盐酸苯达莫司汀的合成工艺研究

目的 盐酸苯达莫司汀的合成.方法 以5-硝基-1-甲基-2-苯并咪唑丁酸乙酯(BD-a)为起始原料,经还原、取代、氯代、水解成盐制备得目标产物盐酸苯达莫司汀,共三步反应.结果 该工艺对文献报道的合成方法中提供的反应条件及终产物的纯化方法等各方面工艺参数作了相当程度的优化更新,终产物粗品的总收率61.5％.结论 本研究获得了一种清洁、操作简单、适于工业化,盐酸苯达莫司汀收率较高和纯度高的合成工艺.     

盐酸苯达莫司汀的电喷雾串联质谱分析

目的:应用电喷雾串联质谱(ESI-MSn)技术对盐酸苯达莫司汀进行质谱裂解分析,通过主要特征碎片离子研究其裂解规律。方法:样品溶液进样后,采用ESI-MS1～4碰撞裂解方式,解析盐酸苯达莫司汀的特征碎片离子。结果:盐酸苯达莫司汀在正离子模式下,采用ESI-MS1获得了分子离子峰[M+H]+m/z 358;采用ESI-MS2获得了离子碎片m/z340;采用ESI-MS3获得m/z304、m/z276等离子碎片;采用ESI-MS4得到的主要离子碎片分别为m/z240、m/z268、m/z276等。负离子模式下采用ESI-MS1获得了[M-H]-m/z356,而采用ESI-MS2～4碰撞裂解,基本无响应。结论:盐酸苯达莫司汀在正离子模式下,主要通过脱去羧基中的H2O、CO碎片和5位N上的HCl、CH3Cl以及CH2=CHCl碎片的方式进行裂解。负离子模式下,只有一级电离产生的分子离子峰[M-H]-m/z356有一定响应。说明盐酸苯达莫司汀在负离子模式下比较稳定,而在正离子模式下容易裂解产生一系列碎片。本研究为盐酸苯达莫司汀的结构修饰及其代谢产物结构判断提供参考依据。     

HPLC法测定盐酸苯达莫司汀含量及有关物质

目的:建立盐酸苯达莫司汀含量及有关物质的分析方法.方法:采用色谱柱:C18(4.6 mm×250 mm,5 μm);流动相:十二烷基硫酸钠溶液(取十二烷基硫酸钠0.5 g置1000 mL水中使溶解,用磷酸调节pH值至4.0)-乙腈 (105∶95);检测波长:234 nm.结果:在选定的色谱条件下,有关物质与主药分离效果好,盐酸苯达莫司汀在84.91 μg～424.6 μg·mL-1范围内线性关系良好(r=0.9999).结论:方法可用于盐酸苯达莫司汀含量及有关物质的测定.     

盐酸苯达莫司汀（bendamustine hydrochloride）

盐酸苯达莫司汀（bendamustine hydrochloride）由Ozegowski及其同事于1963年在德国耶拿的微生物试验协会研制成功。1971~1992年由Jenapharm公司以Cytostasan的商品名生产。1993年后,由Ribosepharm公司以Ribomustine的商品名在德国上市销售用于治疗乳腺癌、慢性淋巴细胞性白血病。2008年3月20日,由Cephalon公司开发的盐酸苯达莫司汀经美国FDA批准用于治疗慢性淋巴细胞白血病(CLL),商品名为Treanda;同年10月31日该药又获准用于惰性B细胞非霍奇金淋巴瘤(NHL)的治疗。 盐酸苯达莫司汀的中文化学名称：4-[5-[双(2-氯乙基)氨基]-1-甲基苯并咪唑-2-基]丁酸盐酸盐;英文化学名称：4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)buta- noic acid hydrochloride;分子式为C16H22Cl3N3O2;相对分子质量为394.73;CAS登记号为3543-75-7     

