Synthesis of 3-( o -stilbenyl)sydnone and 3-( o -stilbenyl)-4-substituted- sydnone derivatives and their antitumor evaluation

A series of novel stilbene-sydnone derivatives were synthesized by the following sequence of reactions: starting from methyl anthranilate via glycine-and nitrosoglycine derivatives the corresponding 3-( o -carbomethoxyphenyl)-4-H/Me/Ph-sydnones were prepared and transformed to 3-( o -formylphenyl)-4-H/Me/Ph-sydnones, starting materials for Wittig reaction with various phosphonium salts to stilbenylsydnone derivatives. Final products were evaluated for their cytotoxic properties on five cancer cell lines, whereby the cis -4-methyl-3-[2-[2-(4-methylphenyl)ethenyl]phenyl]sydnone 5 and cis -4-phenyl-3-[2-[2-(4-chlorophenyl)ethenyl]- phenyl]sydnone 10 showed the most pronounced activity.


Introduction
Sydnones 1 are five-membered heterocycles.They belong to a class of dipolar compounds known as "mesoionic" and can be represented as hybrids of a number of mesomeric ionic forms (Figure 1).Since their first synthesis 2 diverse substituted sydnones have been synthesized and many of them have shown useful biological properties 3 (e.g., as antibacterial, 4 antineoplastic 5 and antiinflammatory 6 agents).In an effort to prepare new sydnones of potential pharmacological activity, especially the combination of the sydnone moiety with some other pharmacophore, we turned our attention to the preparation of stilbene substituted sydnones.Stilbene-based compounds are widespread in nature and have become of particular interest to chemists and biologists because of their wide range of biological activities. 7Stilbene itself does not occur in nature, but hydroxylated stilbenes 8,9 have been found in many medicinal plants.We expected that the structures obtained by combining the stilbene and sydnone moieties might possess valuable biological activity (Figure 2).
The first step involves the reaction of methyl anthranilate with halogenoacetic acids to give N-(o-carbomethoxy)-aminoacids 13-15.The resultant aminoacids were treated with sodium nitrite and the crude N-nitroso derivatives submitted to dehydration with acetic acid anhydride to give N-(o-carbomethoxyphenyl)sydnones 16, 11 17 and 18.The sydnone derivatives were purified by column chromatography and isolated in 46% 16, 59% 17 and 21% 18 yields.After reduction with NaBH4 in methanol/t-butanol to hydroxymethyl derivatives 19-21, followed by oxidation with MnO2 and filtration through the column with silica gel, the formyl derivatives 22-24 were obtained in 61-68% yield.The resulting 3-(2-formylphenyl)sydnone 22, 3-(2-formylphenyl)-4methylsydnone 23 and 3-(2-formylphenyl)-4-phenylsydnone 24 were the starting compounds for the continuation of the synthesis to stilbene-sydnone derivatives 1-12.The obtained formylphenylsydnone derivatives 22-24 were submitted to Wittig reaction with p-substitutedbenzyltriphenylphosphonium salts 29-32, which were prepared from the corresponding psubstituted-benzylbromides 25-28 and triphenylphosphine in toluene solution, by a standard procedure.The Wittig reactions were performed in ethanol with the addition of sodium ethoxide as a base.All sydnone derivatives were prepared as mixtures of trans-and cis-isomers, which were purified and separated by consecutive column chromatography.
All prepared compounds are identified and characterized by spectroscopic methods.Derivatives 14 and 15 have mass spectral molecular ions at m/z 224 and 286, respectively, and in their IR spectra exhibit two bands at 1694-1702 and 1667-1685 cm -1 , which correspond to acid and ester carbonyl groups.Also, their NMR spectra correspond to those expected for the proposed structures.The N-(o-carbomethoxyphenyl)sydnone derivatives 16 11 -18 have in their IR spectra two bands, which correspond to sydnone carbonyls and ester carbonyls, in the range 1772-1759 and 1732-1726 cm -1 .In their 13 C NMR spectra the signals of the two carbon atoms in the carbonyl groups are observed at 167-169 ppm and the sydnone ring carbon (C-4) appears at 107-109 ppm.The hydroxymethylphenyl derivatives 19-21 have not been analyzed spectroscopically but their oxidation products 22-24 have.The spectroscopic data for 22 correspond to those in the literature 12 and formylphenylsydnone derivatives 23 and 24 show singlets at ca 9.9 ppm in their 1 H NMR spectra, which correspond to aldehyde protons, and the characteristic signals of the sydnone carbon (C-4) appear in their 13 C NMR spectra at 108-110 ppm.
The stilbene-sydnone derivatives 1-12, show, besides the characteristic signals for the carbonyl carbon and sydnone ring carbon, the corresponding signals for stilbene moieties in their 13 C NMR spectra.All carbonyl carbons and sydnone ring carbons of the cis-and trans-isomers appear in a very narrow region at 167-169 ppm and 97-109 ppm, respectively.The cis-1-12 and trans-1-12 isomers are easily recognizable in their 1 H NMR spectra by the coupling constants of the ethylenic protons which have values of ~ 12 Hz or ~ 16 Hz, respectively.In the cases of only 3-substituted sydnone derivatives 1-4 the characteristic singlets for the sydnone ring hydrogens at ~ 6.5 ppm for the trans-isomers and 6.4 ppm for cis-isomers unequivocally confirm the sydnone structures.

