Studies on some biologically active substituted 4(3 H )- quinazolinones. Part 1. Synthesis, characterization and anti-inflammatory # -antimicrobial activity $ of 6,8-disubstituted 2-phenyl- 3-[substituted-benzothiazol-2-yl]-4(3 H )-quinazolinones

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Introduction
Design of agents for fast and effective relief from pain and inflammation in the human being is a major challenge for the medicinal chemists.Non-steroidal anti-inflammatory drugs (NSAID's) are the most common choice for the treatment of a number of inflammatory diseases associated with a number of pathological conditions. 1 The commonly proposed mechanism of action of NSAID's is lowering prostaglandin production through inhibition of cyclo-oxygenase.This key enzyme is involved in the conversion of arachidonic acid into prostaglandins (PG's) and thromboxane. 2The major side effects of NSAID's are their gastrointestinal ulcerogenic activity and bronchospasm. 3,4 his is because of decreased levels of PG's which has dual functions; mediation of inflammation and cytoprotection in the stomach and intestine 5 .Recent reports suggest that cyclo-oxygenase enzyme exist in two isoforms COX-1 and COX-2, which are regulated differently 6 .COX-1 is responsible for the synthesis of gastroprotective PG's in the GI and proaggregatory thromboxane in blood platelets, 7 whereas short lived COX-2 is induced by pro-inflammatory stimuli such as endotoxin, bacterial lipopolysaccharide, growth factors, cytokines, mitogens, and tumor-promoting agents. 8,9 t has been shown that the undesirable side effects of NSAID's are may be due to COX-1 inhibition while the beneficial effects, such as reduction of swelling and analgesia, are related to COX-2 inhibition. 10,11 urther recent studies reveals that compounds having more COX-1 selectivity shows evidence of more gastrointestinal toxicity. 12Hence a potent and selective COX-2 should block the production of prostaglandin in inflammatory cells without affecting in the homeostatic and gastro-protective actions mediated by COX-1. 6The search continues to develop new drugs that have potent anti-inflammatory activity with minimum side effects.Although many NSAID's are in the market, the present therapeutic approach and chemical design of NSAID's are now targeted towards the development of selective COX-2 inhibitors and as a result, several selective COX-2 inhibitors are commercially available. 12he quinazoline nucleus has been found to possess varied pharmacological activities viz.anticonvulsant, 14,15 bronchodilator, 16,17 anti-inflammatory, [18][19][20] antimalarial, 21 antituberculous, 22 anti-HIV, 23 narcotic antagonist, 24 anti-tumor, 25 tyrosinekinase inhibitor, 26 adenosine antagonist, 27 antimicrobial, 28 etc.Similarly various substituted benzothiazoles are known to possess varied pharmacological activities like anti-tumor, 29 antimicrobials, 30,31 anthelmintic, 32 analgesic, 33 antiinflammatory, 34 anticonvulsant, 35,36 and CVS agents. 37he simultaneous use of several drugs to treat inflammatory conditions, associated with some microbial infections may cause health problems especially in patients with impaired liver or kidney functions.Also, from the pharmaco-economic point of view, and for better patient compliance, an anti-inflammatory antimicrobial agent with minimum adverse effects and high safety margin is highly desirable.
In view of the fact that several 4-oxoquinazoline derivatives possess useful antiinflammatory as well as antimicrobial properties, 28,38 we designed and synthesized various derivatives of 2-phenyl-3-(substituted-benzothiazol-2-yl)-4(3H)-quinazolinone.Also with aim of obtaining the novel potent anti-inflammatory antimicrobial agents with fewer side effects, we decided to combine the benzothiazole nucleus with the quinazoline molecule.Moreover, it was considered of interest to substitute various groups on the benzothiazole nucleus to investigate the influence of such structural variation on the anticipated biological activities.In addition to the targeted anti-inflammatory and antimicrobial activities, the ulcerogenic toxicity profiles of newly synthesized compounds were also determined.Thus in the present investigation, twenty six different derivatives of 6,8-disubstituted, 2-phenyl-3-(substituted-benzothiazol-2-yl)-4(3H)quinazolinone were synthesized and evaluated for their anti-inflammatory anti-microbial and ulcerogenic activities.
All of the synthesized compounds were characterized by their physical, analytical and spectral data.They were given separately in experimental section and Tables 1 and 2. The title compounds showed disappearance of bands at 1600-1650 cm -1 (N-H deformation) and 3460-3500 cm -1 (N-H stretching) due to conversion of free amino group into cyclic nitrogen.Halogen containing compounds 3g and 3j and 4a-m showed the absorption bands at 590-760 cm -1 (C-X stretching) and nitro compounds 3k and 4k showed the bands at 1490-1550 cm -1 The EI MS and 1 H NMR spectral data of all the synthesized compounds were in conformity with the structure assigned.
In the EI-MS spectra, molecular ion [M + ] peaks, which appeared at different intensities, confirmed the molecular weights of the examined compounds (3a-m and 4a-m).Molecular ion peaks were the base peaks for the compound 3a-f, 3h, 3i, 3k-m.Appearance of an isotope peak [M + +2] as intense as the molecular ion peak confirmed the presence of halogen atom in compounds 3g, 3j and 4a-m Anti-inflammatory activity Carrageenan-induced paw edema in rats All newly synthesized compounds were tested for their anti-inflammatory activity against carrageenan-induced edema at dose of 50 mg/kg using Indomethacin as reference standard.The percentage protection against inflammation was calculated using the formula given below: (V C -V T ) / V C × 100 where V C is the increase in paw volume of control (in the absence of test compound ) and V T is the increase in paw volume after administration of the test compound.The results are recorded in Table 4.

