A facile microwave assisted one pot synthesis of novel xanthene derivatives as potential anti-inflammatory and analgesic agents

2.1.1 Selection of possible cognition enhancers

Prediction of biological activity spectra was made for about 200 virtually designed variedly substituted structures from the theoretical calculations on the basis of PASS prediction. The compounds screened were the structures formed from the various combinations of R (H, 2-OH, 4-OH, 3-OCH₃-4-OH, 4-Cl and 4-NO₂) groups in the hydroxylated xanthene moiety. On the basis of prediction results from the database analysis, potential xanthenes were selected for testing their anti-inflammatory and analgesic activities. The compounds screened above were further subjected to ‘Lipinski rules of five’. The MIPC (mol inspiration property calculator) program has been utilized (www.molinspiration.com) for calculating the Lipinski descriptors. The log P values and the associated parameters for the compounds under consideration have been provided in Table 1 and the probabilities of the test compounds for being active (Pa) are provided in Table 2. On the basis of the above-mentioned criteria, the chosen compounds were synthesized and experimentally tested as anti-inflammatory and analgesic agents.

2.3.1 Synthesis of 9-aryl-9H-xanthene-3,6-diol derivatives

Resorcinol (2 mM) was reacted with various aromatic aldehydes (1 mM) and sulfamic acid (0.04 mM) as a catalyst in required quantity of water in a two necked round bottom flask (RBF). The mixtures were irradiated in a microwave attached with reflux condenser and constant stirring to avoid risk of high pressure development (Make-Raga’s Scientific) at 350 W (110 °C) till the completion of the reaction. The reaction was monitored by TLC (n-Hexane:Ethyl Acetate 1:1). After completion of the reaction, the reaction mixture was cooled to room temperature and ice was added along with stirring. The mixture was extracted with ethyl acetate two to three times in a separating funnel and the organic layer was evaporated to dryness. The crude products on recrystallisation from hot ethanol gave the pure 9-aryl xanthene derivatives (1a–f). The synthesized compounds were characterized by their melting point and spectral data (IR, ¹H-NMR). Tables 3 and 4 represents structure, properties and spectral data of synthesized 9-aryl xanthenes derivatives.

Table 3 Chemical structures and properties of 9-aryl xanthenes (1a–f).

Close

Export to PPT

Compounds	−Ar	Yield (%) (Reaction time)	Rf valuea	Melting point (°C)b (Recrys. solvent)	Solubility	Molecular formula (Molecular weight)	Analysis (%) Calcd/Found			M+ 70 eV EI
							C	H	N
1a		77.34 (10 min)	0.33	172–175 (Ethanol)	Ethanol	C19H14O3 (290.31)	78.61 78.34	4.86 4.98	–	290
1b		78.52 (10 min)	0.37	132–134 (Ethanol)	Ethanol	C19H14O4 (306.31)	74.50 74.69	4.61 4.68	–	306
1c		80.62 (10 min)	0.38	129–131 (Ethanol)	Ethanol	C19H14O4 (306.31)	74.50 74.76	4.61 4.54	–	306
1d		76.54 (10 min)	0.40	199–202 (Ethanol)	Ethanol	C20H16O5 (336.34)	71.42 71.56	4.79 4.68	–	336
1e		82.00 (8 min)	0.42	>225 (Ethanol)	Ethanol	C19H13ClO3 (324.76)	70.27 70.02	4.03 3.88	–	326
1f		82.74 (8 min)	0.46	>225 (Ethanol)	Ethanol	C19H13NO5 (335.31)	68.06 68.32	3.91 4.14	4.18 4.16	335

a Stationary phase: silica gel G, mobile phase: n-hexane:ethyl acetate (1:1), iodine vapors as visualizing agent.

b All melting points were uncorrected.

2.4.1 Experimental animals

Wistar rats (150–220 g) and Swiss albino mice (20–25 g) of either sex were obtained from National Toxicological Centre (NTC) Pune and Serum India Ltd. Pune (India) respectively for the experimental studies. The animals were housed in polypropylene cages at a room temperature (25 ± 2 °C) with 12 h of light and dark cycles and had free access to standard pellet diet and water ad libitum. On the day before the treatments, food was withdrawn, but the animals were allowed free access of water. The allocation of animals to different groups was randomized and the experiments were carried out under blind conditions. Animals used in the present study were cared for as per guidelines of the Committee for the purpose of control and supervision on experiments on animals (CPCSEA), Dept. of Animal Welfare, Govt. of India procedures involving animals and their care were conducted in conformity with international laws and policies and animal studies accepted by the Institutional Animal Ethics Committee (IAEC Reg. No. DYPIPSR/IEAC/08-09/P-26).

