摘要
病毒性疾病对人类健康和生命造成了莫大威胁,新型抗病毒感染药物的研究一直是热点领域之一。本研究组在以Hsp90为靶点的抗病毒药物研究中,筛选得到了若干抗病毒活性强和抗病毒谱广的格尔德霉素(GM)衍生物GM-AMPL、LD01、 GM-GP和GM-TC等。另外,在进行新核苷衍生物的合成与筛选研究中,得到的替诺福韦长链烷基单酯衍生物ODE-TFV显示有进一步开发研究价值。在前期工作的基础上,为充分阐述抗病毒活性化合物GM-AMPL、LD01、GM-GP和GM-TC的开发应用前景,本论文对其前期成药性进行了综合评价研究。同时,为配合进一步研发工作的需要,对抗病毒候选药物ODE-TFV的分析检测及质量控制方法进行了研究。
第一部分是格尔德霉素衍生物GM-AMPL、LD01、GM-GP和GM-TC的合成及成药性研究。在前期研究的基础上,对上述GM衍生物的合成制备、理化性质与稳定性、体内抗鸭肝炎病毒活性、口服吸收、小鼠急性毒性、对Beagle犬呼吸与心血管系统毒性等进行了前期综合评价研究。
理化性质与稳定性：GM-AMPL、LD01、GM-GP和GM-TC在水及正辛醇中的溶解度分别为0.41及0.78、5.41及8.38、3.30及0.0082和2.62及0.22mg/ml,普遍较先导物GM(0.13及0.041mg/m1)明显提高；GM-AMPL的甲醇溶液经4500Lx光照1h降解不到3%,相同条件下,GM降解接近50%。
体内抗DHBV效果：采用鸭肝炎模型动物,分别灌胃给予不同剂量(0.05、0.025和0.0125mmo1/kg)的GM-AMPL、LD01、 GM-GP和0.2mmol/kg的对照药拉米夫定(3TC),1天2次,连续10天。分别在给药前(T0)、给药后5天(T5)、给药后10天(T10)和停药后3天(P3),取血分离血清,测定药物对鸭血清乙型肝炎病毒DNA(DHBV-DNA)的影响。实验结果显示,与病毒对照组相比,不同实验剂量下,GM-AMPL、LDO1及GM-GP均显示显著的抑制鸭血清DHBV DNA的作用,在0.05mmol/kg剂量下的DHBV抑制活性强于/相当于对照药3TC,停药后3天鸭血清DHBV水平无反跳。测试的GM化合物中,GM-AMPL的体内抑制鸭血清DHBVDNA的效果最强。
口服生物利用度：SD大鼠单次灌胃给药GM-AMPL(25mg/kg)、LD01(25mg/kg)和GM-GP(7.5mg/kg)的生物利用度分别为26.36%、1.72%和8.89%。
小鼠单次给药的急性毒性：对比研究了小鼠静脉和腹腔注射给药GM-AMPL、LD01、GM-GP的急性毒性。GM-AMPL、LD01、GM-GP单次静脉注射给予小鼠的LDso分别为152.6、82.01和19.2mg/kg,单次腹腔注射的LDso分别为295.4、203.8和35.5mg/kg。
对Beagle犬呼吸和心血管系统的毒性：用累计给药方法进行不同剂量试验,给药后5-180min期间连续采集Beagle犬血压、心电、呼吸等各项指标的数据,在药后不同时间点对动物血压、心电、呼吸指标进行了测量、分析和评估。结果显示,GM-AMPL:单次静脉注射剂量分别为5、10、15、20μg/kg和10mg/kg。注射15μg/kg在给药后2min内可引起Beagle犬血压明显下降,呼吸频率加快；注射20μg/kg后,可引起心率减慢,血压明显下降,呼吸频率加快；注射10mg/kg后可引起动物死亡。对Beagle犬呼吸和心血管系统无明显影响的安全剂量小于15.0μg/kg；LD01：单次静脉注射药1.0μg/kg和0.5、2.0、8.0mg/kg,在给药后均可引起Beagle犬心率减慢,血压明显下降,呼吸频率加快；对Beagle犬呼吸和心血管系统无明显影响的安全剂量小于1.0μg/kg；GM-GP:单次静脉注射给予Beagle犬10、11、16、21和25μg/kg,在给药后2min内即可引起心率减慢,血压明显下降,呼吸频率加快；单次静脉注射对Beagle犬呼吸和心血管系统无明显影响的安全剂量小于10μg/kg。
第二部分是非环核苷衍生物ODE-TFV分析检测方法的摸索。为配合进一步研究开发工作需要,论文对抗病毒侯选药物ODE-TFV的纯度及主要有关杂质分析检测方法进行了探索研究,研究内容主要包括：ODE-TFV的理化性质与稳定性、主要有关物质分离分析与纯度检测方法、光学异构体的拆分等。研究结果显示：
ODE-TFV在中性水中难溶,但由于其分子结构中有一游离磷酸羟基呈现较强酸性,因此,其在水中的溶解性与pH有关,随着碱性升高其溶解性增大。ODE-TFV在单一溶剂乙醇或氯仿中加热都可溶解、易溶于乙醇和氯仿(20/80)的混合溶液。
ODE-TFV制备过程中涉及到的中间体及可能副产物的理化性质差别很大,而且,产品具有光学活性,因此,目标药物的纯度检测及主要有关杂质分析方法比较复杂。采用HPLC梯度洗脱法,成功实现了对具有紫外吸收的主要有关物质的分离分析检测。确定了关键中间体TFV的光学拆分条件,用于对目标药物光学纯度的暂时控制测定法。考察了ODE-TFV在加速条件下(高温、高湿)的稳定性,以及在强酸、强碱、强氧化剂和光照条件下的降解情况。研究结果显示,ODE-TFV在60℃高温、相对湿度90%及0.1mol稀盐酸、0.1mol稀NaOH条件下比较稳定,在浓盐酸(0.5mol以上)、浓NaOH(0.5mol以上)和氧化剂(H2O2)中有一定降解,降解方式主要为酯基的水解。ODE-TFV对光照不稳定,降解产物有待鉴定。
论文两部分研究工作均取得明显进展和结果,格尔德霉素衍生物的综合评价研究结果提示,GM-AMPL、LD01和GM-GP等的抗病毒效果确切,但对Beagle犬的心脏毒性很强,具有较高的毒性风险。ODE-TFV的分析检测方法及质量研究结果,初步阐述了侯选化合物ODE-TFV的性质、对ODE-TFV主要有关物质及纯度检测中的关键技术进行了有益探索,为目标药物的进一步研发奠定良好基础。