COX-1 and COX-2 catalyzed prostaglandin biosynthesis assay (in-vitro)
Compounds 3e, 3f, and 3l that showed in-vivo anti-inflammatory activity compared to that of Indomethacin in carrageenan-induced paw edema in rats was further tested for their ability to inhibit COX-1 and COX-2 enzyme in-vitro applying the methodology of Wakitani et al.The results clearly indicated that the tested compounds exhibited very weak inhibitory activity against COX-1 enzyme (IC 50 values between 98 ->100 µmol) when compared with Indomethacin (IC50 values 0.22 µmol).While tested compounds, 3e, 3f and3l revealed superior inhibitory profile against COX-2 enzyme as indicated by their Ic 50 values (1.57, 1.87 and 0.39 µmol respectively), when compared with reference standard Indomethacin.

Ulcerogenic effects
Compounds 3e, 3f and 3l which exhibited moderate to potent anti-inflammatory profiles in the animal model were evaluated for their ulcerogenic potential in rats.All the active compounds revealed a superior GI safety profiles with oral dose of 100 mg/kg/day, when compared with reference standard, Indomethacin; which was found to create 100% ulceration under same conditions.Gross observation of the isolated rat stomachs showed a normal stomach texture for all active compounds.

In vitro antimicrobial activity
Compounds 3a-m and 4a-m have been evaluated for their in vitro antimicrobial activity against Escherichia coli (E. coli ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as an example of Gram negative bacteria, Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212) as examples of Gram positive bacteria, and Candida albicans (ATCC 90018) as a representative of fungi.The microdilution susceptibility test in Muller Hinton Broth (Sigma Aldrich) was used when testing bacterial strain, while Saboureaud Liquid medium (Sigma Aldrich) was used for the determination of antifungal activity.The minimum inhibitory concentrations (MICs in µg/ml) of the tested compounds are recorded in Table 4.
The results revealed that all newly synthesized compounds were exhibited potent antibacterial activities and poor antifungal activity.In general, compounds 4a-m exhibited more pronounced antibacterial potencies than the compound 3a-m, with better activity against both Gram positive and Gram negative bacteria.Among all the compounds tested, 4g, 4j and 4k exhibited remarkable antibacterial activity against the Gram negative Escherichia coli (E.Coli ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as compared with the broad spectrum antibiotic Tobramycin.Compounds 4j and 4k were equipotent as Tobramycin in Pseudomonas aeruginosa (ATCC 27853).It is worth-mentioning that, a compound 4g was more active than Tobramycin, against the same organisms.
On the other hand, compounds 4b, 4d, 4h, 4i and 4m exhibited potent activity against the Gram positive Staphylococcus aureus ATCC 25923 and Enterococcus faecalis (ATCC 29212) as compared with Tobramycin.The antibacterial activities of compounds 4c, 4d, and 4e were 50% lower than, the standard against the Staphylococcus aureus and 4a, 4e, and 4f against Enterococcus faecalis.Moreover, compounds 4l and 4m were moderately active against the same organisms.The rest of the tested compounds were less active against all organisms with MIC values ranging between 14-48 µg/ml.All the tested compounds showed weak antifungal activity against Candida albicans (ATCC 90018), when compared with reference antifungal agent Fluconazole.
