苄基膦酸二乙酯乙酰胺类熊果酸衍生物的合成及其体外抗肝癌活性研究

目的 考察新型熊果酸衍生物的体外抗肝癌活性及其作用机制。方法 依据"Topliss决策法"将熊果酸与不同取代的苄基膦酸二乙酯片段通过取代反应得到目标化合物,并采用MTT法考察其体外抗肝癌活性;通过分子对接试验分析化合物可能的作用靶点并通过Western blotting试验加以验证。结果 目标化合物4a~4e的化学结构经过¹H-NMR﹑ ¹³C-NMR以及HRMS的联合确证;化合物4b及4e对BEL-7402及HepG2 2种肝癌细胞株的抑制活性优于熊果酸及阳性对照药5-氟尿嘧啶,同时对正常肝细胞L02的毒性显著降低;化合物4e呈浓度依赖性下调p-AKT蛋白的表达。结论 化合物4e的体外抗肝癌活性最为理想(针对HepG2的IC₅₀值为2.69 μmol·L^-1),可作为AKT蛋白抑制剂进行深入研究。     

二茂铁杂环类化合物的合成及抗三阴性乳腺癌活性研究

目的 设计、合成杂环二茂铁衍生物,并研究其抗三阴性乳腺癌活性。方法 以二茂铁查耳酮为先导化合物,对其进行结构改造,合成了一系列含有杂环的二茂铁衍生物,并通过CCK8试剂盒测试化合物抗乳腺癌活性。结果 合成了28个二茂铁衍生物,其结构均通过¹H-NMR和MS加以确证。初步的生物活性测试结果表明,所合成的二茂铁衍生物对三阴性乳腺癌MDA-MB-231细胞有较强的选择性和抑制活性,其中咪唑杂环化合物抗肿瘤活性强于相应的吡唑类和嘧啶化合物。尤其是28a[IC₅₀=（1.6±0.23）μmol·L^-1]对MDA-MB-231的抑制活性分别是先导化合物3[IC₅₀=（10.7±1.41）μmol·L^-1]和他莫昔芬[IC₅₀=（13.7±1.17）μmol·L^-1]的6和10倍,同时这些二茂铁衍生物对正常乳腺上皮细胞MCF-10A均没有毒性。结论 本研究为开发具有抗三阴性乳腺癌活性的化合物提供了信息和依据。     

1位芳基取代的青藤碱衍生物的合成和抗炎活性研究

目的 进行1位取代芳基的青藤碱衍生物的合成及体外抗炎活性研究。方法 以青藤碱为先导化合物,首先1位溴取代反应,然后通过Suzuki偶联反应对A环1位进行修饰,制得一系列取代芳基的青藤碱衍生物,所有化合物均经¹H-NMR、¹³C-NMR、MS结构确认;采用报告基因法研究青藤碱衍生物对NF-κB转录活性的影响。结果 Suzuki偶联反应得到的一系列化合物活性均优于对照药青藤碱。结论 筛选到的4f和4g可作为候选药物进行深入研究,对研发治疗风湿性关节炎药物具有重要意义。     

氨基噻唑(噁唑)类化合物的合成及抗K562细胞活性研究

目的 设计、合成一系氨基噻唑（噁唑）类似物,并测试新衍生物对人类慢性粒细胞白血病细胞系K562的体外抑制作用。方法 以dasatinib为先导化合物设计新衍生物,通过亲核取代和关环缩合得到目标化合物,其结构通过¹H-NMR、¹³C-NMR、IR和MS测定。结果 发现5个目标化合物抑制活性高于对照药伊马替尼。4个化合物抗K562细胞活性高于dasatinib。结论 保持药效基团不变,用五元噻唑环取代芳香苯环,活性能得以改善。     

