystallized.
Synthesis of 1-((diethyl amino) methyl)-3-(6-fluoro-7-(substituted phenyl amino) benzo [d] thiazol-2-yl)-2, 3-dihydro-2-methylquinazolin-4(1H)-one18: Compound 3(0.01mol) dissolved in DMF, equimolar quantities of primary or secondary amine (0.01mol) was added and refluxed for 2hrs. The mixture was cooled and poured into crushed ice. The separated solid was filtered, dried and re-crystallized. TLC; mobile phase: n-butanol: ethyl acetate: benzene-1:2:1.
Antimicrobial screening 19
The synthesized compounds were evaluated for antimicrobial activity by disc plate method. The antimicrobial activity was tested on Gram positive bacteria Bacillus subtilis (ATCC 6051), Staphylococcus aureus (ATCC 12600), Gram negative bacteria Klebsiella pneumonia (ATCC 13883), Escherichia coli (ATCC 11775) and fungal strains Aspergillus flavus (NCIM 536) and Aspergillus niger (NCIM 548). Concentrations of synthesised compounds 50,100,150 µg/ml and 150, 200 µg/ml anti bacterial and antifungal activities were compared with standard drugs, Ciprofloxacin and Ketoconazole respectively using solvent control, DMSO. The results were described in Table.3 and 4.
In vitro anti-inflammatory activity20
The Quinazolinone derivatives (4a-4h) were screened for the antiinflammatory activity using inhibition of albumin denaturation technique. The 1mg/ml concentration of standard and test compounds were dissolved in a minimum amount of dimethyl sulphoxide (DMSO). The test solution (1 ml) containing sythesized compound was mixed with 1 ml of egg albumin solution (1% mM in phosphate buffer) and incubated at 370±10C in incubator for 15 min. The denaturation of protein was induced by keeping the reaction mixture in a water bath for 10 min at 600±10C. The turbidity produced after cooling was measured at 660 nm (UV-Visible Spectrophotometer SL-159, Elico India Ltd.). Each experiment was done in triplicate and average was taken. The Diclofenac sodium was used as standard drug. Percentage of inhibition of denaturation was calculated from the formula below.
In vitro antioxidant activity 21
The Quinazolinone derivatives(4a-4h) were evaluated in vitro for antioxidant activity using hydrogen peroxide scavenging method. The concentrations of the synthesized compounds (4a-4h) 1-5µg/ml, the positive control(ascorbic acid) dissolved in DMSO were added to 2 ml hydrogen peroxide(40mM was added and the volume was made to 10 ml with phosphate buffer saline (pH-7.4). A control solution was prepared with DMSO in phosphate buffer saline without drug. The absorbance at 230nm was recorded using U.V spectrophotometer against blank (Phosphate buffer saline). The % inhibition of hydrogen peroxide scavenging activity was calculated using the following formula:
Results and Discussion
All the synthesized compounds were first purified by successive crystallization using appropriate solvents. The physicochemical properties of the synthesized compounds were tabulated in table no.1 and compounds were subjected to spectral analysis such as IR, 1H-NMR.
Spectral analysis
1-((diethylamino)methyl)-3-(7-(2-methoxyphenylamino)-6-fluorobenzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one: IR (KBr, cm-1): 3039.42(Ar-NH), 1306.11(Ar-CH), 1630(C=N), 1196(C-S). 1H-NMR (DMSO, δ in ppm): 6.7-7.2(s, Ar-H, 11H), 2.97(m, N-CH2, 3H), 8.43(s, CONH2, 1H), 3.1(d, NH, 1H), 5.28(s, CH3, 1H).
1-((diethylamino)methyl)-3-(7-(o-tolylamino)-6-fluorobenzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one: IR (KBr, cm-1): 3041.56(Ar-NH), 1229.11(Ar-CH), 1604(C=N), 1643(ArC-O), 1190(C-S). 1H-NMR (DMSO, δ in ppm): 6.7-7.4(m, Ar-H, 11H), 3.8(s, O-CH3, 3H), 8.43(s, CONH2, 1H), 3.31(d, CH-HB, 1H), 5.28(s, CH-Cl, 1H), 3.1(d, Ar-NH2, 2H), 2.93(t, N-CH2-C, 2H).
1-((diethylamino)methyl)-3-(6-fluoro-7-(phenethylamino)benzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one: IR (KBr, cm-1): 3475 (Ar-NH), 1281(C-S), 1645(C=O), 1198(C-S). 1H-NMR (DMSO, δ in ppm): 7-9(s, Ar-H, H), 2.97(m, N-CH2, 3H), 8.43(s, CONH2, 1H), 7.28(m, CO-CH, 1H), 7.48 (d, Ar-NH2, 2H), 8-8.5(d, NH2, 2H).
1-((diethylamino)methyl)-3-(6-fluoro-7-morpholinobenzo[d]thiazol-2-yl)-2,3-dihydro-2-methylquinazolin-4(1H)-one: IR (KBr, cm-1): 3012.56(Ar-CH), 1356.3(Ar-NH), 1595(C=N), 1160(C-S). 1H-NMR (DMSO, δ in ppm): 6.7-7.5(m, Ar-H, 11H), 4.1(s, N-H, 1H), 8.43(d, CONH2, 1H), 2.97(m, N-CH2, 4H)
Table 1
Physical characterization of the synthesized compounds
Table 2
Antibacterial activity of the synthesized compounds
Table 3
Antifungal activity of the synthesized compounds
Table 4
In Vitro anti inflammatory activity of the synthesized compounds
Table 5
In Vitro antioxidant activity of the synthesized compounds
Conclusion
The novel benzothiazole substituted Quinazolinone derivatives (4a-h) synthesized were characterized and structures were confirmed by spectral characterization like FTIR, 1H NMR. The synthesized compounds were evaluated for anti microbial activity, in vitro anti-inflammatory and antioxidant activity. The compounds substituted with p-anisidine, o-toludine and morpholine at 7th position on benzothiazole showed significant antibacterial activity at lower concentration (50 µg/ml) while p-anisidine, p-toludine substituted quinazolinones have significant antifungal activity. In vitro studies reveals that the synthesised compounds have less significant anti-inflammatory activity while the compound subatituted with phenyl ethyl amine showed significant antioxidant activity as that of the positive control. Further evaluation and structural modification of the compounds with significant activity produces a lead with antimicrobial and antioxidant activity.
