氟马西尼对苯二氮（艹卓）类药物的拮抗作用观察

氟马西尼化学名为8-氟-5,6-二氢-5-甲基-6-氧代-4 H-咪唑并-［1,5-α］［1,4］苯并二氮（艹卓）-3-甲酸乙酯。氟马西尼对苯二氮（艹卓）类类（ BDZ）受体有很强的亲和力,可竞争性地拮抗BDZ与受体结合,从而消除BDZ的药理作用［1］。笔者对地西泮（安定）或咪达唑仑（咪唑安定）复合静脉全麻术后的4例患者运用氟马西尼拮抗其麻醉苏醒延迟,观察其效果,现报道如下。     

佐匹克隆

佐匹克隆Zopiclone，由法国罗钠普朗克公司研制，1987年首次在法国上市，商品名Imovan“忆梦返”。佐匹克隆是非苯并二氮杂类药物，为激动GABAA受体，增强GABA抑制作用的吡咯环酮类化合物：作用于苯二氮卓受体，但结合方式不同于苯二氮卓类药物，为速效催眠药，能延长睡眠时间，提高睡眠质量，减少夜间觉醒和早醒次数。     

5-芳基-1,2-二氢-1-吡咯里嗪酮类化合物的合成及抗炎镇痛作用

目的 研究5-芳基-1，2-二氢-1-吡咯里嗪酮类化合物的抗炎镇痛作用构效关系，寻找高效低毒、具有抗炎镇痛活性的新型吡咯里嗪酮衍生物。方法 以吡咯里嗪酮为母体，设计并合成了5-芳基-1，2-二氢-1-吡咯里嗪酮类化合物。用二甲苯致小鼠耳肿胀法和小鼠醋酸扭体法测定了该类化合物的抗炎及镇痛活性。结果 用两条路线合成了10个目标化合物，经1H-NMR和MS确证其结构。小鼠试验表明，一些所合成的化合物具有明显的抗炎和／或镇痛作用。结论 化合物Ⅲ1及Ⅲ5的抗炎活性优于对照药布洛芬；化合物Ⅲ5的镇痛活性优于对照药布洛芬；其中化合物Ⅲ5的抗炎及镇痛活性均强于对照药布洛芬，值得进一步研究。     

5-苯硫基吡哩酮及其异构体的合成研究

目的研究1H-2，3-二氢-1-吡咯里嗪酮类衍生物的构效关系，设计合成了5-苯硫基-2，3-二氢-1-吡咯里嗪酮(1)。方法 以吡咯为原料经Michael加成、苯硫化、分子内Hoesch反应合成目标化合物。结果 在生成目标化合物的同时，生成了其同分异构体7-苯硫基-2，3-二氢-1-吡咯里嗪酮(5)。结论 推测其生成机制为中间体2-苯硫基-1-氰乙基吡咯在酸性下的异构化反应。     

天麻甙元衍生物的合成及作为脑苯二氮受体基的体外筛选

α-第二丁基对羟基苄醇(简称天-018)是中枢苯二氮革受体的新型配基。作者以天-018为先导化合物,设计并合成了17个目标化合物,其中13个未见文献报道。通过体外竞争性放射配基受体结合分析,对所合成的化合物进行筛选,发现有7个化合物选择性地作用于脑苯二氮(艹卓)受体,其亲和力与结构相关。以α-苄基对碘苄醇竞争抑制作用最强,IC_(50)为3.4×10~(-8)mol/L,表明其对该受体的亲和力最高。     

氟马西尼的有关物质B的合成(英文)

我们以7-甲氧基-4-甲基-3H-1,4-苯并二氮杂卓-2,5-(1H,4H)–二酮为原料,经脱甲基、苄基保护、环合和脱苄基反应合成了氟马西尼有关物质B。氟马西尼有关物质B结构经氢谱、碳谱和高分辨质谱确证,总收率为29.3%。该发现有助于合成工艺的优化和氟马西尼的质量控制。     

