Anti-leukemia activity of semi-synthetic phenolic derivatives from Polygonum limbatum Meisn.

Background The present report describes the semi-synthesis of a few O-prenylated phenolic derivatives and their in vitro antitumor activities. These compounds were prepared by modifying two naturally occurring antitumor phenols, 5,7-dihydroxy-3-(1′-hydroxy-1′-phenyl-methyl)-6-methoxy-chroman-4-one (A) and 2′,4′-dihydroxy-3′,6′-dimethoxychalcone (B), previously isolated from Polygonum limbatum Meisn. (Polygonaceae). The structures were elucidated by spectroscopic means and comparison with published data. The cytotoxicity of compounds was determined by using the resazurin assay in the parental drug-sensitive CCRF-CEM cell line and its multidrug-resistant P-glycoprotein-over-expressing subline, CEM/ADR5000. Results We describe in the present paper four new semi-synthetic derivatives of A and B: 5-hydroxy-6-methoxy-7-O-(3′-methylbut-2′-enyl)chroman-4-one (1), trivially named metapchromone, 5-acetoxy-6-methoxy-7-O-[3′-methylbut-2′enyl]chroman-4-one (2), trivially named sargisin, 2′-hydroxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (3) trivially named limbachalcone A, and 2′-acetoxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (4) trivially named tsedengchalcone. Their preliminary cytotoxic activities have been determined. We also report herein the isolation of 1-methylhydantoin (C) and betulinic acid (D) from Polygonum limbatum for the first time. Conclusions The study clearly suggests that semi-synthesis involving O-prenylation and acetylation of chalcones or other chromanones should be avoided in a search for anticancer drugs. This conclusion should be helpful when selecting substituents for the synthesis of potential anticancer drugs.

The isolation of 1-methylhydantoin and betulinic acid from the Polygonum genus and the Polygonaceae family could be an important chemotaxonomic finding.

Results
The structures of natural compounds A, B and C isolated from P. limbatum were elucidated on the basis of spectroscopic data such as IR, 1D and 2D NMR spectra. Comparison of the data with those reported in the literature led to the identification of compounds such as betulinic acid (D) [5] and sitosterol 3-O-β-D-glucopyranoside (E) [6].
Compound C was obtained as brownish needles from n-hexane-EtOAc, mp 155-157°C. It reacted negatively to the FeCl 3 , suggesting the absence of a phenolic group in the molecule. Its molecular formula of C 4 H 6 N 2 O 2 , corresponding to 3 degrees of unsaturation, was determined by EI-MS (M + m/z 114) in conjunction with the NMR spectra. In the 13 C NMR spectrum, signals at δ C 173.8 (C-4) and 159.9 (C-2) for two carbonyl groups, one methylene signal at δ C 29.2 (C-5) and one methyl at δ C 53.9 (N-CH 3 ), were characteristic of 1-methylhydantoin structure [7]. In the 1 H NMR spectrum, two singlets were observed at δ H 3.95 and 2.90, respectively, assignable to these methyl and methylene groups, respectively (Table 1). In the HMBC spectrum, pertinent correlations were observed between H-5 and C-2, CH 3 and C-2 and C-4. The structure of compound C was established as 1-methyldiazolidine-2,4-dione (Fig. 2). It is a natural product from the Polygonum genus and has been fully characterized here for the first time. It has been previously reported as a synthetic compound and was found to be a renal metabolite of dupracetam [7].
Compounds A and B were prenylated under standard basic conditions by exposure to prenyl bromide. As expected, the poorly reactive chelated phenolic groups were not alkylated. In fact, subsequent acetylation of these groups required forcing conditions with the aid of 4-dimethylaminopyridine (DMAP) in a catalytic amount.
Interestingly, the prenylation of chroman-4-one A resulted in decomposition of the molecule with loss of benzaldehyde and formation of a 3-unsubstituted chromanone, namely compound 1. This rearrangement was likely due to a retro-aldol-like reaction following the mechanism shown in Fig. 3.

Discussion
In this study, we determined the cytotoxicity of the natural compound A as well as the semi-synthetic compounds 1-4. We previously reported the cytotoxicity of compound B [3,4], and the data were also reported herein for a better understanding of the structure-activity relationship (SAR). As shown in Fig. 4, the two natural compounds A and B were much more active than the semi-synthetic ones, inducing less than 20 % growth of CCRF-CEM leukaemia cells. This was confirmed in the dose-response assays, as IC 50 values below 20 μM were recorded for the two natural compounds. Among the synthetic compounds (Table 2), 3 displayed the highest activity with IC 50 values below 20 μM on the two tested leukaemia cell lines. Interestingly, the resistant cell line was more sensitive to compound 3 as well as to A and B than to doxorubicin. Nonetheless, the cytotoxicity can be considered moderate [8]. In contrast to doxorubicin, which was about 1000 times less active in multidrugresistant CEM/ADR5000 cells than in parental CCRF-CEM cells, compounds A and B, as well as the semisynthetic compound 3, showed minor cross-resistance in the otherwise highly drug-resistant CEM/ADR5000 cells. Regarding the structure-activity relationship, it clearly appeared that O-prenylation at position C4′ of B to afford 3 considerably reduced the cytotoxic activity. In addition to the O-prenylation, acetylation at C-2′ of 3 to afford 4 or at C5 of 1 to yield 2, further reduced the antiproliferative activity. These data clearly suggest that semi-synthesis involving O-prenylation and acetylation of chalcones or chromones should be avoided in the search for potential anticancer drugs. Usually it has been found that Cprenylation of the flavonoid nucleus increases the cytotoxic activity [9]. Moreover, as an additional benefit, prenylated flavonoids are relatively non-toxic to non-cancer cells [9]. In our case, O-prenylation was accompanied by a decrease in bioactivity, indicating the importance of free phenolic groups. This conclusion is further supported by the observation that, after prenylation, acetylation of the remaining free phenolic groups further reduced the cytotoxicity.

Conclusions
The objective of this study was to modify the structures of antitumor compounds A and B by O-prenylation and acetylation and to evaluate the structure-activity relationship (SAR). The results clearly suggest that O-prenylation and acetylation of chalcones, chromanones and possibly other flavonoids should be avoided in the search for potential anticancer drugs.

Cytotoxicity assay
The resazurin reduction assay [10] was performed to assess the cytotoxicity of compounds and doxorubicin was used as a control drug towards the parental, drug-sensitive CCRF-CEM leukaemia cell line and its multidrug-resistant, P-glycoprotein-over-expressing subline, CEM/ADR5000. The assay is based on the reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells. Non-viable cells rapidly lose their metabolic capacity to reduce resazurin and, thus, do not produce fluorescent signals anymore. Briefly, aliquots of 2 × 10 4 cells per well were seeded in 96-well-plates in a total volume of 100 μL. The studied compound was immediately added at varying concentrations to an additional 100 μL of culture medium to obtain a total volume of 200 μL/well. After 72 h, resazurin (Sigma-Aldrich, Schnelldorf, Germany) (20 μL, 0.01 % w/v) in distilled H 2 O was added to each well and the plates were incubated at 37°C for 4 h. Fluorescence was measured on an Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. A preliminary assay was done with all samples on leukaemia CCRF-CEM cells at 125 μM. Each assay was done at least twice with six replicates each. The viability was evaluated by comparison with untreated cells. IC 50 values represent the compound concentrations required to inhibit 50 % of cell proliferation and were calculated from a calibration curve by linear regression with the aid of Microsoft Excel [11].