WO2020177748A1

WO2020177748A1 - Quaternized modified taxane derivative, and pharmaceutical composition and use thereof

Info

Publication number: WO2020177748A1
Application number: PCT/CN2020/078071
Authority: WO
Inventors: 贺玖明; 阿不力孜再帕尔; 李燕; 张锦; 杜倩倩; 孙成龙; 张瑞萍; 靳洪涛; 宋肖伟; 罗志刚
Original assignee: 中国医学科学院药物研究所
Priority date: 2019-03-05
Filing date: 2020-03-05
Publication date: 2020-09-10
Also published as: CN111662250B; CN111662250A

Abstract

The present invention relates to the technical field of medicines, and specifically disclosed are a quaternized modified taxane derivative, a pharmaceutical composition, and a synthetic pathway and use thereof. The taxane derivative is prepared by taxane and active targeting molecules, i.e., choline or betaine, being subjected to hydroxyl substitution reaction by means of certain linking groups. The taxane derivative has good water solubility and certain tumor targeting, can be metabolized to paclitaxel or docetaxel in a tumor tissue, and as a prodrug strategy, is used as an effective antitumor drug. With respect to paclitaxel, the tumor suppression rate is equivalent at a same dose, but an immune system is not damaged, and toxicity is significantly reduced.

Description

季铵化修饰紫杉烷衍生物、其药物组合物和用途Quaternized modified taxane derivative, its pharmaceutical composition and use

技术领域Technical field

本发明属于医药技术领域，具体涉及一类新的季铵化修饰紫杉烷衍生物、其药物组合物、其合成途径和用途，所述衍生物不仅具有良好的水溶性，同时具有一定的肿瘤靶向性，相比同等剂量紫杉醇，肿瘤抑制率相当，但不破坏免疫***，毒性明显降低，并且对紫杉醇耐药肿瘤敏感性提高，可用作高效低毒的抗肿瘤药物。The present invention belongs to the technical field of medicine, and specifically relates to a new class of quaternized modified taxane derivatives, their pharmaceutical compositions, their synthetic pathways and uses. The derivatives not only have good water solubility, but also have certain tumors. Targeting, compared with the same dose of paclitaxel, the tumor suppression rate is equivalent, but the immune system is not damaged, the toxicity is significantly reduced, and the sensitivity to paclitaxel-resistant tumors is improved, and it can be used as a highly effective and low-toxic anti-tumor drug.

背景技术Background technique

癌症是威胁人类健康的第二大疾病，从全球情况看，近六分之一的死亡由癌症造成，癌症负担不断加重。据世界卫生组织(WHO)发布的《世界癌症报告》，到2035年，全球肿瘤患者可能达到2400万人，在20年间增加近五成，受人口增长和老龄化影响，发展中国家的癌症数量不断攀升，全球60％的病例发生在非洲、亚洲和中美洲及南美洲地区，并且占全世界癌症死亡数的70％，而中国新增癌症病例约占全球的20％，癌症死亡病例约占全球25％。最新的癌症流行病学调查显示，在中国每天约为1万人确诊癌症，平均每分钟就有7人确诊。面临当前日益严峻的癌症发病形势，肿瘤的治疗成为当今疾病治疗领域的世界性难题。Cancer is the second largest disease threatening human health. From a global perspective, nearly one-sixth of all deaths are caused by cancer, and the burden of cancer is increasing. According to the World Cancer Report issued by the World Health Organization (WHO), by 2035, the number of cancer patients in the world may reach 24 million, an increase of nearly 50% in 20 years. Affected by population growth and aging, the number of cancers in developing countries Climbing, 60% of global cases occurred in Africa, Asia and Central America and South America, and accounted for 70% of the world’s cancer deaths, while China’s new cancer cases accounted for about 20% of the world’s total, and cancer deaths accounted for about 20% 25% of the world. The latest cancer epidemiological survey shows that about 10,000 people are diagnosed with cancer every day in China, and an average of 7 people are diagnosed every minute. Facing the current increasingly severe situation of cancer incidence, tumor treatment has become a worldwide problem in the field of disease treatment.

抗肿瘤药物作为癌症治疗的重要方法之一，近年来呈现快速发展趋势，每年各类机构针对不同适应症、不同靶点进行大量的抗肿瘤药物开发。开发疗效高、广谱性强、副作用小的抗肿瘤药物是医药界的新药研发课题的焦点。As one of the important methods of cancer treatment, anti-tumor drugs have shown a rapid development trend in recent years. Every year, various institutions conduct a large number of anti-tumor drug developments for different indications and different targets. The development of anti-tumor drugs with high efficacy, strong broad-spectrum and low side effects is the focus of new drug research and development topics in the medical field.

紫杉醇作为一个具有抗癌活性的二萜生物碱类化合物，1992年底被美国FDA批准用于抗晚期癌症的治疗，是继阿霉素和顺铂之后最成功的抗癌药物，其化学结构新颖复杂、生物活性广泛而显著、作用机制全新独特、自然资源珍贵奇缺，它是目前已发现的最优秀的天然抗癌药物之一，也是近三十年来药物开发最伟大发现之一。紫杉醇抗瘤谱广，疗效显著，广泛用于非小细胞肺癌、乳腺癌、卵巢癌，及其食管癌、头颈部癌、胃癌等的治疗。然而，紫杉醇的低水溶性和高细胞毒性限制了其临床广泛应用。紫杉醇本身的水溶性很差，其在水中的溶解度小于0.004mg/mL，目前临床上使用的紫杉醇注射液是用聚氧乙烯蓖麻油：无水乙醇(1：1)作为溶媒，极易于在体内发生严重的过敏反应；其次，紫杉醇作为一类细胞毒类抗肿瘤药物，具有骨髓抑制、神经毒性、心血管毒性、肝脏损伤等诸多不良反应。紫杉醇上述缺陷为紫杉醇的后续开发留下了广阔的空间。Paclitaxel, as a diterpene alkaloid compound with anticancer activity, was approved by the US FDA for the treatment of advanced cancer at the end of 1992. It is the most successful anticancer drug after adriamycin and cisplatin. Its chemical structure is novel and complex. , The biological activity is extensive and significant, the mechanism of action is new and unique, and the natural resources are rare and scarce. It is one of the best natural anticancer drugs discovered so far, and one of the greatest discoveries in drug development in the past 30 years. Paclitaxel has a broad anti-tumor spectrum and significant curative effect. It is widely used in the treatment of non-small cell lung cancer, breast cancer, ovarian cancer, and esophageal cancer, head and neck cancer, and gastric cancer. However, paclitaxel's low water solubility and high cytotoxicity limit its wide clinical application. The water solubility of paclitaxel itself is very poor, and its solubility in water is less than 0.004mg/mL. Paclitaxel injection currently used clinically uses polyoxyethylene castor oil: absolute ethanol (1:1) as a solvent, which is very easy to Severe allergic reactions occur in the body; secondly, paclitaxel, as a class of cytotoxic anti-tumor drugs, has many adverse reactions such as bone marrow suppression, neurotoxicity, cardiovascular toxicity, and liver damage. The above-mentioned deficiencies of paclitaxel leave a broad space for the subsequent development of paclitaxel.

临床需求是新药研发的核心追求，为了提高紫杉醇的溶解性，减少紫杉醇对病人的毒副作用，国内外就紫杉醇的新技术与新剂型开展了大量研究。研究热点主要分为两个方向：一是合成能改变紫杉醇溶解性，降低其细胞毒性的衍生物；二是制备紫杉醇的新型制剂。Clinical demand is the core pursuit of new drug research and development. In order to improve the solubility of paclitaxel and reduce the toxic and side effects of paclitaxel on patients, a large number of studies have been carried out on new technologies and new dosage forms of paclitaxel at home and abroad. The research focus is mainly divided into two directions: one is the synthesis of derivatives that can change the solubility of paclitaxel and reduce its cytotoxicity; the other is the preparation of new paclitaxel preparations.

目前，在紫杉醇新型给药***方面取得了一定的成果，占据市场的主流产品主要有紫杉醇脂质体

和紫杉醇白蛋白纳米制剂

两种新型制剂在一定程度上克服了水溶性低的难题，并且降低了药物的毒性，提高了抗肿瘤效果。然而，对于紫杉醇脂质体，由于紫杉醇在水相和脂相的溶解度都比较低，长期放置紫杉醇容易从脂质体或脂质纳米粒中析出，继而形成沉淀，使得脂质体和纳米粒制剂缺乏长期稳定性，给临床应用带来不便；对于紫杉醇白蛋白纳米制剂，利用人源性白蛋白作为载体,具有微生物污染及免疫抵抗等风险。因此，为了克服上述障碍，本领域迫切需要开发合成一种既能改变其溶解性，也能降低细胞毒性，抗肿瘤效果更好的紫杉醇衍生物。 At present, certain achievements have been made in the new drug delivery system of paclitaxel. The mainstream products occupying the market mainly include paclitaxel liposomes

And paclitaxel albumin nanoformulations

The two new formulations overcome the problem of low water solubility to a certain extent, reduce the toxicity of the drug, and improve the anti-tumor effect. However, for paclitaxel liposomes, due to the relatively low solubility of paclitaxel in the aqueous and lipid phases, paclitaxel is easy to precipitate from liposomes or lipid nanoparticles after long-term storage, and then precipitates are formed, making liposomes and nanoparticle preparations The lack of long-term stability brings inconvenience to clinical application; for paclitaxel albumin nano preparations, human-derived albumin is used as a carrier, which has risks of microbial contamination and immune resistance. Therefore, in order to overcome the above-mentioned obstacles, there is an urgent need in the art to develop and synthesize a paclitaxel derivative that can not only change its solubility, but also reduce cytotoxicity and have better anti-tumor effects.

胆碱，是一类季铵碱，极性非常大。据文献报道胆碱对于细胞维持正常的生理功能起着至关重要的作用，胆碱的缺乏会诱发机体产生病理变化。它作为人体必需营养素具有如下生理功能：它是构成细胞膜的重要成分，也是甘氨酸、甜菜碱、乙酰胆碱、血小板活化因子等体内重要代谢物的前体物质；参与信号传导，促进脑发育；调控细胞凋亡；胆碱是肝脏分泌极低密度脂蛋白所必需的物质，从而有利于降低血清胆固醇，维护肝胆功能等。胆碱作为甲基供体，还与叶酸、甜菜碱及其他B族维生素一起参与一碳代谢，参与核酸的合成，影响DNA的甲基化。并且在许多文献报道中，胆碱及其衍生物作为肿瘤的潜在生物标志物，较正常组织细胞，肿瘤组织存在高水平的分布，可能由于肿瘤细胞具有快速增殖的特性，往往会从体液中主动大量摄取该营养物质物质以满足其增殖需求，因此，它对胆碱的吸收利用远大于正常细胞。Choline, a type of quaternary ammonium base, is very polar. According to reports in the literature, choline plays a vital role in maintaining normal physiological functions of cells, and the lack of choline can induce pathological changes in the body. As an essential nutrient for the human body, it has the following physiological functions: it is an important component of cell membranes and the precursor of important metabolites in the body such as glycine, betaine, acetylcholine, platelet activating factor, etc.; it participates in signal transduction, promotes brain development, and regulates cell apoptosis. Choline is a substance necessary for the liver to secrete very low-density lipoprotein, which is beneficial to lower serum cholesterol and maintain liver and gallbladder function. As a methyl donor, choline also participates in one-carbon metabolism together with folic acid, betaine and other B vitamins, participates in nucleic acid synthesis, and affects DNA methylation. And in many literature reports, choline and its derivatives, as potential biomarkers of tumors, have a higher level of distribution than normal tissue cells and tumor tissues. It may be due to the rapid proliferation of tumor cells that they tend to take the initiative from body fluids. Ingest a large amount of this nutrient substance to meet its proliferation needs, therefore, its absorption and utilization of choline is much greater than that of normal cells.

发明内容Summary of the invention

本发明解决的技术问题是提供一类具有下列通式(Ⅰ)所示的紫杉烷衍生物及其药学上可接受的盐，其制备方法、药物组合物和在制备抗肿瘤药物中的应用。The technical problem solved by the present invention is to provide a taxane derivative represented by the following general formula (I) and a pharmaceutically acceptable salt thereof, its preparation method, pharmaceutical composition and application in the preparation of antineoplastic drugs .

为解决本发明的技术问题，本发明提供如下技术方案：In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

本发明技术方案的第一方面提供了一类具有下列通式(Ⅰ)所示的紫杉烷衍生物及其药学上可接受的盐：The first aspect of the technical scheme of the present invention provides a class of taxane derivatives and pharmaceutically acceptable salts thereof having the following general formula (I):

(Ⅰ)式中：(Ⅰ) where:

R ₁为苯甲酰基，或叔丁氧酰基，或甜菜碱酰基或取代的甜菜碱酰基，或为碳酸酯键、磷酸酯键、C _2-8二酸酯键、醚键或磺酸酯键连接的胆碱或取代的胆碱；所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基，不同种类的氨基酸。 R ₁ is benzoyl, or tert-butoxy acyl, or betaine acyl or substituted betaine acyl, or carbonate bond, phosphate bond, C _2-8 diacid ester bond, ether bond or sulfonate bond Linked choline or substituted choline; the substituents are each independently selected from halogen atoms, C _1-6 alkyl, C _1-7 acyl, C _3-6 cycloalkyl, hydroxyl, amino, different types Of amino acids.

