Synthesis of Key Fragments of Amphidinolide Q — A Cytotoxic 12-membered Macrolide

β-Hydroxy aldehyde and alkyl ketone moieties were effectively synthesized as key intermediates of amphidinolide Q, a cytotoxic macrolide from the cultured dinoflagellate Amphidinium sp.. The asymmetric center of the former derivative was produced by Sharpless asymmetric epoxidation, followed by E-selective 1,4-addition to give the sp2 methyl group. Derivatization of the L-ascorbic acid derivative by Evans asymmetric alkylation and Peterson olefination provided the latter intermediate. The coupling reaction of the segments was examined.


Introduction
Amphidinolide Q (1, Scheme 1) is a member of the cytotoxic macrolide family isolated from the cultured dinoflagellate Amphidinium sp., and its absolute stereostructure was chemically determined by Kobayashi and co-workers [1,2]. This macrolide exhibits potent cytotoxicity against L1210 murine leukemia cells. Due to its low natural abundance, the detailed structure-activity relationship of 1 has not been established. Although 1 was synthesized by Kobayashi's group in 2009 [3], further increases in synthetic efficiency and examination of related substances based on new synthetic approaches are OPEN ACCESS required to explain the detailed mode of action of 1 by structure-activity relationship studies, and to develop new bioactive substances superior to the mother macrolide. We describe herein an efficient method for synthesis of segments 2 and 3, which will be important intermediates for the construction of 1.

Results and Discussion
As outlined in Scheme 1, retrosynthetic analysis showed that amphidinolide Q (1) could be obtained by successive coupling of 2 and 3, with the former, carrying a stereogenic center and a tri-substituted olefin, being produced from the known allyl alcohol 4 through E-selective 1,4-addition of a methyl group at C3 and Sharpless asymmetric epoxidation. The alkyl ketone 3 carrying three stereogenic centers could be obtained from the ascorbic acid derivative 5 through Evans asymmetric alkylation. Scheme 1. Amphidinolide Q (1) and retrosynthetic analysis.

Synthesis of segment A (2)
The synthesis of 2 commenced with the conversion of 1,3-propanediol (6) into alcohol 4 (Scheme 2) [4]. Incorporation of the asymmetric center at C4 and carbon chain elongation were carried out by Sharpless asymmetric epoxidation, followed by chlorination, alkynylation [5], and final p-methoxybenzyl (PMB) protection of the newly produced OH group under acidic conditions to avoid unexpected removal of the tert-butyldiphenylsilyl (TBDPS) group. Further manipulation of 7 involved introduction of a methoxycarbonyl group at the terminal of the alkynyl carbon to give 8, which upon E-selective alkylation using a PhS group as an auxiliary [6], afforded the α,β-unsaturated ester 9. In this case, the two-step procedure involving preparation of the vinyl sulfide, followed by exchange with a methyl group must be used to afford the α,β-unsaturated ester 9 in good yield (Table 1). In the case of direct methylation (path A), the desired compound 9 was obtained in low yield, even when the reaction temperature was increased to 0 °C (entry 2). Although a number of reagent combinations to control the properties of cuprates are known, the desired product was obtained in good yield in the case of entry 5.
Deprotection and oxidation processes ultimately gave the β-hydroxyl aldehyde 2 (segment A) in good overall yield. Scheme 2. Synthesis of segment A (2). HO

