酞丁安对映体合成及其抗单纯疱疹病毒活性评价

酞丁安(3-酞酰亚胺-2-氧-正丁醛双缩氨硫脲,TDA)是药物研究所创制的抗病毒新药。为了研究其对映异构体(R),(S)-TDA对病毒活性及毒性是否有差异,并与消旋酞丁安(RS)-TDA的抗病毒活性及毒性进行比较,本文分别用已知构型的(R)-与(S)-丙氨酸为原料,通过缩合等6步反应,得到光学活性的(R)-,(S)-TDA,并与外消旋酞丁安比较其抗病毒活性及毒性。三者的抗单纯疱疹病毒活性与对细胞的毒性差别不大,说明消旋酞丁安临床使用是安全有效的。     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N²- and N³-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N²- and N³-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N²-dechloroethylIFA (N2D) and (R)-N³-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Enantioselective synthesis of tri-deuterated (–)-geosmin to be used as internal standard in quantitation assays

For the accurate and sensitive quantitation of the off-flavor compound geosmin, particularly in complex matrices, a stable isotopologue as internal standard is highly advantageous. In this work, we present a versatile synthetic strategy leading from (4aR)-1,4a-dimethyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one to tri-deuterated (–)-geosmin ((4S,4aS,8aR)-4,8a-dimethyl(3,3,4-²H₃)octahydronaphthalen-4a(2H)-ol). The starting material was readily accessible from inexpensive 2-methylcyclohexan-1-one using previously published procedures.     

Enantioselective synthesis of (R)- and (S)-2-methyl-[3,3,2-2H3] alanines

The synthesis of optically pure (R)- and (S)-2-methyl-[3,3,3-²H₃] alanines of biological interest is described. The stereochemistry of the reaction of the lithio derivative of (R)-(©)-2,5-dimethoxy-3-benzyl-3-methyl-3, 6-dihydropyrazine with alkyl and deuterated alkyl iodides is discussed. The configuration of the newly formed center of chirality in (R)- and (S)-2-methyl-[3,3,3-²H₃] alanines is derived from ¹ H NMR.     

Reduced number of α- and β-adrenergic receptors in the myocardium of rats exposed to tobacco smoke

The concentration of α- and β-adrenergic receptors-as measured by specific [³H]WB-4101 and (−)-[³H]-dihydroalprenolol binding-was diminished by 60% below control values in the hearts of hearts of rats exposed to tobacco smoke. These changes in receptor numbers took place almost immediately after tobacco smoke exposure and were rapidly reversible after termination of the exposure. The dissociation constant, K , for [³H]WB-4101 was identical in exposed (K_D = 0.34 ± 0.09 nM) and control (K_D = 0.35 ± 0.07 nM) hearts but was significantly different i in the case of (-)-[³H] dihydroalprenolol binding (exposed, (K_D = 2.83 ± 0.30 nM vs control K_D = 5.22 ± 0.61 nM). For β-receptor binding there was no significant difference between exposed and control animals in the K_i values (−)-epinephrine, (−)-norepinephrine, (−)-alprenolol, (±)-propranolol or timolol. (−)-Isoproterenol, however, was found to bind with lower affinity in exposed compared with control hearts. For α-receptor binding there was no significant difference between control and ‘smoked’ animals in the K_i values for (−)-epinephrine, (−)-norepinephrine or phentolamine. The decrease in α- and β-adrenergic receptor concentration may be related to the phenomenon of receptor desensitization resulting from a release of catecholamines in rats exposed to tobacco smoke.     

Synthesis and antiviral activity of new 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids derivatives

The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (3a-l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) derivatives was achieved with an efficient synthetic route. Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l) (52–82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c) (50–60%), respectively. Subsequent hydrolysis of the esters afforded the corresponding carboxylic acids (3a–l) (86–93%) and (5a–c) in high yield (80–93%). Inhibitory effects of 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids. Derivatives on Herpes simplex virus type 1 (HSV-1), Mayaro virus (MAY) and vesicular stomatitis virus (VSV) were investigated. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against HSV-1, MAY, and VSV virus with EC₅₀ values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of these compounds showed toxicity for Vero cells.     

