巴罗沙星化学结构分析

目的:建立药物巴罗沙星的化学结构确证方法。方法:通过使用元素分析(EA)、热分析(TA)、X射线粉末衍射(XRD)、紫外吸收光谱(UV)、红外吸收光谱(IR)、核磁共振(NMR)以及质谱(MS)等分析手段对巴罗沙星进行了结构测定。结果:证实巴罗沙星的结构为1-环丙基-6-氟-1,4-二氢-8-甲氧基-4-氧代-7-(3-甲氨基-1-哌啶基)-3-喹啉羧酸二水合物。结论:方法测定结果准确全面,可为巴罗沙星的生产和鉴定提供较为全面的参考数据。     

Chemical synthesis and characterization of the epidermal growth factor-like module of human complement protease Clr

Clr is one of the two serine proteases of Cl, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease Cls. This interaction is mediated by the N-terminal region of Clr, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of Clr and evaluate its contribution to calcium binding, this module [Clr(123–175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional ¹H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent K_d of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native Clr. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site. © Munksgaard 1997.     

马来酸氨氯地平的结构分析

目的:建立药物马来酸氨氯地平的化学结构确证方法。方法:通过高效液相色谱(HPLC)测定纯度;热分析(TA)、红外光谱(IR)、核磁共振(NMR)、元素分析(EA)以及X射线粉末衍射(XRD)等分析手段测定结构。结果:证实马来酸氨氯地平的结构为3-乙基-5-甲基-2-(2-氨乙氧甲基)-4-(2-氯苯基)-1,4-二氢-6-甲基-3,5-吡啶二羧酸酯顺丁烯二酸盐。结论:本法测定结果准确,为马来酸氨氯地平的生产和鉴定提供了较为全面的参考数据。     

基于Markov模型对2种治疗高血压联合用药方案的药物经济学评价

目的 通过建立Markov模型,对血管紧张素受体拮抗剂（angiotensin receptor blockage,ARB）联合二氢吡啶类钙通道阻滞剂（calcium channel blockers,CCB）、ARB联合噻嗪类利尿剂2种治疗高血压方案进行药物经济学评价,为临床用药提供循证依据。方法 检索Medline及PubMed英文数据库,以“hypertension、calcium channel blockers、diuretics、angiotensin II receptor blocker、randomized controlled trial”及其对应的MeSH词为关键词进行检索。参考药品价格、治疗成本、健康效用值、状态转移概率建立Markov模型,对2种方案的有效性和经济学进行评价,并进行敏感性分析。结果 ARB+CCB组治疗高血压累计成本为32 780.34元,获得6.73质量调整生命年（quality-adjusted life years,QALYs）;ARB+利尿剂组治疗高血压累计成本为31 781.07元,获得6.81QALYs,相比于ARB+CCB组的增量成本效果比为-12 490.88元/QALYs。敏感性分析与原结果一致。结论 与ARB+CCB组相比,ARB+利尿剂组能获得更长的生存结果,其付出的增量成本–效果低于1倍人均GDP,更具有经济学效益。     

一种天然三萜皂苷的化学合成和抗肿瘤活性研究

目的 合成具有抗炎活性的天然三萜皂苷oleanolic acid 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranoside(1)。方法 以齐墩果酸为起始原料,采用逐步糖苷化策略,应用苄基、异丙亚甲基、乙酰氧基、过原酸酯等保护基方法,合成目标产物。以三萜皂苷β-hederin为阳性对照,采用MTT法测试目标物对肿瘤细胞的抑制活性。结果与结论 经13步反应,合成目标化合物,总收率23.7%,目标化合物结构经MS和1H-NMR确证。初步的体外药理实验结果表明,合成的皂苷产物仍然具有较强的肿瘤细胞毒活性。     

Chemical synthesis of insulin-like growth factor II

Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical.     

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus+

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (¹ H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD₅₀, 3.1μg/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).     

Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3

Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC₅₀ of 57 nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC₅₀ reported for the native toxin (63 nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals.     

丁酸氯维地平成品中杂质的合成与结构鉴定

目的研究丁酸氯维地平主要杂质的结构和产生的因为.方法以3-羟基丙腈为起始原料,经过酯化、缩合、水解、取代反应制备目标化合物.用液-质联用法推导该杂质结构.结果按照设计路线,合成出丁酸氯维地平主要杂质.结论经1H-NMR确证目标化合物结构为4-(2,3-二氯苯基)-1-4-二氢-2-6-二甲基-3,5-吡啶二羧酸双(1-...     

