ANALOGUES OF CARBAZOLO CONDENSED AZEPINONES AND THEIR EVALUATION FOR ANALGESIC ACTIVITY

Extremely simple protocols based on the reactivity of corresponding oxoketenedithioacetal (4), 2(dimethylaminomethylene) ketone (5), β-oxoenolether (6) and α,β-unsaturated ketone (7) derivatives of 7-ethyl-3, 4-dihydroazepino[3,2-b]carbazol-2,5(1H,7H)-dione (3) have been developed to provide an easy access to their pyrimido annulated analogues (8-15) of medicinal interest. The key compound 3 from which, the synthesis proceeded has been realized in two steps from the commercial 3-amino-9-ethyl carbazole (1) on its reaction in the first step with ethyl succinyl chloride followed by cyclocondensation of the resulting ester 2 with PPA. The selected synthesized compounds were screened for in-vivo analgesic activity using acetic acid induced writhing model in mice. Among them, compound 13was found to be most active and found comparable to standard aspirin.


INTRODUCTION
Carbazoles, benzazepines and pyrimidines exhibits a wide array of biological responses in combating a variety of body ailments by virtue of their ability to provide ligands to a number of functionally and structurally discrete receptors.Current demonstrations revealed that some of their derivatives can serve as potential agents in the treatment of cancer and AIDS has stimulated further interest in these molecules from yet another perspective.Literature is replete with examples showing that heterocycles which incorporate carbazoles [1], azepinones [2] and pyrimidines [3] in their molecular framework display wide range of bioefficacies such as anti-cancer [4], anti-leukemic [5], antiproliferative [6], anti-microbial [7], anti-viral [8], anti-malarial [9], antioxidant [10], etc.Ever since, pyrimidine derivatives have been recognized to belong to the class of 'privileged medicinal scaffolds' [11] its potential have been widely exploited in the discovery of such pyrimidine based drugs as 'Etravirine' [12] which has emerged as a highly potent anti-HIV agent, to have found the FDA approval for the treatment of AIDS.The advent of its inherent potential such as this, has led to the interest on the various facets of the chemistry of pyrimidine derivatives has expand exponentially, thereafter.In view of exploring, their biological potential further, we thought that it could be interesting to develop libraries ofsuch materials which contained a carbazole nucleus on one side of the azepinone framework and the bioactive pharmacophore such as the pyrimidine on its other side, on this premise, that their presence in tandem in the same molecular framework could contribute significantly to produce novel series of compounds with enhanced biological activities.In putting this concept into the action we require to develop expedient protocols to obtain the pyrimidine annulated analogues of azepino condensed carbazole derivatives 8-15 using conventional as well as microwave assisted methods.We explored the feasibility of their preparation by exploiting the synthetic potentials of oxoketenedithioacetal, 2-dimethylaminomethylene ketone, β-oxoenolether and α,β-unsaturated ketone derivatives 4-7 in their synthesis through their reaction with the bidentate nucleophiles, indicated in Scheme 1 and Scheme 2. These highly reactive systems having an exceptionally high propensity in reactions towards these nucleophiles were appended on to the adjacent position, of the carbonyl function in 3, in accordance to the procedures reported for their incorporations in the literature on related substrates.The intermediate 3 was in turn realized through the procedure outlined in Scheme 3.

General
Melting points were determined in an open capillary and are uncorrected.The IR spectra were recorded on Schimadzu FTIR-8400S. 1 H-NMR spectra were recorded in CDCl 3 on Bruker DRX-300 MHz spectrometer using TMS as internal reference with their values expressed in δ ppm.Purity of all the synthesized compounds were checked by TLC on silica gel G plates in the solvent system (9:1, benzene: methanol).

Preparation of (E)-4-((dimethylamino) methylene
92 g, 0.01 mol) was dissolved in N,N-dimethylformamide dimethylacetal (15 mL) and the solution was heated under reflux for 4 h.and concentrated.The residue is triturated with hexane, filtered and washed with hexane to give 5 as a powder (2.04 g).

Solution phase microwave assisted method for the preparation of (9)
Equimolar quantities of thiourea (0.152 g, 0.002 mol), sodium ethoxide (0.14 g, 0.002 mol) and 4-(bis(methylthio)methylene)-7-ethyl-3,4-dihydroazepino[3,2-b]carbazol-2,5(1H,7H)-dione (4) (0.79 g, 0.002 mol) were taken in ethanol (5.0 mL) and was placed in a 100 mL borosil flask fitted with a funnel as a loose top.The reaction mixture was subjected to microwave irradiation at 180 W microwave power for 2 min, 360 W for 5 min and then at 720 W for 2 min (with short interval of 1 min.to avoid the excessive evaporation of solvents).The residue was treated with glacial acetic acid (4-5 mL) (just enough to dissolve sodium salt of pyrimidine) and refluxed in a microwave at 180 W microwave power for 1 min.and 360 W for 2 min.The overheating of the solution was avoided.The reaction mixture was poured on crushed ice and precipitated 9 was purified by crystallization with chloroform (400 mg), m.p. 210-212 o C.

Preparation of 2-amino-5H-N-ethyl carbazolo[b]pyrimido
Solution phase microwave assisted method for the preparation of ( 11) 5) (0.115 g, 0.333 mmol) in ethanol was added guanidine carbonate (1.5 g, 66.5 mmol) and sodium acetate (2.6 g, 0.04 mole).The mixture was placed in a 100 mL borosil flask fitted with a funnel as a loose top.The reaction mixture is subjected to microwave irradiation at 360 W for 6 min and then at 720 W for 3 min (with short interval of 1 min to avoid the excessive evaporation of solvents).The reaction mixture was filtered; insoluble material was extracted with chloroform and washed with water.The organic layer was dried over anhydrous MgSO 4 and evaporated to give 11 (75 mg).

