Synthesis of S-(28a-homobetulin-28a-yl) thiophosphate, thiophosphonate, and thiophosphinate

GRAPHICAL ABSTRACT ABSTRACT A concise synthesis of thiophosphate, phenylthiophosphonate, and diphenylthiophosphinate esters bearing a 28a-homolupane residue is reported. The new triterpenes were obtained from the readily available 3-O-acetylichopanol by a nucleophilic substitution of the corresponding mesylate with thiocyanate ion followed by a Michaelis–Arbuzov reaction. These results open the way to new lupane-type derivatives having a thiophosphorus moiety at the lupane core as potential anticancer compounds. Additionally, the cytotoxic activities of the new homolupane compounds were evaluated in vitro.


Introduction
Triterpenes-betulin, lupeol and betulinic acid-isolated from white birch bark (Betula sp.) and their derivatives possess interesting biological properties which have been extensively studied in recent years. [1][2][3][4][5][6][7][8] Recent investigations have demonstrated that even a simple modification of betulin can profoundly influence its anticancer activities. Recently, we presented a method for the preparation of lupanes with an extended C-17 side chain. [9][10][11] The promising cytotoxicity of homobetulin and homothiobetulin derivatives encouraged us to synthesize novel lupane-type triterpenes bearing thiophosphorus substituents at the C-28a homobetulin position and evaluate the cytotoxicity of these compounds. This study has opened up a new research field in lupane chemistry because lupanes bearing a sulfur atom as a part of the core region, are practically unknown. [12][13][14][15] Similarly, only a few betulinyl phosphates have been prepared to date. 15 Herein we report the synthesis of thiophosphoric acid esters bearing a 28a-homobetulin moiety and their anticancer properties determined by an evaluation of their cytotoxicity. This study is a continuation of our interest in the synthesis and utility of lupane type triterpenes modified at the C-17 side chain. [9][10][11]

Results and discussion
Thiocyanates are readily prepared by the nucleophilic thiocyanation of organic halides or sulfonyl esters. Due to a good leaving group ability of the cyanide fragment, their reactions with phosphorus(III) acid esters afford, via a Michaelis-Arbuzov type rearrangement, a number of thiophosphorus compounds. 18, 19 The easily available trimethylsilylated derivatives of the phosphorus(III) acids are mild, selective and highly reactive substitutes for the classical phosphorus(III) acid esters. Although very useful, the above reaction was, however, only very occasionally used in organic synthesis. 20,21 In this paper we want to demonstrate, that the Michaelis-Arbuzov reaction between silylated phosphorus(III) acids and organic thiocyanates may be successfully applied for the synthesis of highly complex thiophosphorus derivatives of lupane-type triterpenes.
3-O-Acetylichopanol (1), 10 readily available by Wittig elongation of the side chain of betulin, was treated with mesyl chloride to afford 3-O-acetylichopanol mesylate (2) in 97% yield. Reaction of mesylate 2 with an excess of KSCN in acetone gave thiocyanate 3 in 86% yield (Scheme 1). Its structure was confirmed by 13 C NMR and IR spectral data; the resonance for the SCN carbon atom was found at 112.4 ppm in the 13 C NMR spectrum, and a weak band at 2154 cm −1 was observed in the IR spectrum.
Initially, the Michaelis-Arbuzov reaction with the thiocyanate 3 was carried out in neat diethyl trimethylsilyl phosphite [(EtO) 2 P-OTMS]. The reaction proceeded smoothly at 40°C and afforded pure thiophosphate 4 in high yield (75%). Hydrolysis of ester 4 to the corresponding thiophosphoric acid 5 was unsuccessful. Both, basic (MeONa in methanol, Et 3 N in dichloromethane) and acidic conditions (TMSI or TMSI / NaI mixture) resulted in a fast decomposition of the starting material. Therefore, we turned our efforts toward the use of tris(trimethylsilyl) phosphite [(TMSO) 3 P] as the starting phosphorus reagent. We expected, that neutral conditions required for the deprotection of trimethylsilyl ester 6 would open the way to the free thiophosphoric acid 5. Reaction of thiocyanate 3 with (TMSO) 3 P at 40°C afforded a crude oily 6 which was immediately treated with methanol. A white precipitate, considered to be the required free acid 5, was practically insoluble in a wide range of standard solvents (hydrocarbons, halogenated hydrocarbons, THF, acetone, alcohols, DMSO, water, etc.). We reasoned that the ammonium salt of 5 should be, at least partially, soluble in organic solvents. However, treatment of 5 with diethylamine caused fast decomposition of the starting material and a product containing no phosphorus atom was isolated. Its NMR spectra showed a single set of signals, whereas mass spectrometry revealed that its molecular mass [M+Na] + = 1049.7410 corresponds to the disulfide 8. No mass peaks matching the possible thiol 7 were identified.
Application of ethyltrimethylsilyl phenylphosphonite 22 [PhP(OEt)(OTMS)] in the reaction with thiocyanate 3 under similar conditions afforded the expected thiophosphonate 9 as an inseparable mixture of diastereoisomers in high yield (84%, approx. 1:1 ratio). Similarly, the use of trimethylsilyl diphenylphosphinite 23 (Ph 2 P-OTMS) gave the expected thiophosphinate 10, again in excellent yield (88%). Both reactions were carried out at room temperature. The structures of all new compounds described in this communication were confirmed by extended NMR experiments, as well as by elemental analysis and HRMS. Observed positions for 31 P NMR signals of 4, 9 and 10 (28.2-44.7 ppm) were in the expected range. 24 Anticancer activities of the studied triterpene derivatives were tested against human BJ fibroblasts and cancer cell lines of various histopathological origins, including T-lymphoblastic leukemia CEM, breast adenocarcinoma MCF7 and cervical carcinoma HeLa lines. The cytotoxicity assay was performed as previously described. 10 Mesylate 2 had an interesting therapeutic index. It was selective against cancer cell lines, although the activity was only moderate (IC 50 : CEM-16.8 ± 0.6 µM; MCF7-41.4 ± 1.7 µM; HeLa -25.1 ± 1.7 µM) without affecting the growth of normal human fibroblasts (BJ >50 µM). At present, only a few natural agents are known to possess the potential ability for selective/preferential elimination of cancer cells without affecting the growth of normal cells. [25][26][27] By comparison, thiocyanate 3 was highly active against all tested cancer cell lines (IC 50 : CEM-13.7 ± 4.9 µM; MCF7-7.8 ± 1.7 µM; HeLa-4.7 ± 0.3 µM), however, it was also toxic against normal cells (BJ-IC 50 18.0 ± 0.8 µM). Thiophosphorus derivatives 4, 9, and 10 were inactive.

