An Original Method for the Synthesis of Partially Deuterated Natural Lembehyne B and the Study of Its Biological Activity ^†

Abstract

An efficient method for the synthesis of a partially deuterated analogue of the natural neuritogenic alkynol, lembehyne B, has been developed for the first time, based on the use of a new reaction of Ti-catalyzed cross-cyclomagnesiation of O-containing 1,2-dienes and terminal aliphatic 1,2-dienes using EtMgBr in high yield. The introduction of two deuterium atoms is carried out at the stage of treatment of the formed in situ magnesacyclopentane with D₂O.

1. Introduction

Acetylene alcohol lembehyne B, isolated in trace amounts from the Indonesian sea sponge Haliclona sp. [1], exhibits neuritogenic activity on Neuro-2A neuroblastoma cells [2], and is also an inducer of early apoptosis of Jurkat, HL-60, and K562 cell cultures [3].

Numerous studies carried out in recent years have shown that partially deuterated analogues of drugs have better pharmacokinetic characteristics, leading to changes in the mechanisms of biotransformation and a decrease in toxicity [4].

Earlier, at the Laboratory of Catalytic Synthesis of the Institute of Petrochemistry and Catalysis of the Ufa Federal Research Center of the Russian Academy of Sciences, a complete synthesis of natural lembehyne B was carried out for the first time [3], using the Ti-catalyzed cross-cyclomagnesiation of terminal allenes at the key stage of the reaction (the Dzhemilev reaction) [5,6,7,8,9,10,11,12,13,14,15,16]. Having obtained positive results of the study of the cytotoxicity of this alkynol, we obtained its deuterated analogue and studied its cytotoxicity in vitro.

2. Results and Discussion

At the first stage, the reaction of cross-cyclomagnesiation of 1,2-nonadecadiene 2 and tetrahydropyran ether 13,14-pentadecadienol 3 was carried out using EtMgBr in the presence of metallic Mg and a catalyst Cp₂TiCl₂ (10 mol%), through the stage of formation of magnesacyclopentane 4, the deuterolysis of which gives tetrahydropyran ether 14,17-Dideutero-(13Z,17Z)-tetraconte-13,17-dienol 5 in 84% yield. Successive reactions of removal of tetrahydropyranyl protection and oxidation of unsaturated deuterated alcohol 6 using Dess–Martin periodinan led to 14,17-Dideutero-(13Z,17Z)-tetrakont-13,17-dienal 7 in ~86% yield. As a result of the reaction of the previously synthesized lithium (trimethylsilyl)acetylenide with aldehyde 7 and removal of the trimethylsilyl protection with TBAF, racemic d₂-lembehyne B is formed in ~92% yield. Oxidation of hydroxyl group 9 with Dess–Martin periodinane gives ketone 10, and subsequent stereoselective reduction leads to the target d₂-lembehyne B 1 (Scheme 1).

For the synthesized compound, the in vitro antitumor activity was assessed on Jurkat, K562, HL-60, and U937 cell lines and fibroblasts, including the determination of IC50 using flow cytometry and multiplex analysis.

3. Conclusions

Thus, we are the first to obtain a partially deuterated analogue of lembehyne B, using the reaction of Ti-catalyzed cross-cyclomagnesiation of 1,2-dienes (Dzhemilev reaction) at the key stage of synthesis, and also studied its antitumor activity using modern methods of flow cytometry and multiplex analysis.

4. Experimental Part

Commercially available reagents (Sigma-Aldrich «Sigma-Aldrich Corparation, PO Box 14508, Saint Louis, MO 63178 USA» and Acros Organics «Janssen-Pharmaceuticalaan 3a, 2440 Geel, Belgium») were used. Reactions with organomagnesium compounds were carried out under dried argon atmosphere. 1,2-dienes was prepared according to the known procedure. Reaction products were analyzed on a Carlo Erba chromatograph (a Hewlett Packard Ultra-1 glass capillary column, 25 m × 0.2 mm, flame ionization detector, operating temperature 50–170 °C, carrier gas helium). TLC was performed on Silufol UV-254 plates. Elemental composition of compounds was determined using a Carlo Erba-1106 instrument. Mass spectra were obtained using a Bruker MALDI-TOF/TOF Autoflex III instrument. Then, 1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (100.62 MHz for 13C and 400.13 MHz for 1H). Chemical shifts of 1H and 13C nuclei (δ) are given relative to the residual signals of the deuterated solvent (δ 7.28 for protons and 77.2 for carbon nuclei).