2.4.2 Acute toxicity determination

The acute toxicity study for the test drugs was carried out in mice according to the OECD guidelines (OECD-423, 2004). Albino mice of either sex weighing between 20 and 25 g were divided into six groups of six animals each. Animals were starved for 24 h with water ad libitum prior to test. On the day of the experiment, test drugs were administered at graded doses up to 2000 mg/kg, p.o. and animals were monitored individually and continuously for 30 min, 2 h and up to 24 h to detect any changes in the autonomic or behavioral responses and also for tremors, convulsions, excessive salivation, diarrhea, lethargy, sleep and coma. The animals were then monitored for any mortality for the following 14 days. As there was no toxic reaction or mortality observed, the test drugs were found to be devoid of any toxicity up to a maximum dose of 2000 mg/kg body weight.

2.4.3 Anti-inflammatory activity

In vivo anti-inflammatory activity was evaluated by carrageenan induced rat paw edema assay model (Winter et al., 1962). Rats were divided into different groups each consisting of 8 animals and administered with 0.5% sodium CMC (10 ml/kg p.o.) as control group, reference standard indomethacin (10 mg/kg p.o.) and test compounds 1a–f were administered at the dose of 30, 60 and 90 mg/kg p.o. One hour after dosing, the rats were challenged by a sub plantar injection of 0.05 ml of 1% solution of carrageenan (Sigma, St. Louis, Missouri, USA) in sterile distilled water which was administered to the left hind footpad of each animal. The total increase in edema volume is measured by mercury displacement technique with the help of the plethysmometer (UGO Basile, Italy) at 1, 2, 3, 4, 5 and 6 h after carrageenan treatment. The percentages of the inhibition in the treated animals versus control group were calculated at each 60 min interval by using the following formula:

Ec = Mean of edema volume of control group.

Et = Mean of edema volume of test group.

2.4.4 Gastric ulcerogenicity

Animals were subjected to acute ulcerogenic effect after administration of control group 0.5% sodium CMC (10 ml/kg p.o.), test drugs (30, 60 and 90 mg/kg p.o.) and standard Indomethacin (10 mg/kg p.o.). Twenty four hours after the dosing, rats under deep ether anesthesia were sacrificed so that the stomach could be removed, opened along the curvature and cleaned gently by dipping in saline. Then the stomachs were examined for lesions under a dissecting microscope. Stomachs exhibiting one or more ulcers were considered positive (Hafez et al., 2010).

2.4.5 Analgesic activity

2.4.6 Statistical analysis

All values in the texts are expressed as means ± standard error of the mean (SEM) for n observations, where n represents number of animals studied. The results were analyzed by one way analysis of variance (ANOVA) followed by Dunnett’s test. ‘p’ values more than 0.05 was considered as non significant, less than 0.05 as significant and less than 0.01 as extremely significant compared with control group.

3.2.1 Anti-inflammatory activity

Carrageenan induced paw edema model is used to study and evaluate the effects of acute inflammation involving various types of inflammatory mediators like histamine, serotonin, bradykinin and prostaglandin (Vinger et al., 1987). The anti-inflammatory activity of synthesized derivatives (1a–f) were assessed from their ability to inhibit the paw edema induced by carrageenan in mice and activity was expressed as “mean increase in paw volume ± SEM”, in terms of millimeters (mm) and percentage inhibition in paw volume by different doses of the compounds. As observed from the results in Table 5, the compounds (1a–f) possessed significant anti-inflammatory activity at second phase (at a time interval between 180 and 360 min) at a dose of 30, 60 and 90 mg/kg dose p.o.

Edema produced by carrageenan is a biphasic event in which the involvement of the cyclooxygenase products of arachidonic acid metabolism and the production of reactive oxygen species are well established. (Smith et al., 1974). If the inhibition is more effective in the first phase of carrageenan-induced edema, it indicates that the anti-inflammatory effect is taking place through the inhibition of the pathway mediated by is histamine and serotonin. In addition, if the inhibition is more effective in the second phase of carrageenan-induced edema, it indicates that the anti-inflammatory effect is taking place through the inhibition of the pathway mediated by prostaglandin (PG) (Holsapple et al., 1980). According to this literature information, it might be stated that the synthesized xanthene derivatives (1a–f) exhibit their anti-inflammatory effect through the PG mediated mechanism (Table 5).

3.2.2 Ulcerogenic activity

In order to determine the extent of these side effects, the compounds were further tested for ulcerogenicity. The ulcerogenic liabilities of compounds (1a–f) were either absent or less than that of indomethacin at all three graded doses. The results indicating% inhibition of inflammation and% of animals found ulcerogenic has been shown in Table 5.

3.2.3 Analgesic activity