General methods
Silica gel HF 254 and Silica gel 230-400 mesh (E. Merck) were used for TLC and column chromatography, respectively. 1 H and 13 C NMR spectra were recorded at 298 K with Varian NMR-vnmrs600 or vnmrs500 spectrometers, using standard experimental conditions and Varian software (ChemPack 4.1). 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). Internal TMS was used as the 1 H and 13 C NMR chemical shift standard. J values are given in Hz. High-resolution mass spectra (HRMS ESI) were acquired with Mariner and Maldi SYNAPT G2-S HDMS (Waters) mass spectrometers. Optical rotations were measured with a Jasco P-2000 automatic polarimeter. IR spectra were recorded on Jasco 6200 FT-IR spectrophotometer.

3β-O-Acetyl-28a-thio-28a-homolup-20(29)-en-28a-yl disulfide (8)
A mixture of (TMSO) 3 P (250 μL, 0.75 mmol) and thiocyanate 3 (66 mg, 0.12 mmol) was heated at 50°C for 18 h under an argon atmosphere in a screw cap tube. Volatile components were removed by evaporation under reduced pressure to afford crude oily ester 6. Immediately, it was dissolved in methanol (3 mL) and kept at r.t. for 24 h. The white precipitate was separated and washed twice with methanol (1 mL) by centrifugation to give crude 5 as insoluble powder (31 mg). It was treated with freshly distilled diethylamine (1 mL) and kept for 1 h at 40°C under an argon atmosphere in a screw cap tube. The excess of amine was evaporated under reduced pressure. The residue was purified by preparative TLC (hexane-ethyl acetate, 9:1) to afford the title compound as foam (16 mg, 25% over the three steps).

Conclusions
In conclusion, a series of thiophosphate, thiophosphonate, and thiophosphinate esters bearing the 28a-homolupane fragment have been successfully synthesized by a Michaelis-Arbuzov reaction of the corresponding homolupane thiocyanate. This is the first report of the incorporation of these functional groups into lupane type triterpenes. All new compounds were evaluated for their cytotoxic activities towards normal and cancer cell lines. It was found that mesylate 2 was selective against cancer lines. Thiocyanate 3 was highly active against all tested cell lines.