Chiral separation and determination of R-(-)- and S-(+)-baclofen in human plasma by high-performance liquid chromatography

The method presented here is a high-performance liquid chromatography (HPLC)-UV detection method for the determination of baclofen R-(-)- and S-(+)-enantiomers in human plasma using a chiral separation technique. Baclofen enantiomers were extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge. The extract was then injected onto a HPLC system with a UV detection system set at 220 nm. The separation was achieved by using a 150x4.6 mm, 5 microm Phenomenex chirex 3216 chiral column with a mobile phase consisting of 0.4 mM CuSO(4) in acetonitrile-20 mM sodium acetate (17:83). The calibration curves were linear for both R-(-)- and S-(+)-enantiomers of baclofen in the concentration range of 20-5000 ng/ml. The average regressions were 0.9980 and 0.9991 for R-(-)- and S-(+)-baclofen, respectively. Inter-day precision was 3.3-5.2% for R-(-)-baclofen and 3.5-3.9% for S-(+)-baclofen at a concentration range of 60-4000 ng/ml. Intra-day precisions were 0.6-4.4 and 0.5-3.5% for R-(-)-baclofen and S-(+)-baclofen, respectively. The average extraction recovery was 81.6% for R-(-)-baclofen, 83.0% for S-(+)-baclofen and 94.0% for the internal standard (p-aminobenzoic acid). The limit of quantitation for both R-(-)- and S-(+)-baclofen in human plasma was 20 ng/ml. The method is simple and easy to operate with accuracy and reproducibility and it is suitable for pharmacokinetic studies.     

生物催化3-(4-氯苯基)-戊二腈去对称性水解合成光学纯巴氯芬的关键前体

研究了利用生物催化剂制备(S)-4-氰基-3-(4-氯苯基)-丁酸.以3-(4-氯苯基)-戊二腈为底物,采用苯酚-次氯酸钠法对实验室保藏的菌株进行筛选,得到一株产物立体选择性较高的菌株赤霉菌Gibberella intermedia WX12,并对其催化特性和发酵条件进行了初步研究.以30 g/L的乳糖和20 g/L的蛋白胨分别为碳、氮源,发酵培养96 h,收集的菌体在50 mmol/L磷酸缓冲液(pH 8.0)中30℃催化反应24 h,将3-(4-氯苯基)-戊二腈转化为4-氰基-3-(4-氯苯基)-丁酸,产率为90％.将产物化学转化为巴氯芬,手性HPLC分析表明水解产物构型是(S),其对映异构体过量值ee＞ 99％.该产物可以用来合成光学纯的(R)-和(S)-巴氯芬.     

Biochemical approaches to the synthesis of ethyl 5-(s)-hydroxyhexanoate and 5-(s)-hydroxyhexanenitrile

Three different biochemical approaches were used for the synthesis of ethyl 5-(S)-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2. In the first approach, ethyl 5-oxo-hexanoate 3 and 5-oxo-hexanenitrile 4 were reduced by Pichia methanolica (SC 16116) to the corresponding (S)-alcohols, ethyl (S)-5-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2, with an 80-90% yield and >95% enantiomeric excess (e.e). In the second approach, racemic 5-hydroxyhexanenitrile 5 was resolved by enzymatic succinylation, leading to the formation of (R)-5-hydroxyhexanenitrile hemisuccinate and leaving the desired alcohol 5-(S)-hydroxyhexanenitrile 2 with a yield of 34% (50% maximum yield) and >99% e.e. In the third approach, enzymatic hydrolysis of racemic 5-acetoxy hexanenitrile 6 resulted in the hydrolysis of the R-isomer to provide 5-(R)-hydroxyhexanenitrile, leaving 5-(S)-acetoxyhexanenitrile 7 with a 42% yield (50% maximum yield) and >99% e.e.     

