Synthesis of 28a-homoselenolupanes and 28a-homoselenolupane saponins†

A practical synthesis of 28a-homo-28a-selenolupane triterpenes and the corresponding selenosaponins containing D-mannose, L-arabinose, L-rhamnose, and D-idose moieties is described. Selenium containing triterpenes were obtained from the readily available 3-O-allyl-homobetulin mesylate by nucleophilic substitution with the selenocyanate ion which upon reduction of the –SeCN group afforded the free selenol. Glycosylation using classical Schmidt donors gave 1,2-trans selenosaponins as the main product as well as minute amounts of 1,2-cis isomers. This is one of the very few examples of the synthesis of selenoglycosides by direct glycosylation of free selenols. The studied selenol showed high resistance to air oxidation resulting in good stability during the synthesis of selenolupane derivatives. Cytotoxic activities of new homoselenolupane derivatives were also evaluated in vitro and revealed that some triterpenes exhibited an interesting profile against human cancer cell lines.


Introduction
In the last few decades, the application of organoselenium compounds, 1 including steroid derivatives, 2 in cancer prevention and treatment became an objective of investigations of many scientific groups. These studies show that organoselenium compounds display a high cytotoxic activity against different cancer cell lines. Moreover, in some cases a positive effect on the cytotoxic properties was achieved by replacing the sulphur atom with selenium without modifying the basic structure of the compound. 3 Additionally, some heterocycles containing selenium possess an interesting anti-inflammatory activity profile with a significant analgesic effect. 4 Furthermore, many organoselenium derivatives demonstrate antimicrobial, antiviral and antifungal activities. 5 Previously, we reported the structure-activity relationships of the cytotoxic activity of ichopanol (28a-homobetulin) and ichopanothiol (28a-homothiobetulin) derivatives and saponins based on a homobetulin scaffold. We found, that the heteroatom (oxygen or sulphur) located on the C-17 side-chain is necessary for the anticancer activity of the studied compounds. [6][7][8] Based on the high biological activity of selenium derivatives, we reasoned that replacing these heteroatoms on the C-17 side-chain of the lupane core with selenium could enhance their anticancer effect. These results will broaden our knowledge regarding the synthesis, reactivity and stability of selenium-containing triterpenes and steroids as they are virtually unknown in the literature. 2,9 Herein, we report the first synthesis of homobetulin and homobetulin based saponins bearing a selenium atom. Furthermore, cytotoxic activities of all new derivatives were investigated.