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyms and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20–100 nmol/5 μl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 μmol/5 μl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-α-methyl-carboxyphenylglycine (MCPG, 1 μmol), and the group1 mGluR antagonist, S-4- carboxyphenylglycine (4CPG, 100 nmol) competely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50–200 nmol/5 μl) induced a dosedependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slowonset potentiation may be mediated by group 1 mGluRs.     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G_α16 was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

VKORC1-3673G>A、CYP2C9*3、CYP4F2 rs2108622和CYP2C19*2基因多态性对中国汉族房颤患者华法林维持剂量的影响

目的 探讨VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2位点基因多态性对中国汉族房颤患者华法林维持剂量的影响。方法 收集107例服用华法林达维持剂量的汉族房颤患者的血样和临床相关资料,应用PCR-RFLP法检测VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2基因型,采用独立样本t检验分析基因型与华法林维持剂量的相关性。多元线性回归建立给药模型,探讨基因多态性对华法林维持剂量的影响。结果 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性和患者年龄、体质量能解释45.2%的华法林维持剂量差异。CYP2C19^*2基因多态性对本研究人群华法林维持剂量无影响。结论 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性显著影响中国汉族房颤患者的华法林维持剂量。     

Ceramides and cerebrosides from the marine bryozoan Bugula neritina inhabiting South China Sea

From the marine bryozoan Bugula neritina inhabiting South China Sea, a new ceramide named (2S,3R,4E)-2-(14′-methyl-pentadecanoylamino)-4-octadecene-l,3-diol (1) and a new cerebroside named 1-O-(β-d-glucopyranosyl)-(2S,3R,4E)-2-(heptadecanoylamino)-4-octadecene-l,3-diol (6), together with one known ceramide (2) and three known cerebrosides (3, 4, and 5), were isolated. Their structures were deduced by extensive spectral analysis and chemical evidences. Compound 1 is branched with a methyl [–CH(CH₃)₂] in the fatty acid moiety, which is a rare structural feature among ceramides. Compound 6 is a new cerebroside with 17 carbons in the fatty acid moiety, while 5 is a new natural product which was isolated from a natural origin for the first time.     

Identification of cytochromes<Emphasis Type="Italic"> P</Emphasis><Subscript>450</Subscript> 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro

Objective This in-vitro study aimed at an identification of cytochrome P₄₅₀ (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites.Methods Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes.Results Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4-, 6- and 7-hydroxylation of both enantiomers with mean K_m1 and K_m2 of 144.5±34.9 and 10.0±6.49 µM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by ~2/3 and ~1/3 using mAb2C8–9-18–19 and TAO, respectively, but (R)-6- and 7-hydroxylation by ~50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (K_m <5 µM), whereas CYP3A4 operated with low affinity (K_m >100 µM).Conclusion CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes.Part of this work was presented at the 6th Congress of the European Association for Clinical Pharmacology and Therapeutics, Istanbul, June 2003.     

Compounds from Gum Ammoniacum with Acetylcholinesterase Inhibitory Activity

The use of herbal medicinal preparations in dementia therapy has been studied based on experience from traditional medicine. A dichloromethane extract of gum ammoniacum, the gum-resin from Dorema ammoniacum D. Don had shown acetylcholinesterase (AChE) inhibitory activity in a previous study. The aim of this study was the isolation and characterization of the active compounds from this resin. The extract was investigated by a respective colorimetric microplate assay and the active zones were identified via TLC bioautography and isolated using several chromatographic techniques. The structures of the active components were characterized by one- and two-dimensional ¹H and ¹³C NMR spectroscopy and mass spectrometry as (2′S,5′S)-2′-ethenyl-5′-(3-hy-droxy-6-methyl-4-oxohept-5-en-2-yl)-7-methoxy-2′-methyl-4H-spiro[chromene-3,1′-cyclopentane]-2,4-dione (1), which is an analogue of doremone A and a new natural compound, and as (2′S,5′R)-2′-ethenyl-5′-[(2R,4R)-4-hydroxy-6-methyl-3-oxohept-5-en-2-yl]-7-methoxy-2′-methyl-4H-spiro[chromene-3,1′-cyclo-pentane]-2,4-dione (2 = doremone A), (4E,8E)-1-(2,4-dihydroxyphenyl)-5,9,13-trimethyltetradeca-4,8,12-trien-1-one (3 = dshamirone), and 4,7-dihydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one (4 = am-moresinol). Dshamirone turned out to be the most active compound with an IC₅₀ value for AChE inhibitory activity of 23.5 μM, whereas the other substances showed weak activity. The concentrations of the analytes in the resin were determined by HPLC as 3.1%, 4.6%, 1.9%, and 9.9%, respectively.     

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-Tetrazolo[1,5-c]quinazolin-5(6H)-ones

Methods of 1-[2-(1H-tetrazol-5-yl)-R¹-phenyl]-3-R²-phenyl(ethyl)ureas and R¹-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, ¹H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg).     