R ₂为氢或甲基或乙酰基； R ₂ is hydrogen or methyl or acetyl;

R ₃为氢或甲基或乙酰基； R ₃ is hydrogen or methyl or acetyl;

R ₄为氢或甲基或乙酰基； R ₄ is hydrogen or methyl or acetyl;

L为碳酸酯键、磷酸酯键、C _2-8二酸酯键、缩酮键、缩醛键、醚键或磺酸酯键连接； L is a carbonate bond, a phosphate bond, a C _2-8 diacid ester bond, a ketal bond, an acetal bond, an ether bond or a sulfonate bond;

R ₅为氢或甜菜碱酰基或为取代的甜菜碱酰基或胆碱或取代胆碱，所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基、不同种类的氨基酸。 R ₅ is hydrogen or betaine acyl group or substituted betaine acyl group or choline or substituted choline, the substituents are each independently selected from halogen atoms, C _1-6 alkyl, C _1-7 acyl, C _3-6 cycloalkyl, hydroxyl, amino, different kinds of amino acids.

进一步的，通式(I)中，所述的氨基酸包括丙氨酸、缬氨酸、苏氨酸、天冬氨酸、丝氨酸等，所述的C _1-6烷基包括甲基、乙基、丙基、丁基等，所述的C _1-7酰基包括甲酰基、乙酰基、丙酰基、丁酰基等，所述的C _3-6环烷基包括环丙烷基、环丁烷基、环戊烷基和环己烷基，所述的卤原子选自F、Cl、Br、I；所述的C _2-8二酸酯键包括乙二酸酯键、丙二酸酯键、丁二酸酯键、丁烯二酸酯键、羟基丁二酸酯键、氨基丁二酸酯键、戊二酸酯键、戊烯二酸酯键，羟基戊二酸酯键、氨基戊二酸酯键、己二酸酯键、己烯二酸酯键，羟基己二酸酯键、氨基己二酸酯键、庚二酸酯键、庚烯二酸酯键，羟基庚二酸酯键、氨基庚二酸酯键、辛二酸酯键、辛烯二酸酯键，羟基辛二酸酯键、氨基辛二酸酯键。C2’位羟基为(RS)-、(S)-、(R)-构型。 Further, in the general formula (I), the amino acids include alanine, valine, threonine, aspartic acid, serine, etc., and the C _1-6 alkyl group includes methyl, ethyl , Propyl, butyl, etc., the C _1-7 acyl group includes formyl, acetyl, propionyl, butyryl, etc., and the C _3-6 cycloalkyl group includes cyclopropyl, cyclobutanyl, Cyclopentyl and cyclohexane, the halogen atom is selected from F, Cl, Br, I; the C _2-8 diacid ester bond includes oxalate bond, malonate bond, butane Diester bond, crotonate bond, hydroxysuccinate bond, amino succinate bond, glutarate bond, glutarate bond, hydroxyglutarate bond, aminoglutaric acid Ester bond, adipate bond, hexenedioate bond, hydroxyadipate bond, aminoadipate bond, pimelate bond, peptenediate bond, hydroxypimelate bond, Aminopimelate bond, suberate bond, octenedoate bond, hydroxysuberate bond, aminosuberate bond. The hydroxyl group at the C2' position is in (RS)-, (S)-, (R)-configuration.

进一步的，优选的化合物结构通式为：Further, the general formula of the preferred compound structure is:

(II)式中：(II) where:

R ₁为苯甲酰基，叔丁氧酰基，甜菜碱酰基，取代的甜菜碱酰基，碳酸酯键、磷酸酯键、C _2-8二酸酯键、醚键或磺酸酯键连接的胆碱或取代的胆碱；所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基、不同种类的氨基酸 R ₁ is benzoyl, tert-butoxy acyl, betaine acyl, substituted betaine acyl, carbonate bond, phosphate bond, C _2-8 diacid ester bond, ether bond or sulfonate bond connected choline Or substituted choline; the substituents are each independently selected from halogen atoms, C _1-6 alkyl, C _1-7 acyl, C _3-6 cycloalkyl, hydroxyl, amino, and different kinds of amino acids

R ₂为氢，甲基或乙酰基； R ₂ is hydrogen, methyl or acetyl;

R ₃为氢，甲基或乙酰基； R ₃ is hydrogen, methyl or acetyl;

R ₄为氢，甲基或乙酰基； R ₄ is hydrogen, methyl or acetyl;

L为碳酸酯键、磷酸酯键、C _2-8二酸酯键、缩酮键、缩醛键、醚键或磺酸酯键连接。 L is a carbonate bond, a phosphate bond, a C _2-8 diacid ester bond, a ketal bond, an acetal bond, an ether bond or a sulfonate bond.

进一步的，通式(II)中，所述的氨基酸包括丙氨酸、缬氨酸、苏氨酸、天冬氨酸、丝氨酸等，所述的C _1-6烷基包括甲基、乙基、丙基、丁基等，所述的C _1-7酰基包括甲酰基、乙酰基、丙酰基、丁酰基等，所述的C _3-6环烷基包括环丙烷基、环丁烷基、环戊烷基和环己烷基，所述的卤原子选自F、Cl、Br、I；所述的C _2-8二酸酯键包括乙二酸酯键、丙二酸酯键、丁二酸酯键、丁烯二酸酯键、羟基丁二酸酯键、氨基丁二酸酯键、戊二酸酯键、戊烯二酸酯键，羟基戊二酸酯键、氨基戊二酸酯键、己二酸酯键、己烯二酸酯键，羟基己二酸酯键、氨基己二酸酯键、庚二酸酯键、庚烯二酸酯键，羟基庚二酸酯键、氨基庚二酸酯键、辛二酸酯键、辛烯二酸酯键，羟基辛二酸酯键、氨基辛二酸酯键。C2’位羟基为(RS)-、(S)-、(R)-构型。 Further, in the general formula (II), the amino acids include alanine, valine, threonine, aspartic acid, serine, etc., and the C _1-6 alkyl group includes methyl, ethyl , Propyl, butyl, etc., the C _1-7 acyl group includes formyl, acetyl, propionyl, butyryl, etc., and the C _3-6 cycloalkyl group includes cyclopropyl, cyclobutanyl, Cyclopentyl and cyclohexane, the halogen atom is selected from F, Cl, Br, I; the C _2-8 diacid ester bond includes oxalate bond, malonate bond, butane Diester bond, crotonate bond, hydroxysuccinate bond, amino succinate bond, glutarate bond, glutarate bond, hydroxyglutarate bond, aminoglutaric acid Ester bond, adipate bond, hexenedioate bond, hydroxyadipate bond, aminoadipate bond, pimelate bond, peptenediate bond, hydroxypimelate bond, Aminopimelate bond, suberate bond, octenedoate bond, hydroxysuberate bond, aminosuberate bond. The hydroxyl group at the C2' position is in (RS)-, (S)-, (R)-configuration.

上述式(II)的紫杉烷衍生物中，优选地，胆碱和甜菜碱化修饰紫杉醇衍生物、多西紫杉醇衍生物为下列结构：Among the taxane derivatives of the above formula (II), preferably, choline and betaine-modified paclitaxel derivatives and docetaxel derivatives have the following structures:

本发明的部分化合物分子中含有季铵基团，可如本专业公认的那样，通过酸处理，转化成药学上可接受的盐。本发明所述的药学上可接受的盐包括无机酸盐或有机酸盐，无机酸盐包括盐酸盐、氢溴酸盐、硫酸盐或硫酸氢盐、硝酸盐、磷酸盐或磷酸氢盐等，有机酸盐包括甲酸盐、乙酸盐、苯甲酸盐、丁二酸盐、富马酸盐、马来酸盐、乳酸盐、柠檬酸盐、酒石酸盐、琥珀酸盐、葡糖酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐等。Some of the compounds of the present invention contain quaternary ammonium groups in their molecules, which can be converted into pharmaceutically acceptable salts by acid treatment as recognized in the art. The pharmaceutically acceptable salts of the present invention include inorganic acid salts or organic acid salts, and inorganic acid salts include hydrochloride, hydrobromide, sulfate or hydrogen sulfate, nitrate, phosphate or hydrogen phosphate, etc. , Organic acid salts include formate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, succinate, glucose Acid salt, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.

本发明技术方案的第二方面是提供了第一方面所述紫杉烷衍生物的制备方法。The second aspect of the technical solution of the present invention provides a method for preparing the taxane derivative described in the first aspect.

其中取代基的定义同第一方面的定义相同。The definition of the substituent is the same as that of the first aspect.

本发明所述的紫杉烷衍生物(化合物(Ⅰ))，胆碱或甜菜碱或取代的胆碱或甜菜碱结合紫杉烷类化合物自由羟基的部位不限于2’位，也可以同紫杉烷的7位羟基进行反应，本领域技术人员都了解，2’位和7位羟基均有一定的亲核性，都可实现与一步反应后得到的酰氯发生取代反应，并且在实验中发现2’位羟基有非常好的的区域选择性，与文献报道一致。若想实现7位与胆碱结合，可将2’位羟基通过硅醚化试剂保护，再进行后续的取代反应。According to the taxane derivative (compound (I)) of the present invention, the position where choline or betaine or substituted choline or betaine binds to the free hydroxyl group of the taxane compound is not limited to the 2'position, and can also be the same as the purple The 7-position hydroxyl group of the taxane reacts. Those skilled in the art know that the 2'-position and 7-position hydroxyl groups have certain nucleophilicity, and both can realize the substitution reaction with the acid chloride obtained after the one-step reaction, and found in experiments The 2'-hydroxyl has a very good regioselectivity, which is consistent with literature reports. If you want to combine the 7 position with choline, you can protect the 2'position hydroxyl group with a silyl etherification reagent, and then perform the subsequent substitution reaction.

本发明所述的紫杉烷类化合物不限于紫杉醇、多西紫杉醇、卡巴他赛，本领域技术人员都了解紫杉烷类化合物有共同的母核骨架结构，具有相类似的抗肿瘤作用，并且均有2’位和7位羟基与胆碱或甜菜碱进行反应，有相应的反应条件和结果。The taxane compounds of the present invention are not limited to paclitaxel, docetaxel, and cabazitaxel. Those skilled in the art know that taxane compounds have a common core skeleton structure and have similar anti-tumor effects, and Both 2'and 7 hydroxyls react with choline or betaine, and there are corresponding reaction conditions and results.

本发明所述的化合物的连接基团不限于碳酸酯键，也可以是饱和的碳链，芳环，磺酰基、磷酸酯、二酸酯和醚键等。从化学角度易于连接和和肿瘤羧酸酯水解酶酶高表达的易于断开考虑，碳酸酯、酰基酯键是最好的选择。The linking group of the compound described in the present invention is not limited to carbonate bonds, but may also be saturated carbon chains, aromatic rings, sulfonyl groups, phosphate esters, diacid esters, and ether bonds. From a chemical point of view, it is easy to connect and easily disconnect from the high expression of tumor carboxylate hydrolase enzyme. Carbonate and acyl ester bonds are the best choice.

优选的化合物的制备如下：The preparation of the preferred compound is as follows:

本发明技术方案的第三方面是提供了一种药物组合物，其特征在于，含有治疗有效量的本发明技术方案第一方面所述的紫杉烷衍生物及其药学上可接受的盐以及药学上可接受的载体。The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, characterized in that it contains a therapeutically effective amount of the taxane derivative and the pharmaceutically acceptable salt thereof according to the first aspect of the technical solution of the present invention, and A pharmaceutically acceptable carrier.

优选的组合物含有作为活性成分的化合物A、B、C、D、E、F、G或其他胆碱化修饰的紫杉烷类化合物、及其药学上可接受的盐以及药学上可接受的载体或赋形剂。A preferred composition contains compounds A, B, C, D, E, F, G or other choline-modified taxanes as active ingredients, and pharmaceutically acceptable salts and pharmaceutically acceptable Carrier or excipient.

该类化合物可根据本领域公知的方法制备不同的非肠道给药剂型，适于人或动物使用。例如，将本发明化合物制成注射用制剂，如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和多种药效学上可接受的载体、稀释剂、防腐剂、表面活性剂、助溶剂、缓冲剂、pH调节剂。这些辅料都是本领域常用的。为达到用药目的，增强治疗效果，本发明药物或药物组合物可用任何公知的给药方法给药。Such compounds can be prepared in different parenteral dosage forms according to methods known in the art, and are suitable for human or animal use. For example, the compound of the present invention is prepared into injection preparations, such as solutions, suspension solutions, emulsions, and lyophilized powder injections. Such preparations may be aqueous or non-aqueous, and may contain one or more pharmacodynamics. Acceptable carriers, diluents, preservatives, surfactants, solubilizers, buffers, and pH regulators. These auxiliary materials are commonly used in this field. In order to achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

本发明的组合物可单独服用，或与其他治疗药物或对症药物合并使用，当本发明的化合物与其他治疗药物存在协同作用时，应根据实际情况调整它的剂量。The composition of the present invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

本发明技术方案的第四方面是提供了第一方面所述的季铵化修饰紫杉烷衍生物及药学上可接受的盐或第三方面所述的药物组合物在制备抗肿瘤药物中的用途，所述的肿瘤包括肺癌、乳腺癌、卵巢癌、肝癌、结肠癌、胃癌等。The fourth aspect of the technical solution of the present invention is to provide the quaternized modified taxane derivative and pharmaceutically acceptable salt of the first aspect or the pharmaceutical composition of the third aspect in the preparation of anti-tumor drugs Uses, said tumors include lung cancer, breast cancer, ovarian cancer, liver cancer, colon cancer, gastric cancer and the like.