高效液相色谱荧光检测法测定人血浆中苯达莫司汀及丫-羟基化苯达莫司汀浓度

目的建立测定人血浆中苯达莫司汀及Y一羟基化苯达莫司汀浓度的高效液相色谱荧光检测法。方法色谱柱为AgilentC18柱（150mm×4．6mm,5μm）;流动相为0．01m01．L-1。磷酸二氢钾水溶液一乙腈（68：32,v／v）;荧光检测器激发波长328nm,发射波长420nm。结果高、中、低3种浓度的苯达莫司汀（16000、5000、500gg．L。）和Y一羟基化苯达莫司汀（800、250、25Pg．L-1）平均相对回收率分别为96．5％、100．3％、103．3％和98．7％、100．1％、101．3％;绝对回收。率分别为54．1％、54．1％、55．6％和62．3％、643％、68．5％;日内、日间差RSD均低于5％（n=5）;最低定量限分别为200μg．L-1和10／ag．L-1;线性范围分别为200-20000μg.L-1“和10-1000μg．L-1结论该方法灵敏、准确、简单、快速,可用于临床血药浓度监测和药动学研究中同时测定苯达莫司汀及y-羟基化苯达莫司汀浓度。     

盐酸苯达莫司汀合成路线图解

盐酸苯达莫司汀是由Merckle公司开发的烷化抗肿瘤药物,目前正处于Ⅲ期临床。现对其合成路线进行解析。     

盐酸苯达莫司汀的合成工艺改进

目的:改进盐酸苯达莫司汀的合成方法。方法:以5-硝基-1-甲基-2-苯并咪唑丁酸乙酯为原料,经还原、取代、氯化、成盐等反应合成目标化合物。结果:合成的目标化合物,其结构经核磁共振氢谱、核磁共振碳谱、质谱及元素分析确证。结论:本方法简化了操作步骤,优化了反应条件,降低了成本,提高了收率。     

石墨炉原子吸收分光光度法测定盐酸苯达莫司汀中钯的残留量

摘 要 目的：建立石墨炉原子吸收分光光度法测定盐酸苯达莫司汀中钯的残留量。方法: 采用石墨炉原子吸收光谱法,供试品经加热破坏处理后测定,测定波长为247.6 nm。结果: 钯质量浓度在20～60 ng·mL^-1的范围内与吸光度线性关系良好(r=0.998 4),平均回收率为102.9%（RSD=1.7%,n=9）。结论:该方法简便、灵敏,可用于盐酸苯达莫司汀中钯的残留量检测。     

Cephalon在美申请Treanda治疗NHL

Cephalon公司已向美国FDA申请其抗肿瘤药Treanda（bendamustine HCl，盐酸苯达莫司汀，从Astellas公司转让）（Ⅰ）的第二适应症——非霍奇金淋巴瘤（NHL）。     

盐酸苯达莫司汀的合成

目的:合成盐酸苯达莫司汀。方法:以2,4-二硝基氯苯为起始原料,经取代、还原、酰化、环合、还原、取代、水解、成盐等9步反应制得目标化合物。结果:目标化合物的质谱、核磁共振氢谱、红外光谱均与文献报道相吻合,总收率为33.5%。结论:本路线操作简单,成本较低,适合工业化生产。     

Renal actions of bendamustin (Cytostasan) in rats

In adult rats influences of single doses of bendamustin (0.5, 1 or 5 mg/100 g b.wt. i.p.) on kidney function were measured (time of experimentation 8 d following administration). Bendamustin administration is followed by an increase of kidney weight caused by a higher water content of kidney tissue. An increase of plasma concentrations of creatinine and of urea following bendamustin (1 or 5 mg/100 g b.wt. i.p.) indicates a reduced excretion capacity of kidney. Bendamustin administration is not connected with a distinct proteinuria. The highest administered does (5 mg bendamustin/100 g b.wt. i.p.) caused an oliguric effect connected with a distinctly reduced renal excretion of osmotically active substances. There is a distinct diminution of renal excretion of sodium following bendamustin administration and a decrease of renal excretion of PAH can be stated. It is most likely that bendamustin can reduce the glomerular function: Following administration of 1 mg bendamustin/100 g b. wt. i.p. CIn is reduced whereas a statistically significant increase of tubular transport capacity for organic anions can be measured (TmPAH).     