Biological evaluation
Stilbene-sydnones were screened for their antiproliferative activity in the Laboratory for Experimental Therapy at the Department for Molecular Medicine of Rudjer Bošković Institute.In vitro studies on 5 cell lines derived from 5 different tumor types: HeLa (cervical carcinoma), MCF-7 (breast carcinoma), SW 620 (colon carcinoma), MiaPaCa-2 (pancreatic carcinoma), and H 460 (lung carcinoma).
The tested compounds showed diverse, but overall very low, antiproliferative effects on the tested cell lines.Interestingly, although many trans-stilbenes, including resveratrol, pterostilbene, piceatannol etc. exhibit high levels of biological activities, 13 in our study transderivatives showed generally lower activity compared to cis-derivatives.Among the latter, the most interesting compounds are cis-5 and cis-10 which showed significantly more pronounced inhibitory effects towards all cell lines.Such activity can be correlated to the resveratrol activity in tumor cells. 14Only cis-2, cis-6 and cis-7 did not show growth inhibition, while from tested trans-derivatives only trans-3 and trans-6 showed low inhibition of cell growth.

Conclusions
We have synthesized cis-and trans-stilbene-sydnones 1-12 using the strategy in which the properly substituted sydnone moiety was made first followed by building the stilbene moiety.This approach is especially convenient in the cases where both stilbene diastereomers are the target molecules.Tested compounds showed diverse antiproliferative effects on tested cell lines that ranged from low to moderate, except cis-5 and cis-10, which showed significantly stronger inhibitory effects towards all cell lines.In general cis-derivatives showed significantly better antiproliferative activity, compared to trans-derivatives, whereby only trans-3 and trans-6 showed low inhibition of cell growth.

Experimental Section
General.The 1 H and 13 C NMR spectra were recorded on a Bruker AV-600 Spectrometer at 300 and 600 MHz.All NMR spectra were measured in CDCl3 or DMSO using tetramethylsilane as reference.UV spectra were measured on a Varian Cary 50 UV/VIS Spectrophotometer.IR spectra were recorded on FTIR-ATR Vertex 70 Bruker or Perkin-Elmer M-297 spectrophotometer.Mass spectra were obtained on Extrel FT MS 2001 DD, Auto Spec Q (VG Analytical Manchester, GB), on Platform LCZ (Micromass, UK) and/or on a Varian Saturn 2200 equipped with Factor Four Capillary Column VF-5ms.Melting points were obtained using an Original Kofler Mikroheitztisch apparatus (Reichert, Wien).Elemental analyses were carried out on a Perkin-Elmer, Series II, CHNS Analyzer 2400 at Rudjer Bošković Institute.Silica gel (Merck 0.063e0.2mm) was used for chromatographic purifications.Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 plates.Solvents were purified by distillation. 15 mixture of methyl anthranilate (2.4 g, 15.9 mmol), chloroacetic acid (1.0 g, 10.6 mmol) and sodium acetate trihydrate (1.4 g, 10.6 mmol) in water (10 mL), was heated for 6 hours at reflux.The reaction mixture was cooled to 0°C (on ice bath), then an aqueous solution of NaOH (10%) was added until pH 8 was reached.After extraction with dichloromethane (3 x 30 mL) the water phase was acidified with hydrochloric acid to pH 3.5 resulting in a white solid precipitate.Filtration and desiccation afforded the title compound as a white solid (621 mg, 28%).