In short, compounds 4g and 4j posses a broad spectrum of antibacterial activities against both, Gram positive and Gram negative bacteria but insignificant antifungal activity.
The different substituents on the aromatic ring exert a significant influence on the biological activity.The presence of electron withdrawing groups (halogen and nitro) on the aromatic ring in general decreases the anti-inflammatory activity of test compounds compared to compounds having electron-donating groups (alkyl).Based upon the results it will also be necessary to optimize the lead compound by substituting series of electron -donating groups on aromatic ring and selectively modifying the quinazoline nucleus.

Experimental Section
General Procedures.Melting points were determined in open capillaries using a Thermonik precision melting point cum boiling point apparatus, Model C-PMB-2 (Mumbai, India) and are uncorrected.Purity of the compounds was checked by precoated TLC plates (E.Merck Kieselgel 60 F254, Mumbai, India).IR spectra were recorded using KBr pellets on a Perkin-Elmer 337 Spectrophotometer from Perkin Elmer International Incorporation, Rorkreuz, Switzerland (v max cm -1 ), 1 H NMR spectra on Bruker W.M. 400 Spectrometer (Bruker AG, Fallanden, Switzerland) at 360 MHz using TMS as internal standard (chemical shift in δ ppm) and mass spectra (EI-MS) were recorded on a Jeol D-300 spectrometer(Jeol Ltd.,Tokyo,Japan).Elemental analyses were carried out at using Heraeus Carlo Erba 1180 CHN analyser (from Heraeus Instrument GmbH, Hanau, Germany).All the chemicals were purchased from Aldrich Company Ltd.Dorset (UK) Synthesis of substituted products of 2-aminobenzothiazole 1a-m.These compounds were synthesized from aniline and substituted aniline using known methods 42 .The product 1a-m on recrystallization from ethanol was obtained in pure form.Synthesis of substituted products of 2-phenyl-4H-3,1-benzoxazin-4-one 2a-m.These compounds were synthesized from anthranilic acid and 3,5-dibromoanthranilic acid using known methods 43 .The products 2a-m on recrystallization from ethanol was obtained in pure form.

Pharmacology
Locally bred Sprague-Dawley rats of either sex weighing between 120 to 160 g, obtained from National Center for Laboratory Animal Sciences, Hyderabad, India, were used in present study.Animals were kept in wire-mesh cages and maintained under constant environmental conditions [23±2°C 12-h light].All animals had free access to standard pellet diet (Hindustan Leaver Ltd.. Mumbai) and water, in a constant light-dark cycle.During the course of experiment, the general behavior of animal was normal.All the experimental protocols were approved by the institutional animal ethical committee and experiments were conducted in accordance with the standard guidelines.