新型含喹啉和噻吩结构的抗真菌化合物的设计与合成

目的 基于喹啉类和噻吩类抗真菌化合物的构效关系,设计合成含喹啉和噻吩结构片段的新型抗真菌化合物,并测试其对白念珠菌的抑制活性。方法 以5-氰基噻吩-2-甲醛或者5-氰基噻吩-3-甲醛为起始原料,经还原氨化、氰基还原、与喹啉甲酸或异喹啉甲酸的酰胺化等反应,合成13个目标化合物,并通过¹H NMR和MS确证其化学结构,以微量液基稀释法测定这些化合物体外抗白念珠菌SC5314活性。结果 目标化合物均具有一定的抗真菌活性,其中化合物6k显示很好的抗真菌活性,MIC₈₀值为0.5 μg/ml,与对照药氟康唑相当。结论 所设计合成的含喹啉和噻吩结构片段的新型化合物具有较好的体外抗真菌活性,值得深入研究。     

新型抗EV71病毒3C蛋白酶抑制药的设计、合成及其活性

摘 要 目的： 寻找抗EV71病毒新化合物。方法: 采用分子模拟技术,以EV71病毒3C蛋白酶活性口袋为靶,设计二肽、三肽,四肽,五肽和六肽等多个系列寡肽化合物,合成对接评分较高的寡肽化合物并进行抗EV71病毒活性筛选。结果: 五肽对接评分高于其他系列寡肽,显示五肽化合物与EV71 病毒3C蛋白酶有更强的键合力。研究中合成了两个评分较高的五肽化合物25和化合物16,体外抗EV71病毒活性实验显示化合物25活性较强（EC₅₀=1.36 μmol·L^-1,CC₅₀=211.94 μmol·L^-1, SI=155.84）,而化合物16则无明显活性。结论:五肽化合物25可作为抗EV71病毒新骨架开展进一步研究。     

苯氧基磷酰氮芥取代的槐定碱衍生物的合成及其抗肿瘤活性研究

目的 设计合成苯氧基磷酰氮芥取代的槐定碱衍生物,并对其进行体外抗肿瘤活性研究。方法 以槐定碱为起始原料,经开环、酯化、磷酰化等反应合成目标化合物2a ～2e ,采用MTT法测定其对S180、H22、K562、MCF-7、SMMC-7721、LoVo肿瘤细胞的抑制活性。结果 设计并合成了5个目标化合物,其结构经¹H-NMR和ESI-MS确证。体外细胞测试结果显示所有化合物对S180和H22细胞株活性较强,且对正常细胞L929毒性小,部分化合物活性高于阳性对照药多柔比星。结论 目标化合物具有较强的抗肿瘤活性,化合物2b 为有潜力的候选化合物。     

苯二醛缩氨基硫脲的合成及其酪氨酸酶抑制活性研究

目的 合成苯二醛单缩和二缩氨基硫脲类化合物,并初步研究其抑制酪氨酸酶的活性和作用机制。方法 以5种苯二醛和氨基硫脲为原料,通过缩合反应合成9个目标化合物;采用蘑菇酪氨酸酶多巴速率氧化法和酶抑制动力学实验,测定目标化合物对酪氨酸酶的抑制活性和作用机制;选择化合物3a和4a进行抑制机制和抑制动力学研究。结果 目标化合物的结构经¹H-NMR、¹³C-NMR及MS确证;所有化合物抑制酪氨酸酶的活性均优于对照药物曲酸;苯二醛二缩氨基硫脲3a~3d的活性明显强于相应的单缩氨基硫脲4a~4d;化合物3a和4a对酪氨酸酶的抑制作用均表现为混合型可逆抑制作用。结论 苯二醛二缩氨基硫脲类化合物具有优异的抑制酪氨酸酶的活性,值得进一步深入研究。     