Cross-cyclomagnesiation of nonadeca-1,2-diene (2) and 2-(pentadeca-13,14-dien-1-yloxy)tetrahydro -2H-pyran (3) with EtMgBr in the presence of Mg metal and Cp₂TiCl₂ catalyst. Diethyl ether (30 mL), nonadeca-1,2-diene (2) (1.27 g, 4.8 mmol), 2-(pentadeca-13,14-dien-1-yloxy) tetrahydro-2H-pyran (3) (1.23 g, 4.0 mmol), EtMgBr (16.0 mmol) (as 1.5 M solution in Et₂O), Mg powder (0.29 g, 12.0 mmol), and Cp₂TiCl₂ (0.1 g, 0.4 mmol) were placed in a glass reactor with stirring under argon (~0 °C). The reaction mixture was warmed up to room temperature (20–22 °C) and stirred for 6 h. The reaction mixture was treated with D₂O (20 mL) and extracted with diethyl ether (2 × 100 mL). The combined organic phases were dried over MgSO₄ and filtered, and the solvents were removed under reduced pressure. Silica gel column chromatography (hexane/EtOAc (35/1)) of the residue gave compound 5 (1.98 g, 88 %) as a pale yellow oily liquid.

14,17-Dideutero-2-[(13Z,17Z)-tetratriaconta-13,17-dien-1-yloxy]tetrahydro-2H-pyran (5). Yeld 84%. R_f = 0.40. IR (film) υ_max 724, 815, 1075, 1110, 1254, 1303, 1360, 1384, 1468, 2853, 2924 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.87 (3H, t, J = 6 Hz, CH₃), 1.25–1.70 (48H, m, CH₂), 1.78–1.85 (6H, m, CH₂), 1.95–2.05 (8H, m, CH₂), 3.32–3.87 (4H, m, CH₂-O), 4.54–4.56 (1H, m, CH-O), 5.32–5.39 (2H, m, CH=). ¹³C NMR (100.62 MHz, CDCl₃) δ: 14.07, 19.56, 22.68, 25.54, 26.27, 27.20, 27.28, 29.21, 29.25, 29.32, 29.38, 29.51, 29.56, 29.62, 29.72, 29.76, 30.73, 31.94, 61.99, 67.53, 98.61, 130.06. MS (MALDI-TOF), m/z: 577 [M]⁺. C₃₉H₇₂D₂O₂. Found (%): C, 81.26; H, 12.90. Calc. for C₃₉H₇₂D₂O₂ (%):C, 81.18; H, 13.27.

THP-deprotection of ether (5) was carried out with p-TsOH in CH₂Cl₂/MeOH using known method [17]. 14,17-Dideutero-(13Z,17Z)-tetratriaconta-13,17-dien-1-ol (6). Yeld 77%. R_f = 0.43 (hexane/EtOAc—5:1). IR (film) υ_max 674, 729, 1034, 1078, 1124, 1159, 1180, 1204, 1354, 1382, 1441, 1662, 2853, 2924 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.91 (3H, t, J = 6 Hz, CH₃), 1.26–1.37 (44H, m, CH₂), 1.55–1.61 (2H, m, CH₂), 1.98–2.10 (8H, m, CH₂), 3.64–3.68 (2H, m, CH₂-O), 5.40–5.42 (4H, m, CH=). ¹³C NMR (100.62 MHz, CDCl₃) δ: 14.12, 22.70, 25.76, 27.24, 27.32, 29.28, 29.34, 29.41, 29.47, 29.55, 29.58, 29.62, 29.65, 29.71, 29.75, 31.94, 32.83, 63.09, 130.24. MS (MALDI-TOF), m/z: 492 [M]⁺. C₃₄H₆₄D₂O. Found (%): C, 82.11; H, 13.44. Calc. for C₃₄H₆₄D₂O (%): C, 82.85; H, 13.90.