独一味总环烯醚萜苷分离纯化工艺的优化

目的 优化独一味总环烯醚萜苷的制备工艺,获取纯度相对较高的总环烯醚萜苷。方法 以甲醇-水为流动相,梯度洗脱方式建立分离分析独一味总环烯醚萜苷的高效液相色谱方法,实现各组分的最佳分离,统计、记录其三维吸收图谱,并结合紫外全波长扫描,对前期制备的独一味总环烯醚萜苷中影响纯度的杂质特征进行分析,根据杂质特性优化大孔树脂结合聚酰胺柱制备过程,紫外实时监测,剔除杂质,产品溶液经冷冻干燥获得冻干粉,并以HPLC对比前后制备工艺产品成分差异。结果 工艺优化前产品中环烯醚萜苷含量为69.06%,优化后含量为90.60%,含量提升21.54%;本次制备总产率为16.70%（以水提取物的质量为参照）,总环烯醚萜苷转移率为74.72%（相对于水提取物中总环烯醚萜苷的质量）。结论 优化过程简单,操作步骤可控,易于实现,纯化效果显著。     

牛胰蛋白酶制备工艺的改进及其对基因工程人胰岛素前体的酶切作用

目的对牛胰蛋白酶制备工艺进行改进,制备高纯度牛胰蛋白酶,并对其酶切基因工程人胰岛素前体的作用进行考察。方法采用粗制后先色谱分离再活化的方法对传统工艺进行改进,对传统工艺制备样品和改进工艺制备样品中的胰蛋白酶和胰凝乳蛋白酶的比活力进行测定和比较;进一步采用这2种样品对基因工程人胰岛素前体进行酶切,对酶切作用进行比较。结果传统工艺制备样品中胰蛋白酶比活力为212.5 U·mg-1,胰凝乳蛋白酶比活力为20.4 U·mg-1;而改进工艺样品中胰蛋白酶比活力为240.4 U·mg-1,胰凝乳蛋白酶比活力为0.14 U·mg-1。传统工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为16.3%,改进工艺制备样品酶切基因工程人胰岛素前体目标产物纯度质量分数为32.2%。结论粗制后先进行SP色谱可实现胰蛋白酶原和胰凝乳蛋白酶原较好的分离,该方法适合于高纯度胰蛋白酶的制备,制备的胰蛋白酶适合作为工具酶进行基因工程人胰岛素前体的酶切。     

猪小肠黏膜脱氧核糖核酸的提取及质量研究

目的以肠衣生产的副产物肠黏膜为原料,进行大分子DNA的提取,并进行质量研究。方法用酶解-超滤法提取DNA,工艺过程中监控主要组分的分离及终产品质量。结果酶解法释放基因组DNA、肝素,蛋白质被水解为寡肽及氨基酸,DNA被超滤截留,钙盐沉淀法去除大分子肝素。终产品在含量及纯度上达到国外同类产品水平。结论从肠黏膜提取DNA切实可行,对猪小肠综合利用有着重要的现实意义。     

Hepatobiliary elimination of bendamustin (Cytostasan) in rats.

Biliary excretion of bendamustin (Cytostasan, 5-[bis(2-chloroethyl)amino]-i-methylbenzimidazole-2-butyric acid; 1) and its metabolites was studied in rats after i.v. administration of 14C-1. The most significant finding was the rapid excretion of 1 related radioactivity in the bile occurring shortly after injection. While radioactivity eliminated by bile within 2 h was 41.8%, in the course of subsequent 22 h it amounted only to 3.2%. Bile samples analyzed by TLC indicated that the total amount of radioactivity was excreted in the form of conjugates and two hydroxy metabolites. A significant amount of radioactivity was excreted in urine. The diversion of bile by cannulation of the bile duct led to a significant decrease of elimination by feces.     

Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye

In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.     

Selective synthesis of alpha monoglycerides by a clean method: Techno-economic and environmental assessment