β-咔咻-3-羧酸酯的合成

苯二氮(艹卓)(Ⅰ)的药理作用是通过中枢神经系统特殊的结合部位传递的。“苯二氮(艹卓)受体”对于药理和临床上具有活性的苯二氮(艹卓)类药物有很高的专一性。和Ⅰ不同而能与那些受体结合的一组化合物是β-咔啉类化合物。其中β-咔啉-3-羧酸酯类比母体酸的亲合力大三个数量级(低纳摩尔范围),使得β-咔啉衍生物成为研究苯二氮(艹卓)受体机能的重要工具。再者其结构能成为设计苯二氮(艹卓)类新药的重要基础。     

1,4-苯并二氮-3-取代氨基乙酸酯类化合物的合成

本文报道了一系列1,4-苯并二氮(艹卓)-3-取代氮基乙酸酯类化合物的合成。A_3与取代氨基乙酸直接酯化制备最终产物,用DCC作缩合剂时只分离到反应的中间体;改用活性更大的1,1′-碳酰二咪唑作缩合剂,反应获得成功。合成的新化合物能溶于水。放射配体-受体抑制试验表明,这些化合物与苯并二氮革受体的亲和力较强,IC_(50)在10~(-8)～10~(-10)之间,其中A_9与苯并二氮(艹卓)受体的亲和力比氟安定高一个数量级,比安定高两个数量级。     

2-芳基咪唑并[1,2-a]吡啶-3-乙酰胺衍生物的合成与抗焦虑活性研究

目的寻找水溶性较好、抗焦虑活性较强的新型化合物。方法设计合成2-芳基咪唑并[1,2-a]吡啶-3-乙酰胺类化合物,通过体外苯二氮受体竞争结合实验及小鼠高架迷宫行为实验,评价化合物的体内外抗焦虑活性,分析化合物的构效关系。结果与结论共合成28个新型化合物,通过1H-NMR和MS确证其结构,其中I8和I10水溶性良好。苯二氮受体竞争结合实验结果表明,化合物Ⅰ1、Ⅰ8、Ⅰ10、Ⅰ13、Ⅰ19与苯二氮受体的亲和力与阳性对照药物Ro5-4864相当,在浓度为100nmol/L时,其对放射性配体与受体结合的抑制率分别为87%、89%、85%、89%和76%,而Ro5-4864为82%。对水溶性及活性均较好的化合物Ⅰ和Ⅰ进行小鼠抗焦虑活性评价结果表明,其具有明显的体内抗焦虑作用。     

作用于外周苯二氮卓受体的新型抗焦虑剂

外周苯二氮卓受体位于线粒体上，调节胆固醇在线粒体膜上的转运速率，而胆固醇是线粒体合成类固醇的原料，合成的类固醇能调节GABAA受体的行为。外周苯二卓受体和适当的配体结合后，通过调节类固醇的合成间接地调节GABAA受体的行为，表现出明显的抗焦虑作用。此类化合物没有传统抗焦虑剂的肌松、镇静等副作用。本文介绍了外周苯二氮卓受体的生理生化机制及其在抗焦虑中的作用以及作用于外周苯二卓受体的几类新的抗焦虑化合物。     

Differences in pharmacological profiles of a new generation of benzodiazepine and non-benzodiazepine hypnotics

The hypnotics, quazepam (a benzodiazepine), brotizolam (a thienotriazolodiazepine), zopiclone (a cyclopyrrolone) and zolpidem (an imidazopyridine) have a common ability to bind to the benzodiazepine recognition site (omega receptor) within the GABAA receptor. For this reason we compared their pharmacological profiles in mice. All compounds shared anticonvulsant and central depressant effects. However, the sedative activity of zolpidem appeared at much lower doses than did the anticonvulsant and myorelaxant effects but the opposite was observed with the other hypnotics. In contrast to brotizolam, quazepam and zopiclone, zolpidem did not increase food intake in mice placed in a novel environment, indicating that this drug lacks disinhibitory activity. Moreover the efficacy of zolpidem at the GABAA receptor, as indicated by its activity against convulsions induced by the GABA synthesis inhibitor, isoniazid, was much greater than that of other hypnotics. These results suggest that the hypnoselective properties observed with zolpidem might be related to a high selectivity for the omega 1 recognition site of the GABAA receptor coupled with a very high intrinsic activity.     