有益技术效果：Beneficial technical effects:

季铵化修饰紫杉烷衍生物是基于前药修饰策略改造的靶向抗肿瘤药物。Quaternized modified taxane derivatives are targeted antitumor drugs modified based on prodrug modification strategies.

本发明基于前药修饰策略，将胆碱，或取代胆碱，或甜菜碱，或取代甜菜碱，与紫杉烷类化合物结合，不仅提高了紫杉醇的水溶性(如化合物A在水中的溶解度是3.66mg/ml，相比紫杉醇的0.004mg/mL提高了915倍)。此外，胆碱和甜菜碱是体内常见的内源性代谢物，其作为修饰分子具有许多独特的优点，如相对分子量小、无免疫原性、廉价易得、稳定性好、与药物分子之间化学键连接简单易行，靶向应用范围广泛等。The present invention is based on the prodrug modification strategy, combining choline, or replacing choline, or betaine, or replacing betaine with taxane compounds, which not only improves the water solubility of paclitaxel (for example, the solubility of compound A in water is 3.66mg/ml, which is 915 times higher than the 0.004mg/mL paclitaxel). In addition, choline and betaine are common endogenous metabolites in the body, and they have many unique advantages as modified molecules, such as small relative molecular weight, no immunogenicity, cheap and easy to obtain, good stability, and compatibility with drug molecules. The chemical bond connection is simple and easy, and the target application range is wide.

该类紫杉醇衍生物使用碳酸酯键、磷酸酯键和二酸酯键等作为连接部分，将靶向分子胆碱，或甜菜碱，或其类似物和抗肿瘤药物紫杉醇、多西紫杉醇、卡巴他赛部分的自由羟基偶联，得到季铵化修饰的紫杉烷衍生物。本发明提供的季铵化修饰紫杉烷类衍生物，因修饰的位点是紫杉醇的活性部位，使该化合物的细胞毒性明显降低。由于肿瘤细胞对胆碱的吸收利用远大于正常细胞，从而使该衍生物能被肿瘤细胞主动摄取，在肿瘤部位聚集。在肿瘤组织区域，尤其是恶性增殖的肿瘤组织区域，往往会有羧酸酯酶的更高表达，因而在酶的作用下衍生物进一步水解释放紫杉醇，进而到达抗肿瘤的效果同时减弱对正常器官的毒性，因而可以制成更低毒和有效的抗肿瘤药物。本发明提供的季铵化修饰紫杉烷类衍生物及其抗肿瘤用途是本发明人的创新，首次公开发表。This kind of paclitaxel derivatives use carbonate bonds, phosphate bonds and diacid ester bonds as the connecting part, and will target the molecule choline, or betaine, or its analogues and anti-tumor drugs paclitaxel, docetaxel, carbata The free hydroxyl group of the race part is coupled to obtain a quaternized modified taxane derivative. The quaternized modified taxane derivatives provided by the present invention, because the modified site is the active site of paclitaxel, the cytotoxicity of the compound is significantly reduced. Because the absorption and utilization of choline by tumor cells is much greater than that of normal cells, the derivative can be actively taken up by tumor cells and accumulate at the tumor site. In the tumor tissue area, especially the tumor tissue area with malignant proliferation, there is often higher expression of carboxylesterase. Therefore, under the action of the enzyme, the derivative is further hydrolyzed to release paclitaxel, thereby achieving the anti-tumor effect and weakening the effect on normal organs. Therefore, it can be made into less toxic and effective anti-tumor drugs. The quaternized modified taxane derivatives and their anti-tumor use provided by the present invention are innovations of the present inventors and published for the first time.

附图说明Description of the drawings

图1紫杉醇衍生物A在水中的溶解性Figure 1 Solubility of paclitaxel derivative A in water

图2羧酸酯酶抑制剂(BNPP)对紫杉醇衍生物A在血浆中代谢的作用Figure 2 The effect of carboxylesterase inhibitor (BNPP) on the metabolism of paclitaxel derivative A in plasma

图3胆碱转运体抑制剂(HC-3)对肺癌A549细胞摄取紫杉醇衍生物A的作用Figure 3 The effect of choline transporter inhibitor (HC-3) on the uptake of paclitaxel derivative A by lung cancer A549 cells

图4紫杉醇衍生物A对小鼠C26结肠癌的生长抑制作用Figure 4 Paclitaxel derivative A inhibits the growth of mouse C26 colon cancer

图5紫杉醇衍生物A对C26结肠癌小鼠免疫***的影响Figure 5 The effect of paclitaxel derivative A on the immune system of C26 colon cancer mice

图6紫杉醇衍生物A对小鼠EMT6乳腺癌的生长抑制作用Figure 6 Paclitaxel derivative A inhibits the growth of mouse EMT6 breast cancer

图7紫杉醇衍生物A对EMT6乳腺癌小鼠免疫***的影响Figure 7 The effect of paclitaxel derivative A on the immune system of EMT6 breast cancer mice

图8紫杉醇衍生物A对小鼠Lewis肺癌的生长抑制作用Figure 8 Paclitaxel derivative A inhibits growth of Lewis lung cancer in mice

图9紫杉醇衍生物A和紫杉醇衍生物F对小鼠Lewis肺癌的生长抑制作用Figure 9 Paclitaxel derivative A and paclitaxel derivative F inhibit growth of Lewis lung cancer in mice

图10紫杉醇衍生物A(前药)在整体动物(A)及肿瘤组织(B)的成像分布图；Figure 10 The imaging distribution map of paclitaxel derivative A (prodrug) in whole animals (A) and tumor tissues (B);

图11三个不同治疗组(PTX injection，紫杉醇注射液；紫杉醇脂质体注射液，PTX-Liposomes，PTX-R，紫杉醇衍生物A在整体动物(A)及肿瘤组织(B)的成像分布图；紫杉醇衍生物A(PTX-R)在不同组织器官的分布情况统计(C)；三个不同治疗组中在不同组织器官紫杉醇暴露情况统计(D)；紫杉醇衍生物A与紫杉醇脂质体注射液(PTX-Liposomes)的靶向性比较分析(E)Figure 11 The imaging distribution of three different treatment groups (PTX injection, paclitaxel injection; paclitaxel liposome injection, PTX-Liposomes, PTX-R, paclitaxel derivative A in whole animals (A) and tumor tissues (B) Statistics of the distribution of paclitaxel derivative A (PTX-R) in different tissues and organs (C); statistics of paclitaxel exposure in different tissues and organs in three different treatment groups (D); Paclitaxel derivative A and paclitaxel liposome injection Comparative analysis of targeting of PTX-Liposomes (E)

具体实施方式detailed description

缩写词：abbreviation:

TEA：三乙胺TEA: Triethylamine

DCM：二氯甲烷DCM: Dichloromethane

本发明公开了一类季铵化修饰的紫杉烷衍生物，含有它们的盐、溶剂合物、前药与药物组合物的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The present invention discloses a class of quaternary ammonium modified taxane derivatives, containing their salts, solvates, prodrugs, and applications of pharmaceutical compositions. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

下面结合具体实施方式，进一步阐述本发明：In the following, the present invention will be further explained in combination with specific embodiments:

实施例1Example 1

胆碱化紫杉醇衍生物A的合成路线Synthetic route of cholined taxol derivative A

步骤1 2-((氯甲酰基)氧基)-N,N,N-三甲基-1-铵的制备 Step 1 Preparation of 2-((chloroformyl)oxy)-N,N,N-trimethyl-1-ammonium

在2-羟基-N,N,N-三甲基-1-铵(10.0g，96.15mmol)的二氯甲烷(150ml)溶液中加入三乙醇胺(24.2g，240mmol)。在0℃和N ₂的条件下，加入碳酸三氯甲基酯(14.23g，48.075mmol)的二氯甲烷溶液(100ml)，搅拌45分钟进行充分的反应。浓缩混合物，不经进一步纯化直接使用。 Triethanolamine (24.2g, 240mmol) was added to a solution of 2-hydroxy-N,N,N-trimethyl-1-ammonium (10.0g, 96.15mmol) in dichloromethane (150ml). Under the conditions of 0° C. and N ₂ , a dichloromethane solution (100 ml) of trichloromethyl carbonate (14.23 g, 48.075 mmol) was added, and the mixture was stirred for 45 minutes for a sufficient reaction. The mixture was concentrated and used without further purification.

步骤2胆碱化紫杉醇衍生物(紫杉醇衍生物A)的制备Step 2 Preparation of Cholined Taxol Derivative (Paclitaxel Derivative A)

在0℃条件下在2-((氯甲酰基)氧基)-N,N,N-三甲基-1-铵(348mg，2.1mmol)的二氯甲烷溶液(10ml)溶液中加入紫杉醇(1.8g，2.1 mmol)。在N ₂条件下，将混合物在室温下搅拌12小时，反应完成后，过滤混合物。浓缩滤液，通过制备型HPLC纯化，得到500mg白色固体。(本发明中，紫杉醇衍生物A即为紫杉醇衍生物PTX-R) ¹H NMR(400MHz,DMSO-d6)δppm:9.87(s,1H),8.55(s,0.56H),8.02-7.93(m,4H),7.79-7.73(m,1H),7.69(t,J＝7.4Hz,2H),7.56(t,J＝7.3Hz,1H),7.53-7.43(m,6H),7.17(ddd,J＝8.5,5.9,2.6Hz,1H),6.29(s,1H),5.83(t,J＝8.8Hz,1H),5.48(t,J＝7.0Hz,2H),5.41(d,J＝7.2Hz,1H),4.90(d,J＝10.1Hz,2H),4.62(s,3H),4.09(dd,J＝10.7,6.8Hz,1H),4.00(q,J＝8.4Hz,2H),3.81(dt,J＝9.2,4.2Hz,1H),3.70(dt,J＝8.8,4.2Hz,1H),3.55(d,J＝7.2Hz,1H),3.12(s,9H),2.31(dt,J＝21.5,7.8Hz,1H),2.21(s,3H),2.15-2.07(m,3H),1.81(d,J＝11.4Hz,3H),1.70(ddd,J＝34.0,20.5,9.3Hz,2H),1.49(s,3H),1.40(dd,J＝15.5,9.1Hz,1H),1.12-0.94(m,6H). Add paclitaxel (((chloroformyl)oxy)-N,N,N-trimethyl-1-ammonium (348mg, 2.1mmol) in dichloromethane solution (10ml) at 0℃ 1.8g, 2.1 mmol). Under N ₂ conditions, the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was filtered. The filtrate was concentrated and purified by preparative HPLC to obtain 500 mg of white solid. (In the present invention, paclitaxel derivative A is paclitaxel derivative PTX-R) ¹ H NMR (400MHz, DMSO-d6) δppm: 9.87 (s, 1H), 8.55 (s, 0.56H), 8.02-7.93 (m ,4H),7.79-7.73(m,1H),7.69(t,J=7.4Hz,2H),7.56(t,J=7.3Hz,1H),7.53-7.43(m,6H),7.17(ddd, J = 8.5, 5.9, 2.6 Hz, 1H), 6.29 (s, 1H), 5.83 (t, J = 8.8 Hz, 1H), 5.48 (t, J = 7.0 Hz, 2H), 5.41 (d, J = 7.2 Hz, 1H), 4.90 (d, J = 10.1 Hz, 2H), 4.62 (s, 3H), 4.09 (dd, J = 10.7, 6.8 Hz, 1H), 4.00 (q, J = 8.4 Hz, 2H), 3.81(dt,J=9.2,4.2Hz,1H),3.70(dt,J=8.8,4.2Hz,1H),3.55(d,J=7.2Hz,1H),3.12(s,9H),2.31(dt ,J=21.5,7.8Hz,1H),2.21(s,3H),2.15-2.07(m,3H),1.81(d,J=11.4Hz,3H),1.70(ddd,J=34.0,20.5,9.3 Hz, 2H), 1.49 (s, 3H), 1.40 (dd, J = 15.5, 9.1 Hz, 1H), 1.12 to 0.94 (m, 6H).

实施例2Example 2

甜菜碱化紫杉烷衍生物E的合成路线Synthetic route of betaineated taxane derivative E

实验过程experiment procedure

步骤AStep A

N-2-氯羰基甲基-N，N，N-三甲基氯化铵(2)N-2-chlorocarbonylmethyl-N,N,N-trimethylammonium chloride (2)

将干燥好的甜菜碱盐酸盐加入到(450mg，3.1mmol)加入到氯化亚砜(0.6mL，8mmol)中并加热至75℃。停止放气之后得到黄色粘稠液体。加入热甲苯(80℃，3mL)，充分搅拌溶液，倒出上层甲苯相。为了除去氯化亚砜，重复该过程六次，最后的甲苯悬浮液用于下一步反应。The dried betaine hydrochloride (450 mg, 3.1 mmol) was added to thionyl chloride (0.6 mL, 8 mmol) and heated to 75°C. After stopping gassing, a yellow viscous liquid was obtained. Add hot toluene (80°C, 3 mL), stir the solution thoroughly, and pour the upper toluene phase. To remove thionyl chloride, the process was repeated six times, and the final toluene suspension was used in the next reaction.