This work proposes an alternative green and selective biocatalytic route for Glycerin Monostearate (α-monostearin) production. The conventional method of production uses an elevated temperature. Apart from the high energy consumption, such high temperatures darken the final product's color, lead to random reactions, and produce high orders of diglycerides and triglycerides instead of monoglycerides. The proposed production process was performed by esterifying stearic acid with glycerin in an organic medium using Candida antarctica lipase (Novozym 435) at a mild temperature. The reaction conditions were optimized using the response surface methodology (RSM): optimum conditions were a temperature of 60 °C, glycerin to stearic acid molar ratio of 8:1, and Novozym 435 amount of 6% w/w. The solvent addition remarkably improved the α-monostearin yield to nearly 80% without the need for the energy-intensive distillation step. The conventional autocatalytic esterification (AUT) process was also performed to investigate the comparative monoglyceride yield, and it was found to be 22.5%. Proton nuclear magnetic resonance and gas-chromatography confirmed that α-monostearin could be produced with the highest purity using the proposed enzymatic method (ENZ). Economic and environmental analyses were also conducted for the proposed ENZ process, and the results were compared with those of the AUT process. The total capital investment of α-monostearin production, considering a projected capacity of 4950 t year^?1 and 11% interest for the proposed ENZ process, was favorably 2.5 times lower than that of the AUT process, suggesting a promising investment opportunity. However, the total production costs showed unfavorable negative net present value (NPV) and return on investment (ROI) for the ENZ process and favorable positive NPV and ROI for the AUT process, indicating that the proposed venture is not profitable for α-monostearin production. However, the process can be profitable at improved operational stability of Novozym 435 up to 1 kg per 3-ton product. The carbon footprint was calculated on the basis of the given capacity and conditions of 50 and 656 t CO₂ eq./year for the ENZ and AUT processes, respectively. The synthesis of α-monostearin using the proposed route can be considered a building block toward a cleaner large-scale production of α-monoglycerides.     

4-羟基-2-(1H-吡唑-1-基)嘧啶-5-甲酸的合成工艺改进研究

目的对4-羟基-2-(1H-吡唑-1-基)嘧啶-5-甲酸的合成工艺条件进行优化。方法以1-脒基吡唑盐酸盐和乙氧基甲叉基丙二酸二乙酯为起始原料,在三乙胺催化下环合得到中间体4-羟基-2-(1H-吡唑-1-基)嘧啶-5-甲酸乙酯,再经过氢氧化锂在四氢呋喃–水混合溶剂中水解得到目标化合物。结果合成了目标化合物,经MS、1H-NMR确证了结构,质量分数为99.5%,本合成工艺的总收率为87%。结论该合成工艺具有操作简单、反应条件温和、成本低、产率和纯度较高等优点,适合工业化生产。     

高速逆流色谱法分离纯化长春花中长春新碱

目的采用高速逆流色谱(HSCCC)分离纯化长春花中长春新碱,建立相关工艺条件,为工业生产提供参考数据。方法采用高速逆流色谱仪,氯仿-甲醇-0.3mol.L-1 HCl(4∶3∶2)溶剂体系,上相为固定相,下相为流动相,流动相流速为2.0mL.min-1,紫外检测波长为280nm,主机转速为800r.min-1,恒温循环温度为28℃。结果 HPLC检测HSCCC纯化后长春新碱质量分数为79.7%。结论高速逆流色谱纯化长春花中长春新碱容量大、分离纯度和效率高,避免了有机溶剂的大量使用,适用于工业生产高纯度产品。     

Biotransformation of mogrosides from Siraitia grosvenorii by Ganoderma lucidum mycelium and the purification of mogroside III E by macroporous resins

Mogrosides are the major triterpenoidal saponins found in swingle, the fruit of Siraitia grosvenorii, which have recently been widely used throughout the world as natural food sweeteners. Among this class of compounds, mogroside III E (MG III E) exhibits the most intense sweetness, and it was also found to effectively regulate blood glucose levels. However, the relative abundance of naturally occurring MG III E is low compared to other mogrosides. Therefore, the purpose of this study was to enrich MG III E through biotransformation of fruit extracts and to develop a reliable method for its purification. We used HPLC coupled with mass spectrometry and nuclear magnetic resonance spectroscopy for metabolite analysis and identified MG III E as a major metabolite of Ganoderma lucidum mycelium. This organism converts the most abundant mogroside, mogroside V, to MG III E via a deglycosylation reaction; high levels of β-glucosidase activities were also detected. In addition, we established an efficient purification method for MG III E using HP-20 macroporous resin. Optimization of the method was accomplished by kinetic model fitting, dynamic adsorption studies, and desorption experiments. The purity of MG III E was increased from 11.71% to 54.19%, with a 70%–76% recovery rate, and the scaled-up purification process allowed us to harvest 17.38 g of MG III E with 55.14% purity and a 74.71% of recovery rate. Therefore, our low cost, time-saving, easy to scale-up procedure for isolating MG III E could be applicable in industrial processes.