Synthesis of the Aspidosperma Alkaloid Na-Formyl-16-α- Hydroxyaspidospermidine

The first total synthesis of Na-formyl-16α-hydroxyaspidospermidine and its isomer via (°)-vincadifformine is described and their structure elucidation using different methods of analysis is reported.


Introduction
The indole alkaloid strictanine (1b) was recently isolated from the fruit of Rhazia stricta Decsne [1] which is an indigenous medicinal plant abundantly found in Pakistan. This medicinal plant has long been used for the treatment of various diseases [2,3]. We report here the first total synthesis of this compound starting from (±)-vincadifformine (2), which was previously prepared from tryptamine hydrochloride by an adaptation of Kuehne's biomimetic synthesis [4,5]. This prompted us to extend it to more oxygenated alkaloids such as spegazzinine (10a) and spegazzinidine (10b) [6].

Results and Discussion
The existing literature method for the conversion of the readily available (±)-vincadifformine (2) into the key intermediate 16-hydroxyindolenine (3) showed that the latter compound is particularly ( 1 ) a. 16B -OH unstable [7,8]. Similarly, photo-oxygenation [9] of 2 in the presence of Rose Bengal and sodium thiosulphate as reducing agent to give 16-hydroxyindolenine (3) resulted in low yields of the desired compound. The other product was mainly recovered starting material. This is undoubtely due to the different tungsten lamps used (2x150W). Le Men and co-workers [10] also achieved this convertion in vitro through a multi-step procedure involving the prior oxidation with peroxyacids to afford the 16hydroxy-1,2-dehydrovincadifformine-N b -oxide (4) in which the N b centre was blocked to avoid any spontaneous rearrangement.
Reaction of vincadifformine (2) with m-chloroperbenzoic acid in dry benzene gave, after evaporation of the solvent, a variable yield of 16-hydroxyindolenine-N b -oxide derivative 4. However, an essentially quantitative yield of 4 was obtained after solvent removal under reduced pressure (t bath < 40°C) when vincadifformine was treated with 3 equivalents of m-chloroperbenzoic acid in the dark for 36h under a nitrogen atmosphere.
The molecular ion was observed at m/z 370, in agreement with the empirical formula C 21 H 26 N 2 O 4 which was also supported by microanalysis. The UV spectrum in methanol gave maxima at 223 and 270 nm and the IR spectrum indicated a non conjugated ester at 1738 cm -1 (in contrast with the absorptions at ν max 1670 and 1610 cm -1 in the starting material) and a hydroxy group at 3450 cm -1 . The 1 H-NMR spectrum showed a multiplet at 8 ppm (OH), a doublet due to the C-9 proton at 7.6 ppm, a multiplet at 7.5-7.1 (3 ArH), the methoxycarbonylmethyl group at 3.95 ppm and the C-18 methyl group as a triplet at 0.5 ppm. Subsequent hydrolysis with 1.25M sodium hydroxide solution, followed by decarboxylation under acidic conditions (pH = 1) at 100°C for 20 min, produced the desired product 5a as a yellow amorphous solid in 98% yield. The presence of the carbonyl function in 5a was confirmed by the long wavelength absorption observed in the UV spectrum [λ max 218, 243 (sh), 300 nm] typical of the indolinic compound and by the absorption in the IR spectrum at 1720 cm -1 (CO). The molecular ion in the mass spectrum was observed at m/z 312 and was consistent with the molecular formula C 19 H 24 N 2 O 2 . Attempts to reduce the N-oxide group with palladium-hydrogen gave a low yield (32%) of 5b whereas Adam's catalyst (PtO 2 ) and hydrogen at atmospheric pressure afforded a good yield (80%). However, the major mass spectral fragment of 5b was not observed at m/z 124 as for vincadifformine and its derivatives. Instead, the molecule exhibits a M + -28 peak at m/z 268 and shows its most intense peak at m/z = 138, which is also observed in the 16-dehydrospegazzinidine dimethyl ether 8 [11]. Consequently, the retro Diels-Alder fragmentation is less important in compound such as 5b with a carbonyl group at C-16 which thus alters the typical aspidospermine mass spectral fragmentation pattern. The molecular ion decomposes by expulsion of carbon monoxide to give species b (m /z=268). Subsequent fission of the C-5, C-6 bond therefore produces not the anticipated ion at m/z 124, but rather the ion c (m/z 138, 100%) as the major fragment (Scheme 1).
16-Oxoaspidospermidine 5b, when subjected to reduction with sodium borohydride in ethanol, followed by heating, gave a mixture of C-16 epimers, which were separated by column chromatography on kieselgel G using chloroform -methanol (9:1) as eluent. The major product (less polar, 72.5%) was 16α-H,16β-OH aspidospermidine 6a, obtained as colourless plates (m.p. 55 °C), which exhibited UV absorptions at λ max 212, 244 and 300 nm. The exact structure of this compound was deduced from its 1 H-NMR spectrum which exhibited signals clearly indicating that a coupling constant (J = 4 Hz) of H -2 (d, 3.77 ppm) and H -16 (m, 4.85 ppm) that is compatible with a cis configuration (2α -H, 16α-H). Irradiation of 16-H gave C-2 as a singlet at 3.75 ppm. Therefore the configuration of the hydroxyl group is β. The 13 C-NMR spectrum showed nineteen resonances with the C-16 atom giving rise to the resonance at δ 67.65 ppm. The more polar product (6b, 12.5%) was found to be epimeric at C-16 and was compared with that also obtained by Le Men et al. [10] from the indolenine 9. Reaction of 16β-hydroxyaspidospermidine 6a with formic acid and acetic anhydride afforded the N,O-diformyl derivative 7a in 90% yield as colorless plates (m.p.70-72 °C). The infrared spectrum indicated the presence of two characteristic absorptions at 1720 (OCHO) and 1670 cm -1 (NCHO) with no free NH group. In the 1 H-NMR spectrum, the two singlets which correspond to the two formyl functions (OCHO) and (NCHO) were observed at 8.9 and 8.6 ppm respectively. Data from the 13 C-NMR spectra are reported in Table 1 for comparison. When sodium carbonate was added to a solution of the diformyl 7a compound in methanol the reaction produced N a -formyl-16β-hydroxyaspidospermidine (1a) in 98% yield (m.p. 66-68 °C). The spectroscopic data suggested that the correct relative stereochemistry had been obtained. In the IR spectrum a broad absorption was seen at 3400 cm -1 (OH) and a strong absorption at 1680 cm -1 which was assigned to the N a -formyl group. The UV spectrum in methanol is found to be essentially identical to that reported by Atta -Ur-Rahman [1] for the alkaloid strictanine (1b). The mass spectrum shows the anticipated fragmentation pattern (Scheme 2). In the 1 H-NMR spectrum the N-formyl proton (NCHO) was seen as two singlets (relative intensity 7:1) at 9 and 8.72 ppm respectively. Saturation of one peak caused the disappearance of the other one. These are the two s-cis/s-trans geometrical isomers resulting from restricted rotation, well-known for amides. The H-12 proton was observed as a doublet at 8.03 ppm due to the ortho-coupling to H-11