步骤BStep B

(2aR,4aS,6R,9S,11S,12S,12bS)-12b-乙酰氧基-9-(((2R)-3-氨基-2-羟基-3-苯基丙酰基)氧基)-4,6,11三羟基-4a，8,13,13-四甲基 -5-氧代-2a,3,4,4a，5,6,9,10,11,12,12a,12b-十二-1氢-7,11-甲环癸[3-4]苯并[1,2-b]氧杂-12-苯甲酸酯(4)：(2aR,4aS,6R,9S,11S,12S,12bS)-12b-acetoxy-9-(((2R)-3-amino-2-hydroxy-3-phenylpropionyl)oxy)-4 ,6,11Trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-twelve -1Hydro-7,11-methylcyclodecane[3-4]benzo[1,2-b]oxa-12-benzoate (4):

将(2aR,4aS,6R,9S,11S,12S,12bS)-12b乙酰氧基-9-(((2R)-3-((叔丁氧基羰基)氨基)-2-羟基-3-苯基丙酰基)氧基)-4,6,11三羟基甲基-4a，8,13,13四甲基-5-氧代-2a,3,4,4a,5,6,9,10,11,12,12a,12b-十二-1氢-7,11-甲环癸[3,4]苯并[1,2-b]氧杂-12-苯甲酸酯(2.34g,2.9mmol)溶解在25mL的浓甲酸中，将溶液在室温下搅拌4小时。接下来，减压蒸馏出甲酸，得到(2aR,4aS,6R,9S,11S,12S,12bS)-12b乙酰氧基-9-((2R)-3-氨基-2-羟基-3-苯基丙酰基)氧基)-4,6,11-三羟基-4a,8,13,13四甲基-5-氧代-2a,3,4,4a,5,6,9,10,11,12,12a,12b-十二-1氢-7,11-甲环癸[3-,4]苯并[1,2-b]氧杂-12-苯甲酸酯2.35g白色固体。Add (2aR, 4aS, 6R, 9S, 11S, 12S, 12bS)-12b acetoxy-9-(((2R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-3-benzene Propionyl)oxy)-4,6,11trihydroxymethyl-4a,8,13,13tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10, 11,12,12a,12b-Dodeca-1 hydrogen-7,11-methylcyclodecane[3,4]benzo[1,2-b]oxa-12-benzoate (2.34g,2.9mmol ) Was dissolved in 25 mL of concentrated formic acid, and the solution was stirred at room temperature for 4 hours. Next, the formic acid is distilled off under reduced pressure to obtain (2aR, 4aS, 6R, 9S, 11S, 12S, 12bS)-12b acetoxy-9-((2R)-3-amino-2-hydroxy-3-phenyl Propionyl)oxy)-4,6,11-trihydroxy-4a,8,13,13 tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11, 12,12a,12b-Dodeca-1hydro-7,11-methylcyclodecane[3-,4]benzo[1,2-b]oxa-12-benzoate 2.35g white solid.

步骤CStep C

在氮气冰浴的条件下，向N-2-氯羰基甲基-N，N，N-三甲基氯化铵(2.35g，粗品)的二氯甲烷溶液(10mL)中加入(2aR,4aS,6R,9S,11S,12S,12bS)-12b乙酰氧基-9-((2R)-3-氨基-2-羟基-3-苯基丙酰基)氧基)-4,6,11-三羟基-4a,8,13,13四甲基-5-氧代-2a,3,4,4a,5,6,9,10,11,12,12a,12b-十二-1氢-7,11-甲环癸[3-,4]苯并[1,2-b]氧杂-12-苯甲酸酯，和4-(二甲氨基)吡啶(878mg,8.7mmol)，将混合物在室温下搅拌12小时后过滤混合物，浓缩滤液，通过制备型HPLC纯化，得到所需产物400mg白色固体。Under nitrogen ice bath, to the dichloromethane solution (10mL) of N-2-chlorocarbonylmethyl-N,N,N-trimethylammonium chloride (2.35g, crude product) was added (2aR,4aS ,6R,9S,11S,12S,12bS)-12b acetoxy-9-((2R)-3-amino-2-hydroxy-3-phenylpropionyl)oxy)-4,6,11-tri Hydroxy-4a,8,13,13tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodeca-1 hydrogen-7, 11-Methylcyclodecane[3-,4]benzo[1,2-b]oxa-12-benzoate, and 4-(dimethylamino)pyridine (878mg, 8.7mmol), the mixture was kept at room temperature After stirring for 12 hours, the mixture was filtered, and the filtrate was concentrated and purified by preparative HPLC to obtain 400 mg of the desired product as a white solid.

质谱(ESI)m/z＝807.3(M+).Mass spectrum (ESI) m/z=807.3 (M+).

1H NMR(400MHz,DMSO-d6):δ9.30(d,J＝8.9Hz,1H),7.99(d,J＝7.4Hz,2H),7.70(t,J＝7.3Hz,1H),7.61(t,J＝7.6Hz,2H),7.45-7.33(m,4H),7.28(t,J＝7.2Hz,1H),6.29(d,J＝5.0Hz,1H),5.92(t,J＝8.9Hz,1H),5.43(d,J＝7.2Hz,1H),5.32(dd,J＝8.8,5.6Hz,1H),5.10(s,1H),5.07-4.85(m,3H),4.57(s,1H),4.44(s,1H),4.17(t,J＝9.1Hz,2H),4.03(q,J＝8.3Hz,3H),3.69(d,J＝7.1Hz,1H),3.17(s,11H),2.37-2.20(m,5H),2.01(dd,J＝15.2,9.4Hz,1H),1.83(dd,J＝15.2,8.9Hz,1H),1.78-1.61(m,5H),1.51(d,J＝14.4Hz,3H),1.07-0.95(m,6H).1H NMR(400MHz,DMSO-d6):δ9.30(d,J=8.9Hz,1H),7.99(d,J=7.4Hz,2H),7.70(t,J=7.3Hz,1H), 7.61( t,J=7.6Hz,2H),7.45-7.33(m,4H),7.28(t,J=7.2Hz,1H),6.29(d,J=5.0Hz,1H),5.92(t,J=8.9 Hz, 1H), 5.43 (d, J = 7.2 Hz, 1H), 5.32 (dd, J = 8.8, 5.6 Hz, 1H), 5.10 (s, 1H), 5.07-4.85 (m, 3H), 4.57 (s ,1H),4.44(s,1H),4.17(t,J=9.1Hz,2H),4.03(q,J=8.3Hz,3H), 3.69(d,J=7.1Hz,1H), 3.17(s ,11H),2.37-2.20(m,5H),2.01(dd,J=15.2,9.4Hz,1H),1.83(dd,J=15.2,8.9Hz,1H),1.78-1.61(m,5H), 1.51(d,J=14.4Hz,3H),1.07-0.95(m,6H).

实施例3Example 3

丁二酸酯连接胆碱化紫杉烷衍生物F(PTX-SA-R)的合成路线Synthetic route of succinate-linked cholined taxane derivative F (PTX-SA-R)

步骤A:Step A:

2-(3-羧基丙酰基)酯乙基-N,N,N-三甲基，1-氯化铵(3)2-(3-carboxypropionyl) ester ethyl-N,N,N-trimethyl, 1-ammonium chloride (3)

盐酸胆碱(2.0g,14.3mmol)和丁二酸酐(7.15g,71.5mmol)混合物在150℃的35mL高压釜中搅拌反应3h.冷却到室温，反应混合物用丙酮/***(9/1,v/v)(150mL x 2)清洗两次，除去过剩的酸酐。固体溶解于60℃甲醇,后缓慢加入***结晶。室温下静置17小时。过滤，乙酸乙酯清洗后真空干燥获得化合物3。The mixture of choline hydrochloride (2.0g, 14.3mmol) and succinic anhydride (7.15g, 71.5mmol) was stirred and reacted in a 35mL autoclave at 150°C for 3h. After cooling to room temperature, the reaction mixture was mixed with acetone/ether (9/1, v /v) (150mL x 2) Wash twice to remove excess acid anhydride. The solid was dissolved in methanol at 60℃, and ether was slowly added to crystallize. Let stand at room temperature for 17 hours. Filtered, washed with ethyl acetate and dried in vacuum to obtain compound 3.

质谱(ESI)m/z＝204.1(M+H ⁺). Mass spectrum (ESI) m/z=204.1 (M+H ⁺ ).

步骤B:Step B:

2-(4-氯-4-酰基丁酰)酯乙基-N,N,N-三甲基，1-氯化铵(4)2-(4-Chloro-4-acylbutyryl) ester ethyl-N,N,N-trimethyl, 1-ammonium chloride (4)

化合物3(2.0g,8.3mmol)在室温下搅拌2h溶解于10ml二氯亚砜.真空干燥出去溶剂得到化合物4粗品(2.2g)，无纯化，直接用于后续反应.Compound 3 (2.0g, 8.3mmol) was stirred at room temperature for 2h and dissolved in 10ml of thionyl chloride. The solvent was dried under vacuum to obtain crude compound 4 (2.2g), which was used directly in subsequent reactions without purification.

质谱(ESI)m/z＝218.1(M+H ⁺). Mass spectrum (ESI) m/z=218.1 (M+H ⁺ ).

步骤C:Step C:

目标化合物F(PTX-SA-CH)Target compound F (PTX-SA-CH)

化合物4(1.24g,4.8mmol)溶解于40ml DCM，然后在0℃和氮气保护下加紫杉醇(4.12g,4.8mmol)和DMAP(2.34g,19.2mmol).混合物在室温下搅拌反应12h.反应结束后过滤，滤液浓缩后经制备色谱获得终产物PTX-SA-CH的白色固体(2.1g,42％产率).Compound 4 (1.24g, 4.8mmol) was dissolved in 40ml of DCM, and then paclitaxel (4.12g, 4.8mmol) and DMAP (2.34g, 19.2mmol) were added under the protection of nitrogen at 0°C. The mixture was stirred at room temperature for 12h. Reaction After the completion of filtration, the filtrate is concentrated and subjected to preparative chromatography to obtain the final product PTX-SA-CH as a white solid (2.1g, 42% yield).

质谱(ESI)m/z＝1039.4(M+H ⁺). Mass spectrum (ESI) m/z=1039.4 (M+H ⁺ ).

¹H NMR(400MHz,DMSO-d6)δ9.56(brs,1H),8.50-8.49(m,1H),7.99-7.97(m,2H),7.91-7.88(m,2H),7.77-7.73(m,1H),7.70-7.66(m,2H),7.59-7.56(m,1H),7.52-7.48(m,2H),7.46(d,J＝4.8Hz,4H),7.19-7.15(m,1H),6.28(s,1H),5.80(t,J＝9.2Hz,1H),5.51(t,J＝8.8Hz,1H),5.42-5.35(m,2H),4.91(d,J＝12.0Hz,1H),4.66(brs,1H),4.41(s,2H),4.11-4.07(m,1H),4.03-3.98(m,2H),3.65(s,3H),3.55(d,J＝7.2Hz,2H),3.12(s,9H),2.73-2.70(m,2H),2.65-2.62(m,2H),2.54(s,1H),2.35-2.27(m,1H),2.22(s,3H),2.11(s,3H),1.76(s,3H), 1.74-1.61(m,2H),1.50(s,3H),1.46-1.40(m,1H),1.03(m,3H),0.99(s,3H). ¹ H NMR (400MHz, DMSO-d6) δ9.56 (brs, 1H), 8.50-8.49 (m, 1H), 7.99-7.97 (m, 2H), 7.91-7.88 (m, 2H), 7.77-7.73 ( m,1H),7.70-7.66(m,2H),7.59-7.56(m,1H),7.52-7.48(m,2H),7.46(d,J=4.8Hz,4H),7.19-7.15(m, 1H), 6.28 (s, 1H), 5.80 (t, J = 9.2 Hz, 1H), 5.51 (t, J = 8.8 Hz, 1H), 5.42-5.35 (m, 2H), 4.91 (d, J = 12.0 Hz, 1H), 4.66 (brs, 1H), 4.41 (s, 2H), 4.11-4.07 (m, 1H), 4.03-3.98 (m, 2H), 3.65 (s, 3H), 3.55 (d, J = 7.2Hz, 2H), 3.12(s, 9H), 2.73-2.70(m, 2H), 2.65-2.62(m, 2H), 2.54(s, 1H), 2.35-2.27(m, 1H), 2.22(s ,3H),2.11(s,3H),1.76(s,3H), 1.74-1.61(m,2H),1.50(s,3H),1.46-1.40(m,1H),1.03(m,3H), 0.99(s, 3H).

体外实验In vitro experiments

实验例1紫杉醇衍生物A在水中的溶解度Experimental example 1 Solubility of paclitaxel derivative A in water

取紫杉醇衍生物A适量(使适当过量)置于10mL棕色储液瓶中，加适量纯水，涡旋混合30s置于超声仪中进行超声，分别于5，15，30min，1，2，4，6h取出一定体积的液体经孔径为0.45μm微孔滤膜滤过，其续滤液经稀释10000倍后，通过LC-MS/MS方法测定紫杉醇衍生物的浓度，绘制紫杉醇衍生物的溶解平衡曲线。结果如图1所示，紫杉醇衍生物经超声1h后达到溶解平衡，其平衡溶解度为3.66mg/mL。据文献报道，紫杉醇本身的溶解度很差，其在水中的溶解度小于0.004mg/mL，与其相比，紫杉醇衍生物溶解度提高倍数大约为1000倍。Take an appropriate amount of paclitaxel derivative A (make an appropriate excess) and place it in a 10mL brown storage bottle, add an appropriate amount of pure water, vortex and mix for 30s and place it in an ultrasonic instrument for sonication, respectively for 5, 15, 30 min, 1, 2, 4 After 6 hours, a certain volume of liquid was taken out and filtered through a 0.45μm microporous membrane. After the subsequent filtrate was diluted 10,000 times, the concentration of paclitaxel derivatives was determined by LC-MS/MS method, and the dissolution equilibrium curve of paclitaxel derivatives was drawn . The results are shown in Figure 1. The paclitaxel derivative reached a dissolution equilibrium after being sonicated for 1 h, and its equilibrium solubility was 3.66 mg/mL. According to reports in the literature, the solubility of paclitaxel itself is very poor, and its solubility in water is less than 0.004mg/mL. Compared with it, the solubility of paclitaxel derivatives is increased by about 1000 times.