Synthesis of segment B (3)
Synthesis of alkyl ketone 3 was initiated by transformation of the ascorbic acid derivative 5 into diol 10 by a known procedure [7]  Selective asymmetric induction at C9 by the Evans protocol effected the desired introduction of a methyl group to yield 12. Removal of the chiral auxiliary by LiBH 4 and manipulation of protecting groups in five steps gave the alcohol 14 via 13. After Parikh-Doering oxidation of 14, the aldehyde generated was reacted with Horner-Wadsworth-Emmons reagent 16 [8] to yield 15, which on hydrogenation followed by addition of a methyl group produced 17. After removal of the auxiliary by LiBH 4 , an ethyl ketone function was constructed (compound 18), and the exo-methylene function was introduced by Peterson olefination, followed by basic treatment, whereas other methods, such as the Wittig and Petasis reactions did not produce the desired compound, probably due to the low reactivity of the ethylketone moiety. Synthesis of segment B (3) was accomplished by Birch reduction, followed by alkylation similar to that described the case of 18 and Parikh-Doering oxidation and Grignard reaction. The synthetic strategies used for both segments may enable the synthesis of analogs. The stereogenic centers were introduced via asymmetric reactions, which can produce another enantiomer by exchanging the auxiliary group or catalyst, with the exception of the C11 center: in this case the opposite (R)-enantiomer would be produced using (R)-glyceraldehyde acetonide, derived from D-mannitol [9].
Aldol coupling of both segments was attempted, as shown in Scheme 4. Whereas Lewis acidic conditions such as method B [10] caused undesirable elimination of 2 and 20 [11] to give the corresponding α,β,δ,γ-unsaturated ester, basic conditions (method A) provided 20, equipped with the complete carbon framework, in moderate yield,. Detailed studies of the diastereomer distribution to improve the coupling yields are currently in progress in our laboratory.