The study of antiviral study has become a hotspot because viral diseases give a great threat on human health and lives. During the screening of antiviral drug acting on Hsp90as a target in our group, we obtained a number of geldanamycin (GM) derivatives exhibiting stronger and broad spectrum antiviral activities, typical representative derivatives were GM-AMPL, LD01, GM-GP and GM-TC. In addition, in the process of synthesis and screening of new nucleoside derivatives, the obtained ODE-TFV, a tenofovir (TFV) derivative with a long chain alky mono-ester, exhibited a good value for further research and development. On the base of previous studies, we conducted a comprehensive evaluation on their pre-druggability to predict the development and application prospects of the four compounds with antiviral activities:GM-AMPL, LD01, GM-GP and GM-TC. Meanwhile, the analysis method and qualitative research of antiviral candidate, ODE-TFV, were carried out to meet the needs of further research and development.
The first part is the synthesis and druggability evaluation of GM derivatives (GM-AMPL, LD01, GM-GP and GM-TC):on the basis of preliminary studies in our laboratory, the above GM derivatives were synthesized and their physical and chemical properties, the stability, the activities against duck Hepatitis B Virus in vivo, oral absorption, acute toxicity in mice, the toxicities of cardiovascular and respiratory systems in beagle dogs, etc., were comprehensively evaluated.
The physical and chemical properties and stability:the solubility of GM-AMPL, LD01, GM-GP and GM-TC in the water and n-octanol were0.41and0.78,5.41and8.38,3.30and0.0082,2.62and0.22mg/ml, respectively, which significantly increased compared with that of lead compound GM (0.13and0.041mg/ml); The light stability of GM-AMPL has significantly improved, the degradation of GM-AMPL in methanol was less than3%when exposed the light of4500Lx for1hour, but the degradation of GM was about50%under the same conditions.
Anti-DHBV-DNA activity in vivo:three different dosages (0.05,0.025and0.0125mmol/kg, respectively) of GM-AMPL, LD01and GM-GP, and one dosage of the positive control Lamivudine (3TC,0.2mmol/kg) were given per ora (p.o.) twice daily for10consecutive days. Blood samples were taken on the before-treatment day (TO), the fifth day (T5), the tenth day (T10) during treatment, and the third day (P3) after the cessation of treatment and serum was prepared for the analysis of DHBV-DNA levels to monitor the effects of the compounds on infected ducks. The results showed that GM-AMPL, LD01and GM-GP, at all different treatment regimens, exhibited the inhibition of the DHBV-DNA level in infected duckling serum compared with that of the virus control group. The activity of three compounds at0.05mmol/kg was similar or better than that of3TC, and there was not rebound at day P3. The effect of GM-AMPL on DHBV-DNA was the strongest in three compounds.