实验例2紫杉醇衍生物A和紫杉醇衍生物F的注射液制备称取适量紫杉醇衍生物A和紫杉醇衍生物F，加入所需药液体积5％的DMSO完全溶解药物，再加入所需药液体积5％的95％乙醇，涡旋30s混合均匀，加入所需剩余体积的生理盐水，超声5min即可配置完成紫杉醇衍生物注射液。Experimental Example 2 Preparation of Paclitaxel Derivative A and Paclitaxel Derivative F Injection Weigh an appropriate amount of Paclitaxel Derivative A and Paclitaxel Derivative F, add 5% of the required liquid volume of DMSO to completely dissolve the drug, and then add the required liquid volume Mix well with 5% 95% ethanol, vortex for 30s, add the required remaining volume of normal saline, and ultrasound for 5 minutes to complete the paclitaxel derivative injection.

实验例3紫杉醇衍生物A的特异性羧酸酯酶水解释放紫杉醇本实验目的是通过羧酸酯酶抑制剂考察该酶对紫杉醇衍生物A代谢的影响。在90uL SD大鼠血浆中分别加入10uL的500mM羧酸酯酶抑制剂(双-P-硝基苯基磷酸盐，BNPP)和溶剂作为抑制剂组和对照组，涡旋混匀，置于37℃水浴槽中孵育10min来抑制羧酸酯酶蛋白的活性,两组分别均加入紫杉醇衍生物A，涡旋混匀，置于37℃水浴槽中分别孵育5,15,30min,1,2,4,8h,每个时间点重复三个样本，孵育结束后将样本置于冰浴中，加入300uL冰甲醇(0.1％甲酸)，涡旋3min，4℃下12000rpm离心10min，分取上清液吹干，加入100μL的复溶溶剂乙腈/水(90:10,v/v)进行复溶，涡旋1min，4℃下12000rpm离心5min，分取上清液，进行LC-MS/MS分析。结果如图2所示，BNPP能显著抑制紫杉醇衍生物A在血浆中的代谢。Experimental Example 3 The specific carboxylesterase hydrolysis of paclitaxel derivative A releases paclitaxel. The purpose of this experiment is to investigate the effect of this enzyme on the metabolism of paclitaxel derivative A through a carboxylesterase inhibitor. Add 10uL of 500mM carboxylesterase inhibitor (bis-P-nitrophenyl phosphate, BNPP) and solvent to 90uL SD rat plasma as the inhibitor group and the control group, vortex and mix well, place 37 Incubate in a water bath at ℃ for 10 minutes to inhibit the activity of carboxylesterase protein. Paclitaxel derivative A was added to the two groups respectively, vortexed to mix, and incubated in a water bath at 37℃ for 5, 15, 30 min, 1, 2, 4,8h, repeat three samples at each time point. After incubation, place the samples in an ice bath, add 300uL ice methanol (0.1% formic acid), vortex for 3min, centrifuge at 12000rpm at 4℃ for 10min, and divide the supernatant Blow dry, add 100 μL of reconstituted solvent acetonitrile/water (90:10, v/v) for reconstitution, vortex for 1 min, centrifuge at 12000 rpm at 4°C for 5 min, divide the supernatant, and perform LC-MS/MS analysis. The results are shown in Figure 2, BNPP can significantly inhibit the metabolism of paclitaxel derivative A in plasma.

实验例4紫杉醇衍生物A的肿瘤细胞特性摄取实验Experimental Example 4 Tumor cell characteristic uptake experiment of paclitaxel derivative A

本实验目的是通过胆碱转运体抑制剂考察胆碱转运体对细胞摄取紫杉醇衍生物A的影响。将肺癌A549细胞以2*10 ⁵密度均匀种在6孔板内，培养箱内培养约48小时。将6孔板置于37℃水浴槽中，前三个孔弃去培养基，以37℃PBS(每孔2mL)清洗一次弃去，再加37℃PBS温育10min(每孔2mL)，弃去PBS，加紫杉醇衍生物A1mL，5min后弃去药物，加入冷PBS(每孔2mL)清洗两次，弃去PBS，每孔加1ml升冰甲醇(0.1％甲酸)盖好盖子静置30min，将甲醇及其基质完全转移至EP管。后三个孔按上述操作，加入药物和胆碱转运体抑制剂(HC-3,100)混合物，进行相应的处理。将收集的含有基质的甲醇溶液于4℃下12000rpm离心10min，分取上清液吹干，加入100μL的复溶溶剂乙腈/水(90:10,v/v)进行复溶，涡旋1min，4℃下12000rpm离心5min，分取上清液，进行LC-MS/MS分析。结果如图3所示，肺癌A549细胞在一定程度上通过胆碱转运体对紫杉醇衍生物A进行主动运输。 The purpose of this experiment is to investigate the effect of choline transporter on the uptake of paclitaxel derivative A by cells through choline transporter inhibitors. Lung cancer A549 cells at a density of 2 * ¹⁰⁵ evenly grown in 6-well plates, culture incubator for about 48 hours. Place the 6-well plate in a 37°C water bath, discard the culture medium in the first three wells, wash it once with 37°C PBS (2 mL per well), add 37°C PBS and incubate for 10 min (2 mL per well), discard Remove PBS, add 1mL of paclitaxel derivative A, 5min later, discard the drug, add cold PBS (2mL per well) to wash twice, discard PBS, add 1ml of ice methanol (0.1% formic acid) to each well, cover the lid and let stand for 30min. Transfer methanol and its matrix completely to the EP tube. Add the mixture of drugs and choline transporter inhibitor (HC-3,100) to the last three holes according to the above operation, and perform corresponding treatments. Centrifuge the collected methanol solution containing the matrix at 12000 rpm at 4°C for 10 min, aliquot the supernatant and blow dry, add 100 μL of the reconstitution solvent acetonitrile/water (90:10, v/v) for reconstitution, vortex for 1 min, Centrifuge at 12000 rpm at 4°C for 5 min, and aliquot the supernatant for LC-MS/MS analysis. The results are shown in Figure 3, lung cancer A549 cells actively transport paclitaxel derivative A to a certain extent through choline transporter.

毒理实验Toxicology Experiment

实验例1紫杉醇衍生物A(PTX-R)大鼠2周重复给药毒性研究Experimental Example 1 Toxicity Study of Paclitaxel Derivative A (PTX-R) Repeated Dosing in Rats for 2 Weeks

本试验目的是通过SD大鼠分别静脉注射反复给予PTX-R及紫杉醇注射液(PTX)，观察SD大鼠可能出现的毒性反应性质和程度，毒性反应的发展和恢复情况；确定毒性剂量效应关系；确定毒性作用的靶器官；同时比较PTX-R与PTX的毒性情况。The purpose of this test is to repeatedly administer PTX-R and paclitaxel injection (PTX) to SD rats by intravenous injection respectively, to observe the nature and extent of toxic reactions that may occur in SD rats, the development and recovery of toxic reactions; to determine the toxic dose-effect relationship ; Determine the target organ for toxicity; compare the toxicity of PTX-R and PTX at the same time.

本试验设溶媒对照组(15％磺丁基-β-环糊精溶液)、PTX-R低(6mg/kg)、高剂量组(12mg/kg)和PTX组(5mg/kg)。每组12只，雌雄各半。采用尾静脉注射给药，每3天给药1次，共给药4次。给药结束及恢复期结束时每组每性别剖杀3只，检测指标包括动物一般状况、体重、血液常规、血清生化、电解质、凝血指标、尿指标、大体解剖等。This experiment set up a solvent control group (15% sulfobutyl-β-cyclodextrin solution), low PTX-R (6mg/kg), high dose group (12mg/kg) and PTX group (5mg/kg). There are 12 animals in each group, half male and half female. It was administered by tail vein injection, once every 3 days, for a total of 4 administrations. At the end of the administration and the end of the recovery period, 3 animals of each gender were dissected in each group. The detection indicators included general animal condition, body weight, blood routine, serum biochemistry, electrolytes, coagulation indicators, urine indicators, gross anatomy, etc.

1.一般生理指标观察1. Observation of general physiological indicators

1.1笼旁观察1.1 Observation beside the cage

本试验过程中，所有受试动物均未见明显毒性反应，无动物死亡。During the experiment, all the tested animals had no obvious toxic reaction and no animal died.

1.2体重1.2 Weight

整个试验期间，所有动物体重均逐渐增加。但与溶媒对照组相比，PTX给药组雌雄鼠D8、D16及恢复期结束体重均显著降低；PTX-R低、高剂量组动物体重也呈现出降低趋势，且有量效关系，提示更长期试验时关注体重指标。Throughout the experiment period, all animals' body weights gradually increased. However, compared with the vehicle control group, the body weights of female and male mice D8, D16 and the end of the recovery period in the PTX administration group were significantly reduced; the body weight of the animals in the PTX-R low and high dose groups also showed a decreasing trend, and there was a dose-effect relationship, suggesting more Pay attention to body weight indicators during long-term trials.

表1.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雌鼠体重变化

Table 1. Paclitaxel derivative PTX-R rats repeated administration toxicity study for 2 weeks, female rats weight change

注：与溶媒对照组比较，**P＜0.01。Note: Compared with the vehicle control group, **P＜0.01.

表2.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠体重变化

Table 2. Toxicity study of paclitaxel derivative PTX-R rats after repeated administration for 2 weeks, male weight change

2.临床病理学检查2. Clinicopathological examination

2.1血液学2.1 Hematology

给药结束时，与溶媒对照组相比，PTX给药组雌雄鼠RBC、HGB、HCT均显著降低，考虑为与药物相关的贫血表现；恢复期结束可恢复。PTX给药组雌雄鼠MPV显著升高；PTX组雄鼠白细胞WBC、Reti也显著降低。At the end of the administration, compared with the vehicle control group, the RBC, HGB, and HCT of the male and female rats in the PTX administration group were significantly reduced, which is considered to be a drug-related anemia; the recovery period can be restored. The MPV of male and female mice in the PTX administration group increased significantly; the WBC and Reti of the male mice in the PTX group also decreased significantly.

给药结束时，PTX-R低、高剂量组雄鼠网织红细胞计数Reti与溶媒对照组相比显著增加，但无剂量效应关系，无毒理学意义。At the end of the administration, the male rat reticulocyte count Reti in the PTX-R low and high dose groups increased significantly compared with the vehicle control group, but there was no dose-effect relationship and no toxicological significance.

表3.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雌鼠血液学指标变化

Table 3. Paclitaxel derivative PTX-R rats repeated administration toxicity study for 2 weeks, female rats hematological index changes

注：与溶媒对照组比较，*P＜0.05，**P＜0.01。Note: Compared with the vehicle control group, *P＜0.05, **P＜0.01.

表4.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠血液学指标变化

Table 4. Paclitaxel derivative PTX-R rats repeated administration toxicity study for 2 weeks, male rats hematological index changes

2.2血清生化、电解质2.2 Serum biochemistry, electrolytes

给药结束时，与溶媒对照组相比，PTX给药组雄鼠ALT、AST均显著升高；恢复期结束可恢复。给药结束时，与溶媒对照组相比，PTX-R低、高剂量组血清生化指标及电解质均未见明显差异。At the end of the administration, compared with the vehicle control group, the ALT and AST of the male rats in the PTX administration group increased significantly; the recovery period can be restored after the end of the recovery period. At the end of the administration, compared with the vehicle control group, there were no significant differences in serum biochemical indicators and electrolytes in the PTX-R low and high dose groups.

表5.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠生化指标变化

Table 5. Toxicity study of paclitaxel derivative PTX-R rats repeated administration for 2 weeks. Changes of biochemical indexes in male rats

2.3凝血指标2.3 coagulation index

给药结束时，与溶媒对照组相比，PTX给药组雄鼠PT延长；恢复期可恢复。给药结束时，与溶媒对照组相比，PTX-R低、高剂量组凝血指标均未见明显差异。At the end of the administration, compared with the vehicle control group, the PT of the male rats in the PTX administration group was prolonged; the recovery period could be restored. At the end of the administration, compared with the vehicle control group, there was no significant difference in coagulation indexes between the low and high dose groups of PTX-R.

表6紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雌鼠凝血指标变化

Table 6 Toxicity study of paclitaxel derivative PTX-R rats after repeated administration for 2 weeks. Changes of coagulation indexes in female rats

表7紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠凝血指标变化

Table 7: Toxicity study of paclitaxel derivative PTX-R rats after 2 weeks of repeated administration

注：与溶媒对照组比较，*P＜0.05。Note: Compared with the vehicle control group, *P＜0.05.

2.4尿指标2.4 Urine Index

给药结束及恢复期结束时，与溶媒对照组相比，各给药组动物尿指标均未明显差异。At the end of the administration and the end of the recovery period, compared with the vehicle control group, there was no significant difference in the urine indicators of the animals in each administration group.

