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Melting points of synthesized compounds were determined in Thermonik melting point apparatus and are uncorrected. UV spectra were recorded on Thermospectronic (Rochester, NY, USA) and IR spectra were recorded on Thermo Nicolet IR200 FT-IR Spectrometer (Madison, WI, USA) by using KBr pellets. The 1HNMR was recorded on Bruker AVANCE 300 (Bruker, Rheinstetten/Karlsruhe, Germany) using CDCl3/ DMSO-d6 as solvent. Chemical shifts are reported in δ ppm units with respect to TMS as internal standard. Mass spectra were recorded on Autospec Mass Spectrometer under the electron impact at 70 eV. The elemental analysis (C, H and N) of the compounds was performing on Heraus CHN rapid analyzer. Results were within ±0.4% of the theoretical values of elemental analysis. Purity of the compounds was checked on TLC plates using silica gel G as stationary phase and iodine vapors as visualizing agent.
The title compounds were synthesized as given in scheme 1. It is apparent from the scheme that the new heterocyclic compounds possess a coumarinyl moiety along with a 1H-pyrazole, 2-pyrazoline and 4H-pyran unit. Numerous synthetic routes to 3-substituted coumarins from 2-hydroxyarylaldehydes or 2-hydroxyarylketones have been reported including synthesis requiring the use of noxious phosphorylating agents such as POCl3, bases such as piperidine or solvents such as DMF. Synthesis of 3-acetylcoumarin 1 was carried out by reacting salicylaldehyde with ethylacetoacetate in the presence of catalytic amount of piperidine at room temperature. The reaction is an example of the Knoevenagel reaction,116 in which the active methylene compound reacts with 2-hydroxybenzaldehyde, has been extensively used as the first step in the synthesis of 3-acetylcoumarins 1.
The key intermediates 3-aryl-1-(3-coumarinyl)prop-2-en-1-ones 2a-k were obtained by Claisen-Schmidt condensation of 3-acetylcoumarin 1 with various substituted benzaldehydes in the presence of a mixture of piperidine and n-butanol.6 Bromination of key intermediates191 2a-k was carried out in chloroform with bromine in chloroform to yield dibromo compounds 3a-k, which were cyclized with phenyl hydrazine in the presence of triethylamine in absolute ethanol to afford the 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one 4a-k.
Furthermore, intermediates 2a-k were converted to 3-[5-(substituted phenyl)-1-(phenylcarbonyl)-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen-2-one 5a-k by refluxing with benzohydrazide in hot pyridine. Key intermediates 2a-k were cyclized with malononitrile in presence of piperidine in absolute ethanol to afford 2-amino-6-(2-oxo-2H-chromen-3-yl)-4(substituted phenyl)-4H-pyran-3-carbonitrile 6a-k.
184.108.40.206 General procedure of preparation of 3-acetyl coumarin (1)
To a mixture of salicylaldehyde (1.8gm, 0.02 M) and ethylacetoacetate (2.5gm, 0.02 M), piperidine (2ml) was added by quick stirring. After 20 minute the yellowish solid separated was filtered off and washed with ethanol. It was recrystallised from ethanol, it melts at 120°C (lit mp 120-122°C) and yield was 83.55%.
UV (CHCl3): λmax 295 (ε 7296)
IR (KBr cm-1): Characteristics peak at 1740.12 (lactone of coumarin); 1677.07 (Ketone C=O). Spectrum No-1.
1HNMR (CDCl3): δ 2.73 (s, 3H, -CH3); δ 7.32-7.68(m, 4H, -ArH) and δ 8.51 (s, 1H, C4 of coumarin). Spectrum No-2.
13C NMR (CDCl3): δ 30.65 (C of CH3), 116.78, 118.34, 124.61, 125.06, 130.31, 134.48, 147.56, 155.41 (aromatic carbons), 159.32 (C=O of ketonic carbon of α pyrone), 195.58 (C=O of acetyl) ppm. Spectrum No-3.
Mass spectrum: M+ ion 188. Spectrum No- 4.
220.127.116.11 General procedure of preparation of 3-aryl-1-(3-coumarinyl)prop-2-en-1-ones (2a-k)
A mixture of 3-acetyl coumarin (1, 0.01 mol) and the various substituted aromatic aldehydes (0.012 mol) was dissolved in 10 ml of n-butanol under heating; then 0.3 ml of glacial acetic acid and the same quantity of piperidine were added. The reaction mixture was refluxed for 4h and then the solvent was removed in vacuum. The residue was triturated with 10 ml of ethanol until a precipitate formed. The precipitate was filtered off and crystallized from appropriate solvents. The physical data and spectral data of the synthesized compounds 2a-k are depicted in Table 1 and Table 2.
