Synthesis of 3-phenyl-2 H ,5 H -pyrano[3,2-c ]chromen-2-one derivatives and their antineoplastic activity

Reaction of 4-oxo-4 H -chromen-3-carbaldehydes 1 with phenylacetic acids 2 under mild conditions or microwave irradiation led to 3-phenyl-2-oxo-2 H ,5 H -pyrano[3,2-c ]chromen-5-yl acetates 3 . At stronger conditions by-products 5-(2-hydroxybenzoyl)-3-phenyl-2 H -pyran-2-ones 4 and 5-hydroxy - 2 H ,10a H -pyrano[2,3-b ]chromen-2-ones 5 were also obtained. 5-Hydroxy-and 5-alkyloxy-2 H, 5 H -pyrano[3,2-c ]chromen-2-ones 6 and 7 , respectively were easily prepared by reaction of 3 with water or alcohols. Twelve synthesized compounds were evaluated on their antineoplastic activities on 60 human tumour cell lines panels in NCI USA. According to the screening results 3-phenyl-2 H ,5 H -pyrano[3,2-c ]chromen-2-one was discovered as a new leading skeleton suitable for further development. Some SAR conclusions were made. Antitubuline mechanism for the most active compound 3g has been proposed.


Introduction
Synthetic exploitation of 4-oxo-4H-chromen-3-carbaldehyde 1 and its derivatives results from their reactivity towards nucleophiles.The wide variety of heterocycles can be synthesized due to the presence of three electrophilic centres in the molecule of 1 and its ability to open or retain pyran-4-one ring. 1 Many of chromene derivatives exhibit significant biological activity, such as antitumour, 2,3 antimycobacterial 4 or antiviral. 5f ethanol liberated from ethyl acetate eluent.Compound 8 can be also prepared from 5b by its heating in EtOH in the presence of catalytic amount of p-TsOH (Scheme 2).Scheme 1.The conditions for the synthesis of compounds 3a -8.
We have observed that the acetyloxy group at C (5) in the compounds 3 easily undergoes nucleophilic substitution.The acetates 3 were partially converted into their alcohols 6 during flash chromatography purification on SiO2.Therefore we have investigated the conditions for hydrolytic removing of AcO-group from 3 to prepare alcohols 6. Acid catalyzed reaction of 3a in the mixture of dioxane-water (3 : 1) at 50 °C within 2 h gave 74% of alcohol 6a.Stirring of 3 with water in THF for 15 -20 h at room temperature gave alcohols 6 in 26 -98% yields (Scheme 2).Alkyloxy derivatives 7 were prepared by heating of 3 in alcohol (prop-2-yn-1-ol or 2-thioethanol) in the presence of catalytic amount of p-toluenesulfonic acid.The reaction heated at 60 °C for 3 h gave 65 -75% of 7 compared to only 10 min microwave-assisted reaction that gives the yields 68 -77% (Scheme 2).Scheme 2. The conditions for the synthesis of compounds 5a -7d.

Antineoplastic activity
Twelve from our prepared compounds were proposed to NCI USA for biological evaluation for anticancer properties (Table 1). 17ased on structures and obtained activities we can conclude that para substitution at Ph-C(3) in compounds 3 or 6 decreases antitumour properties with no relation to the electronic properties of substituents.Therefore in this case unsubstituted compounds are the most suitable (R 1 : H or 4-F) 3g, 3h, 3b and 6a.Two methyl substituents in 3g, 3h lolcated in the skeleton on C(8) and C(9) seems to be important for the increase of antitumour activity.a,b exceptionally two compounds have been pre-screened by an older method on three selected cell lines at single conc.10 -4 M. In this case to pass to further screenings at least one growth % should be below 32%: a NCI H460 -NSCLC lung cancer (growth 33%), MCF7 -breast cancer (96%), SF-268 -CNS cancer (107%); b NCI H460 (2%), MCF7 (17%), SF-268 (25%).Mean Growth (MID): represents average of growth % through all 60 human tumour cell lines at initial compound concentration 10 -5 M compared to 100% for untreated tumour cell lines (controls).The Best inhibition activities are the border growth % observed in individual the most sensitive human tumour cell lines at One-dose compound concentration 10 -5 M; N: no further screening indicated; 60CC: means selection for screening on 60 human tumour cell line panel at five different concentrations from 10 -4 M to 10 -9 M. Negative values in the table represent cytotoxic influence of screened compound.
The most promising compounds 3b, 3g, 3h and 6a, derived from the results of One-dose prescreening, were further evaluated at five different concentrations (10 -5 to 10 -9 M) on 60 human tumour cell line panels (Table 2).Compound 3g was shown to be the most active in slowing down tumour cell division compare to the control.Its structure contains unsubstituted phenyl group at C(3) and two methyl substituents at C(8) and C(9) (Table 1).This compound possesses submicromolar mean growth inhibition activity over all 60 cancer cell lines panel (GI50: 447 nM).Individual tumour cell lines (e.g.Leukemia, NSCLC, Melanoma) have shown even nanomolar sensitivities for this compound (GI50: 31 -63 nM, not stated in this paper).
The exact mechanism of antitumour activity of compound 3g is not known.We believe that 3g interacts with tubulines.The proposed mechanism is based on comparison of GI50 activity profile for individual tumour cell lines (kind of finger print) with profile of standard antitumour compounds having known antitumour mechanism.Compare analysis software is accessible online and it is free of charge. 18The compare analysis has been done with 3g inhibitor experimental GI50 values determined for all 60 human tumour cell lines (CC60 assays).These data were compared to sets of GI50 values of STANDARD_AGENTS taken from NCI database.A correlation between our most active compound 3g (NSC:745523) with compounds possessing typical antitubuline interactions has been obtained: e.g.rhizoxin with NCI code NSC:S332598 (correlation coefficient, 0.597), maytansine NSC:S153858 (0.438), paclitaxel NSC:S125973 (0.400), S-trityl-L-cysteine NSC:S83265 (0.374), vinblastine sulfate NSC:S49842 (0.358).The list of compounds with known antitubuline mechanism is published in NCI databases. 19

Table 1 .
The One-dose pre-screening assay results performed on 60 human tumour cell lines incubated with experimental compounds at single concentration 10 -5 M for 48 h

Table 2 .
The results of five-Dose assay performed on 60 human tumour cell lines