表8紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雌鼠尿液颜色及澄明度Table 8 Toxicity study of paclitaxel derivative PTX-R rats after repeated administration for 2 weeks. Color and clarity of urine in female rats

表9紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠尿液颜色及澄明度Table 9 Toxicity study of paclitaxel derivative PTX-R rats after repeated administration for 2 weeks. Male urine color and clarity

3.大体尸检及脏器重量3. Gross autopsy and organ weight

3.1大体尸检3.1 Gross autopsy

给药结束及恢复期结束时，所有受试动物大体尸检均未见明显异常。At the end of the administration and the end of the recovery period, no obvious abnormalities were found in the autopsy of all the tested animals.

3.2脏器重量3.2 Organ weight

给药结束时，与溶媒对照组相比，PTX给药组雌鼠体重、肺脏重量、肾脏重量、胸腺重量均显著降低；PTX给药组雄鼠体重、心脏重量、胸腺重量均显著降低。PTX-R低、高剂量组动物脏器重量与溶媒对照组相比均未见明显异常。At the end of the administration, compared with the vehicle control group, the weight of female rats in the PTX administration group, lung weight, kidney weight, and thymus weight were significantly reduced; the weight of male rats in the PTX administration group, heart weight, and thymus weight were significantly reduced. Compared with the vehicle control group, the organ weights of animals in the low and high dose groups of PTX-R were not significantly abnormal.

恢复期结束时，PTX给药组雌鼠肺脏重量、肾脏重量、胸腺重量与溶媒对照组相比均显著降低。PTX给药组雄鼠脏器重量基本可恢复至对照组水平。At the end of the recovery period, the lung weight, kidney weight, and thymus weight of the female rats in the PTX administration group were significantly reduced compared with the vehicle control group. The organ weight of the male rats in the PTX administration group can basically be restored to the level of the control group.

表10.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雌鼠脏器重量变化

Table 10. Toxicity study of paclitaxel derivative PTX-R rats after repeated administration for 2 weeks

表11.紫杉醇衍生物PTX-R大鼠2周重复给药毒性研究雄鼠脏器重量变化

Table 11. Toxicity study of paclitaxel derivative PTX-R rats after 2 weeks of repeated administration

药理实验Pharmacological experiment

实验例1紫杉醇衍生物A体外抗肿瘤检测Experimental Example 1 In vitro anti-tumor detection of paclitaxel derivative A

MTT法测定肿瘤细胞存活率MTT method to measure tumor cell survival rate

将对数生长期的细胞用胰酶消化后，配制成一定浓度的单细胞悬液，根据细胞生长速度的差异，按1500-3000个/孔接种于96孔板，每孔加入细胞悬液100μl。次日加入含不同浓度药物及相应溶剂对照的新鲜培养基，每孔加100μl(DMSO终浓度<0.1％)，每种受试化合物设4个剂量组(0.05，0.5，5，50μmol/L)每组设三个平行孔。于37℃，5％CO ₂继续培养96h后弃上清，每孔加入200μL新鲜配制的含0.5mg/mL MTT的无血清培养基。继续培养4h，弃上清，每孔加入200μL DMSO溶解MTT甲簪沉淀，微型振荡器振荡混匀后，酶标仪在检测波长570nm条件下测定光密度值(OD)，以溶剂对照处理的肿瘤细胞为对照组，按下列公式计算药物对肿瘤细胞的抑制率，并按中效方程计算IC ₅₀： After the cells in the logarithmic growth phase are digested with trypsin, they are prepared into a single cell suspension of a certain concentration. According to the difference of cell growth rate, 1500-3000 cells/well are seeded in a 96-well plate, and 100μl of cell suspension is added to each well . The next day, add fresh medium containing different concentrations of drugs and corresponding solvent controls, add 100μl to each well (final concentration of DMSO<0.1%), each test compound has 4 dose groups (0.05, 0.5, 5, 50μmol/L) There are three parallel holes in each group. Incubate at 37°C and 5% CO _{2 for} 96 hours, then discard the supernatant, and add 200 μL of freshly prepared serum-free medium containing 0.5 mg/mL MTT to each well. Continue to incubate for 4 hours, discard the supernatant, add 200μL of DMSO to each well to dissolve the MTT formazan precipitation, shake and mix with a micro-shaker, measure the optical density (OD) with a microplate reader at a detection wavelength of 570nm, and treat the tumor with a solvent control The cells are the control group. The inhibitory rate of the drug on the tumor cells is calculated according to the following formula, and the IC _{50 is} calculated according to the intermediate effect equation:

紫杉醇衍生物A体外抗肿瘤活性结果见表12、表13、表14The results of in vitro anti-tumor activity of paclitaxel derivative A are shown in Table 12, Table 13, and Table 14.

表12紫杉醇衍生物A MTT筛选结果-1Table 12 Paclitaxel derivative A MTT screening results-1

Taxol-Choline：紫杉醇衍生物A；MGC-803，BGC823，HGC27：人胃癌细胞株；NCI-H1975，NCI-H1650，A549：人非小细胞肺癌细胞株；NCI-H460：人大细胞肺癌细胞株；A549/Taxol：人耐紫素细胞非小细胞肺癌细胞株；U-87MG，T98G：人神经胶质瘤细胞株Taxol-Choline: Taxol derivative A; MGC-803, BGC823, HGC27: human gastric cancer cell line; NCI-H1975, NCI-H1650, A549: human non-small cell lung cancer cell line; NCI-H460: human large cell lung cancer cell line; A549/Taxol: Human purpurin-resistant non-small cell lung cancer cell line; U-87MG, T98G: Human glioma cell line

表13紫杉醇衍生物A MTT筛选结果-2Table 13 Paclitaxel derivative A MTT screening results-2

Taxol-Choline：紫杉醇衍生物A；SW1990，Mia-PaCa2，Capan2，BxPC3：人胰腺癌细胞株；HCT15，HCT-8，DLD-1：人结直肠癌细胞株；SW48，HCT116：人结肠癌细胞株；A2780：人卵巢癌细胞株Taxol-Choline: Taxol derivative A; SW1990, Mia-PaCa2, Capan2, BxPC3: human pancreatic cancer cell line; HCT15, HCT-8, DLD-1: human colorectal cancer cell line; SW48, HCT116: human colon cancer cell line Strain; A2780: human ovarian cancer cell line

表14紫杉醇衍生物A MTT筛选结果-3Table 14 Paclitaxel derivative A MTT screening results-3

Taxol-Choline：紫杉醇衍生物A；A498，ACHN：人肾癌细胞株；HK2：人肾近曲小管上皮细胞株；LO2：人正常肝细胞株；MCF-7，MDA-MB-231：人乳腺癌细胞株；MCF-7/Taxol：耐紫素的人乳腺癌细胞株；Bel7402，Huh-7，HepG2：人肝癌细胞株；Bel7402/5-FU：耐五氟尿嘧啶的人肝癌细胞Taxol-Choline: Taxol derivative A; A498, ACHN: human renal cancer cell line; HK2: human renal proximal tubule epithelial cell line; LO2: human normal liver cell line; MCF-7, MDA-MB-231: human breast Cancer cell line; MCF-7/Taxol: purpurin-resistant human breast cancer cell line; Bel7402, Huh-7, HepG2: human liver cancer cell line; Bel7402/5-FU: pentafluorouracil-resistant human liver cancer cell line

实验例2紫杉醇衍生物A体内抗小鼠结肠癌C26效果Experimental Example 2 The effect of paclitaxel derivative A against mouse colon cancer C26 in vivo

小鼠结肠癌移植瘤C26实验Experiment on transplanted tumor C26 of mouse colon cancer

KM小鼠(16～18g)，雄性，无菌条件下取传代接种后7d、生长良好的腹水肿瘤细胞，用无菌生理盐水稀释，调整细胞密度至5×10 ⁷个/ml，取0.2ml接种于小鼠腋窝皮下。次日随机分组并给药。对照组注射生理盐水，体积与时间周期同给药组；Taxol(紫素)配制成2.5mg/ml，每20g小鼠腹腔注射给药0.2ml，每3日给药1次；受试药(紫杉醇衍生物A)组分别配制成1.44,2.88,4.32mg/ml浓度的药液，每20g小鼠腹腔注射给药0.2ml，每3日给药一次，连续给药10日。实验结束时将小鼠脱臼处死，然后剥离肿瘤，称重并拍照。最后计算肿瘤抑制率。 KM mice (16～18g), male, under aseptic conditions, take the well-growing ascites tumor cells 7 days after passaging and dilute with sterile normal saline to adjust the cell density to 5×10 ⁷ cells/ml, take 0.2ml Inoculated subcutaneously in the armpit of mice. Randomly group and administer the drugs the next day. The control group was injected with physiological saline with the same volume and time period as the administration group; Taxol (purpurin) was formulated to be 2.5mg/ml, 0.2ml per 20g mouse was injected intraperitoneally, once every 3 days; the test drug ( The paclitaxel derivative A) group was formulated into a drug solution with a concentration of 1.44, 2.88, and 4.32 mg/ml, and was administered by intraperitoneal injection of 0.2 ml per 20 g mouse, once every 3 days, for 10 consecutive days. At the end of the experiment, the mice were dislocated and sacrificed, then the tumors were peeled off, weighed and photographed. Finally, the tumor inhibition rate was calculated.

体内抗肿瘤活性效果及对免疫***和血细胞的影响见表15-19和图4-5。The anti-tumor activity in vivo and its effect on the immune system and blood cells are shown in Table 15-19 and Figure 4-5.

表15紫杉醇衍生物A对小鼠C26结肠癌的生长抑制作用Table 15 Paclitaxel derivative A inhibits the growth of mouse C26 colon cancer

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△△p<0.01，△△△p<0.001，与紫素组比较。紫杉醇衍生物A：28.8mg/kg与紫素25.0mg/kg等摩尔浓度。*p<0.05, **p<0.01, ***p<0.001, compared with the solvent control group; △△p<0.01, △△△p<0.001, compared with the purpurin group. Paclitaxel derivative A: 28.8mg/kg equimolar concentration with 25.0mg/kg purin.

表16紫杉醇衍生物A对C26结肠癌小鼠脾指数及胸腺指数的影响Table 16 Effect of taxol derivative A on spleen index and thymus index of C26 colon cancer mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△△△p<0.001，与紫素组比较。*p<0.05, **p<0.01, ***p<0.001, compared with the solvent control group; △△△p<0.001, compared with the purpurin group.

表17紫杉醇衍生物A对C26结肠癌小鼠外周血计数的影响Table 17 Effect of Taxol Derivative A on Peripheral Blood Count of C26 Colon Cancer Mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△p<0.05，△△△p<0.001，与紫素组比较。*p<0.05, **p<0.01, ***p<0.001, compared with the solvent control group; △p<0.05, △△△p<0.001, compared with the purpurin group.

WBC：白细胞计数LYM％：淋巴细胞比率MON％：单核细胞比率NEUT％：中性粒细胞比率WBC: White blood cell count LYM%: Lymphocyte ratio MON%: Monocyte ratio NEUT%: Neutrophil ratio

表18紫杉醇衍生物A对C26结肠癌小鼠外周血计数的影响Table 18 Effect of Taxol Derivative A on Peripheral Blood Count of C26 Colon Cancer Mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△p<0.05，△△p<0.01，与紫素组比较。*p<0.05, **p<0.01, ***p<0.001, compared with the solvent control group; △p<0.05, △△p<0.01, compared with the purpurin group.

RBC：红细胞计数HGB：血红蛋白HCT：红细胞比容MCV：红细胞平均容积MCH：红细胞平均血红蛋白含量MCHC：红细胞平均血红蛋白浓度RBC: Red blood cell count HGB: Hemoglobin HCT: Hematocrit MCV: Average red blood cell volume MCH: Average red blood cell hemoglobin content MCHC: Average red blood cell hemoglobin concentration

RDW：红细胞分布宽度RDW: Red blood cell distribution width

表19紫杉醇衍生物A对C26结肠癌小鼠外周血计数的影响Table 19 Effect of Taxol Derivative A on Peripheral Blood Count of C26 Colon Cancer Mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△p<0.05，与紫素组比较。*p<0.05, **p<0.01, ***p<0.001, compared with solvent control group; △p<0.05, compared with purpurin group.

PLT：血小板计数PCT：血小板比积MPV：血小板平均体积PDW：血小板体积分布宽度PLT: platelet count PCT: platelet specific product MPV: average platelet volume PDW: platelet volume distribution width

实验例3紫杉醇衍生物A体内抗小鼠乳腺癌EMT6效果Experimental example 3 The effect of paclitaxel derivative A against mouse breast cancer EMT6 in vivo

小鼠乳腺癌移植瘤EMT6实验EMT6 experiment on mouse breast cancer transplantation tumor

KM小鼠(19～21g)，雄性，无菌条件下取传代接种后7d、生长良好的腹水肿瘤细胞，用无菌生理盐水稀释，调整细胞密度至5×107个/ml，取0.2ml接种于小鼠腋窝皮下。次日随机分组并给药，对照组注射生理盐水，体积与时间周期同给药组；Taxol(紫素)配制成2.5mg/ml，每20g小鼠腹腔注射给药0.2ml，每3日给药1次；受试药(紫杉醇衍生物A)组分别配制成1.44,2.88,4.32mg/ml浓度的药液，每20g小鼠腹腔注射给药0.2ml，每3日给药一次，连续给药10日。实验结束时将小鼠脱臼处死，然后剥离肿瘤，称重并拍照。最后计算肿瘤抑制率。KM mice (19-21g), male, aseptically take the well-growing ascites tumor cells 7 days after passaging and dilute with sterile normal saline, adjust the cell density to 5×107 cells/ml, and inoculate 0.2ml Under the skin of the mouse armpit. The next day was randomly divided into groups and administered. The control group was injected with physiological saline with the same volume and time period as the administration group; Taxol (purpur) was formulated to be 2.5mg/ml, and 0.2ml per 20g mouse was given by intraperitoneal injection, every 3 days The drug is administered once; the test drug (paclitaxel derivative A) group is formulated into a drug solution with a concentration of 1.44, 2.88, and 4.32 mg/ml, 0.2 ml per 20g mouse intraperitoneal injection, once every 3 days, continuous administration Medicine on the 10th. At the end of the experiment, the mice were dislocated and sacrificed, then the tumors were peeled off, weighed and photographed. Finally, the tumor suppression rate is calculated.

