Synthesis and bioactivity of some 2-oxo-5-aryl-3-hydrazone and 2-oxo-5-aryl-4-hydrazone pyrrolidine derivatives

2-Aryl-4,5-dioxopyrrolidine-3-carboxylate and 5-aryl-2,4-dioxopyrrolidine-3-carboxylate derivatives were successfully synthesized via carbonyl-based multiple component reaction and Dieckmann cyclization, respectively. Successive functional group transformations which include decarboxylation and hydrazonation afforded 2-oxo-5-aryl-3-hydrazone and 2-oxo-5-aryl-4hydrazone derivatives. Compound 3d exhibited activity against human histiocytic lymphoma (U937) and neuroblastoma (SH-SY5Y) cell lines while compound 6 showed neuroprotective ability from oxidative stress medium induced with H2O2.


Introduction
][8][9][10][11][12][13] Recognizing the importance of such compounds in drug discovery has prompted us to investigate potential anticancer and neuroprotection activities of the synthesized novel compounds.The pyrrolidone ring system has been the subject of research for more than three decades.In the early 1970s, piracetam was the first pyrrolidone to come to the attention of clinicians, and since then there has been much pharmaceutical interest in a broad range of indications and in new compounds.Modes of action of the pyrrolidones have revealed various pharmacological effects, as reviewed by Shorvon. 6There are evidence that the pyrrolidones exert a number of biological effects and that there is unlikely to be no single predominant mode of action that is common to all pyrrolidone compounds. 6tructural modification on the pyrrolidine ring to produce more bioactive compounds has attracted many research groups.Tsou and co-workers have reported on synthesizing natural product-based small molecule libraries containing 2-aryl pyrrolidine skeleton. 14Interestingly, the structural modification via hydrazonation reactions of the 2,3-or 2,4-dioxoarylpyrrolidinones has not been reported before.8] Consequently, novel 2-oxo-5-aryl-3hydrazone and 2-oxo-5-aryl-4-hydrazone derivatives have been synthesized in our laboratory and their bioactivities are reported in this paper.

Chemistry
Previously we have reported 5 that the synthetic intermediate 2,3-dioxo-5-(hetero) arylpyrrolidines, 1a-d, can be formed by refluxing equimolar diethyl oxalacetate, aromatic aldehyde and amine via carbonyl based on Dehaen's multi component reaction protocol. 16In addition, we have also prepared some new compounds of 2,3-dioxo-5-(hetero) arylpyrrolidines 1e-1j utilizing different aldehydes and amines (in the case of 1e, ammonia is used) through the one pot reaction (Table 1).Although the yield of this reaction was not excellent, the expected products however, could easily be isolated without the need of any column chromatography. 5t is interesting to note that as racemization is expected to occur at C-5 of the pyrrolidinone ring during the cyclisation process, we found that only one sharp singlet peak of C5-H (δ 4.95 -5.00) was observed in the proton spectra.This suggested that the cyclisation reaction might take place in a stereo controlled manner of which the group at C-5 dictates the stereochemistry of the five membered ring system.For the record, similar observation of the stereochemistry at C-5 of the cyclized products was also reported by Dehaen et al. 16 In order to validate this, compound 1c was subjected to decarboxyethylation with concomitant carbonyl reduction to give 3-hydroxyl 5-aryl pyrrolidine ring.Treatment of 1c with Boc anhydride gave 2-oxo-3-OBoc 5-aryl pyrrolidine ring, of which the structure was proved by X-ray analysis (Scheme 2, Figure 1).Scheme 1.One pot reaction of 2,3-dioxo-5-(hetero) arylpyrrolidine.The similarity of the carbon skeleton of compound 1c to that of the natural compound (-)codonopsinine [14][15] prompted us to further explore the chemistry of this compound.For example, when 1c was refluxed in 10% HCl, the decarboxylated 2,3-diketo 7 was formed with a yield of 68%.Subsequently, hydrazonation of 7 with various hydrazine salts furnished hydrazone derivatives of 2-oxo-5-aryl-3-hydrazone 3a-d (Scheme 3).Wang et al. reported that pyrrolidine ring bearing hydrazones functionality were shown to display a variety of interesting biological activities. 17In another development, 2,4-dioxo-3-alkoxy-5-aryl-pyrrolidines were synthesized initially by reacting D-4-hydroxyphenylglycine 8 with thionyl chloride to give a known compound glycine methyl ester hydrochloride salt 9 in a quantitative yield. 18Treatment of 9 with methyl malonate potassium salt in the presence of dicyclohexylcarbodiimide (DCC) furnished compound 10. 19Dieckmann cyclisation reaction of 10 using excess sodium methoxide in methanol 20 gave the cyclised product 2,4-dioxo-3-carboxy-5-hydroxyphenyl pyrrolidine 4 in good yield.Since both the N and OH groups of compound 10 were not protected the cyclisation reaction must have occurred via the formation of a trianion intermediate.
All compounds were inactive except 3d which showed activity against U937 and SH-SY5Y cell lines with IC50 values of 137 and 227 µM, respectively.The present findings showed that concentration ranges from 1nM to 1mM of compounds 4 and 6 did not show neurotoxicity effects to differentiated SH-SY5Y cells.Compound 6 significantly protected cells from H2O2 insults at 100nM to 1mM.The above results did not show any potential for cancer treatment, but .HCl give us insights for therapeutic application in neurodegenerative, that may have potentials in clinical utility.