The Enantiomers of the Valproic Acid Analogue 2-n-Propyl-4-pentynoic Acid (4-yn-VPA): Asymmetric Synthesis and Highly Stereoselective Teratogenicity in Mice

The teratogenic activities of R( +)- and S( – )-2-n-propyl-4-pentynoic acid (R and S-4-yn-VPA), the enantiomers of the highly teratogenic valproic acid (VPA) analogues (±)-4-yn-VPA, were investigated in mice. The enantiomers were prepared via asymmetric synthesis, each in three steps employing the chiral auxiliaries (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone and S-4-benzyl-2-oxazolidinone. The determination of the absolute configurations and the optical purities is described. R( + )-4-yn-VPA contained 7%, and S( – )-4-yn-VPA 8%, of the respective antipodes. The aqueous solutions of the sodium salts of R- and S-4-yn-VPA were administered as single i.p. injections during early organogenesis in the mouse (day 8 of gestation) using the induction of exencephaly as the teratological end point. Dose/exencephaly curves indicated that S-4-yn-VPA is 7.5 times more teratogenic than its antipode, 1.9 times more teratogenic than (±)-4-yn-VPA and 3.9 times more teratogenic than the parent drug VPA. In contrast, the neurotoxicity (maternal toxicity) of the 4-yn-VPA enantiomers was found to be independent of the stereo-chemical configuration and lower than achieved after VPA administration. Due to its low neurotoxicity and highly Stereoselective neural tube-inducing activity, S-4-yn-VPA should prove an important tool for the investigation of molecular mechanism of the teratogenic action in this class of compounds; R-4-yn-VPA could act as the negative control in these studies.     

(R)-(+)-脱水芽子碱甲酯的合成工艺研究及结构表征

目的对(R)-(+)-脱水芽子碱甲酯的合成工艺进行研究和结构表征。方法以托品酮为原料,经克莱森缩合、拆分、还原和消除得到目标产物,并以成盐的方式得到其富马酸盐,以溶剂挥发法培养单晶,采用X射线单晶衍射对结晶形态进行表征。结果优化了合成工艺,总收率为36%。测试结果表明不对称单位化学计量式为C_(14)H_(19)NO_6,相对分子质量为297.30,晶体密度为1.359 g/cm~3,该晶胞属于正交晶系,空间群为P2_12_12_1。结论本合成工艺简化了实验操作,更适合大规模制备,且为脱水芽子碱的结构确证提供了新的依据。     

Atomoxetine occupies the norepinephrine transporter in a dose-dependent fashion: a PET study in nonhuman primate brain using (S,S)-[18F]FMeNER-D2

Rationale Atomoxetine is a potent and selective norepinephrine transporter (NET) reuptake inhibitor acting as a nonstimulant for the treatment of attention-deficit/hyperactivity disorder (ADHD). Previous positron emission tomography (PET) studies had failed to demonstrate the feasibility of measuring a dose-dependent and saturable NET occupancy in human brain using [¹¹C]MeNER.Objectives To determine if atomoxetine occupies NET in a dose-dependent fashion using (S,S)-[¹⁸F]FMeNER-D₂ in nonhuman primate brain.Methods A total of eight PET measurements were performed in two cynomolgus monkeys. Each monkey was examined four times with PET: under baseline conditions and after steady-state infusion with 0.03, 0.06, or 0.12 mg/kg/h of atomoxetine. A prolonged intravenous (i.v.) infusion design was developed rather than an i.v. bolus to better mimic an oral absorption profile and to reach plasma steady state.Results During baseline conditions, (S,S)-[¹⁸F]FMeNER-D₂ uptake was highest in the locus coeruleus, thalamus, mesencephalon, and the cingulate gyrus, whereas the radioactivity in the caudate was low. Peak equilibrium measurements were achieved using (S,S)-[¹⁸F]FMeNER-D₂ in contrast to the previously reported data for [¹¹C]MeNER. After administration of atomoxetine, a dose-dependent occupancy from 38 to 82% was observed for various brain regions known to contain high densities of NET.Conclusions This is the first in vivo PET study to successfully demonstrate the ability to measure a dose-dependent change in NET occupancy in brain using (S,S)-[¹⁸F]FMeNER-D₂. Furthermore, an asymptotic relationship between N-desmethylatomoxetine plasma concentration and NET occupancy was established. In total, these data encourage further PET studies using (S,S)-[¹⁸F]FMeNER-D₂ in humans.     

5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through G_i type G proteins. In the present study, 5-HT-stimulated [³⁵S]GTPγS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl₂ and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 μM GDP, 5 mM MgCl₂ and 100 mM NaCl. The specific [³⁵S]GTPγS binding was stimulated by several compounds that had been shown to be agonists at 5-HT_1B/1D receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC₅₀) for these agonists were significantly correlated with their pK _i’s reported in the previous study of 5-HT_1B receptor binding in rat frontal cortical membranes. The increase in specific [³⁵S]GTPγS binding in response to 1 μM 5-HT was potently inhibited by several 5-HT_1B/1D receptor antagonists as well as β-adrenoceptor antagonists such as S(−)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT_1D receptors, was inactive as an antagonist at least up to 1 μM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(−)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [³⁵S]GTPγS binding was reduced by 1′-methyl-5-([2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4′-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(−)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT_1B receptors in the present functional assay system. 5-HT-stimulated [³⁵S]GTPγS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT_1B receptors and their coupled G proteins in this brain region.