体内抗肿瘤活性效果及对免疫***和血细胞的影响见表20-24和图6-7。The anti-tumor activity in vivo and its effect on the immune system and blood cells are shown in Table 20-24 and Figure 6-7.

表20紫杉醇衍生物A对小鼠EMT6乳腺癌的生长抑制作用Table 20 The inhibitory effect of taxol derivative A on mouse EMT6 breast cancer

*p<0.05，与溶剂对照组比较；△△p<0.01，△△△p<0.001，与紫素组比较。*p<0.05, compared with the solvent control group; △△p<0.01, △△△p<0.001, compared with the purpurin group.

紫杉醇衍生物A 28.8mg/kg与紫素25.0mg/kg等摩尔浓度。Paclitaxel derivative A 28.8mg/kg and purin 25.0mg/kg equimolar concentration.

表21紫杉醇衍生物A对EMT6乳腺癌小鼠脾指数及胸腺指数的影响Table 21 Effect of paclitaxel derivative A on spleen index and thymus index of EMT6 breast cancer mice

**p<0.01，***p<0.001，与溶剂对照组比较；△△p<0.01，△△△p<0.001，与紫素组比较。**p<0.01, ***p<0.001, compared with the solvent control group; △△p<0.01, △△△p<0.001, compared with the purpurin group.

表22紫杉醇衍生物A对EMT6乳腺癌小鼠外周血计数的影响Table 22 Effect of paclitaxel derivative A on peripheral blood counts of EMT6 breast cancer mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；△p<0.05，△△p<0.01，△△△p<0.001，与紫素组比较。*p<0.05, **p<0.01, ***p<0.001, compared with the solvent control group; △p<0.05, △△p<0.01, △△△p<0.001, compared with the purpurin group.

表23紫杉醇衍生物A对EMT6乳腺癌小鼠外周血计数的影响Table 23 Effect of paclitaxel derivative A on peripheral blood counts of EMT6 breast cancer mice

*p<0.05，**p<0.01，与溶剂对照组比较；△p<0.05，与紫素组比较。*p<0.05, **p<0.01, compared with the solvent control group; △p<0.05, compared with the purpurin group.

RDW：红细胞分布宽度RDW: Red blood cell distribution width

表24紫杉醇衍生物A对EMT6乳腺癌小鼠外周血计数的影响Table 24 Effect of paclitaxel derivative A on peripheral blood counts of EMT6 breast cancer mice

*p<0.05，***p<0.001，与溶剂对照组比较；△p<0.05，△△p<0.01，△△△p<0.001，与紫素组比较。*p<0.05, ***p<0.001, compared with the solvent control group; △p<0.05, △△p<0.01, △△△p<0.001, compared with the purpurin group.

实验例4紫杉醇衍生物A体内抗小鼠肺癌Lewis效果Experimental Example 4 The effect of paclitaxel derivative A against mouse lung cancer Lewis in vivo

小鼠肺癌移植瘤Lewis实验Lewis experiment on transplanted lung cancer in mice

KM小鼠(18～20g)，雄性，无菌条件下取传代接种后7d、生长良好的腹水肿瘤细胞，用无菌生理盐水稀释，调整细胞密度至5×10 ⁷个/ml，取0.2ml接种于小鼠腋窝皮下。次日随机分组并给药，对照组注射生理盐水，体积与时间周期同给药组；Taxol(紫素)配制成2.5mg/ml，每20g小鼠腹腔注射给药0.2ml，每3日给药1次；受试药组(紫杉醇衍生物A)分别配制成1.44,2.88,5.76mg/ml浓度的药液，每20g小鼠腹腔注射给药0.2ml，每3日给药一次，连续给药7日。实验结束时将小鼠脱臼处死，然后剥离肿瘤，称重并拍照。最后计算肿瘤抑制率。 KM mice (18-20g), male, aseptically take the well-growing ascites tumor cells 7 days after passaging and dilute with sterile saline to adjust the cell density to 5×10 ⁷ cells/ml, take 0.2ml Inoculated subcutaneously in the armpit of mice. The next day was randomly divided into groups and administered. The control group was injected with physiological saline with the same volume and time period as the administration group; Taxol (purpur) was formulated to be 2.5mg/ml, and 0.2ml per 20g mouse was given by intraperitoneal injection, every 3 days The drug is administered once; the test drug group (paclitaxel derivative A) is formulated into a drug solution with a concentration of 1.44, 2.88, and 5.76 mg/ml, and 0.2 ml is administered by intraperitoneal injection per 20g mouse, once every 3 days, continuously Medicine 7th. At the end of the experiment, the mice were dislocated and sacrificed, then the tumors were peeled off, weighed and photographed. Finally, the tumor inhibition rate was calculated.

体内抗肿瘤活性效果及对免疫***和血细胞的影响见表25-29和图8。The anti-tumor activity in vivo and its effect on the immune system and blood cells are shown in Table 25-29 and Figure 8.

表25紫杉醇衍生物A对小鼠Lewis肺癌的生长抑制作用Table 25 The inhibitory effect of paclitaxel derivative A on the growth of Lewis lung cancer in mice

**p<0.01，***p<0.001，与溶剂对照组比较。紫杉醇衍生物A 28.8mg/kg与紫素25.0mg/kg等摩尔浓度。**p<0.01, ***p<0.001, compared with the solvent control group. Paclitaxel derivative A 28.8mg/kg and purin 25.0mg/kg equimolar concentration.

表26紫杉醇衍生物A对Lewis肺癌小鼠脾指数及胸腺指数的影响Table 26 Effect of paclitaxel derivative A on spleen index and thymus index of Lewis lung cancer mice

*p<0.05，**p<0.01，***p<0.001，与溶剂对照组比较；*p<0.05, **p<0.01, ***p<0.001, compared with solvent control group;

表27紫杉醇衍生物A对Lewis肺癌小鼠外周血计数的影响Table 27 The effect of paclitaxel derivative A on peripheral blood counts in mice with Lewis lung cancer

表28紫杉醇衍生物A对Lewis肺癌小鼠外周血计数的影响Table 28 The effect of paclitaxel derivative A on peripheral blood counts in mice with Lewis lung cancer

RDW：红细胞分布宽度RDW: Red blood cell distribution width

表29紫杉醇衍生物A对Lewis肺癌小鼠外周血计数的影响Table 29 Effect of paclitaxel derivative A on peripheral blood counts in mice with Lewis lung cancer

PLT：血小板计数PCT：血小板比积MPV：血小板平均体积PDW：血小板体积分布PLT: Platelet count PCT: Platelet specific product MPV: Average platelet volume PDW: Platelet volume distribution

实验例5紫杉醇衍生物A(PTX-R)与紫杉醇衍生物F(PTX-SA-R)的体内抗小鼠肺癌Lewis效果Experimental Example 5: The Lewis effect of paclitaxel derivative A (PTX-R) and paclitaxel derivative F (PTX-SA-R) against lung cancer in mice in vivo

KM小鼠(18～20g)，雄性，无菌条件下取传代接种后7d、生长良好的腹水肿瘤细胞，用无菌生理盐水稀释，调整细胞密度至5×10 ⁷个/ml，取0.2ml接种于小鼠腋窝皮下。次日随机分组并给药。 KM mice (18-20g), male, aseptically take the well-growing ascites tumor cells 7 days after passaging and dilute with sterile saline to adjust the cell density to 5×10 ⁷ cells/ml, take 0.2ml Inoculated subcutaneously in the armpit of mice. Randomly group and administer the drugs the next day.

1.阳性对照药：吸取1500μL紫杉醇注射液(6mg/ml),加入2100μL生理盐水，超声5-10分钟，配制成2.5mg/ml PTX的溶液，按0.1ml/10g给小鼠尾静脉注射。1. Positive control drug: Draw 1500μL of paclitaxel injection (6mg/ml), add 2100μL of normal saline, ultrasound for 5-10 minutes, prepare a 2.5mg/ml PTX solution, and inject 0.1ml/10g into the tail vein of mice.

2.PTX-R：称取适量PTX-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，加入5％药液体积的吐温80，震荡并超声30s,加入剩余体积的生理盐水，超声5-10分钟，配制成4.32mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射。2. PTX-R: Weigh an appropriate amount of PTX-R, add 5% DMSO of the required liquid volume to completely dissolve the drug, add 5% of the liquid volume of 95% ethanol, and add 5% of the volume of liquid Tween 80, Shake and ultrasound for 30s, add the remaining volume of normal saline, ultrasound for 5-10 minutes to prepare a 4.32mg/ml PTX solution, and inject the mouse into the abdominal cavity of 0.1ml/10g.

3.PTX-SA-R(低剂量)：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的生理盐水，超声5-10分钟，配制成1.53mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射。3. PTX-SA-R (low dose): Weigh an appropriate amount of PTX-SA-R, add 5% DMSO of the required volume of the drug solution to completely dissolve the drug, add 5% of the volume of the drug solution, 95% ethanol, shake and ultrasound For 30 seconds, add the remaining volume of normal saline, ultrasound for 5-10 minutes to prepare a solution of 1.53mg/ml PTX, and inject the mouse into the abdominal cavity of 0.1ml/10g.

4.PTX-SA-R(中剂量)：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的生理盐水，超声5-10分钟，配制成3.05mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射。4. PTX-SA-R (medium dose): Weigh a proper amount of PTX-SA-R, add 5% DMSO of the required volume of the drug solution to completely dissolve the drug, add 5% of the volume of the drug solution, 95% ethanol, shake and ultrasound For 30 seconds, add the remaining volume of normal saline, ultrasound for 5-10 minutes to prepare a 3.05mg/ml PTX solution, and inject it into the mouse's abdominal cavity at 0.1ml/10g.

5.PTX-SA-R(高剂量)：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的生理盐水，超声5-10分钟，配制成4.58mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射。5. PTX-SA-R (high dose): Weigh an appropriate amount of PTX-SA-R, add 5% DMSO of the required volume of the drug solution to completely dissolve the drug, add 5% of the volume of the drug solution, 95% ethanol, shake and ultrasound For 30 seconds, add the remaining volume of normal saline, ultrasound for 5-10 minutes to prepare a 4.58mg/ml PTX solution, and inject the mouse into the abdominal cavity of 0.1ml/10g.

6.PTX-SA-R(高剂量)-磺丁基环糊精：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的15％磺丁基环糊精，超声5-10分钟，配制成4.58mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射。6. PTX-SA-R (high dose)-sulfobutyl cyclodextrin: Weigh a proper amount of PTX-SA-R, add 5% DMSO of the required liquid volume to completely dissolve the drug, and add 95% of the 5% liquid volume Ethanol, shake and ultrasound for 30 seconds, add the remaining volume of 15% sulfobutyl cyclodextrin, ultrasound for 5-10 minutes to prepare a 4.58mg/ml PTX solution, and inject it into the mouse abdominal cavity at 0.1ml/10g.

7.PTX-SA-R(高剂量)-羟丙基环糊精：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的15％羟丙基环糊精，超声5-10分钟，配制成4.58mg/ml PTX的溶液，按0.1ml/10g给小鼠腹腔注射7. PTX-SA-R (high dose)-hydroxypropyl cyclodextrin: Weigh an appropriate amount of PTX-SA-R, add 5% DMSO of the required volume of the drug solution to completely dissolve the drug, and add 5% of the volume of the drug solution 95% ethanol, shake and sonicate for 30s, add the remaining volume of 15% hydroxypropyl cyclodextrin, sonicate for 5-10 minutes to prepare a 4.58mg/ml PTX solution, and give the mouse an intraperitoneal injection of 0.1ml/10g

8.PTX-SA-R(低剂量-iv)：称取适量PTX-SA-R，加入所需药液体积的5％DMSO使药物完全溶解，加入5％药液体积的95％乙醇，震荡并超声30s,加入剩余体积的生理盐水，超声5-10分钟，配制成1.53mg/ml PTX的溶液，经0.22μm滤膜过滤后，按0.1ml/10g给小鼠尾静脉注射。8. PTX-SA-R (low dose-iv): Weigh an appropriate amount of PTX-SA-R, add 5% DMSO of the required liquid volume to completely dissolve the drug, add 5% of the liquid volume of 95% ethanol, and shake Ultrasound for 30 seconds, add the remaining volume of normal saline, sonicate for 5-10 minutes to prepare a 1.53mg/ml PTX solution, filter through a 0.22μm filter membrane, and inject 0.1ml/10g into the tail vein of the mouse.