General
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. IR spectra were recorded on either a Perkin-Elmer 1420 or a 1310 spectrophotometer. UV absorption spectra were obtained on a Unicam PU 8800 spectrometer. NMR spectra were recorded on either a JEOL FX90QFT (90 MHz for 1 H and 13 C), a GE QE 300 (300 MHz for 1 H and 13 C) or a Bruker 400 MHz spectrometer (400 MHz for 1 H and 13 C). Solutions in deuteriochloroform, with tetramethylsilane as internal standard were used, unless otherwise stated. J values are given in Hz. Mass spectra were recorded on a Kratos MS 25 instrument; accurate mass measurements were carried out on a AEI/Kratos MS 902/50 spectrometer.

Preparation of aldimine
Cyclohexylamine (49.59 g, 0.5 mmol) was placed in 250mL three-necked, round bottomed flask fitted with a thermometer, mechanical stirrer, and an addition funnel, then cooled to -5 °C in an icesalt bath. Butyraldehyde (36.05 g, 0.5 mol) was carefully added dropwise at such a rate that the temperature remained below 5° C. Stirring was continued for 30 minutes. The solution was then allowed to warm to room temperature, poured into a separatory funnel, and the water produced during the reaction was removed. The reaction was allowed to go to completion by leaving it standing for 24h in the presence of K 2 CO 3 (16 g) and KOH pellets (16 g). Filtration followed by distillation gave the desired aldimine (65 g, 68%) as a colorless liquid, b.p. 80-84 °C / 20 mm Hg; 1 H-NMR: δ H (CDCl 3 , 90 Mz) 7.7 (1H, t, J=5.14 Hz, =CH-), 3 -1.2 (14 H, m), and 1 ppm (3 H, t, J: 7.4 Hz, -CH 3 ).

Lithium salt of the imine
Lithium diethylamide solution in dry tetrahydrofuran (15 mL) was cooled to -60 °C and the imine (15.3 g , 0.1 mol) was added via a syringe at such a rate that the temperature remained between -60°C and -65 °C during the addition. After 30 min stirring the reaction mixture was allowed to warm to -10°C for 2 h.