18.104.22.168 General procedure for preparation of 3-[2,3-dibromo-3-(substituted phenyl)propanoyl]-2H-chromen-2-one derivatives (3a-k)
A 3-aryl-1-(3-coumarinyl)prop-2-en-1-ones (2a-k, 0.01 mol) was dissolved in chloroform (100 ml) and bromine (0.01 mol) in chloroform was added drop wise with constant stirring. After the complete addition of bromine solution, the reaction mixture was stirred for 12 h. Excess of chloroform was distilled off under reduced pressure. Thus obtained solid was filtered, dried and washed from hot ethanol. The physico-chemical and spectral data of synthesized compounds 3a-k is depicted in Table 3 and 4 respectively.
22.214.171.124 General procedure for synthesis of 3-[3-(substituted phenyl)-1-phenyl-1H-pyrazol-5-yl]-2H-chromen-2-one (4a-k)
A mixture of 3-[2,3-dibromo-3-(substituted phenyl) propanoyl]-2H-chromen-2-one (3a-k, 0.01 mol) and phenyl hydrazine (0.01 mol) was dissolved in ethanol (150 ml) and triethylamine (10 ml). The reaction mixture was heated under reflux for 12 h. Excess of ethanol was distilled off under reduced pressure and residue was triturated with ice-cold water. The precipitated solid was filtered, dried and purified by column chromatography using silica gel (60-120) as stationary phase and chloroform:methanol (10:1) as mobile phase. The physico-chemical and spectral data of synthesized compounds 4a-k is summarized in Table 5 and 6 respectively.
126.96.36.199 General procedure for the synthesis of 3-[5-(substituted phenyl)-1-(phenylcarbonyl)-4,5-dihydro-1H-pyrazol-3-yl]-2H-chromen-2-one (5a-k)
3-aryl-1-(3-coumarinyl)prop-2-en-1-ones (2a-k, 0.05 M) and benzohydrazide (0.2 M) were dissolved in pyridine (30 ml) and refluxed for 6 h. Reaction mixture was poured onto the crushed ice and neutralized with 2 N hydrochloric acid. The precipitated solid was filtered, dried and recrystallised from appropriated solvent to afford the title compounds (3a-k). The physicochemical, spectral and elemental analysis data of the synthesized compounds are depicted in Table 7 and 8 respectively.
188.8.131.52 General procedure for the synthesis of 2-amino-6-(2-oxo-2H-chromen-3-yl)-4(substituted phenyl)-4H-pyran-3-carbonitrile (6a-k)
A mixture of 3-aryl-1-(3-coumarinyl)prop-2-en-1-ones (2a-k, 0.01M) and malononitrile (0.002M) were dissolved in absolute ethanol (10 ml) and then piperidine (2ml) was added dropwise. The reaction mixture was stirred at room temperature for 3 hrs. After stirring, excess of solvent was removed by vacuum. The cold water was added; crude solid was separated by filtration and recrystalize by suitable solvent. The physico-chemical and spectral data of the synthesized compounds are given in Table 9 to 10 respectively.
5.1.4 Docking Study
The structure COX-1 and COX-2 receptors were retrieved from Protein Data Bank (PDB). All synthesized compounds 4a-k, 5a-k and 6a-k as well as the bound ligand of the protein were docked by using the software Auto Dock and the score values are predicted. The protein ligand interactions were also studied. All molecules were drawn using ChemDraw Ultra 8.0 tool and energy minimized using Chem 3D Ultra 8.0 software. The results are depicted in Table 11, 12 and 13.
5.2 Pharmacological evaluation
Acute toxicity study was performed out on Albino mice of either sex weighing 20-25 g. Male adult rats (albino) weighing 150-230 g were used for various pharmacological screenings. Animals were procured from Department of Pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Tamilnadu, India, (1012/C/06/CPCSEA) and housed individually in cages (polypropylene), maintained under standard conditions of alternating 12 h light and dark cycles at a constant temperature (25±20C and 35-60% relative humidity). Animals were kept with standard rat pellet diet and water ad libitum.
5.2.2 Acute toxicity235
Guidelines of Organization for Economic Cooperation and Development (OECD) were used for the acute toxicity test. Acute toxicity study was used to establish the effective dose of test compounds after obtaining ethical clearance from Animal Ethics Committee of RVS College of Pharmaceutical Sciences, Sulur (India). Albino mice of either sex weighing between 20 and 25 g were grouped starved for 24 h with water ad libitum prior to test. Animals were administered with different compounds to different groups in an increasing dose of 10, 20, 100, 200, 1000 and 2000 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 finally for next 24 h or till death.