Anti-inflammatory activity
The Anti-inflammatory activity of the synthesized compounds 3a-m and 4a-m were evaluated by carrageenan induced rat paw edema model 39 .All the test compounds were suspended in 0.5% carboxy methyl cellulose and administered either orally or intraperitoneally (50 mg/kg) 60 min prior the injection of 0.1 ml of freshly prepared solution of carrageenan (1%) in physiological saline solution (154mM NaCl) into the sub-planter tissue of hind paw of each rat.The same volume of saline solution was injected into hind paw of the control.The volume was measured by water plethysmometer prior to the administration of carrageenan (sigma) and; 1h and 3hrs.after the injection of carrageenan.The increase in volume of the paw was adopted as a measure of oedema.The antiedmatous effects of the compounds were estimated as percentage inhibition in comparison with control.

COX-1 and COX-2 catalyzed prostaglandin biosynthesis assay
Compounds 3e, 3f and 3l that showed in vivo anti-inflammatory activity comparable to that of Indomethacin in carrageenan induced paw edema in rats were further tested for their ability to inhibit COX-1 and COX-2 enzymes by using the procedure described by Wakitani et.al. 40 .In brief, Human COX-1(0.3 mg protein/assay) or COX-2 (1mg protein/assay) was suspended in 0.2 ml of 100mMol Tris-HCl buffer (pH 8) containing cofactors, Tryptofan (5mMol) and Hematin (and 2µl µMol).The reaction mixture was pre-incubated with each test compound (indifferent concentration) for 5 min.at 24°C.To it [ 14 C] arachidonic acid (100.00dpm,30µMol) was added and the reaction mixture again incubated for 2 (COX-1) or 45 min (COX-2) at 24°C.The reaction was terminated by the addition of 400 µl stock solution containing diethyl ethermethanol-1M citric acid (30:4:1 v/v).The reaction mixture was then centrifuged at 1700×g for 5 min at 4 °C.50µ of the upper phase of the centrifuged mixture were applied to a thin-layer chromatography (TLC) plate.TLC was performed at 4 °C by using diethyl ether-methanolacetic acid (90:2:0.1 v/v) as mobile phase.The COX enzyme inhibitory activity was calculated from the percent conversion of arachidonic acid to PGH 2 and its decomposition product, using a radiometric photographic system.The concentration of the compound causing 50% enzyme inhibition (Ic 50 ) was calculated.The results were given in Table 5.

Gastrointestinal ulceration studies 44
Compounds were 3e, 3f and 3l that exhibited moderate to potent anti-inflammatory profiles in the animal models were also evaluated for their ulcerogenic effects in rat.Rats were fasted for 24 h (with water ad libitum).The test compounds were suspended in a carboxymethylcellulose vehicle and administered orally by gavage at 100-mg/kg/day dose for 5 days in a volume of 0.5ml/100 g of body weight.The animals were sacrificed with diethyl ether inhalation, their stomachs removed by cutting along the greater curvature, washed under running water and fixed in 5% formalin solution.The stomachs were then examined for lesions under a dissecting microscope.

In vitro antimicrobial activity
Compounds 3a-m and 4a-m have been evaluated for their in vitro antimicrobial and antifungal activity.The microdilution susceptibility test in Muller Hinton Broth (Sigma Aldrich) was used when testing bacterial strain, while Saboureaud Liquid medium (Sigma Aldrich) was used for the determination of antifungal activity.The inoculum densities were 5×10 5 cfu/ml and 0.5×10 3 cfu/ml for bacteria and fungi respectively.The minimum inhibitory concentrations (MICs in µg/ml) of the tested compounds are recorded in Table 4.
T., not tested.b S.E.M. denotes the standard error of the mean.c All data are significantly different from control ( P> 0.005).

Table 1 .
Physical and analytical data of compounds 3a-m and 4a-m

Table 2 .
Elemental analysis of compound 3a-m and 4a-m

Table 3 .
The anti-inflammatory activity and gastric ulceration effect of compounds 3a-m and 4a-m

Table 5 .
In vitro human COX-2 b and COX-1 a enzymes inhibitory activity of compounds 3e, 3f and 3l b Human COX-1 enzyme from human platelet.c Values are means of at least four experiments.