酮洛芬丹皮酚酯及其类似物的合成及抗炎活性研究

目的 合成5种酮洛芬酯类化合物,并研究其抗炎活性和胃肠道不良反应。方法 以丹皮酚及结构类似物和酮洛芬为原料,通过碳二亚胺法合成5种化合物;采用二甲苯致小鼠耳廓肿胀模型和角叉菜胶致大鼠足趾肿胀试验对化合物抗炎活性做了初步的药理学筛选;同时考察化合物对胃肠道不良反应的影响。结果 化合物的结构经¹H-NMR及MS得到确证;5个目标化合物均能显著地减轻小鼠的耳肿胀和大鼠的足趾肿胀程度,与其相应的阳性和阴性对照组相比,都具有良好的抗炎效果;同时,5个目标化合物对胃肠损伤面积明显小于阳性组和阴性组,能够明显地减轻胃肠道的不良反应。结论 合成了5种酮洛芬酯类化合物;抗炎活性均明显强于酮洛芬;且对胃肠道的不良反应明显地减轻。     

新型三唑类化合物的设计、合成和抗真菌活性研究

目的 基于唑类药物合理优化的分子设计模型,设计新型三唑类化合物,并测试其对常见致病真菌的抑制活性。方法 采用环氧化物开环法合成目标化合物,通过¹H NMR和MS确证其化学结构,经微量液基稀释法测试体外抗真菌活性。结果 合成了2个含三唑酮侧链的新型唑类化合物,它们均显示了优秀的广谱抗真菌活性。结论 目标化合物对白色念珠菌的活性优于对照药氟康唑和酮康唑,值得进一步深入构效关系研究。     

Anti-proliferative and antibacterial in vitro evaluation of the polyurethane nanostructures incorporating pentacyclic triterpenes

Context: Oleanolic and ursolic acids are antitumor and antibacterial agents which are extensively studied. Their major disadvantage is the poor water solubility which limits their applications.

Objectives: Oleanolic and ursolic acid were encapsulated into polyurethane nanostructures that act as drug carriers. In order to evaluate the effectiveness of the particles, anti-microbial and anti-proliferative activity compared to un-encapsulated active compounds was tested.

Materials and methods: Using an interfacial polycondensation technique, combined with spontaneous emulsification, structures with nanoscale dimensions were obtained. Scanning electron microscopy, differential scanning calorimetry and X-ray assays confirmed the encapsulation process. Concentrations of 10 and 30?μM particles and un-encapsulated compounds were tested by MTT viability assay for several breast cancer lines, with an exposure time of 72?h. For the antibacterial studies, the dilution method with MIC determination was used.

Results: Ursolic acid had an excellent inhibitory effect with IC₅₀ value of 2.47, 1.20, 1.26 and 1.34?μM on MCF7, T47D, MDA-MB-231 and MDA-MB-361, respectively. Oleanolic acid did not show anti-proliferative activity. The pure compounds showed their antibacterial activity only against Bacillus species and Candida albicans, but MIC values were too high to be considered efficient antimicrobial agents (2280 and 4570?μg mL?^??¹, respectively). Polyurethane nanoparticles which incorporated the agents did not show any biological activity.

Discussion and conclusion: Although the active compounds did not fully exert their anti-proliferative activity following encapsulation inside polymeric nanoparticles, in vivo evaluation is needed in order to obtain an exhaustive conclusion, as the active compounds could be released as a result of metabolic activity.     

Ionized Prodrugs of Dehydroepiandrosterone for Transdermal lontophoretic Delivery

Purpose. The aim of this work was to synthesize ionized dehydroepiandrosterone (DHEA) prodrugs with higher water solubility, useful for iontophoretic transdermal application. Methods. The synthesized derivatives were characterized and tested for sensitivity to chemical and enzymatic hydrolysis. Solid state and solution stability was also determined. Transdermal iontophoretic anodal transport in vitro was studied using excised rabbit skin. Results. Two DHEA ionized prodrugs were synthesized: PRO1, a primary amine derivative, and PRO2, a quaternary ammonium salt. The two derivatives possess higher water solubility and lower octanol/saline partition coefficients than DHEA. Prodrugs were sensitive to enzymatic hydrolysis; in particular the primary amine was hydrolyzed faster than the quaternary salt by esterase from porcine liver in vitro. Transdermal flux of the two prodrugs was slightly higher than the parent drug. In the case of passive diffusion, only DHEA was found in the receptor compartment, indicating the complete breakdown of the prodrug in the skin. Current application gave higher drug flux and a significant amount of prodrug was found in the receptor. Conclusions. The use of ionized prodrugs of DHEA can increase the flux attainable during transdermal anodal iontophoresis by up to 7 times, but they are useful for passive transport as well.     