Results and discussion
Mesylate 1, readily prepared via the reaction of 3-O-allyl-ichopanol and mesyl chloride, 8 was used for the synthesis of selenolupane triterpenes. Standard synthesis of selenocyanate 3 involves the nucleophilic replacement of sulfonyl ester with potassium selenocyanate. Our first attempt using acetone as a solvent failed giving only selenide 2. However, changing the solvent from acetone to DMF resulted in the formation of the desired product 3 in 87% yield; a small amount of by-product selenol 4 (7%) was also isolated. Reduction of 3 with LiAlH 4 gave the corresponding selenol 4 in 93% yield. Compound 5, needed for SAR studies, was prepared in 69% yield from allyl ether 3 by treatment with palladium(II) chloride (Scheme 1).
Synthesis of selenoglycosides by direct glycosylation of selenols is rarely found in the literature. Due to high susceptibility of selenols to oxidation the introduction of selenium at the anomeric position of a monosaccharide is achieved by the treatment of the sugar component with alkyl or aryl selenolates usually generated in situ from the corresponding diselenides in the presence of a reducing agent. 10 Free selenols were used occasionally to react with anomeric halogenides, 11 acetates, 11b,12 orthoesters, 13 or Schmidt donors. 11a,14 For the synthesis of lupane-type selenoglycosides we chose the Schmidt methodology. TMSOTf promoted glycosylation was carried out by the treatment of selenol 4 with perbenzoylated glycosyl trichloroacetimidates 6, 15 7, 16 8, 16 and 9, 17 which gave the desired selenoglycosides in 29-51% yield. A significant amount of unreacted starting selenol 4 was also recovered (13-40%). Although yields were rather moderate, further manipulation of the reaction conditions did not improve the yields of the selenoglycosides. As expected, the presence of the benzoyl protecting groups in the donor molecules directed the anomeric selectivity of glycosylation. 18 Regioselectivity was, however, slightly lower than that in the case of the corresponding sulfur analogues. 8 With the exception of α-D-mannopyranoside 10, mixtures of α and β anomers 11-16 of L-arabino, L-rhamno, and D-ido derivatives were obtained as specified in Scheme 2.
Selenols are considered highly susceptible to air oxidation leading to diselenides, however, selenol 4 was relatively resistant to oxidation. Recovered selenol 4 was reused for glycosylation without any significant decrease in the reaction yields. It must be noted, however, that prolonged storage slowly decreased its reactivity, although the physicochemical properties of 4 did not change. Considering the above observations which are crucial criteria in the synthesis of selenolupane derivatives, a study examining the stability of selenol 4 was undertaken. First, the independent synthesis of diselenide 27, the expected product of the air oxidation of 4, was conducted. The homogeneous product was prepared by the reaction of mesylate 1 with dilithium diselenide (Li 2 Se 2 ) 19 affording compound 27 in 88% yield (Scheme 3). The same compound containing inseparable unknown byproducts, may also be obtained by the oxidation of selenol 4 with hydrogen peroxide in the presence of NaOH (79%) and from the equimolar mixture of selenocyanide 3 and selenol 4 in the presence of a base (94%) according to Krief's method. 20 Surprisingly, in all cases, the physichochemical properties of 27, including 77 Se NMR, were identical as found for selenol 4 but differences in reactivity were observed. In contrast to compound 4, compound 27 did not react with Schmidt donors. On the other hand, both derivatives were alkylated by treatment with methyl iodide to afford selenide 28. However, in the case of the reaction of compound 27 with methyl iodide a longer reaction time was required (5 days, 70%), whereas the alkylation of freshly prepared selenol 4 in the presence of sodium hydride was complete within a few hours giving compound 28 in 74% yield (Scheme 3).
There are two possible explanations for such unusual behaviour. The first is that both compounds (4 and 27) have identical spectroscopic properties but they are different compounds. This speculation may be supported by the result of alkylation described above. In the first case (considering diselenide 27) sodium hydride acting first as a reducing agent (breaking Se-Se bond) and then as a base was required for alkylation. 21 It significantly prolonged the reaction time. On the other hand, when the formation of the Se-Se bond was not possible due to high steric hindrance, selenol was formed in both cases. However, reaction conditions and/or long storage time promoted the aggregation of selenol and the formation of relatively unreactive polymeric forms which caused a decrease in reactivity. Similar aggregation of betulin derivatives was described in the literature. 22 Attempts to remove the allyl block from 3-O-allylselenosaponins with palladium(II) chloride were unsuccessful and caused decomposition of the starting materials. Therefore, deallylation was performed under neutral conditions by isomerisation of the allyl double bond in the presence of the hydrogen activated iridium complex [Ir(COD)(PMePh 2 )]PF 6 followed by hydrolysis of 1-propenyl ether in the presence of p-TsOH. 17 Deallylated saponins 17-21 were obtained in high yields (78-93%) with the exception of 19 which was isolated in slightly lower yield (63%). Debenzoylation with potassium carbonate in methanol gave free saponins 22-26 in good yields (61-88%, Scheme 2).
Antiproliferative activities of the studied derivatives of homobetulin were tested in vitro. Activity against normal human BJ fibroblasts was compared with cytotoxicity on cancer cell lines of various histopathological origins, including T-lymphoblastic leukaemia (CEM), breast adenocarcinoma (MCF7), malignant melanoma (G-361) and cervical carcinoma (HeLa) lines. The cells of all these lines were exposed to six serial 3-fold dilutions of each drug for 72 h, the proportions of surviving cells were then estimated and IC 50 values (50% inhibitory concentrations) were calculated. A detailed procedure for the cytotoxicity assay was described previously. 7 The results obtained from Calcein AM assays are presented in Table 1.
Selenosaponins were practically inactive, except L-rhamnoside 25 which showed low activity (IC 50 32.5-45.4 µM). Similar results were obtained for oxygen 7 and sulphur analogs 8 which suggest that homobetulin saponins do not affect the growth of the cancer lines. Compound 30 was selective against malignant melanoma and leukemia cell lines (IC 50 10.2 or 15.3 µM) without affecting the growth of normal human fibroblasts and other cancer cells which suggested a high therapeutic index. By comparison, selenocyanate 5 showed the highest cytotoxic activity of all compounds tested (IC 50 2.0-5.2 µM). Its cytotoxicity was also higher than that measured for the appropriate sulphur analogue 29, and much higher than those observed for ichopanol (30). Sulphur analogue 29 showed an interesting profile as much more higher activity was determined for G-361 melanoma cells than for other cancer cell lines and fibroblasts. Both compounds, however, exhibit a relatively low therapeutic index.