The oxidation of the alcohol (6) with Dess-Martin periodinane was carried out according known procedure [18]. 14,17-Dideutero-(13Z,17Z)-tetratriaconta-13,17-dien-1-al (7). Yeld 86%. IR (film) υ_max 721, 910, 1091, 1466, 1729, 2852, 2922, 3009 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, CH₃, J = 6 Hz), 1.27–1.35 (44H, m, CH₂), 1.60–1.67 (3H, m), 2.00–2.08 (8H, m, CH₂), 2.40–2.44 (2H, m, CH₂), 5.36–5.43 (4H, m, =CH), 9.76–9.77 (1H, t, O=CH, J = 6 Hz). ¹³C NMR (100.62 MHz, CDCl₃) δ: 14.10, 22.09, 22.70, 27.23, 27.30, 29.29, 29.37, 29.44, 29.52, 29.56, 29.59, 29.62, 29.68, 29.72, 29.76, 31.94, 43.91, 130.16, 130.19, 202.24. MS (MALDI-TOF), m/z: 490 [M]⁺. C₃₄H₆₂D₂O. Found (%): C, 83.44; H, 13.23. Calc. for C₃₄H₆₂D₂O (%): C, 83.19; H, 13.55.

Procedure for preparation of alkyne (8). To a solution of 0.58 g (6 mmol) of trimethylsilyl acetylene, THF (10 mL) was added dropwise a solution of 4 ml n-BuLi (1.5 M in hexane) at −40 °C. The solution was stirred for 1 h at −40 to 0 °C. Then, the solution was added dropwise to THF solution of 1.5 g (3.08 mmol) dienal (7) at −10 °C. The reaction mixture was warmed up to room temperature (20–22 °C) and stirred for 3 days. The reaction mixture was treated with a 5% solution of NH₄Cl in H₂O (20 mL) and extracted with diethyl ether (2 × 100 mL). The combined organic phases were dried over MgSO₄ and filtered, and the solvents were removed under reduced pressure. Silica gel column chromatography of the residue gave compound 8 (1.64 g, 91 %) as a pale yellow oily liquid.

16,19-Dideutero-(15Z,19Z)-1-(trimethylsilyl)hexatriaconta-15,19-dien-1-yn-3-ol (8). Yeld 91%. IR (film) υ_max 550, 627, 655, 720, 781, 808, 890, 909, 965, 1022, 1306, 1377, 1464, 1654, 2116, 2835, 2924, 3008, 3313 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.18 (9H, т, CH₃), 0.90 (3H, t, CH₃, J = 6 Hz), 1.28–1.75 (49H, m, CH₂), 1.91–2.14 (8H, m, CH₂), 4.35 (1H, t, O-CH, J = 5 Hz), 5.36–5.43 (2H, m, =CH). ¹³C NMR (100.62 MHz, CDCl₃) δ:−0.11, 14.13, 22.72, 25.14, 27.24, 27.32, 29.28–29.78, 31.97, 37.69, 62.76, 89.01, 107.19, 130.16. MS (MALDI-TOF), m/z: 590 [M]⁺. C₃₉H₇₄OSiD₂. Found (%): C, 79.68; H, 12.85. Calc. for C₃₉H₇₄OSiD₂ (%): C, 79.51; H, 13.00.

Procedure for preparation of 16,19-Dideutero-(15Z,19Z)-dimethylhexatriaconta-15,19-dien-1 -yn-3-ol (9). To a solution of 1.17 g (2 mmol) of alkyne (8), THF (10 mL) was added TBAF (1M in THF, 1.2 equv.) at 0 °C, and then the solution was stirred for 6 h at room temperature. Then, the solution was added dropwise to THF solution of 1.5 g (3.08 mmol) dienal (7) at −10 °C. The reaction mixture was treated with saturated aq. NaCl and extracted with diethyl ether (2 × 50 mL). The combined organic phases were dried over MgSO₄ and filtered, and the solvents were removed under reduced pressure. Silica gel column chromatography of the residue gave compound 1 (1.07 g, 99 %) as a colorless powder.