Solution phase microwave assisted method for the preparation of (12)
The solution of 7-ethyl-4-(hydroxymethylene)-3,4-dihyroazepino[3,2-b]carbazol-2,5(1H,7H)dione (6) (1.74 g, 0.005 mol), malononitrile (0.33 g, 0.005 mol) and ammonium acetate (0.04 mol) in ethanol (10 mL) was placed in a 100 mL borosil flask fitted with a funnel as a loose top.The reaction mixture was subjected to microwave irradiation at 180 W microwave power for 3 min, 360 W for 4 min and then at 720 W for 3 min (with a short interval of 1 min to avoid the excessive evaporation of solvents).The reaction mixture was cooled and poured on the crushed ice with constant stirring.The solid mass thus obtained was washed with water and ethanol and then to this mixture of this solid mass (0.01 mol) and urea (0.02 mol) and heated in a microwave at 180 W microwave power for 1 min, 360 W for 2 min and then at 720 W for 1 min (with short interval of 1 min.to avoid the excessive evaporation of solvents).On cooling the product solidified, which was recrystallized from DMF-EtOH mixture (1:2) to give 12 (1.08 g).
Solution phase microwave assisted method for the preparation of (13) The mixture of 7-ethyl-4-(hydroxymethylene)-3,4-dihyroazepino[3,2-b]carbazole-2,5(1H,7H)dione (6) (1.74 g, 0.005 mol), malononitrile (0.33 g, 0.005 mol) and ammonium acetate (0.04 mol) in ethanol (10 mL) was placed in a 100 mL borosil flask fitted with a funnel as a loose top.The reaction mixture was subjected to microwave irradiation at 180 W microwave power for 3 min.,360 W for 4 min and then at 720 W for 3 min (with short interval of 1 min to avoid the excessive evaporation of solvents).The reaction mixture was cooled and poured on the crushed ice with constant stirring.The solid mass thus obtained was washed with water and ethanol and then to this mixture was added thiourea (0.02 mol) and heated to microwave at 180 W microwave power for 1 min., 360 W for 2 min and then at 720 W for 1 min (with a short interval of 1 min to avoid the excessive evaporation of solvents).On cooling the product solidified, which was recrystallized from DMF-EtOH mixture (1:2) to give 13 (1.06 g).

Evaluation of analgesic activity
Acute toxicity studies were carried out in mice in accordance to OECD-420 guidelines [13].Albino mice (20-25 g) either sex were divided into six groups of containing six animals each.Animals were starved for 24 h with water ad libitum prior to test.On the day of the experiment, animals were administered with different test compounds to different groups in graded doses of 10-1000 mg/kg body weight orally.The animals were then observed continuously for 3 h for general behavioral, neurological, autonomic profiles and then every 30 min for next 3 h and also for incidences of mortality if any.

Peripheral analgesic activity -acetic acid induced writhing test in mice
Mice were divided into six groups containing six each mice.The control group received normal saline (2 mL/kg, I.P.).The test groups were treated with compounds (10 mg/kg I.P.) while the second group received aspirin at the dose of 10 mg/kg I.P.After 30 min of compound administration, the mice were challenged with 0.6% acetic acid intraperitoneally (10 mL kg -1 ) [14].Five minutes after acetic acid injection, mice were placed in individual cage and the number of abdominal contractions was counted for each mouse for a period of 10 min after 5 min latency, and the percentage inhibition of writhing was calculated.
The percentage inhibition was calculated by using the formula: Where N T is average number of writhing in treated group and N C is average number of writhing in control group.

Physical, analtical and spectral data of compounds
The abdominal constriction response induced by glacial acetic acid is a sensitive procedure to establish peripherally acting analgesics.This response is thought to involve local peritoneal receptors.Acetic acid causes inflammatory pain by inducing capillary permeability and liberating endogenous substances that excite pain nerve ending.Acetic acid is also known to increase PGE 1 and PGE 2 peripherally [19].In acetic acid induced writhing model, all the compounds (10 mg/kg) exhibited impressive analgesic activity via reduction of writhes in mice.It is noteworthy, that compound 13 caused maximum inhibition (80.1%, p < 0.05) and was comparable to standard (Table 2).NSAIDs can inhibit COX in peripheral tissues and therefore interfere with the mechanism of transduction of primary afferent nociceptors [20].The mechanism of analgesic activity of compounds could be probably due to the blockade of the effect or the release of endogenous substances that excite pain nerve endings similar to that of aspirin and NSAIDs.Thus, the reduction in the number of writhing by the test compounds indicates that compounds might exert analgesic activity by inhibition of liberating endogenous substances like prostaglandin and other cytokines that excite pain nerve ending and induces pain or via inhibition of arachidonic acid pathway or their key enzymes like cyclooxygenases.Thus results obtained in this study indicate that test compounds possess analgesic properties probably via peripheral inhibitory mechanisms.

CONCLUSION
In summary, several elegant protocols have been developed to provide an easy access to the novel pyrimido annulated analogues of carbazolo condensed azepinone derivatives (8)(9)(10)(11)(12)(13)(14)(15) from the corresponding oxoketenedithioacetal, 2-(dimethylaminomethylene) ketone, β-oxo-enolether and α, β-unsaturated ketones (4, 5, 6 and 7), respectively, in high yield and purity.Among all test compounds, compound 13 displayed most profound analgesic effect and found comparable to standard.Further studies in depth toxicity studies and PK-PD studies could to be helpful in designing a more potent analgesic for therapeutic use in the field of medical science.

Table 1 .
). Showing the difference between conventionally and microwave assisted reactions.

Table 2 .
Effect of synthesized compounds on acetic acid induced writhing in mice (n = 6).