Synthesis and Transformations of di-endo-3-Aminobicyclo-[2.2.2]oct-5-ene-2-carboxylic Acid Derivatives

all-endo-3-amino-5-hydroxybicyclo[2.2.2]octane-2-carboxylic acid (13) and all-endo-5-amino-6-(hydroxymethyl)bicyclo[2.2.2]octan-2-ol (10) were prepared via dihydro-1,3-oxazine or γ-lactone intermediates by the stereoselective functionalization of an N-protected derivative of endo-3-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acid (2). Ring closure of β-amino ester 4 resulted in tricyclic pyrimidinones 15 and 16. The structures, stereochemistry and relative configurations of the synthesized compounds were determined by IR and NMR.


Introduction
The synthesis of non-natural α-amino acids is currently an important synthetic challenge in view of their increasing role in chemistry and biology. Among them, bicyclic amino acids exhibit biological activity; as an example, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) blocks the transport of nonpolar amino acids across cell membranes, acts as an insulin-releasing factor and also inhibits the flavoprotein amino acid oxidases [1]. Straub et al. determined whether protein acylation plays a part in the action of glucose on insulin-secreting β-cells. They reported that BCH, a non-metabolizable analog OPEN ACCESS of leucine that mimics the stimulatory effect of glucose on insulin secretion, increased the incorporation of 3 H-palmitic acid into protein [2]. Maechler et al. examined the whether activation of glutamate dehydrogenase (a mitochondrial enzyme playing a key role in the control of insulin secretion) by BCH enhances glutamine oxidation and insulin secretion [3]. BHC is a model compound for the study of amino acid transporters, as it is an L-selective inhibitor that at suitable concentration can induce the suppression of cell growth and cancer cell apoptosis. [4,5] The interest in synthetic amino acids possessing a bicycle [2.2.2]octane structure is highlighted by a number of investigations relating to their biological action. Dihydroxylated derivatives of 4-aminobicyclo[2.2.2]octane-1carboxylic acid have been used as scaffolds for antiviral agents [6,7], and 2-aminobicyclo[2.2.2]octane-2-carboxylic acid selectively disturbs levels of neutral amino acids in the cerebral cortex [8,9]. Although of less biological importance than their α-analogs, some bicyclic β-amino acid derivatives exert biological activity [10,11], and are also present in peptides [10,12]. For example, a series of cyclic β-amino acid dipeptide derivatives have been investigated as VLA-4 antagonists in various inflammatory and autoimmune disease states [13].
During the past 20 years, a number of bicyclic β-amino acid derivatives have been synthesized, some of them with useful pharmacological effects [4], and they are widely used for the preparation of saturated 1,3-heterocycles. The synthesis and stereochemical aspects of the diexo-and diendo-fused norbornane-and norbornene-1,3-heterocycles have been thoroughly studied [14]. To date, only a few bicyclo [2.2.2]octene-fused heterocycles have been prepared [15][16][17][18][19]. Because of their therapeutic interest, the syntheses of cycloalkane-fused pyrimidinones have been studied [14], but syntheses of their bicyclo [2.2.2]octene-condensed derivatives have not yet been reported.
cis-and trans-3-Aminobicyclo [2.2.2]octane-2-carboxylic acid were prepared some years ago [20][21][22], but their partially saturated analogs and further functionalized derivatives have not yet been described. Our work was focused on the syntheses of di-endo-3-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acid and its hydroxyl-substituted derivatives by stereoselective and regioselective functionalization of the double bond via 1,3-oxazine or γ-lactone intermediates. A further aim was a study of the ring-closure reactions of amino esters, and the retro-Diels-Alder reactions of the synthesized tricyclic pyrimidinones.
The importance of this retro Diels-Alder procedure (cycloreversion) lies in the fact that 3-substituted 2-thiouracyl derivative of type 17 can be synthesized in this way [36,37]. When 15 was boiled in chlorobenzene, or heated at the melting point, or heated under MW-irradiation, the reaction mixture turned deep-brown, but the formation of 17 was not observed, the starting thioxopyrimidinone derivative 15 was not undergone any transformation.