GABAB antagonists: Resolution,absolute stereochemistry,and pharmacology of (R)- and (S)-phaclofen

Phaclofen, which is the phosphonic acid analogue of the GABA_B agonist (RS)-3-(4-chlorophenyl)-4-aminobutyric acid (baclofen), is a GABA_B antagonist. As part of our studies on the structural requirements for activation and blockade of GABA_B receptors, we have resolved phaclofen using chiral chromatographic techniques. The absolute stereochemistry of (?)-(R)-phaclofen was established by X-ray crystallographic analysis. (?)-(R)-Phaclofen was shown to inhibit the binding of [³H]-(R)-baclofen to GABA_B receptor sites on rat cerebellar membranes (IC₅₀ = 76 ± 13 μM), whereas (+)-(S)-phaclofen was inactive in this binding assay (IC₅₀ > 1000 μM). (?)-(R)-Phaclofen (200 μM) was equipotent with (RS)-phaclofen (400 μM) in antagonizing the action of baclofen in rat cerebral cortical slices, while (+)-(S)-phaclofen (200 μM) was inactive. The structural similarity of the agonist (R)-baclofen and the antagonist (?)-(R)-phaclofen suggests that these ligands interact with the GABA_B receptor sites in a similar manner. Thus, it may be concluded that the different pharmacological effects of these compounds essentially result from the different spatial and proteolytic properties of their acid groups. © 1994 Wiley-Liss, Inc.     

Synthesis of (25R)-26-hydroxycholesterol

We describe the synthesis of (25R)-cholest-5-en-3beta,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall, yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.     

Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Stereoselective synthesis of chirally deuterated (S)-D-(6-(2)H(1))glucose

Chirally deuterated (S)-D-(6-(2)H(1))glucose has been prepared in good overall yield from d-(6,6'-(2)H(2))glucose by a short, five-step synthesis from D-(6,6-(2)H(2))glucose utilizing (R)-(+)-Alpine-Borane [(R)-9-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-9-borabicyclo[3.3.1]nonane]. Suitably protected methyl 2,3,4-tri-O-benzyl-D-(6,6-(2)H(2))glucopyranoside was prepared and the deuterated O-6 primary alcohol was oxidized to an aldehyde by Swern oxidation. Stereoselective reduction with nondeuterated (R)-(+)-Alpine-Borane gave methyl 2,3,4-tri-O-benzyl-(6S)-D-(6-(2)H(1))glucopyranoside, which was deprotected under standard conditions to afford the title compound. The key stereoselective reduction step was achieved in 90% yield. The preparation uses economical, commercially available starting materials and will be useful for elucidating biosynthetic mechanisms.     

Bioconversion of ethyl (R)-4-cyano-3-hydroxybutyate into (R)-ethyl-3-hydroxyglutarate via an indirect pathway by Rhodococcus boritolerans

(R)-Ethyl-3-hydroxyglutarate, (R)-3, is an intermediate in the synthesis of the statin side chain. Here, a new two-step, indirect biotransformation pathway involving the formation of ethyl (R)-4-carbamoyl-3-hydroxybutanoate, (R)-2, as an intermediate for (R)-3 production was developed using Rhodococcus boritolerans with ethyl (R)-4-cyano-3-hydroxybutyate, (R)-1, as substrate. Maximum conversion was with 10?g (R)-1/l, 7?g cells/l (dry wt), pH 7.5 and 25°C. A yield of 98?±?0.5% (w/w) was attained within 8?h.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.     

The positive allosteric modulator GS39783 enhances GABA(B) receptor-mediated inhibition of cyclic AMP formation in rat striatum in vivo

We studied the effects of the positive allosteric modulator GS39783 on GABA(B) receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABA(B) receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABA(B) agonist R(-)-baclofen inhibited cyclic AMP formation stimulated by a water-soluble forskolin analogue in a concentration-dependent manner (EC50 7.3 microM, maximal inhibition 40%). The selective GABA(B) antagonist CGP56999 reversed R(-)-baclofen-induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(-)-baclofen (1 microM), significantly decreased cyclic AMP formation in a dose-dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABA(B) receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(-)-baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABA(B) receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein-coupled receptor-positive allosteric modulator.     

A new synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (AraF-G)

Interesting and very promising antisense properties of 2'-deoxy-2'-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2'-Deoxy-2'-fluoroarabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625-3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1-4.15.21) (2'F-ANA) has encouraged our research group to optimize the synthetic procedures for 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (beta-FIAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (beta-FMAU). J. Org. Chem. 1985, 50, 3644-3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85-88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966-970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. Synthesis of 2'-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2'F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1-1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861-1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-beta-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150 degrees C, 6 h) in 67% yield.     

