Synthesis of metabolites of cis and trans apovincamine derivatives

Synthesis of oxidative metabolites of ethyl cis - and hydroxyethyl trans -apovincaminates have been described. For cis -metabolite 8 the functionalization of the 10-hydroxy group was established through key intermediate 8-methoxy-indolopyranoquinolizine 16 , for trans - metabolite 10 the crucial 19-oxo-intermediate 24 was constructed from methyl 4-oxo-octahydroindoloquinolizine propionate 21a


Introduction
Over the past decades clinical and non-clinical research on vincamine and its semi-synthetic derivatives 1−3 has confirmed their beneficial cerebrovascular effect, including a neuroprotective action (Scheme 1). 1 -3 Previous SAR studies focused on structural modifications involving C-14 and the C/D/E ring junctions. 4,5In order to find a potent antiamnesic agent, further changes in the structure of compound 3 have been considered.To that end, a new series of substituted-alkyl esters of (3S,16R)-and (3R,16S)-trans-apovincaminic acid was synthesized. 6From the combined results of the data obtained from in vitro and in vivo tests and metabolism studies, 2'-hydroxyethyl (3R,16S)-apovincaminate (4b, RGH-10885) was identified as the most promising compound, owing to its potent neuroprotective and antiamnesic activities. 7ompounds 3 and 4b can be synthesized from the common intermediate 5.The direction of stereoselectivity could be modified by variation of the reaction conditions: cis-diester 6 8 or transdiester 7 7 were isolated from the reduction, wich was further transformed to the targeted endproducts (Scheme 2).

Scheme 2
The metabolism of compounds 1-3 has been established earlier.Apovincaminic acid is the major metabolite of vinpocetine (3).Ethyl vincaminate and a compound hydroxylated on the aromatic ring were also reported as being minor metabolites of 3. 9 In recent studies the latter A quite different pattern of metabolism was observed for the trans hydroxyethylester 4b.In contrast to the cis ethyl ester 3 and trans ethyl ester 4a, 4b does not appear to be affected by the esterase enzyme, but by the CYP enzymes.A series of oxidative metabolites were isolated.As a major metabolite the 19-oxo-derivative 10 was identified.Production of minor metabolites 10hydroxy-4b (11), 6α-and 6β-hydroxy-4b (12a,b) as well as 18α-and 18β-hydroxy-4b (13a,b) were also observed (Scheme 3). 11e synthesized cis metabolite 8, and the major metabolite of 4a trans-apovincaminate, 10 for the purpose of structural confirmation and for further studies of the biological activity.

Results and Discussion
A simple transformation of 3 into N-oxide 9 10 was accomplished with magnesium monoperoxyphtalate in DMF.
ARKAT USA, Inc.In conclusion, different strategies were employed to synthesize oxidative metabolites of apovincaminic acid esters.The AB(C)D→ABCDE strategy (C 17 N 2 + C 3 ) was applied by alkylation of the 9-methoxy-Wenkert-enamine 15 to prepare 10-hydroxy metabolite 8 of ethyl cis-apovincaminate.The skeleton of 19-oxo trans-metabolite 10 was constructed in a reaction of tryptamine and dimethyl formylpimelate (C 10 N + C 10 , AB→ABCDE approach).The subsequent steps to the end-products was carried out by using standard methods. 15RKAT USA, Inc.

Experimental Section
General Procedures.General procedures followed during the course of the work detailed herein were similar to those reported elsewhere. 4,8IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer using KBr pellets.NMR spectra were recorded on a Varian VNMRS-400 spectrometer (400 MHz for 1 H detection). 2D NMR experiments (COSY, HSQC, HMBC, NOESY) experiments were recorded by using the standard spectrometer software package; 0.75 s mixing time was used in the NOESY experiments.Mass spectrometric (highresolution /HRMS/) measurements were performed on a LTQ FT Ultra (Thermo Finnigan, San Jose, CA) system.The ionization method was ESI (275 °C was the ion transfer capillary temperature and 4.2 kV was the capillary voltage).For CID experiment helium was used as the collision gas, and normalized collision energy (expressed in percentage), which is a measure of the amplitude of the resonance excitation RF voltage applied to the endcaps of the linear ion trap, was used to bring about fragmentation.14) (20 g, 0.105 mol) and ethyl 3-hydroxypropylmalonic acid 16 (23.2g, 0.12 mol) in chlorobenzene (220 mL) was refluxed for 2 h, then 35 g (21 mL, 0.23 mol) phosphorus oxychloride was added at 50 °C and refluxed for 2 h.The reaction mixture was cooled to 70 °C and ethanol (40 mL) was added.The mixture was added dropwise to sodium hydroxide (31.2 g) in water (150 mL) at 70 °C.After stirring an additional 0.5 h, the separated organic phase was dried (MgSO 4 ), filtered and concentrated under reduced pressure to give a red oil.It was solved in ethanol (50 mL), then diethyl malonate (24 g, 0.15 mol), paraformaldehyde ( 9 g, 0.3 mol) and triethylamine (1 mL) was charged.The reaction mixture was stirred for 2 h at 50 °C, then 2 h for 5 °C.The separated yellow crystals were filtered, washed with ethanol, to give 18.  (14).Mass accuracy was between -0.87 and -0.21 ppm for the fragment ions.The protonated molecular ion peak can be detected at m/z 485.26455 (delta: -0.13 ppm), calculated value for C 27 H 37 O 6 N 2 : 485.26461.