Anxiolytic cyclopyrrolones zopiclone and suriclone bind to a novel site linked allosterically to benzodiazepine receptors

The interactions of zopiclone and suriclone, representatives of nonbenzodiazepine cyclopyrrolone anxiolytics, with central-type benzodiazepine receptors have been characterized in rat and bovine brain. While zopiclone potently (IC50 approximately 50 nM) inhibits [3H]Ro-15-1788 binding in an apparent mass action fashion, suriclone and its metabolite 35,489 RP are extremely potent (IC50 approximately 350 pM and 1 nM, respectively) and display Hill coefficients of approximately 2.0. Like classical benzodiazepines, none of the cyclopyrrolones studied display selectivity for type I or type II benzodiazepine receptors. Using [3H]suriclone, saturable high affinity sites for cyclopyrrolone anxiolytics were directly labeled in rat and bovine brain. The regional distribution and pharmacologic specificity of [3H]suriclone and [3H]Ro-15-1788 binding sites are similar, suggesting that [3H]suriclone recognition sites reside on the benzodiazepine receptor complex. Unlike classical benzodiazepine agonists, such as diazepam, the binding of [3H]suriclone is not modulated by GABA, Cl-, pentobartibal, or tracazolate. Unlike those of [3H]diazepam, [3H]suriclone-binding sites are only minimally affected by photoaffinity labeling with flunitrazepam. Whereas the binding affinities of [3H]Ro-15-1788, [3H]flunitrazepam, and [3H]ethyl beta-carboline 3-carboxylate increase at lower temperatures, [3H]suriclone binds with higher affinity at higher temperatures. Scatchard analysis of [3H]flunitrazepam, [3H]ethyl beta-carboline 3-carboxylate, and [3H]Ro-15-1788 binding in the presence of all cyclopyrrolones studied reveals an apparent noncompetitive pattern of inhibition of binding in each case; by contrast, inhibition of [3H]suriclone binding by Ro-15-1788 flunitrazepam, methyl beta-carboline 3-carboxylate and all of the cyclopyrrolones studied appears competitive. The dissociation kinetics of [3H]Ro-15-1788 indicate that cyclopyrrolones, but not benzodiazepines, increase the dissociation rate of [3H]Ro-15-1788 from its membrane receptors; the converse is true for [3H]suriclone dissociation kinetics. The association kinetics of [3H]suriclone suggest that suriclone induces a conformational change upon binding to receptors. Taken together, these results indicate that [3H]suriclone labels a site on the benzodiazepine receptor complex allosteric to the recognition site for benzodiazepines. A model is proposed to describe the interaction between benzodiazepines and cyclopyrrolones.     

The effect of cyclopyrrolones on GABAA receptor function is different from that of benzodiazepines

The effects of the cyclopyrrolones zopiclone and suriclone on the function of the central -aminobutyric acid type A (GABA_AA) receptor complex in mouse brain were evaluated both in vitro and in vivo. Added in vitro to mouse cerebral cortical membranes, these compounds potently inhibited [³H]flumazenil binding with IC₅₀ (50% inhibitory concentration) values of 35.8 nM (zopiclone) and 1.1 nM (suriclone). Similar results were obtained with cerebellar membranes, indicating that these drugs do not discriminate between putative type I and type II benzodiazepine receptors. The interaction of cyclopyrrolones with recognition sites present at the level of the GABA receptor complex appears to be competitive, because zopiclone decreased the affinity of the receptors for [³H]flumazenil without affecting the maximal number of binding sites. Moreover, zopiclone and suriclone did not affect the rate of dissociation of [³H]flumazenil from benzodiazepine receptors. The in vitro efficacy of zopiclone appeared different from that of suriclone and the benzodiazepines diazepam and flunitrazepam. Thus, zopiclone failed to affect muscimol-stimulated ³⁶Cl^– uptake and only slightly inhibited t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding. In contrast, like diazepam and flunitrazepam, suriclone increased muscimol-stimulated ³⁶Cl^– uptake and markedly inhibited [³⁵S]TBPS binding. On the other hand, suriclone, like zopiclone, did not modify [³H]muscimol binding to mouse cerebral cortical membranes. Moreover, zopiclone antagonized the reduction in [³⁵S]TBPS binding elicited by the benzodiazepine receptor full agonist diazepam. Consistent with its low efficacy in vitro, oral administration of zopiclone (2.5 to 100 mg/kg, p.o.) in mice failed to modify [³⁵S]TBPS binding subsequently measured in cerebral cortical membranes ex vivo. In contrast, suriclone (10 to 20 mg/kg, p.o.), like diazepam, decreased [³⁵S]TBPS binding measured ex vivo. Moreover, both zopiclone (50 to 100 mg/kg, p.o.) and suriclone (1 to 10 mg/kg, p.o.) abolished the increase in [³⁵S]TBPS binding induced by isoniazid (200 mg/kg, s.c.). These results suggest that suriclone may enhance GABAergic transmission with an efficacy similar to that of diazepam. In contrast, the low efficacy of zopiclone both in vitro and in vivo suggests that this drug may act as a partial agonist at benzodiazepine receptors.Abbreviations GABA -aminobutyric acid - TBPS t-butylbicyclophosphorothionate - ANOVA analysis of variance Correspondence to: A. Concas at the above address     