General
All reactions were carried out under an argon atmosphere unless otherwise noted. When necessary, solvents were dried prior to use. Dry THF, dry Et 2 O and dry CH 2 Cl 2 were purchased from Kanto Chemical Co., Inc. Optical rotations were measured on a JASCO P-2200 digital polarimeter with a sodium (D line) lamp. IR spectra were recorded on a Jasco Model A-202 spectrophotometer. 1 H-NMR spectra and 13 C-NMR spectra were obtained on JEOL JNM-EX270, JNM-GX400, JNM-α400, JNM-AL400 and JNM-ECX400 spectrometers in deuterated solvent using tetramethylsilane as an internal standard. Deuteriochloroform was used as a solvent, unless otherwise stated. Optical purity was determined by HPLC using an OJ-H column. High-resolution mass spectra were obtained on a Waters LCT Piemier XE (ESI) or JEOL JMS-700 (FAB). Preparative and analytical TLC were carried out on silica gel plate (Kieselgel 60 F254, E. Merck AG., Germany) using UV light and/or 5% molybdophosphoric acid in ethanol for detection. Kanto silica 60N (spherical, neutral, 105-210 μm) was used for column chromatography.
(R)-tert-Butyl(3-(4-methoxybenzyloxy)pent-4-ynyloxy)diphenylsilane (7). To a suspension of Ti(OiPr) 4 (4.28 mL, 14.6 mmol) and MS4A (4.9 g) in CH 2 Cl 2 (100 mL) was added (-)-DET (3.32 mL, 19.5 mmol) at −25 °C. To the mixture were added 4 (3.31 g, 9.73 mmol) and t-BuOOH (3.17 mL, 29.2 mmol); the solution was stirred overnight. The reaction was quenched by L-(+)-tartaric acid (12.4 g, 82.6 mmol) aqueous and iron (II) sulfate heptahydrate (12.2 g, 43.9 mmol). The mixture was extracted with Et 2 O, and the organic extracts were dried (Na 2 SO 4 ), and concentrated in vacuo. The crude product was purified by silica gel column chromatography using hexane-ethyl acetate (3:1) to give an alcohol (3.38  A stirred mixture of the alcohol (3.38 g, 9.49 mmol), PPh 3 (7.47 g, 28.5 mmol), and NaHCO 3 (0.80 g, 9.5 mmol) in CCl 4 (100 mL) was refluxed under argon atmosphere overnight. After completion of the reaction, CCl 4 was removed under reduced pressure, and the residue was purified by silica gel column chromatography (hexane-ethyl acetate 40:1) to furnish an epoxy chloride (3.04 g, 86%) as a colorless oil: To a stirred solution of the epoxy chloride (0.23 g, 0.63 mmol) in dry THF (7 mL) was added n-BuLi (2.4 mL, 1.6 M solution in n-hexane, 3.79 mmol) dropwise at −40 °C under argon atmosphere; and the mixture was stirred for an additional 2 h. The mixture was quenched with saturated aqueous NH 4 Cl, and the mixture was extracted with ethyl acetate. The organic extracts were dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexaneethyl acetate 7:1) to give an alcohol (0.18 g, 87%) as a colorless oil: To a solution of the alcohol (0.10 g, 0.30 mmol) in anhydrous CH 2 Cl 2 (7 mL) were added 4-methoxybenzyl trichloroacetimidate (0.493 g, 1.74 mmol) and TfOH (cat.) at 0 ºC, and the mixture was stirred overnight. The reaction was quenched with saturated aqueous NH 4 Cl, and extracted with ethyl acetate. The organic extracts were dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 7:1) to give 7 (0.12 g, 90%) as a colorless oil: [ (8). To a solution of 7 (50.8 mg, 0.11 mmol) in THF (1.5 mL) was added dropwise n-BuLi (0.3 mL, 1.6 M solution in hexane, 0.48 mmol) at −78 °C, and the mixture was stirred at the same temperature for 1 h. Methyl chloroformate (0.09 mL, 1.11 mmol) was added dropwise to the mixture; the resulting mixture was stirred at 0 °C for 2.5 h. After being quenched with saturated aqueous NH 4 Cl, the mixture extracted with ethyl acetate. The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 9:1) to give 8 (56. 4 (9). To a solution of 8 (90.8 mg, 0.18 mmol) in MeOH was added PhSH (36 μL, 0.35 mmol) and NaOMe (0.18 mL, 0.02 mmol) at room temperature. After being stirred at the same temperature for 3 h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 9:1) to give a methyl ester (102.1 mg, 93%) as a colorless oil:   A, 2). To a solution of 9 (0.33 g, 0.62 mmol) in THF (6 mL) was added reagent (TBAF-AcOH-H 2 O= 1:1:5 0.1 M in THF, 0.3 mmol) at room temperature; the mixture was stirred at the same temperature overnight. After the addition of cold water, the mixture was extracted with Et 2 O. The organic layer was dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 1:1) to give an alcohol (0.16 g, 88%) as a colorless oil:  (11). To a solution of NaIO 4 (3.78 g, 17.7 mmol) in CH 2 Cl 2 (60 mL) and H 2 O (30 mL) was added 10 (1.91 g, 11.8 mmol) at 0 °C; the mixture was stirred at room temperature for 1.5 h. After the addition of Ph 3 PCHCO 2 Me (7.89 g, 23.6 mmol) at 0 °C, the mixture was stirred at room temperature overnight. The organic layer was separated and the aqueous layer was extracted with CHCl 3 three times. The combined organic layers were washed with brine, dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 3:1) to give an α,β-unsaturated ester (1.84 g, 84%, E:Z = 1:3) as a colorless oil.

(R,E)-Methyl 4-(4-methoxybenzyloxy)-3-methyl-6-oxohex-2-enoate (segment
The ester (1.02 g, 5.48 mmol) was dissolved in EtOH (55 mL) in the presence of Raney Ni W-4. The mixture was stirred at room temperature under hydrogen atmosphere overnight. After filtration, the filtrate was concentrated at 110 °C to afford the corresponding ester as a crude oil. To a solution of the ester in THF (17 mL) was added 1.5 M LiOH aqueous (17 mL) at 0 °C. The mixture was stirred at 0 °C for 3 h. The mixture was acidified to pH 4 with 10% aqueous citric acid and extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried (Na 2 SO 4 ), and concentrated in vacuo to give 11 (0.935 g, 98%) as a colorless oil:

