Synthesis and transformation of sphingosine analogue pinane-based 2-amino-1,3-diols

A library of pinane-based 2-amino-1,3-diols, analogues of biologically active sphingosine, was synthesised in a stereoselective manner. Isopinocarveol prepared from (–)-α-pinene was converted to condensed oxazolidin-2-one in two steps by carbamate formation followed by a stereoselective aminohydroxylation process. The relative stereochemistry of the pinane-fused oxazolidin-2-one was determined by 2D NMR and X-ray technics. The regioisomeric spiro-oxazolidin-2-one was prepared in a similar way starting from commercially available (1R)-(–)-myrtenol (8). Reduction or

Due to the lack of a readily available natural source and the high biological importance of sphingolipid analogues, their synthesis has been the subject of several studies [17].

5
The relative configuration of 7 was determined by 2D NMR technics. Clear NOE signals were observed between the H-7a and Me-10 as well as the Ha-9 and Me-10 protons.
Beside NOESY experiments, the structure was also elucidated by X-ray crystallography ( Figure 2). To synthesise regioisomeric spiro-oxazolidinone derivative 10, (1R)-(-)-myrtenol (8) was chosen as starting material (Scheme 2). The synthesis method was similar to that mentioned above for (-)-isopinocarveol. In the first step, carbamate 9 was prepared, [36] then aminohydroxylation was carried out catalysed by potassium osmate(VI), which led to the formation of spiro-oxazolidine-2-one 10 in a highly regioand stereoselective manner. Based on the NMR measurements of the crude product, spiro derivative 10 was obtained exclusively with relative configuration depicted on Scheme 2. Beside 2D NMR studies, the relative configuration of 10 was determined by the transformation of 10 to the corresponding aminodiols and comparing the products with those obtained from regioisomer 7 (discussed on Scheme 3).

Synthesis and transformations of pinane-based 2-amino-1,3-diols
To obtain a library of pinane-based 2-amino-1,3-diols, oxazolidine-2-ones 7 and 10 were applied as starting materials. Alkaline hydrolysis of both 7 and 10 resulted in the same primary aminodiol 11. [37] According to the NMR spectra and other physical and chemical properties, there was no difference between the products of the two reactions. Since the relative configuration of compound 7 was clarified by NMR and Xray crystallographic results, we were able to assign the stereochemistry of spiro derivate 10 too. In a similar manner, LiAlH4 (LAH) reduction of both 7 and 10 gave the same N-methylaminodiol 12 with modest yield (Scheme 3).  [38,39] Since this finding is quite 7 unusual in the case of Schiff bases, we decided to study the ring/chain tautomeric mixture (13A-E) in the reaction of 7 with benzaldehyde by 1 H-NMR spectroscopy.
When a time-dependent 1 H-NMR measurement was accomplished, we observed that the equilibrium composition was established rapidly without any significant change in the ratio of the tautomers. The equilibrium shifting strongly to product 13E can account for the difficulty of the reduction process and the necessity to use a stronger reducing agent and more sever conditions. The reduction step, therefore, was performed by applying LAH a stronger reducing agent and an elongated time of reflux resulting in 14 (Scheme 4).
Clear NOE signals were observed between the H-7a and Me-10 as well as the Ha-9 8 and Me-10 protons. In addition to NOESY experiments, the structure was also elucidated by X-ray crystallography ( Figure 3).
When 11 was reacted with phenylisothiocyanate, thiourea 18 was obtained, which underwent regioselective ring closure resulting in 19A. It is important to mention that this regioselectivity is the opposite to that observed in the reaction of aminodiols 11 and 14 with aldehydes (see Schemes 4 and 5), but it is similar to that observed in our earlier study with pinane-based 3-amino-1,2-diols. [40] During the NMR study of 19A in CDCl3 for 30 days, an unknown slow ring-ring tautomerisation was observed, 9 forming a 1:1 mixture of two regioisomers 19A and 19B. Compound 19B could be isolated from the mixture by column chromatography in pure form.
Pinane-condensed or spiro-oxazolidin-2-ones were formed in three steps by a stereoselective hydroxyamination process. The relative stereochemistry of new compounds was determined by 2D NMR and X-ray technics. The resulting primary and secondary 2-amino-1,3-diols underwent regioselective ring closure with formaldehyde and benzaldehyde producing pinane-condensed oxazolidines. In the case of 2phenyliminooxazolidine, interesting ring-ring tautomerism was observed in CDCl3.