5.2.3 Acute anti-inflammatory activity
The acute anti-inflammatory activity of synthesized compounds was carried out by using carrageenan-induced rat paw edema method. This is one of the most commonly employed methods which having ability of inhibition of edema in the hind paw of the rat after injection of Carrageenin [a phlogistic agent]. The effect can be measured in terms of volume of the injected paw; which is measured before and after application of carrageenin and the volume of paw of the treated animals is compared to the controls.
In vivo acute anti-inflammatory activity was evaluated using carrageenan-induced rat paw edema assay model of inflammation by adopting the method of Winter et al236 for the synthesized compounds 4a-k, 5a-k and 6a-k. This method is based on the plethysmographic measurement of edema, produced by sub planter injection of of carrageenan (0.05 ml, 1%) in the right hind paw of rats, using water plethysmograph.
Male albino rats (170-220 g) were fasted with free access to water at least 12 h prior to experiments and were divided randomly into different groups of six each. Control group received 1 ml of 0.5% sodium carboxymethyl cellulose (sodium CMC), standard group received 13.5 mg/kg of diclofenac and test groups received 200 mg/kg (1/10th of the maximum tolerated dose) of synthesized compounds. The rats were dosed orally, 1 h later; a subplantar injection of 0.05 ml of 1% solution of carrageenan in sterile distilled water was administered to the left hind footpad of each animal. The paw edema volume will be measured with plethysmometer at 0, 1, 2, 3, 4, 5h after carrageenan injection. Paw edema volume will be compared with vehicle control group and percent reduction was calculated as
Percentage inhibition = 100(1-Vt / Vc)
Where Vt = Volume of the edema in treated group.
Vc = Volume of the edema in control group.
5.2.3 Chronic anti-inflammatory activity
Chronic anti-inflammatory activity was tested for those compounds which had shown promising activity in acute anti-inflammatory model. In vivo chronic anti-inflammatory activity was assessed using adjuvant-induced arthritis assay model of inflammation by utilizing the earlier reported method of Newbould. 237 Arthritis was induced by subcutaneous injection of Freund's adjuvant into planter surface of rat hind paw. Measurement of paw volume was monitored over a period of 19 days.
Normally fed male albino rats weighing between 150 and 230 g were used for the experiment. On day 1, 0.1 ml of heat-killed Mycobacterium tuberculosis (Freund's adjuvant complete) was injected into the left hind footpad of each rat. The rats were kept in cages for 15 days. On day 15, all animals with 'developed' arthritis were used for the study and were divided into six groups of six rats each for various treatments. Control group received 1 ml of 0.5% sodium CMC, standard group received 13.5 mg/kg b.w. of diclofenac. Test groups received 200 mg/kg b.w. of 4c, 4d, 4h, 4i, 5c, 5d, 5h, 5i, 6c, 6d, 6h and 6i. All compounds were administered orally from day 16 to 19. The hind paw volumes, body weight and degree of secondary lesions were recorded daily from day 16 to 19. The decrease or increase, in mean paw volume per group per day was calculated as a percent inhibition from day 15 onwards.
5.2.4 Ulcerogenic Activity
The major drawback of NSAIDs is their gastric ulcer formation due to gastric irritation. Gastric irritation properties of orally administered compounds are evaluated in fasted rats. Following treatment the animals are sacrificed after predetermined time intervals. The stomachs are removed and inspected for irritation and ulcers.
The extent of ulcerogenic effect was evaluated for compounds 4d, 5d and 6h in rat stress model238 at the therapeutic dose (i.e. 200 mg/kg b.w.). The gastric ulcerogenic potential was evaluated by calculating the ulcer index in treated and control animals. Albino rats of either sex were divided into control, standard and different test groups of six animals each group (170-250 g). They were starved for 48 h (water ad libitum) prior to drug administration. Control group received only 0.5% sodium CMC solution, standard group was orally administered with acetylsalicylic acid in sodium CMC solution and test compounds 4d, 5d and 6h were administered orally at the dose of 200 mg/kg b.w. All animals were sacrificed after 7 h of drug administration. Stomach was removed and placed on saline-soaked filter paper until inspection. A longitudinal incision along the greater curvature was made with fine scissors. The stomach was everted over the index finger and the presence or absence of gastric irritation was determined. The ulcer index for each group was determined according to a previously reported method by counting the number of lesions (x) in each of five size classes (y). The classes were defined as y=1 (pinpoint lesion), y= 2 (lesions <1mm diameter), y= 3 (lesions 1-2 mm diameter), y=4 (lesions 2-4mm diameter) and y=5 (lesions>4 mm diameter). The ulcer index was calculated using ∑5i=1 xiyi
5.2.5 Antioxidant Activity
Antioxidant Activity of synthesized compounds were evaluated by using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical239 and Hydrogen peroxide-scavenging method.240
184.108.40.206 DPPH radical-scavenging activity
Free radical scavenging activity of compounds was determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical. Briefly, 2 mL samples of various concentrations (10-100ïg/ml) were added to 2 mL of 100 μM DPPH solution. After 20 minute incubation at room temperature, the absorbance was read against a blank at 517nm. The change in absorbance with respect to the control (containing DPPH only without sample, expressed as 100% free radicals) was calculated as percentage scavenging using following the equation:
(A517blank - A517sample) ÷ A517blank ï‚´ 100%.