IR and NMR Results
The presumed structures of the new compounds (2, 4-7, 8a,b and 9-16) follow straightforwardly from the spectral data [Tables 1 and 2; to facilitate comparison of the analogs' spectroscopic data, the IUPAC numbering for 13 (Scheme 3) is used in this section and in Tables 1 and 2

General
The chemicals were purchased from Aldrich or Fluka. Melting points were determined on a Kofler micro melting point apparatus. Elemental analyses were performed with a Perkin-Elmer CHNS-2400 Ser II Elemental Analyser; Merck Kieselgel 60F 254 plates were used for TLC: the eluent was 4:1 toluene-MeOH. Products were purified by column chromatography on Merck 0.063-0.2 mm silica gel; the elution mixtures were determined case by case. Microwave reactions were performed in a CEM Discover LabMate MW reactor. The 1 H-and 13 C-NMR spectra were recorded in CDCl 3 or DMSO-d 6 solution in 5 mm tubes at room temperature, on a Bruker DRX 500 spectrometer at 500 ( 1 H) and 125 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard. The standard Bruker microprogram NOEMULT.AU to generate NOE was used. DEPT spectra were run in a standard manner, using only the Θ = 135° pulse to separate CH/CH 3 and CH 2 lines phased "up" and "down", respectively. The 2D-HSC spectra were obtained by using the standard Bruker pulse program HXCO.AU. (2): di-endo-Bicyclo[2.2.2]oct-5-ene-2,3dicarboxylic acid anhydride (1, 6,4 g, 30 mmol) was added in portions to dilute NH 4 OH (50 mL, 6%) at 0 °C. The mixture was stirred for 30 min, and 2 M NaOH (60 mL) was then added dropwise at 0 °C over a period of 30 min, after which the excess of NH 3 was removed under reduced pressure at 40 °C. The residue was cooled to 0 °C and 1 M NaClO solution (40 mL) was added dropwise with stirring, the temperature being maintained at 0 °C throughout. The mixture was stirred at the same temperature for 1 h, held at 70-75 °C for 10 min, then cooled to ambient temperature, adjusted with 10 M HCl to pH 7 and evaporated to dryness. The residue was extracted with three 150 mL portions of hot MeOH, and the extract was evaporated. The residue was dissolved in a small amount of water and the HCl was removed by means of a Dowex 50 ion-exchange column (acid cycle). Elution was effected with 1 M NH 4 OH solution. Each fraction was evaporated and the dry residue was dissolved in water, acetone was added until turbidity appeared, and the mixture was then allowed to stand in a refrigerator. The solid crystals were filtered off. Yield (5): 1 M NaOH (20 mL) was added to a solution of 3-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acid (2, 3.34 g, 20 mmol) in a 2:1 dioxane/H 2 O mixture (60 mL). The solution was cooled to 0 °C in an ice bath and di-tert-butyl dicarbonate (4.8 g, 22 mmol) was added slowly. The mixture was stirred at 0 °C for 30 min and then warmed to room temperature and stirred for 4 h. The solvent was concentrated to 20 mL, the pH was then adjusted to 2.5 with 10% H 2 SO 4 , and the resulting solution was extracted with EtOAc (3 × 50 mL). The combined extracts were dried (Na 2 SO 4 ) and evaporated, to give 5 as a white solid, which was recrystallized from iPr 2 O. Yield 3.4 g (63%); m.p. 117-120 °C C 14 -1,t-3,t-6,c-7,t-10)-10-tert-Butoxycarbonylamino-4-oxatricyclo[4.3.1.0 3,7 ]decan-5-one (7): Bu 3 SnH (4.8 mL, 18 mmol) was added to a solution of iodolactone 6 (3.53 g, 9 mmol) in dry CH 2 Cl 2 (65 mL) under Ar. After stirring at 40 °C for 20 h, the solvent was evaporated off, and the residue was crystallized from n-hexane