(2R,4R)-5-(Benzyloxy)-4-methylpentane-1,2-diol
To a suspension of LiCl (0.09 g, 2.2 mmol) in anhydrous CH 3 CN (8 mL) were added 16 (0.29 g, 0.81 mmol), DIPEA (0.3 mL, 1.65 mmol), and the aldehyde (0.18 g, 0.55 mmol) at room temperature; the mixture was stirred at room temperature for overnight. The reaction was quenched with saturated aqueous NH 4 Cl, and the mixture was partitioned between Et 2 O and H 2 O. The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography To a solution of the alcohol (2.5 mg, 6.6 μmol) in CH 2 Cl 2 (0.05 mL) and DMSO (0.05 mL) were added Et 3 N (3 μL, 19.7 μmol) and SO 3 -pyridine (3 mg, 19.7 μmol) complex at 0 °C. After 1.5 h, the reaction was quenched with saturated aqueous NH 4 Cl, and the mixture was partitioned between Et 2 O and H 2 O. The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by PLC (hexane-ethyl acetate 3:1) to give an aldehyde as a colorless oil.
To a solution of the aldehyde in THF (0.1 mL) was added EtMgBr (0.02 mL, 1.0 M in THF, 19.7 μmol) at 0 °C; the mixture was stirred at the same temperature for 15 min. The reaction mixture was quenched with saturated aqueous NH 4 Cl, and extracted with CH 2 Cl 2 . The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by PLC (hexane-ethyl acetate (5:1)) to give an alcohol as a colorless oil.
To a solution of the alcohol in CH 2 Cl 2 (0.05 mL) and DMSO (0.05 mL) were added Et 3 N (3 μL, 19.7 μmol) and SO 3 (19). To a solution of ethyl ketone 18 (60.0 mg, 0.12 mmol) in THF (1 mL) was added (trimethylsilylmethyl) lithium (0.5 mL, 1.0 M in pentane, 0.5 mmol) at −78 °C; the mixture was stirred at the same temperature for 20 min. The reaction mixture was quenched with saturated aqueous NH 4 Cl, and extracted with ethyl acetate. The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by PLC (hexane-ethyl acetate 9:1) to give an alcohol as a colorless oil.

((2R,4S,6R)-1-(Benzyloxy)-2,6-dimethyl-7-methylenenonan-4-yloxy)(tert-butyl)dimethylsilane
To a solution of the alcohol in THF (2 mL) was added NaH (60% dispersion in mineral oil, 50 mg, 0.1 mmol) at room temperature; the mixture was stirred at reflux temperature overnight. The reaction was quenched with saturated aqueous NH 4 Cl, and the mixture was extracted with ethyl acetate. The organic layer was dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was purified by PLC (hexane-ethyl acetate 9:1) to give 19 as a colorless oil (48 mg, 86% in 2 steps):  (segment B, 3). To a solution of lithium (12.4 mg, 1.9 mmol) in liquid NH 3 (10 mL) was added 19 (46.4 mg, 0.11 mmol) in dry THF (3 mL) at −78 °C. The reaction mixture was stirred for 15 min at the same temperature; and quenched with solid NH 4 Cl. The mixture was extracted with ethyl acetate, and the organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 5:1) to give an alcohol (35.6 mg, 99%) as a colorless oil: To a solution of the alcohol (0.35 g, 1.10 mmol) in CH 2 Cl 2 (6 mL) and DMSO (6 mL) were added Et 3 N (0.5 mL, 3.29 mmol) and SO 3 -pyridine (0.52 g, 3.29 mmol) complex at 0 °C. After 1 h, the reaction was quenched with saturated aqueous NH 4 Cl, and the mixture was partitioned between Et 2 O and H 2 O. The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate 7:1) to give an aldehyde as a colorless oil.
To a solution of the aldehyde in THF (10 mL) was added EtMgBr (3 mL, 1.0 M in THF, 2.95 mmol) at 0 °C; the mixture was stirred at the same temperature for 30 min. The reaction was quenched with saturated aqueous NH 4 Cl, and the mixture was extracted with CH 2 Cl 2 . The organic layer was dried (Na 2 SO 4 ), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane-ethyl acetate (9:1)) to give an alcohol as a colorless oil.