The oral bioavailability:After a single intragastric (i.g.) administration to rats, the oral bioavailability of GM-AMPL (25mg/kg), LD01(25mg/kg) and GM-GP (7.5mg/kg) was26.36%,1.72%and8.89%, respectively.
Acute toxicity in mice after single dose:The acute toxicities of GM-AMPL, LD01and GM-GP in mice were investigated after single intravenous (i.v.) injection and intraperitoneal (i.p.) injection, respectively. The median lethal dose (LD50) values of GM-AMPL, LD01and GM-GP after i.v. administration were152.6,82.0and19.2mg/kg, and the LD50values of them after single i.p. administration were295.4,203.8and35.5mg/kg, respectively.
The toxicities of cardiovascular and respiratory systems in beagle dogs:The tests were carried out by the cumulative administration at different dosages. The cardiac index, blood pressure index and respiratory index were continuously recorded5-180min after dosing, and were measured, analysed and evaluated at various time points. The results were showed as following.
GM-AMPL:single dosage by i.v. administration,5,10,15,20μg/kg and10mg/kg of it was given to beagle dogs, respectively. Given15μg/kg, dogs caused a significant decrease in blood pressure and an accelerated rate of respiration within2min; if20μg/kg was given, the symptom of slow heart rate, drop in blood pressure and acceleration of the respiratory rate appeared. Giving10mg/kg to dogs could cause animal death. The safety dose of GM-AMPL which had no effect on the cardiovascular and respiratory systems of beagle dogs was<15μg/kg. LD01:after a single dosage of it at1.0ug/kg and0.5,2.0and8.0mg/kg to beagle dogs by i.v. administration, respectively, the symptoms of drop in blood pressure, acceleration of respiratory rate and slow heart rate occurred, and the safety dose of LD01was<1.0μg/kg. GM-GP:the symptoms of drop in blood pressure, acceleration of respiratory rate and slow heart rate appeared after a single dosage of it at10,11,16,21and25μ g/kg to beagle dogs by i.v. administration, respectively. The safety dose of it was<10μg/kg.
The second part is the analytical method and quality research of acyclic nucleoside derivative ODE-TFV. The analytical and detection methods of content and related substances were explored and studied to assist the further research and development in this paper, the studies included its physical and chemical properties, the stability, the separation and analysis of related substances and detemination methods, the separation of enantiomers, etc. The results were showed as following.
It is difficult for ODE-TFV to dissolve in water. As its free hydroxyl group of phosphate in molecular structure was existing in the form of acid, its solubility was associated with pH values of water and increased with the increase of pH value. ODE-TFV could dissolve in ethanol or chloroform when heated, easily dissolve in the mixed solvent of ethanol/chloroform (20/80v/v).
The physical and chemical properties of intermediates and possible by-product deriving from the synthesis process of ODE-TFV varies considerably, and the target product showed the optical activity. Therefore, the determination of targets compound's content and the analysis methods of relative impurities were complicated. The separation, analysis and detection of relative substances with UV absorption were achieved by the gradient elution of HPLC method. The separation of optical isomers of TFV, a key intermediate, was achieved to control and monitored effectively the optical purity of target product on temporary.
The stabilities of ODE-TFV under the accelerated conditions (high temperature and humidity) were examined, and the degradation under the acid, alkalis and oxidant were also examined. The results showed that ODE-TFV was relatively stable on the condition of the temperature of60℃, the humidity of90%,0.1mol/L hydrochloric acid and0.1mol/L sodium hydroxide, however, some degradations were observed in hydrochloric acid (>0.5mol/L), sodium hydroxide (>0.5mol/L) and oxidant (H2O2), the main form of degradation was esterolysis. ODE-TFV was instability when exposed to the light, and the degradation products need to be identified.
Both parts of the paper make great progress. The comprehensive evaluation of GM derivatives suggest that GM-AMPL, LD01and GM-GP, etc., exhibited exact antiviral activities, but there was the high toxicity on cardiovascular and respiratory systems of beagle with high-risk. The analytical and detection methods of ODE-TFV and quality research illustrate preliminarily the properties of ODE-TFV, and the key technologies of the content of ODE-TFV and impurities are explored usefully to establish a good foundation for further research and development.
引文
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