and recrystallized from iPr 2 O-EtOAc. Yield [4.3.1.0 3,7 ]decan-5-one trifluoroacetate (8a) and hydrochloride (8b) 8a: Trifluoroacetic acid (20 mL) was added to a solution of Boc-lactone derivative 7 (0.35 g, 13 mmol) in a 9:1 THF:H 2 O mixture (60 mL) and the solution was stirred at room temperature for 10 h. The solvent was next evaporated off and the residue was crystallized from Et 2 (9): To a stirred suspension of LiAlH 4 (1 g, 26 mmol) in dry THF (60 mL) was added a solution of Boc-lactone 7 (0.5 g, 1.9 mmol) in dry THF (20 mL). The resulting suspension was refluxed for 4 h and then decomposed by the addition of a mixture of water (2 mL) and THF (10 mL). The inorganic material was filtered off and washed with THF (3 × 50 mL). After drying (Na 2 SO 4 ) and filtration, the solvent was evaporated off to give a pale oil, which was purified by column chromatography (toluene-MeOH = 4:1) Yield 0.41 g (81%) C 14 (12): A solution of 11 (2.42 g, 10.21 mmol) in CH 2 Cl 2 (80 mL) was treated with NIS (2.3 g, 10.21 mmol) and subsequently stirred for 14 h at room temperature. When the reaction was completed, the mixture was washed with 10% NaOH solution (3 × 10 mL). The aqueous solution was extracted with CH 2 Cl 2 (3 × 40 mL) and the organic phase was dried (Na 2 SO 4 ) and evaporated. The oily dihydroiodooxazine product was sensitive to air and it was therefore used without purification in the next step. Bu 3 SnH (4 mL) was added to a solution of oily dihydroiodooxazine (2.5 g) in dry CH 2 Cl 2 (65 mL) under Ar. After stirring for 20 h at 40 °C, the solvent was evaporated off and the residue was purified by column chromatography on silica gel (n-hexane:EtOAc 10:1) to afford the dihydrooxazine derivative as a colorless oil (1.05 g, 64%). This oily product was also sensitive to air; it was therefore used immediately. A solution of oily dihydrooxazine derivative (1.05 g) in 20% aqueous HCl (20 mL) was stirred for 2 h. The solvent was then evaporated off to afford crude 12, which was recrystallized from H 2 O-acetone. Total yield 0.75 g (33%); m.p. 211-218 °C (with decomposition) C 11 (14): To a magnetically stirred toluene solution of amino ester base 4 (0.7 g, 3.6 mmol in 20 mL), one equivalent of PhNCS in toluene (0.5 g, 20 mL) was added dropwise [the free base was obtained from the hydrochloride 4 by treatment with aqueous NaOH and extraction with CHCl 3 , followed by drying (Na 2 SO 4 ) and evaporation]. The mixture was refluxed for 10 h, the reaction mixture was then evaporated and the oily product was crystallized from n-hexane and recrystallized from iPr 2 O-EtOAc. Yield 0.68 g (57%); m.p. 110-112 °C. C 18 (16): To a magnetically stirred toluene solution of amino ester base 4 (0.7 g, 3.6 mmol in 20 mL), one equivalent of ethyl p-chlorobenzimidate in toluene (0.7 g, 20 mL) and a catalytic amount of p-toluenesulfonic acid was added [the free base was obtained from the hydrochloride 4 by treatment with aqueous NaOH and extraction with CHCl 3 , followed by drying (Na 2 SO 4 ) and evaporation]. The mixture was refluxed for 12 h, the reaction mixture was next evaporated and the residue was recrystallized from EtOH. Yield 0.65 g (63%); m.p. 210-215 °C. C 16

Conclusions
In summary, we have successfully synthetized di-endo-3-aminobicyclo[2.2.2]oct-5-ene-2carboxylic acid derivatives, can be used for further valuable transformations, and are good starting materials for which the syntheses of hydroxy-substituted β-amino acids, aminodiols and heterocycles with potential biological activity.