Intramolecular reductive cyclization strategy to the synthesis of (-)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid, (+)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol and their glycosidase inhibitory activity

The first stereoselective synthesis of (2S,3R,6S)-6-methyl-3-hydroxy-piperidine-2-carboxylic acid (-)-6 and (2R,3R,6S)-6-methyl-(2-hydroxymethyl)-piperidine-3-ol (+)-7 was achieved starting from readily available d-glucose in 14 steps with 17% overall yield for both the compounds. The key feature of the present strategy includes the Wittig-olefination for the preparation of required conjugated keto-azide 9 and construction of 2,3,6-trisubstituted piperidine skeleton 11 by applying intramolecular reductive cyclization of conjugated keto-azide intermediate. The glycosidase inhibitory activity of compounds 6 and 7 towards several glycosidases has been evaluated.     

Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide as an imaging probe for 5-HT6 receptors

The serotonin receptor 6 (5-HT(6)) is implicated in the pathophysiology of cognitive diseases, schizophrenia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the pathophysiology of these disorders. Therefore, N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide (SB399885), a selective and high affinity (pK(i)=9.11) 5-HT(6) antagonist, has been radiolabeled with carbon-11 by O-methylation of the corresponding desmethyl analogue with [(11)C]MeOTf in order to determine the suitability of [(11)C]SB399885 to quantify 5-HT(6)R in living brain using PET. Desmethyl-SB399885 was prepared, starting from 1-(2-methoxyphenyl) piperazine hydrochloride, in excellent yield. The yield obtained for radiolabeling of [(11)C]SB399885 was 30±5% (EOS) and the total synthesis time was 30min at EOB. PET studies with [(11)C]SB399885 in baboon showed fast uptake followed by rapid clearance in the brain. Highest uptake of radioactivity of [(11)C]SB399885 in baboon brain were found in temporal cortex, parahippocampal gyrus, pareital cortex, amygdala, and hippocampus. Poor brain entry and inconsistent brain uptake of [(11)C]SB399885 compared to known 5-HT(6)R distribution limits its usefulness for the in vivo quantification of 5-HT(6)R with PET.     

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.     

Metathesis-mediated synthesis of (R)-10-methyl-2-tridecanone, the southern corn rootworm pheromone

(R)-10-Methyl-2-tridecanone, the female sex pheromone of the southern corn rootworm (Diabrotica undecimpunctata howardi Barber), was synthesized in 9 steps from methyl (S)-3-hydroxy-2-methylpropanoate in a 15.7% overall yield. Olefin cross metathesis between (R)-6-methyl-1-nonene and 5-hexen-2-one employing Grubbs' first-generation catalyst was the key step of the synthesis.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

Diastereoselective reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to Ezetimibe by the whole cell catalyst Rhodococcus fascians MO22

The asymmetric microbial reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) by Rhodococcus fascians MO22 is described. The catalytic capability of the microorganism for reduction has been examined also with protected ketone, an intermediate from chemical synthesis of Ezetimibe. Various parameters of the bioreduction have been optimized: the strain converted 94.8% of ketone and 63% of protected ketone into Ezetimibe with the same de of 99.9%. In the later case, two chemical steps are replaced with a single biotransformation.     

Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.     

Chemoenzymatic synthesis and properties of Schiff bases containing (R)-1-(9-anthryl)ethylamine

Racemic 1-(9-anthryl)ethylamine (10), obtained in 70% overall yield from commercial 9-cyanoanthracene, was kinetically resolved by the Candida antarctica A lipase-catalyzed acetylation with isopropyl acetate as acyl donor, affording (R)-(+)-10 with 95.8% enantiomeric excess (e.e.) (E-value 43.5), which afforded Schiff bases (R)-4 and(R)-8. (1)H-NMR, CD, and MM2 calculations offer a consistent picture of the conformational properties of these potential ligands and an explanation for the limited enhancement of enantioselectivity in cyclopropanation of styrene by their Cu(I) complexes, as compared with previously studied ligands in this series.