Synthesis and receptor binding characteristics of [d-Ala2, cysteamine5]enkephalin,a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog, [d -Ala², cysteamine⁵]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.     

ATP敏感性钾通道分子结构和功能的组织特异性研究进展

ＡＴＰ敏感性钾通道（ＫＡＴＰ）通过与细胞代谢和生物电活动相偶联,在许多组织发挥着重要的生理和病理生理学功能。ＫＡＴＰ由内向整流钾通道（Ｋｉｒ）和ＡＴＰ结合蛋白两部分组成,前者形成离子通道,后者决定着ＫＡＴＰ的功能。Ｋｉｒ和ＡＴＰ结合蛋白又分多种亚型,两者不同亚型间的相互偶联导致了不同组织ＫＡＴＰ分子结构的多样性,分子结构的不同又决定了不同组织ＫＡＴＰ特性和功能的复杂性。本文对不同组织ＫＡＴＰ的分子结构、生理学功能、病理生理学和药理学特性进行了综述。     

Single-pill combination of amlodipine and valsartan in the management of hypertension

Combination therapy is increasingly recommended for selected patients with hypertension to facilitate prompt attainment and maintenance of goal blood pressure (BP). Single-pill combination therapy simplifies treatment and optimizes long-term compliance. Amlodipine, a dihydropyridine calcium antagonist, and valsartan, an angiotensin receptor blocker, are well-established antihypertensive agents with complementary mechanisms of action. This combination lowers BP significantly more than either of its components, and valsartan reduces the incidence of dose-related amlodipine-induced edema. Rigorous clinical trial data have proven the BP-lowering efficacy and high tolerability of the amlodipine/valsartan combination in patients with moderate to severe hypertension as well as other difficult-to-treat populations. Amlodipine/valsartan is indicated as initial therapy in patients who are unlikely to be controlled with a single drug and as second-line therapy in patients not responding adequately to monotherapy.     

Neuropeptide Y: identification of the binding site

Based on the hypothetical 3D structure of neuropeptide Y (NPY), NPY 1-4-Aca-25-36, a 17 amino acid analogue, has been synthesized replacing the sequence NPY 5-24 by ε-aminocaproic acid (Aca). This low-molecular weight deletion analogue showed nearly comparable receptor affinity to NPY. In order to elucidate the structural requirements for receptor recognition each amino acid of 1-4-Aca-25-36 was exchanged by its D-enantiomer, glycine and L-alanine. In addition distinct amino acids were replaced by closely related residues. Multiple peptide synthesis was applied using Fmoc-strategy and BOP activation. Binding assay was performed on rabbit kidney membrane preparations. The results of structure affinity studies suggest that the C-terminal tetrapeptide NPY 33-36 is essential for receptor recognition.     

Comparison between the antiproteinuric effects of the calcium channel blockers benidipine and cilnidipine in combination with angiotensin receptor blockers in hypertensive patients with chronic kidney disease

Aims: Benidipine, an L-/T-type calcium channel blocker, dilates renal efferent and afferent arterioles and reduces glomerular pressure; therefore, it may exert renoprotective effects. We conducted an open-labeled randomized trial to compare the effects of benidipine with cilnidipine in hypertensive patients with chronic kidney disease (CKD).

Methods: The patients who were already being treated with angiotensin receptor blockers (ARBs) received one of the following treatment regimens: benidipine at a dose of 2 mg/day that was increased up to a dose of 8 mg/day (benidipine group; n = 118) or cilnidipine at a dose of 5 mg/day that was increased up to a dose of 20 mg/day (cilnidipine group; n = 115).

Results: After 12 months of treatment, we observed a significant and comparable reduction in the systolic and diastolic blood pressure in both groups. The urinary protein:creatinine ratio was significantly decreased in both groups after 3 months of treatment and thereafter; however, the difference between both groups was not significant after 12 months of treatment. Benidipine exerted an antiproteinuric effect to a greater extent than cilnidipine in patients with diabetes.

Conclusion: The addition of benidipine as well as cilnidipine reduces urinary protein excretion in hypertensive patients with CKD who are already being administered ARBs.     