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors.

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.     

Ethyl 8-fluoro-6-(3-nitrophenyl)-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate as novel, highly potent, and safe antianxiety agent

Ethyl 8-fluoro-6-(4-nitrophenyl)- and ethyl 8-fluoro-6-(3-nitrophenyl)-4 H-imidazo[1,5-a][1,4]benzodiazepine 3-carboxylate 6 and 7 were synthesized as central benzodiazepine receptor (CBR) ligands and tested for their ability to displace [(3)H]flumazenil from bovine and human cortical brain membranes. Both compounds showed high affinity for bovine and human CBR. In particular, compound 7 emerged as the most interesting compound, having a partial agonist profile in vitro while possessing useful activity in various animal models of anxiety. In accordance with its partial agonist profile, compound 7 was devoid of typical benzodiazepine side effects. The homology model of the GABA A receptor developed by Cromer et al. was used to assess the binding modes of ligands 6 and 7. From our docking results, the partial agonist activity elicited by compound 7 is likely to be due to the 3'-nitro substituent, which is in the appropriate position to interact with Thr193 of the gamma 2-subunit by means of a hydrogen bond.     

Specific inhibition of benzodiazepine receptor binding by some N-(indol-3-ylglyoxylyl)amino acid derivatives: stereoselective interactions

Several optically active N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. IC50 values were measured and revealed that the D form of the amino acid moiety of the compounds was more potent than both the L form and racemic form, suggesting a key role of the amino acid stereochemistry on the affinity to the benzodiazepine receptors. GABA ratio and proconvulsant/convulsant data reported for the most active compounds reveal they behave as inverse agonists at the benzodiazepine receptor.     

Synthesis and benzodiazepine receptor binding activity of 2, 9-disubstituted quinolino[2', 3'-5, 4](3-pyrazolino)[3, 2-b]purin-4-ones

2, 9-Disubstituted quinolino[2', 3'-5, 4](3-pyrazolino)pyrimidin-2-ones and purin-4-ones were synthesized and their benzodiazepine receptor activity was evaluated for their ability to displace [(3)H]R015-1788 from its specific binding in bovine brain membranes. Compound 5c caused 83 +/- 8 %inhibition in [(3)H]R015-1788 specific benzodiazepine receptor binding followed by compounds 5f, 5h, and 5i while other analogs were inactive at 10 microM concentration.     

Pharmacological properties and SAR of new 1,4-disubstituted piperazine derivatives with hypnotic-sedative activity