General method for the aminohydroxylation process
To a solution of allylic carbamate 6 or 9 (2.00 g, 10.2 mmol) in i-PrOH (180 mL) freshly prepared 0.33% aqueous solution of NaOH (80 mL) was added. The solution was allowed to stir for 5 min, then freshly prepared t-BuOCl (1.026 mL, 10.3 mmol) was added. After stirring for 5 min, N,N-diisopropylethylamine (59 mg, 76.5 μL, 0.46 mmol) and potassium osmate(VI) dihydrate (135 mg, 0.37 mmol) were added to the solution in one portion. After the addition of 0.33% aqueous NaOH solution of (20 mL) the mixture was stirred for 24 h (TLC monitoring) then quenched with Na2SO3 (500 mg) and allowed to stir for 30 min. The mixture was extracted with EtOAc (3 × 50 mL), and the organic layer was washed with brine (1 × 50 mL), dried (sicc. Na2SO4), filtered and concentrated under reduced pressure to yield the crude product, which was purified by column chromatography on silica gel (n-hexane/EtOAc = 1/2).

General method for the LAH reduction of oxazolidinones 7 and 10
To the stirred suspension of LiAlH4 (0.25 g, 6.6 mmol) in dry THF (5 mL) the solution of 7 or 10 (0.35 g, 1.67 mmol) in dry THF (5 mL) was added dropwise under ice cooling.
The reaction mixture was treated under reflux conditions for 2 h, then the mixture of H2O (0.50 mL) and THF (5 mL) was added dropwise with cooling. After 30 min stirring, the inorganic material was filtered off and washed with THF (3 × 30 mL). The filtrate was dried (sicc. Na2SO4), filtered and evaporated. The obtained crude product was purified by column chromatography on silica gel (toluene/EtOH = 1/1).

2-yl)-3-phenylthiourea (18)
To a solution of aminodiol 11 (70 mg, 0.377 mmol) in toluene (30 mL), 1.05 eq. of phenyl isothiocyanate (80 mg, 70 µl, 0.396 mmol) was added and the reaction mixture was stirred for 12 h at room temperature. After evaporation, the crude product was purified by column chromatography on silica gel (CHCl3/MeOH = 19/1). Thiourea 18 (56 mg, 0.174 mmol) was dissolved and stirred in the solution of methanol (5 mL) and MeI (5.5 eq., 63 µl, 0.957 mmol) at room temperature. After a 3-h stirring the solvent was evaporated and the residue was redissolved and stirred in 2 mL of 2.5 N methanolic potassium hydroxide for 12 h at room temperature. After evaporation of the solvent, the residue was dissolved in the mixture of H2O (10 mL) and CHCl3 (10 mL) and the aqueous phase was extracted with CHCl3 (2 × 10 mL) and the organic

X-Ray structure determinations
The crystals of 7 and 15 were immersed in cryo-oil, mounted in a loop, and measured at a temperature of 120 K. The X-ray diffraction data were collected on a Rigaku Oxford Diffraction Supernova diffractometer using Cu Kα radiation. The CrysAlisPro [41] software package was used for cell refinements and data reductions. A multi-scan (7) or an analytical absorption correction (15) was applied to the intensities before structure solutions by using CrysAlisPro [41] software. The structures were solved by intrinsic phasing (SHELXT[42]) method. Structural refinements were carried out using SHELXL [43] software with SHELXLE [44] graphical user interface. The NH and OH hydrogen atoms were located from the difference Fourier map and refined isotropically.
All other hydrogen atoms were positioned geometrically and constrained to ride on their parent atoms, with C-H = 0.95-1.00 Å and Uiso = 1.2-1.5 Ueq(parent atom). The crystallographic details are summarised in Table S1 (Supporting Information, S33).

Supporting Information
Supporting Information File 1: analytical data, NMR spectra and X-data of the prepared compounds.