16,19-Dideutero-(15Z,19Z)-dimethylhexatriaconta-15,19-dien-1-yn-3-ol (9). Yeld 92%. ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ +0.43 (c 0.3, CHCl₃). IR (film) υ_max 551, 627, 655, 721, 781, 809, 890, 909, 965, 1022, 1307, 1377, 1464, 1654, 2116, 2835, 2924, 3008, 3311 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.90 (3H, t, CH₃, J = 6.7 Hz), 1.30–1.50 (44H, m, CH₂), 1.66–1.79 (2H, m, CH₂), 1.88–2.16 (8H, m, CH₂), 2.48 (1H, d, CH), 4.39 (1H, td, J = 7.0, 2.0 Hz), 5.40 (2H, t, =CH, J = 6.7 Hz). ¹³C NMR (100.62 MHz, CDCl₃) δ: 14.14, 22.71, 25.03, 27.25, 27.32, 29.22, 29.26, 29.35, 29.39, 29.54, 29.59, 29.65, 29.68, 29.77, 31.95, 37.68, 62.37, 72.82, 85.03, 130.24. MS (MALDI-TOF), m/z: 516 [M]⁺. C₃₆D₂H₆₄O. Found (%): C, 84.30; H, 12.57. Calc. for C₃₆D₂H₆₄O (%): C, 84.11; H, 12.78.

The oxidation of the alcohol (9) with Dess-Martin periodinane was carried out according [18]. (15Z,19Z)-16,19-Dideutero-(15Z,19Z)-hexaconta-15,19-dien-1-yl-3-one (10). Yield 86%. IR (film) υ_max 553, 627, 655, 720, 786, 807, 890, 909, 965, 1022, 1306, 1377, 1461, 1654, 2116, 2835, 2924, 3008, 3312 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.90 (3H, t, CH₃, J = 6 Hz), 1.23–1.48 (44H, m, CH₂), 1.64–1.72 (2H, m, CH₂), 2.04–2.10 (8H, m, CH₂), 2.58–2.62 (2H, m, CH₂), 3.22 (1H, s, CH), 5.39–5.42 (4H, m, =CH). ¹³C NMR (100.62 MHz, CDCl₃) δ: 187.5, 130.4, 130.3, 129.1, 81.5, 78.2, 45.5, 31.9, 29.3–29.7 (signals of 19C), 28.9, 27.4, 27.3, 22.7, 23.8, 14.1. MS (MALDI-TOF), m/z: 514 [M]⁺. C₃₆D₂H₆₂O.Found (%): C, 84.14; H, 12.80. Calc. for C₃₆D₂H₆₂O (%): C, 84.19; H, 12.82.

The stereoselective reduction of ketone 10 with B-3-pinanyl-9-borabicyclo[3.3.1]nonane (Alpine-borane reagent) was carried out accordingprocedure [19]. d₂-Lembehyne B (1). Yield 89% (95% ee). ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ +0.43 (c 0.3, CHCl₃). IR (film) υ_max 550, 627, 655, 721, 781, 809, 890, 909, 965, 1022, 1307, 1377, 1464, 1654, 2116, 2835, 2924, 3008, 3310 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ: 0.90 (3H, t, CH₃, J = 7 Hz), 1.23–1.54 (44H, m, CH₂), 1.71–1.75 (2H, m, CH₂), 2.04–2.10 (8H, m, CH₂), 4.39 (1H, td, J = 7.0, 2.0 Hz), 5.39–5.41 (4H, m, =CH). ¹³C NMR (100.62 MHz, CDCl₃) δ: 130.4, 129.2, 85.0, 72.8, 62.4, 37.7, 31.9, 29.3–29.7 (signals of 19C), 29.2, 27.4, 27.3, 25.0, 22.7, 14.1.