Conclusions
In conclusion, a series of 28a-homo-28a-selenolupane triterpenes and the corresponding homoselenosaponins bearing D-mannose, L-arabinose, L-rhamnose, and D-idose moieties were synthesised. Required selenol was obtained by a nucleo-philic substitution of the corresponding mesylate with potassium selenocyanate followed by the reduction of the -SeCN group. Its unusual stability to air oxidation was discussed. Further glycosylation of selenol with Schmidt donors under the standard conditions followed by deprotection afforded homoselenosaponins. All new compounds were evaluated for their cytotoxic activities towards normal and cancer cell lines. Homobetulin selenocyanate was the most cytotoxic compound, whereas the corresponding saponins were mostly inactive. To our best knowledge this is the first synthesis of lupane-type triterpenes containing selenium as the core component.

General procedures
Silica gel HF 254 and silica gel 230-400 mesh (E. Merck) were used for TLC and column chromatography, respectively. 1 H, 13 C and 77 Se NMR spectra were recorded at 298 K with a Varian NMR-vnmrs600 or vnmrs500 spectrometer, using standard experimental conditions and Varian software (ChemPack 4.1). Configurational assignments were based on the NMR measurements, generated using two-dimensional techniques like COSY and 1 H-13 C gradient selected HSQC (g-HSQC), as well as 1 H-13 C gradient selected HMBC (g-HMBC). TMS was used as the internal standard for determining 1 H and 13 C NMR chemical shifts. J values are given in hertz. High-resolution mass spectra (HRMS-ESI) were acquired with Mariner and MaldiSYNAPT G2-S HDMS (Waters) mass spectrometers. Optical rotations were measured with a Jasco P-2000 automatic polarimeter. IR spectra were recorded on a Jasco 6200 FT-IR spectrophotometer.
Single crystal X-ray diffraction measurements were carried out on an Agilent Supernova diffractometer, at 100 K with monochromated Cu Kα radiation (1.54184 Å). The data reduction was made by using CrysAlisPRO software. 25 The structures were solved by direct methods and refined on F 2 by full-matrix least-squares by using SHELXS97 and SHELXL97. 26 All non-hydrogen atoms were refined anisotropically while hydrogen atoms were placed in calculated positions, and refined in riding mode. Crystals of 3 suitable for X-ray analysis were obtained by cooling of a hot methanol solution: monoclinic, P2 1 , a = 13.729 (2) and 0.1157 for all data, wR 2 = 0.2644, S = 1.046. Crystallographic data for the structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary material (deposition numbers: CCDC 1422676).
Data for 11.
Data for 13.
Data for 15.