Experimental Section
General.Melting points were determined on an automatic FP62 melting point apparatus from Mettler Toledo and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on Varian NMR Spectrometer instrument operating at 300 MHz at room temperature, in CDCl3 or DMSO solutions.Chemical shifts are given in δ units (ppm) relative to TMS as internal standard.Elemental analyses were performed on Flash Elemental Analyzer 110 series.IR spectra (4000-400 cm -1) were recorded on Varian Excalibur 3100 FT-IR spectrometer, using ATR.Mass spectra were recorded on Agilent 1200 LCMS-QTOF instrument.The progress of the reactions was routinely monitored by thin layer chromatography (TLC) on silica gel GF254 and the products were visualized with an ultraviolet lamp (254 and 365 nm).All reagents and starting materials were purchased from Sigma-Aldrich Co. and Merck Chemical Co.

General procedure. Synthesis of 2,3-dioxo-5-(hetero)arylpyrrolidines (1)
To a stirred solution of diethyl oxalate (47.6 mmol) in ethanol (100 ml), amine (47.6 mmol) and aromatic aldehyde (47.6 mmol) were added.The reaction mixture was heated under reflux for 1 h.After cooling, ice-water was added to the mixture and then acidified with HCl.The precipitate formed was filtered, washed with water and diethyl ether to remove traces of aldehyde to furnish the product.
Assay for cytotoxic activity.Cells (1 × 10 5 cells/ml) were seeded in 96-well plates in specific media with 1% non-essential amino acids (100 ×), 1% L-glutamine (200 mM), 1% gentamicin (10 mg/mL), and supplemented with 10% fetal bovine serum (FBS).All cell lines were maintained in an incubator at 37ºC in a 5% CO2 atmosphere with 95% humidity.Cell viability was determined after 24 h at 37ºC by CellTiter 96 ® AQueous Assay uses the novel tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt; MTS assay) using Glomax multi detection system and read at 490 nm. 25 Neurotoxicity and neuroprotection analysis.Retinoic acid will induce the differentiation of the neuroblastoma cells to behave like neuron-phenotypic cells.Compounds 4 and 6 were tested for their neurotoxicity effects.The differentiated cells were exposed to the concentration ranging from 1nM to 1mM.The neurotoxicity effects were determined by MTS assay.In case of neuroprotection, those compounds were added to each well at final concentrations ranging from 1 nM to 1 mM and incubated for 2 h.Then, treated cultures were exposed to 98 µM hydrogen peroxide (H2O2, 30%), which caused 50% reduction in cell viability (cell's IC50 value).The cultures were further incubated for 24 h, and then cell viability test was performed by MTS assay.