Chemical Stability of Esters of Nicotinic Acid Intended for Pulmonary Administration by Liquid Ventilation

Purpose. It has been suggested that fluorocarbon liquid may be a unique vehicle for the delivery of drugs directly to the acutely injured lung. A prodrug approach was used as a means of enhancing the solubility of a model drug (nicotinic acid) in the fluorocarbon. The solubility, the chemical stability of the putative prodrugs, and the sensitivity to enzymatic hydrolysis was investigated. Methods. The solubility of each nicotinic acid ester was determined in buffer as a function of pH and in perflubron. The octanol/buffer partition coefficient was determined at pH 7.4. The chemical stability of the putative prodrugs was determined as a function of pH, temperature, buffer content, and ionic strength. In addition, sensitivity of the esters to enzymatic degradation was evaluated. Results. Compared with nicotinic acid, the solubility in perflubron of the esters was significantly enhanced. In aqueous buffers, the esters exhibited pseudo-first order degradation kinetics, with both acid and base catalyzed loss. Studies of the fluorobutyl ester indicate quantitative loss of the putative prodrug and release of the parent nicotinic acid. Porcine esterase accelerated the loss of fluorobutyl ester by a factor of over 200 compared with chemical hydrolysis at pH 7.4. Conclusions. The properties of the fluorinated esters suggest that they may be suitable candidates for further testing as possible prodrugs of nicotinic acid based upon higher solubility in perflubron, rapid release of the parent drug after simple hydrolysis, and sensitivity to the presence of a model esterase enzyme.     

In Vivo Evaluation of Acyclovir Prodrug Penetration and Metabolism Through Rat Skin Using a Diffusion/Bioconversion Model

Purpose. In order to evaluate the in vivo penetration of prodrugs which undergo metabolism in skin, we analyzed thein vivo penetration profiles of acyclovir prodrugs based on a two-layer skin diffusion model in consideration of metabolic process. Methods. Acyclovir prodrugs (e.g., valerate, isovalerate and pivarate) were used as model prodrugs and the amounts excreted in urine were measured after percutaneous application. In vivo penetration profiles were then estimated by employing a deconvolution method and the penetration of acyclovir prodrugs was analyzed using a diffusion model. Subsequently, diffusion, partitioning and metabolic parameters were compared under in vitro and in vivo conditions. Results. Although total penetration amounts at the end of the experiment were similar for the three prodrugs, the ratio of intact prodrug to total penetration amount differed significantly. Moreover, the excretion and absorption profiles were also very different for each prodrug. Enzymatic hydrolysis rate constants calculated under in vivo conditions were considerably larger than those obtained in the skin homogenate and in vitro penetration experiments. Conclusions. The present skin diffusion/bioconversion model combined with computer analysis enables us to comprehensively account for diffusion, partitioning and metabolism during in vivo percutaneous absorption. Nevertheless, different enzymatic hydrolysis rate constants obtained under bothin vivo and in vitro conditions demonstrate the difficulty of obtaining accurate values for in vivo enzymatic activity from related in vitro experiments.     

Enhancement of the Systemic and CNS Specific Delivery of L-Dopa by the Nasal Administration of Its Water Soluble Prodrugs