扩张型心肌病大鼠心脏左室内皮素受体及其亚型分析

目的 观察扩张型心肌病大鼠左室ETR及其亚型的变化.方法用125I-ET-1建立内皮素受体及其亚型的放射配基分析法,进行饱和结合试验,对扩张型心肌病大鼠左室心肌ETR及其亚型的含量进行分析.结果(1) 放射配基分析法检测ETR的基本实验条件为37 ℃孵育 60 min,膜蛋白投放量以20～40 μg 为佳; (2) ET-1及非选择性拮抗剂bosentan、选择性拮抗剂BQ123、BQ788等均能竞争抑制125I-ET-1与内皮素受体的结合,而去甲肾上腺素则不能抑制; (3) 正常大鼠左室内皮素受体数量为 (92.21±34.34) nmol·     

Solution structure of the B-chain of insulin as determined by 1H NMR spectroscopy Comparison with the crystal structure of the insulin hexamer and with the solution structure of the insulin monomer

The solution structure of the isolated B-chain of bovine insulin has been determined by ¹H NMR spectroscopy combined with simulated annealing calculations. Complete sequence-specific assignments for the proton resonances are reported together with a set of 309 NOES used in the structure calculations. Chemical-shift variations from random coil values provide support for the existence of helical regions in the polypeptide chain, as do a characteristic series of d_αp(i,i+ 3) NOES from residues B8 to B17. While there is some evidence for a limited degree of conformational averaging over the helical region, in general the helix is relatively well defined and corresponds closely to the helical region seen in the X-ray crystal structure of the insulin hexamer. Other similarities with the crystal structure include turn-like conformations at the carboxy terminal end of the helix and extended strands at both the amino and carboxy termini of the peptide. These similarities between the crystal structure and the isolated B-chain suggest that this peptide has intrinsic folding properties, which allow it to adopt its characteristic structure in intact insulin without the need for extensive cooperative interactions with the A-chain. Despite these general similarities, an important difference between the isolated B-chain and the intact protein occurs in the carboxy terminal region. This region appears significantly more mobile in the isolated B-chain. As a conformational change involving the carboxy terminus has been implicated in receptor binding, the current study of the isolated B-chain provides valuable information on the extent of this region's intrinsic mobility. © Munksgaard 1995.     

4-苯甲酰胺基苯磺酰胺类化合物的合成及其抗炎活性

目的 设计并合成4-苯甲酰胺基苯磺酰胺类化合物，研究其抗炎活性和构效关系。方法 以取代苯甲酸为原料合成目标化合物，并以二甲苯致小鼠耳肿胀模型测试目标化合物的抗炎活性。结果 共合成7个化合物(其中6个化合物未见CA报道)，经红外光谱、核磁共振氢谱和质谱确证其结构，部分化合物对二甲苯致耳肿胀具有较强的抑制作用，并初步总结了构效关系。结论 4-苯甲酰胺基苯磺酰胺类化合物具有较强的抗炎活性。     

Inhibition of [3H]-U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 ((5a,7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benzeneacetamide), a specific κ-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine.
2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ-receptor stimulation by a specific κ-receptor agonist, U50,488H (trans-(±-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective κ-receptor antagonist, nor-BNI (nor-binaltorphimine,17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-binorphinan-3,4′,14, 14′-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED₅₀ values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca²⁺ influx in the cardiac muscle.
3. Measurement of [Ca²⁺]_i in the absence of free extracellular Ca²⁺ by a spectrofluorometric method, with fura-2 as Ca²⁺ indicator, showed that U50,488H 5×10⁻⁵ M slowly increased [Ca²⁺]_i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 μM), or ryanodine (5 μM). Verapamil 1 and 10 μM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca²⁺ transient.
4. The observations suggest that CCBs may inhibit the actions of κ-receptor stimulation at the level of the κ-receptor.

     

Chemical synthesis and purification of proteins: a methodology

Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity. The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence. We have developed a highly effective protocol for stepwise SPPS and‘one-step’purification of small proteins. To demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpnl0) using three different chemical protocols. Highly homogeneous Rat Cpnl0 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxy-carbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6%. Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively. The spontaneous folding of Rat Cpnl0 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained. The degree of homogeneity was also reflected in the ability of purified Rat Cpnl0 to facilitate the refolding of yeast enolase. © Munksgaard 1996.