接种后的第1,4,7,10给药，为保证受试动物存活至实验结束，需要对紫杉醇注射液组及其PTX-SA-R高剂量组的受试动物予以生理状态重点关注，当发现受试动物出现明显体重下降(>15％)或小便潴留等现象后，于当次的给药剂量减半或者停止给药一次。实验结束时将小鼠脱臼处死，然后剥离肿瘤，称重并拍照。最后计算肿瘤抑制率。In order to ensure the survival of the test animals to the end of the experiment, it is necessary to pay special attention to the physiological status of the test animals in the paclitaxel injection group and the PTX-SA-R high-dose group. When the test animal is found to have significant weight loss (>15%) or urinary retention, the dose administered at that time is halved or the administration is stopped once. At the end of the experiment, the mice were dislocated and sacrificed, then the tumors were peeled off, weighed and photographed. Finally, the tumor inhibition rate was calculated.

体内抗肿瘤活性效果及对免疫***和血细胞的影响见表30-33和图9。See Table 30-33 and Figure 9 for the anti-tumor activity in vivo and its effect on the immune system and blood cells.

表30化合物对小鼠Lewis肺癌的生长抑制作用Table 30 Compounds inhibit growth of Lewis lung cancer in mice

*p<0.05，**p<0.01，***p<0.001，与模型组比较；#p<0.05，##p<0.01，###p<0.001，与PTX组比较；*p<0.05, **p<0.01, ***p<0.001, compared with the model group; #p<0.05, ##p<0.01, ###p<0.001, compared with the PTX group;

表31化合物对Lewis肺癌小鼠外周血计数的影响Table 31: Effects of compounds on peripheral blood counts in mice with Lewis lung cancer

***p<0.001，与模型组比较；#p<0.05，##p<0.01，###p<0.001，与PTX组比较。***p<0.001, compared with the model group; #p<0.05, ##p<0.01, ###p<0.001, compared with the PTX group.

WBC：白细胞计数，LYM％：淋巴细胞比率，MON％：单核细胞比率，NEUT％：中性粒细胞比率WBC: white blood cell count, LYM%: lymphocyte ratio, MON%: monocyte ratio, NEUT%: neutrophil ratio

表32化合物对Lewis肺癌小鼠外周血计数的影响Table 32 Effects of compounds on peripheral blood counts of mice with Lewis lung cancer

*p<0.05，**p<0.01，***p<0.001，与模型组比较；#p<0.05，##p<0.01，###p<0.001，与PTX组比较。*p<0.05, **p<0.01, ***p<0.001, compared with the model group; #p<0.05, ##p<0.01, ###p<0.001, compared with the PTX group.

RBC：红细胞计数，HGB：血红蛋白，HCT：红细胞比容，MCV：红细胞平均容积，MCH：红细胞平均血红蛋白含量，MCHC：红细胞平均血红蛋白浓度，RDW：红细胞分布宽度RBC: red blood cell count, HGB: hemoglobin, HCT: hematocrit, MCV: average red blood cell volume, MCH: average red blood cell hemoglobin content, MCHC: average red blood cell hemoglobin concentration, RDW: red blood cell distribution width

表33化合物对Lewis肺癌小鼠外周血计数的影响Table 33 Effects of compounds on peripheral blood counts in mice with Lewis lung cancer

PLT：血小板计数，PCT：血小板比积，MPV：血小板平均体积，PDW：血小板体积分布宽度PLT: platelet count, PCT: platelet specific product, MPV: mean platelet volume, PDW: platelet volume distribution width

药代实验Pharmacokinetic experiment

实验例1紫杉醇衍生物A肿瘤模型动物体内的组织分布和靶向性评价Experimental Example 1 Tissue distribution and targeting evaluation of paclitaxel derivative A in tumor model animals

药物在特定组织器官的暴露或作用部位是其发挥药效或产生毒性的物质基础，因此研发具有明显肿瘤组织靶向性的高效低毒的新型抗肿瘤靶向药物具有重要意义。本研究采用定量质谱成像(VC-QMSI)方法，针对紫杉醇衍生物A的肿瘤靶向性评价开展了***研究，通过对紫杉醇注射液、紫杉醇脂质体、紫杉醇衍生物三个治疗组的荷瘤裸鼠(移植瘤模型)进行整体动物体内药物的定量成像分析，***评价了它们在不同组织区域的分布特征。如图10所示的结果表明紫杉醇注射液和紫杉醇脂质体中的紫杉醇化合物广泛分布于各个组织器官，而紫杉醇衍生物A(作为前药)中经代谢产生的紫杉醇主要分布在肿瘤组织中；以紫杉醇在组织器官的暴露量为参比，相对于紫杉醇脂质体(PTX-Liposomes)而言，紫杉醇衍生物A的肿瘤组织靶向性提高了约50倍(图11)。此外，值得指出的是,紫杉醇衍生物A在肿瘤坏死区与胶原组织高富集，表明其具有良好的肿瘤浸润能力。该研究进一步表明，紫杉醇衍生物A具有较好的器官选择性分布特征和肿瘤靶向性。The exposure or site of action of drugs in specific tissues and organs is the material basis for their efficacy or toxicity. Therefore, it is of great significance to develop new anti-tumor targeted drugs with obvious tumor tissue targeting with high efficiency and low toxicity. In this study, quantitative mass spectrometry imaging (VC-QMSI) was used to conduct a systematic study on the tumor targeting evaluation of paclitaxel derivative A. The tumor-bearing treatment groups of paclitaxel injection, paclitaxel liposomes, and paclitaxel derivatives were tested. Nude mice (xenograft tumor model) performed quantitative imaging analysis of drugs in whole animals, and systematically evaluated their distribution characteristics in different tissue regions. The results shown in Figure 10 show that the paclitaxel compounds in paclitaxel injection and paclitaxel liposomes are widely distributed in various tissues and organs, and paclitaxel produced by the metabolism of paclitaxel derivative A (as a prodrug) is mainly distributed in tumor tissues; Taking paclitaxel exposure in tissues and organs as a reference, compared with paclitaxel liposomes (PTX-Liposomes), the tumor tissue targeting of paclitaxel derivative A increased by about 50 times (Figure 11). In addition, it is worth pointing out that the taxol derivative A is highly enriched in tumor necrosis and collagen tissue, indicating that it has good tumor infiltration ability. The study further shows that taxol derivative A has better organ selective distribution characteristics and tumor targeting.

Claims

一类具有下列通式(Ⅰ)所示的紫杉烷衍生物及其药学上可接受的盐：One class has the taxane derivatives represented by the following general formula (I) and their pharmaceutically acceptable salts:

(Ⅰ)式中：(Ⅰ) where:

R ₁为苯甲酰基，叔丁氧酰基，甜菜碱酰基，取代的甜菜碱酰基，碳酸酯键、磷酸酯键、C _2-8二酸酯键、醚键或磺酸酯键连接的胆碱或取代的胆碱；所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基、不同种类的氨基酸； R ₁ is benzoyl, tert-butoxy acyl, betaine acyl, substituted betaine acyl, carbonate bond, phosphate bond, C _2-8 diacid ester bond, ether bond or sulfonate bond connected choline Or substituted choline; the substituents are each independently selected from halogen atoms, C _1-6 alkyl, C _1-7 acyl, C _3-6 cycloalkyl, hydroxyl, amino, and different kinds of amino acids;

R ₂为氢，甲基或乙酰基； R ₂ is hydrogen, methyl or acetyl;

R ₃为氢，甲基或乙酰基； R ₃ is hydrogen, methyl or acetyl;

R ₄为氢，甲基或乙酰基； R ₄ is hydrogen, methyl or acetyl;

L为碳酸酯键、磷酸酯键、C _2-8二酸酯键、缩酮键、缩醛键、醚键或磺酸酯键连接； L is a carbonate bond, a phosphate bond, a C _2-8 diacid ester bond, a ketal bond, an acetal bond, an ether bond or a sulfonate bond;

R ₅为甜菜碱酰基，取代的甜菜碱酰基，胆碱，或取代的胆碱；所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基，不同种类的氨基酸。 R ₅ is a betaine acyl group, a substituted betaine acyl group, choline, or substituted choline; the substituents are each independently selected from halogen atoms, C _1-6 alkyl groups, C _1-7 acyl groups, and C _{3 -6} cycloalkyl, hydroxyl, amino, different kinds of amino acids.
根据权利要求1的紫杉烷衍生物及其药学上可接受的盐，其特征在于，所述的化合物如式(II)所示：The taxane derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is represented by formula (II):

其中，R ₁为苯甲酰基，叔丁氧酰基，甜菜碱酰基，取代的甜菜碱酰基，碳酸酯键、磷酸酯键、C _2-8二酸酯键、醚键或磺酸酯键连接的胆碱或取代的胆碱；所述的取代基各自独立的选自卤原子，C _1-6烷基，C _1-7酰基，C _3-6环烷基，羟基，氨基、不同种类的氨基酸 Among them, R ₁ is benzoyl, tert-butoxy acyl, betaine acyl, substituted betaine acyl, carbonate bond, phosphate bond, C _2-8 diacid ester bond, ether bond or sulfonate bond connected Choline or substituted choline; the substituents are each independently selected from halogen atoms, C _1-6 alkyl, C _1-7 acyl, C _3-6 cycloalkyl, hydroxyl, amino, and different kinds of amino acids

R ₂为氢，甲基或乙酰基； R ₂ is hydrogen, methyl or acetyl;

R ₃为氢，甲基或乙酰基； R ₃ is hydrogen, methyl or acetyl;

R ₄为氢，甲基或乙酰基； R ₄ is hydrogen, methyl or acetyl;

L为碳酸酯键、磷酸酯键、C _2-8二酸酯键、缩酮键、缩醛键、醚键或磺酸酯键连接。 L is a carbonate bond, a phosphate bond, a C _2-8 diacid ester bond, a ketal bond, an acetal bond, an ether bond or a sulfonate bond.
根据权利要求1-2任一项的紫杉烷衍生物及其药学上可接受的盐，其特征在于，所述的氨基酸包括丙氨酸、缬氨酸、苏氨酸、天冬氨酸、丝氨酸，所述的C _1-6烷基包括甲基、乙基、丙基、丁基，所述的C _1-7酰基包括甲酰基、乙酰基、丙酰基、丁酰基，所述的C _3-6环烷基包括环丙烷基、环丁烷基、环戊烷基、环己烷基，所述的卤原子选自F、Cl、Br、I；所述的C _2-8二酸酯键包括乙二酸酯键、丙二酸酯键、丁二酸酯键、丁烯二酸酯键、羟基丁二酸酯键、氨基丁二酸酯键、戊二酸酯键、戊烯二酸酯键，羟基戊二酸酯键、氨基戊二酸酯键、己二酸酯键、己烯二酸酯键，羟基己二酸酯键、氨基己二酸酯键、庚二酸酯键、庚烯二酸酯键，羟基庚二酸酯键、氨基庚二酸酯键、辛二酸酯键、辛烯二酸酯键，羟基辛二酸酯键、氨基辛二酸酯键。 The taxane derivative and pharmaceutically acceptable salt thereof according to any one of claims 1-2, wherein the amino acids include alanine, valine, threonine, aspartic acid, Serine, the C _1-6 alkyl group includes methyl, ethyl, propyl, and butyl, the C _1-7 acyl group includes formyl, acetyl, propionyl, butyryl, and the C _{3 -6} cycloalkyl includes cyclopropanyl, cyclobutanyl, cyclopentyl, cyclohexane, and the halogen atom is selected from F, Cl, Br, I; the C _2-8 diacid ester Bonds include oxalate bond, malonate bond, succinate bond, crotonate bond, hydroxysuccinate bond, amino succinate bond, glutarate bond, pentenedi Ester linkage, hydroxyglutarate linkage, aminoglutarate linkage, adipate linkage, hexenedioate linkage, hydroxyadipate linkage, aminoadipate linkage, pimelate linkage , Peptenedioate linkage, hydroxypimelate linkage, aminopimelate linkage, suberate linkage, octenedioate linkage, hydroxysuberate linkage, amino suberate linkage.
根据权利要求1-2任一项的紫杉烷衍生物及其药学上可接受的盐，其特征在于，C2’位羟基为(RS)-、(S)-、(R)-构型。The taxane derivative and pharmaceutically acceptable salt thereof according to any one of claims 1-2, wherein the hydroxyl group at the C2' position is in the (RS)-, (S)-, (R)-configuration.
根据权利要求1的紫杉烷衍生物及其药学上可接受的盐，其特征在于，所述的化合物为如下化合物：The taxane derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is the following compound:
一种药物组合物，其特征在于，所述的药物组合物含有治疗有效量的权利要求1-5任一项所述的紫杉烷衍生物及其药学上可接受的盐以及药学上可接受的载体或赋形剂。A pharmaceutical composition, characterized in that the pharmaceutical composition contains a therapeutically effective amount of the taxane derivative according to any one of claims 1 to 5, and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable The carrier or excipient.
权利要求1-5任一项所述的紫杉烷衍生物及其药学上可接受的盐或权利要求6所述的药物组合物在制备抗肿瘤药物中的应用。Use of the taxane derivative and pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.
根据权利要求7的应用，其特征在于，所述的肿瘤包括肺癌、卵巢癌、乳腺癌、结肠癌或肝癌。The use according to claim 7, characterized in that the tumor comprises lung cancer, ovarian cancer, breast cancer, colon cancer or liver cancer.