Synthesis of ester sydnone derivatives (16) 11 and (17)
To a suspension of the corresponding glycine 13 or 14 (9.2 mmol) in water (30 mL) cooled on an ice bath, was added hydrochloric acid (17%, 9.2 mmol) dropwise and the reaction mixture was stirred for 3 hours.Then a solution of sodium nitrite (952 mg, 13.8 mmol) in water (5 mL) was added dropwise during 2 hours with cooling on the ice bath.The reaction mixture was stirred for an additional one hour then dichloromethane was added and stirring was continued for an additional 30 minutes.After extraction, drying on anhydrous MgSO4, filtration and evaporation, the nitroso-derivative was isolated.Without any further purification the nitroso-product was dissolved in quintuple the amount of acetic anhydride and the reaction mixture was put in a dark place for 7 days.Then the reaction mixture was poured onto cold water (100 mL) with cooling and vigorous stirring.Neutralization with sodium bicarbonate, extraction with dichloromethane (3×30 mL), drying, filtration and evaporation afford the crude sydnone derivative. 11From crude mixture isolated by column chromatography on silica-gel as adsorbent and dichloromethane as eluent.Pale yellow crystals, yield 46%, 940 mg, mp 102-104C (lit. 11104-106 C).

Synthesis of alcohol sydnone derivatives (19-21)
The reaction mixture of the corresponding sydnone 16-18 (1.9 mmol ) and NaBH4 (3.8 mmol) in t-BuOH (12 mL) was heated to reflux and MeOH (1mL) was added dropwise during 1 hour.After cooling to room temperature, water (15 mL) was added and the mixture was evaporated under reduced pressure to remove t-BuOH/MeOH as an azeotrope.The product was extracted from water with dichloromethane (3×25 mL).The organic phase was dried over anhydrous MgSO4, filtered and evaporated to afford the crude product which was used in the next reaction without any further purification 19 (oily matter, yield 59%, 217 mg), 20 (oily matter, yield 58%, 395 mg), 21 (oily matter, yield 93%, 478 mg).

General procedure for the synthesis of stilbenylsydnones (1-12)
The previously prepared p-substituted benzyltriphenylphosphonium bromide 29-32 (1.6 mmol) was dissolved in absolute ethanol (10 mL) under argon and a solution of sodium ethoxide in ethanol, prepared by reaction of Na (36 mg, 1.6 mmol) in absolute ethanol (5 mL), was added dropwise.The reaction mixture was stirred for 15 minutes and a solution of the corresponding aldehyde 22-24 (1.0 mmol in 15 mL of ethanol) was added.The reaction mixture was stirred and heated at 40 °C for 1 hour.After solvent evaporation, water (20 mL) and benzene (15mL) were added to the residue.The water phase was washed with benzene (3x15 mL), the organic layers were collected, dried over anhydrous MgSO4, filtrated and evaporated, affording crude product.The mixtures of the trans-and cis-stilbenylsydnones were separated by column chromatography on silica-gel as adsorbent and dichloromethane as eluent.Further column chromatography with dichloromethane and/or dichloromethane/ether (0-3%) afforded the pure trans-and pure cisisomers.