Preparation, pharmacological properties and structure-activity relationships of new pyrimidyl-piperazine derivatives, exhibiting sedative and hypnotic activity in mice, are reported. The hypnotic activity of the compounds was comparable with that of zopiclone (the known hypnotic-sedative agent), their interaction with ethanol, however, being much lower. The obtained results suggested that zopiclone and pyrimidylpiperazines 2, 4 and 5 exerted their pharmacological activity through a different mechanism - zopiclone through the interaction with benzodiazepine receptors and compounds 2, 4 and 5 through an unidentified molecular target. The pharmacological properties of compound 3 could be the result of a mixed mechanism of action, combining the properties of zopiclone and those of compounds 2, 4 and 5. A common feature of zopiclone and compounds 2 and 3 was that, after their systemic administration, independently of mechanism of action, together with the hypnotic effect a reduction of the 5-HT turnover in the mouse brain was observed. Minimum structural requirements for the hypnotic activity were formulated. Structural considerations have shown that removing the alpha-carbonyl group did not influence the drug's ability to inhibit the locomotor activity. However, it did influence its ability to disturb motor coordination or abolish the righting reflex within non-lethal doses.     

Azirino[1, 2-d][1, 4]benzodiazepine derivatives and related 1,4-benzodiazepines as anticonvulsant agents in DBA/2 mice

• 1.1. The behavioral and anticonvulsant effects of several 1,4-benzodiazepine (BDZ) and azirino[1,2-d] [1, 4]benzodiazepine (ABDZ) derivatives were studied after intraperitoneal administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures.
• 2.2. The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB, 12–16 kHz) in animals placed singly under a Perspex dome.
• 3.3. The 1,4-benzodiazepines were generally more potent than the related azirino[1,2-d][1,4]benzodiazepine derivatives which, however, showed a remarkable anticonvulsant activity. The rank order of potency for anticonvulsant activity was flunitrazepam > diazepam > pinazepam > ABDZ5 > ABDZ4 > prazepam > halazepam > ABDZ1 > ABDZ3 > camazepam > ABDZ6 > ABDZ2.
• 4.4. The impairment of locomotor performance following intraperitoneal (IP) administration of the aforementioned derivatives was also evaluated by means of rotarod test. The rank order of potency for impairment of coordinated motor movements was pinazepam > flunitrazepam > diazepam > ABDZ5 > prazepam > halazepam > ABDZ4 > ABDZ3 > ABDZ1 > camazepam > ABDZ2 = ABDZ6.
• 5.5. A hypothermic activity was observed after the highest doses of the benzodiazepines studied.
• 6.6. The potency of various 1,4-benzodiazepines and azirino[1,2-d][1,4]benzodiazepines as inhibitors of specific [³H]flumazenil binding to membranes from cerebellum or cortex was evaluated. In general, they inhibited [³H]flumazenil binding at the micromolar range. However, some ABDZ derivatives, although active as anticonvulsants, failed to displace [³H]flumazenil.
• 7.7. The azirino[1,2-d] [1,4]benzodiazepine derivatives are more lipophilic than the related benzodiazepines, but the different degree of anticonvulsant activity and impairment of coordinated motor movements cannot be directly related to the lipophilicity of the compounds studied.
• 8.8. The pharmacologic actions of ABDZ4 and ABDZ5, which appeared as the most potent anticonvulsants of the azirino[1,2-d] [1,4]benzodiazepine derivatives, were significantly reduced by treatment with flumazenil (8.24μmol/kg IP) suggesting a clear involvement of benzodiazepine mechanisms in the anticonvulsant activity of these compounds or their metabolites.
• 9.9. The anticonvulsant activity of ABDZ4 and ABDZ5 was also evaluated against seizures induced by the two β-carbolines, methylβ-carboline-3-carboxylate (β-CCM) and methyl6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), in DBA/2 mice. Both ABDZ4 and ABDZ5 gave better protection against the seizures induced by β-CCM than DMCM, suggesting a preferential action on BDZ1 receptors.

     

In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry

Typically, the dose‐occupancy curves for GABA‐A receptor ligands are determined using in vivo binding of [³H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA‐A ligands zolpidem, (RS)‐zopiclone, L‐838417 and flumazenil, to demonstrate affinity and saturation of the filter‐retained, membrane‐bound fraction. The in vivo binding of flumazenil and L‐838417 both saturated around 200 nm , at a similar level to the specific binding of (S)‐zopiclone after doses of the racemic zopiclone, using (R)‐zopiclone to estimate non‐specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA‐A receptors in vivo (200 nm ). Dose‐occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)‐isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright © 2009 John Wiley & Sons, Ltd.