Synthesis, anti-inflammatory activity of picen-13-ylmethylene derivatives

A series of picene-13-ylmethylene derivatives (11-17) were synthesized by Knoevenagel condensation of active methylene compounds with picene-13-carbaldehyde


Introduction
Picene or benzo[a]chrysene, is well documented for use in material chemistry. 1,2Picene derivatives were found to be present in abundance as pentacyclic triterpenes and triterpenoids which are often bioactive and present a huge therapeutic potential.Many pentacyclic triterpenes namely oleane, oleanolic acid and ursane are reported to possess promising antitumor, [3][4][5] antiviral, 6 antidiabetic, 7 anti-inflammatory 8 activities.Also a derivative of picene, namely octadecahydro-picene-2,3,14,15-tetranone isolated and purified from the root bark of Zizyphus nummularia, reported to possess anti-cancer and anti-inflammatory activities. 9,10any polycyclic aromatic compounds such as flavone, quercetin, chrysin and pyrimido [4,5-b]quinolines were reported to possess anti-inflammatory or antioxidant properties. 11,12Because of the close structural similarity with the reported polycyclic compounds, we have envisaged to explore the anti-inflammatory activity of picene analogues.Thus, our interest is to conjugate two naphthalene moieties by cyclization which results in the formation of a picene moiety.Substitution at its alpha position with various esters or amides leads to novel picene methylene derivatives in order to evaluate their biological properties.In order to accomplish our objective, we used a Knoevenagel condensation for the synthesis of novel structures incorporating both the picene moiety and several active methylene compounds, namely, ethyl cyanoacetate, malononitrile, cyanoacetamide, diethyl malonate, ethyl acetoacetate, acetylacetone and 5,6 dimethoxy-1indanone with picene-13-carbaldehyde.The synthesized picen-13-ylmethylene derivatives were characterized by NMR, IR, mass spectra, elemental analysis.The structures were confirmed by single crystal XRD of a selected example.The compounds were evaluated by in vitro biological tests for their anti-inflammatory properties.

Results and Discussion
In the present work, we synthesized a new series of picen-13-ylmethylene derivatives 11-17 by condensing active methylene groups with picene-13-carbaldehyde as shown in Scheme 1 and 2. The first step in the Scheme 1 was the condensation of 1-naphthaldehyde 1 with 1-naphthyl acetic acid 2 in the presence of triethylamine and acetic anhydride produced 2,3-di(naphthalen-1-yl)acrylic acid 3 13,14 with 62% yield after recrystallization from ethyl acetate.Compound 3 was then converted to the corresponding ester 4 & 5 using ethanol or methanol and a catalytic amount of sulphuric acid.Esterification of compound 3 with ethanol and sulphuric acid under reflux condition over 3 days gave 50% yield of compound 4. As the starting compound 3 was insoluble in 25 volumes of ethanol under reflux condition, we conducted the experiment using toluene as solvent under Dean Stark conditions.Under continuous removal of water, the esterification reaction was completed in 10 hours with 91% yield of compound 4 without further purification.(Table 1) The ester was cyclized to ethyl or methyl picene-13-carboxylate 6 & 7 [15][16][17][18][19] under irradiation with UV light at 365 nm in the presence of iodine.We studied the oxidative cyclisation of compound 4 with several reactants under different reaction conditions and the results are summarized in Table 2. Treatment of compound 4 with aluminium chloride; or a mixture containing aluminium chloride and sodium chloride 20 at 140 °C; or aluminium chloride and stannic chloride 21 ;or irradiation with 254 nm in the presence of iodine gave no product.Irradiation of compound 4 with 365 nm light without stirring condition gave 30% yield.The previously reported process 22 of vanadium trifluoride oxide in trifluoroacetic acid afforded picene-13-carboxylic acid methyl ester 7 with a yield of 47%.In our present study, it was found that 1 mole of iodine and irradiation with light of 365nm in benzene under stirring are necessary to produce compound 6 & 7 in good yield (86% & 87% respectively) without chromatographic purification.The picene-13-carboxylic acid ethyl ester 6 was hydrolyzed with sodium hydroxide produced picene-13carboxylic acid 8 in 88% yield.
Reduction of picene-13-carboxylic acid methyl ester with lithium aluminium hydride has been reported for the preparation of compound 9 with a yield of 97%. 23In our study, mild reducing agents like di-isobutyl aluminium hydride [DIBAL (1M in Toluene)] was used to reduce picene-13-carboxylic acid ethyl ester 6 to picen-13-ylmethanol 9 24 in 90% yield.It was found that the oxidation of compound 9 with manganese dioxide 11 in dichloromethane was not completed even under reflux condition.Therefore, we tried pyridinium chlorochromate as an oxidising agent in dichloromethane at 25-30 °C and obtained the compound 10 25,26 in 87% yield without further purification.The picene-13-carbaldehyde 10 was condensed with active methylene compounds using piperidine as a base to give the title compounds 11-17. 27,28 Synthetic route for Picene-13-carboxylic acid (8) and Picene-13-carbaldehyde (10).
All the synthesized compounds were characterized by 1 H NMR, 13 C NMR, IR spectroscopy, Mass and elemental analysis.The structure of 11 was confirmed by single crystal X-ray diffractogram (CCDC No. CCDC 1400968).Based on the single crystal structure, the configuration of the compound was confirmed as the Eisomer.The crystal parameters for compound 11 are given in Table 3 and the ORTEP diagram is shown in Figure 1.Anti-inflammatory activity.The synthesized compounds were screened for in vitro anti-inflammatory activity using the inhibition of bovine serum albumin denaturation method.From the activity data, the antiinflammatory activity screening revealed that compounds containing the active methylene groups such as diethylmalonate 14, acetylacetone 16, and 5,6 dimethoxy-1-indanone 17 attached to a picene-13carbaldehyde moiety exhibited the biggest potent anti-inflammatory activity comparable to diclofenac sodium as a reference standard.In addition the data indicated that, the cyano ester 11 or cynamide 13, malononitrile 12 and ethyl acetoacetate ester 15 of picene-13-ylmethylene are comparatively less potent than its malonate 14 or dione 16 or indanone 17 derivatives and moderate activity in comparison to diclofenac sodium (Table 4).