The reading was taken in triplicate and mean used for calculation of IC50. The IC50 (mean ± SEM) stand for the concentration required for 50% inhibition of DPPH radicals and was calculated from ORIGIN PC version 6.0 software.
220.127.116.11 Scavenging of hydrogen peroxide
Hydrogen peroxide scavenging activity of synthesized compounds was determined by the Nabavi et al method.240 Briefly, 2 mL samples of various concentrations (10-100ïg/ml) of synthesized compounds were added to 0.6 mL of 40 mM H2O2 solution (previously prepared in phosphate buffer, pH 7.4). After 10 minute incubation at room temperature, the absorbance was read against a blank at 230nm. The change in absorbance with respect to the control (containing H2O2 only without sample, expressed as 100% free radicals) was calculated as percentage of hydrogen peroxide scavenging using following the equation:
(A230blank - A230sample) ÷ A230blank ï‚´ 100.
The reading was taken in triplicate and mean used for calculation of IC50. The IC50 (mean ± SEM) stand for the concentration required for 50% inhibition of H2O2 radicals and was calculated from ORIGIN PC version 6.0 software.
5.2.6 Antibacterial Activity
18.104.22.168 Cup-plate-agar-diffusion method241
The Cup-plate method depends upon diffusion of the antibiotics from a vertical cylinder in the course of a solidified agar layer in a Petri dish to an extent such that growth of the added microorganism is prevented entirely in zone around the cylinder containing a solution of the antibiotic.
The solid media Mullere Hinton agar (MHA; beef infusion 300 g/L, casein acid hydrolysate 17.5 g/L, starch 1.5 g/L, agar 17 g/L and distilled water 1000 mL, adjusted to pH 7.4) was used for the antibacterial activity.
22.214.171.124.2 Test microorganisms
Two gram-positive bacteria namely, Staphylococcus aureus (ATCC-25923), Bacillus subtilis (ATCC 6633) and two gram-negative bacteria namely, Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-27853) were used for the antibacterial activity.
The measured quantity of inoculums of test microorganisms (0.5 mL) were added to each heated (ï¾550C) Mueller Hinton agar media tubes. After that the tubes were shaken well and then media were poured on to petridishes which was previously sterilized and then the petridishes were allowed to set in a refrigerator (4-80C). The test solutions of synthesized compounds of 100μg/ml were prepared in a DMSO. Cups of 7 mm diameter were cut in culture media using sterilized cork borer. The solutions of each test compounds (100μg/ml) were added separately in cups. Ciprofloxacin was used as standard reference drug and DMSO as a control. The petridishes were kept in the refrigerator for maintaining temperature ï¾100C for ï¾1 hr to allow diffusion of the solution. Then the petridishes were transferred to an incubator and incubator was maintained at 370C for 24 hrs. The zones of inhibition formed were measured by using calipers. The control of DMSO showed no activity.
126.96.36.199 Minimum inhibitory concentration242
The in-vitro antibacterial activity for newly synthesized compounds was evaluated using the conventional agar-dilution method. Twofold serial dilutions of the compounds and reference drug (ciprofloxacin) were prepared in MHA. Drugs (10.0 mg) were dissolved in DMSO (1 mL) and the solution was diluted with water (9 mL). Further progressive double dilution with melted MHA was performed to obtain the required concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.05 µg/mL. The bacterial inocula were prepared by suspending 24 h old bacterial colonies from MHA media in 0.85% saline. Perti dishes were spot-inoculated with 1µL of each prepared bacterial suspension and incubated at 370C for 24 h. At the end of the incubation period, MIC was determined, which is the lowest concentration of the test compound that resulted in no visible growth on the plate. A Control test was also performed with test medium supplemented with DMSO at the same dilutions as used in the experiment in order to ensure that the solvent had no influence on bacterial growth.