Purpose. To study the utility of the nasal route for thesystemic delivery of L-dopa using water soluble prodrugs of L-dopa and toexamine if this delivery method will result in preferential delivery to theCNS. Methods. Several alkyl ester prodrugs of L-dopa wereprepared and their physicochemical properties were determined. Invitro hydrolysis rate constants in buffer, rat plasma, rat brainhomogenate, rat CSF, and rat nasal berfusate were determined by HPLC. Invivo nasal experiments were carried out in rats. Levels of L-dopa anddopamine in plasma, CSF, and olfactory bulb were determined using HPLCmethod with electrochemical detection. Results. All the prodrugs showed improved solubility andlipophilicity with relatively fast in vitro conversion in ratplasma. Absorption was fast following nasal delivery of the prodrugs withbioavailability around 90%. Dopamine plasma levels did not changesignificantly following nasal administration of the butyl ester prodrug.Olfactory bulb and CSF L-dopa concentration were higher following nasaldelivery of the butyl ester prodrug compared to an equivalent intravenousdose. Conclusions. Utilization of water soluble prodrugs ofL-dopa via the nasal route in the treatment of Parkinson's disease may havetherapeutic advantages such as improved bioavailability, decreased sideeffects, and potentially enhanced CNS delivery.     

Nanoparticles as delivery carriers for anticancer prodrugs

Introduction: Prodrugs are inactive compounds which are metabolized in the body to produce parent active agents. It has been shown that prodrugs hold some advantages over conventional drugs, such as increased solubility, improved permeability and bioavailability, reduced adverse effects and prolonged half-lives. Optimization of the vehicles used is very important in order to employ the advantages of prodrugs. Nanocarriers are currently being widely used as prodrug vehicles because of their ability to enhance storage stability, modulate prodrug release and tumor-targeted delivery and protect against enzymatic attack. This combined approach of prodrugs and nanoparticles has a particular attraction for developing anticancer therapies.

Areas covered: This paper discusses liposomes, polymeric nanoparticles and lipid nanoparticles, which are all carriers commonly used for prodrug encapsulation. Macromolecular prodrugs can spontaneously form self-assembled nanoparticles with no intervention of other additives. This review also describes recent developments in prodrug delivery using nanoparticulate strategies. Pharmacokinetic, pharmacodynamic and cytotoxicity evaluations of anticancer prodrugs are systematically elucidated in this review.

Expert opinion: More profiles involved in animal and clinical studies will encourage the future applicability of prodrug nanocarrier therapy. The possible toxicity associated with nanoparticles is a concern for development of prodrug delivery.     

Metabolism and Pharmacokinetics of Novel Oral Prodrugs of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in Dogs

Purpose. A series of prodrugs designed to enhance the oral bioavailability of the antiretroviral agent 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA; 1) have been synthesized, including a bis-(acyloxymethyl) ester 2 and a series of bis-(alkoxycarbonyloxymethyl) esters 3-9. The in vitro biological stability andin vivo pharmacokinetics of these prodrugs were evaluated to support selection of a prodrug candidate for clinical evaluation. Methods. The in vitrobiological stability of the prodrugs was examined in dog tissues (intestinal homogenate, plasma and liver homogenate). The apparent half-lives were determined based on the disappearance of prodrug using reverse-phase HPLC with UV detection. Oral bioavailability of PMPA from each prodrug was determined in fasted beagle dogs. Concentrations of PMPA in plasma were determined by HPLC following fluorescence derivatization. Data for prodrugs were compared to historical data for intravenous PMPA. Results. All prodrugs were rapidly hydrolyzed in dog plasma and tissues (t_1/2 < 60 min). In fasted beagle dogs, bis-[(pivaloyloxy)methyl] PMPA (bis-POM PMPA) 2 had the highest oral bioavailability as PMPA (37.8 ± 5.1%). The oral bioavailabilities of PMPA from bis-(alkoxycarbonyloxymethyl) esters ranged from 16.0% to 30.7% and PMPA was the major metabolite formed. Conclusions. There was a correlation between oral bioavailability and intestinal stability of bis-(alkoxycarbonyloxymethyl) ester prodrugs (r² = 0.96). Lipophilicity (log P) was not a good predictor of oral bioavailability. The most labile prodrugs in dog intestinal homogenates, bis-(n-butyloxycarbonyloxymethyl) PMPA 5 and bis-(neo-pentyloxycarbonyloxymethyl) PMPA 8 (t_1/2 < 5 min) had the lowest oral bioavailabilities. Based on good oral bioavailability (30.1%), chemical and intestinal stability bis-(isopropyloxycarbonyloxymethyl) PMPA (bis-POC PMPA) 4 was selected as a candidate for clinical evaluation.     