Conclusions
A new versatile method with improved yield for the photocyclisation of 2,3-di(naphthalen-1-yl)acrylic acid ester was reported using mild conditions i.e., UV irradiation.A new series of picene-13-ylmethylene derivatives were synthesized, characterized and evaluated for in vitro anti-inflammatory activity.Among the picene-13-ylmethylene derivatives, compounds 14, 16 and 17 possess promising anti-inflammatory activity compared to the reference drug diclofenac sodium.

Experimental Section
General.All the chemicals and reagents used were lab grade material procured from Alfa aesar.The melting points were determined using Buchi apparatus by the open capillary tube method.The IR spectra were recorded in Perkin-Elmer series 2000 FTIR spectrophotometer using KBr pellet. 1 H NMR and 13 C NMR spectra were obtained in CDCl3, DMSO-d6 on a Bruker spectrometer at 400 and 100 MHz, respectively.The chemical shifts are reported in ppm (δ) relative to tetramethylsilane as internal standard, coupling constants (J) are in hertz (Hz).Mass were recorded on ESI -Perkin Elmer Sciex, API 3000 mass spectrometer.Pre-coated silica gel GF254 plates from Merck were used for thin layer chromatography.The elemental analyses were recorded in Thermo Finnigan Flash EA 1112 elemental analyser.
The solvent was evaporated and triturated with cyclohexane (5 mL), afforded the title compound (

General procedure for the synthesis of Picen-13-ylmethylene derivatives (11-17).
A mixture of compound 10 (0.001 mole), active methylene compound scheme 2 (a-h) (0.001 mole) in ethanol (5 mL) was heated under reflux for 3 hours using piperidine (0.5 mL) as a catalyst.The progress of the reaction was monitored using TLC (hexane: ethyl acetate 7:3).The reaction mass was cooled to 25-30 °C and the product was collected by filtration and recrystallized from ethanol afforded compounds the temperature was increased to keep the samples at 57 °C for 30 minutes.After cooling to room temperature, the turbidity was measured using UV-Visible spectrophotometer at 660 nm following addition of phosphate buffered saline.The control represents 100% protein denaturation.The results were compared with reference drug Diclofenac sodium.The percentage inhibition of protein denaturation by the drug was calculated by using the following formula.Percentage Inhibition = 100-[(optical density of test solution-optical density of product control) ÷ (optical density of test control)] x 100

Table 1 .
Reaction conditions and yield of 3 for the esterification using an alcohol

Table 2 .
Mole ratio of reactants, reaction conditions and yield for the oxidative cyclisation of Compound 4

Table 3 .
Crystal data and structure refinement for Compound 11