Paracetamol (acetaminophen) esters of some non-steroidal anti-inflammatory carboxylic acids as mutual prodrugs with improved therapeutic index

Paracetamol (acetaminophen) esters [4a-f] of some acidic NSAIDs were synthesized and evaluated as mutual prodrug forms with the aim of improving the therapeutic index through prevention of the gastrointestinal toxicity. The structures of the synthesized esters were confirmed by IR and ¹H-NMR spectroscopy and their purity was established by elemental analyses and TLC. In-vitro stability studies revealed that the synthesized ester prodrugs 4a-f are sufficiently chemically stable in non-enzymatic simulated gastric fluid (hydrochloric acid buffer of pH 1.3 (t _1/2 ∼ 15–45 h)) and in phosphate buffer of pH 7.4 (t _1/2 ∼ 4–40 h). In 80% human plasma and 10% rat liver homogenate, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis releasing the corresponding NSAID and paracetamol at relatively faster rates (t _1/2 ≈ 15–385 min and 1–140 min, respectively). Calculated log P values indicated that the prodrugs 4a-f are more lipophilic than the parent drugs. In-vivo experiments in rabbits showed higher plasma levels of ibuprofen after oral administration of its ester prodrug 4b compared with those resulting from an equivalent amount of the corresponding physical mixture. Moreover, significant improvement in latency of pain threshold in mice has been observed up to 4 h after po administration of 0.02 mmol/kg of the prodrugs, compared with the corresponding physical mixtures. Gross observations and scanning electromicrographs of the stomach showed that the prodrugs induced very little irritancy in the gastric mucosa of mice after oral administration for 4 days. These results suggest that the synthesized mutual ester prodrugs were characterized by a better therapeutic index than the parent drugs.     

Hydroxypropyl-β-Cyclodextrin Increases the Aqueous Solubility and Stability of Pilocarpine Prodrugs

Purpose. The effects of 2-hydroxypropyl--cyclodextrin (HP--CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. Methods. The solubility of prodrugs was studied by phase-solubility method (0–72.5 mM HP--CD). The stability of one of the prodrugs was investigated as a function of temperature (40°C–70°C) and HP--CD concentration (0–72.5 mM). The apparent rate constants (k ₁, k ₂) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K _1:1, K _l:2) were calculated by the curve-fitting method. Results. The phase-solubility diagrams were classified as A_p-type and the apparent stability constants (K _l:l, K _l:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143–815 M^–l and 29–825 M^–1, respectively. The stability of prodrug increased as a function of HP--CD concentration over the studied temperature range. The shelf-life (t _90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP--CD solution increased 5.1-fold and 6.1-fold at 25°C and 4°C, respectively. Conclusions. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the l:2-complex than in the l:l-complex and the relative amounts of complex species were found to be dependent on CD concentration.     

Prodrugs of Gestodene for Matrix-Type Transdermal Drug Delivery Systems

Purpose. The aim of this study was to enhance the transdermal absorption of the highly active progestin gestodene from matrix type transdermal delivery systems (TDDS) by formation of prodrugs with improved matrix solubility. Methods. Gestodene esters were synthesized via acylation of the drug with the respective carboxylic anhydrides. Subsequently TDDS were produced using the solvent cast method. Selected formulations were examined with in vitro diffusion experiments using skin of nude mice. Results. One prodrug, gestodene caproate proved to be an oil at ambient temperature and showed a very high solubilty of over 10.5% in the TDDS matrix. Within in vitro penetration studies using those systems the prodrug exhibited a significantly higher transdermal penetration rate than gestodene from reference systems. Furthermore, the prodrug was hydrolyzed to the parent drug to a high extent during the passage of the skin. Conclusions. Designing prodrugs to the requirements of matrix TDDS is an efficient way of enhancing the transdermal drug flux rate.