CN1966547B - Double-chain structured polyethylene glycol derivative preparation and its combination with pharmaceutical molecule - Google Patents

Abstract

Translated fromChinese

众多具有生物活性的药物分子，尤其是蛋白质和肽类等生物大分子，已在治疗中广泛应用，并获得成功。但这些生物大分子在临床应用中也有许多不利之处，例如易产生免疫排斥反应、稳定性及可溶性较差、清除速率较快。聚乙二醇化技术是近年来发展起来的一种非常有效的改善生物大分子药代动力学性质的方法。本发明涉及一种新型的以硫辛酸作为连接体，具有双链结构的聚乙二醇活性衍生物、其制备方法以及与药物分子的结合，所述衍生物可用于连接药物分子特别是蛋白质和多肽等生物大分子的氨基、巯基等活性基团从而改善药物分子的药代动力学性质，本发明还涉及包含所述结合物的药物组合物。Numerous biologically active drug molecules, especially biomacromolecules such as proteins and peptides, have been widely used in therapy and achieved success. However, these biomacromolecules also have many disadvantages in clinical application, such as prone to immune rejection, poor stability and solubility, and fast clearance rate. Pegylation technology is a very effective method developed in recent years to improve the pharmacokinetic properties of biomacromolecules. The present invention relates to a novel active derivative of polyethylene glycol with a double-chain structure using lipoic acid as a linker, its preparation method and its combination with drug molecules. The derivatives can be used to connect drug molecules, especially proteins and Active groups such as amino groups and sulfhydryl groups of biomacromolecules such as polypeptides can improve the pharmacokinetic properties of drug molecules, and the invention also relates to pharmaceutical compositions containing the conjugates.

Description

Translated fromChinese

双链结构的聚乙二醇衍生物的制备及其与药物分子的结合Preparation of Polyethylene Glycol Derivatives with Double-Strand Structure and Their Combination with Drug Molecules

                                  技术领域                            

本发明涉及具有双链结构的聚乙二醇活性衍生物、其制备方法以及与药物分子的结合物，所述药物分子特别是蛋白质和多肽等大分子，本发明还涉及包含所述结合物的药物组合物。The present invention relates to active derivatives of polyethylene glycol with a double-chain structure, a preparation method thereof, and a conjugate with drug molecules, the drug molecules are especially macromolecules such as proteins and polypeptides, and the present invention also relates to a compound comprising the conjugate pharmaceutical composition.

                                  背景技术 Background technique

众多具有生物活性的药物分子，尤其是蛋白质和肽类等生物大分子，已在治疗中广泛应用，并获得成功。但这些生物大分子在临床应用中也有许多不利之处，例如易产生免疫排斥反应、稳定性及可溶性较差、清除速率较快。因此，人们采用了各种方法消除上述不利因素，其中聚乙二醇化技术是近年来发展起来的一种非常有效的改善生物大分子药代动力学性质的方法。Numerous biologically active drug molecules, especially biomacromolecules such as proteins and peptides, have been widely used in therapy and achieved success. However, these biomacromolecules also have many disadvantages in clinical application, such as prone to immune rejection, poor stability and solubility, and fast clearance rate. Therefore, people have adopted various methods to eliminate the above-mentioned unfavorable factors, among which PEGylation technology is a very effective method developed in recent years to improve the pharmacokinetic properties of biomacromolecules.

聚乙二醇化技术(PEGylation PEG化)又称为化学修饰，是目前分子变构化学(moleculealtering structure chemistry，MASC)中最重要的技术之一。PEG化技术的研究始于20世纪80年代，Abuchowski等第一次将聚乙二醇共价结合到蛋白质上，以保护蛋白质免遭破坏，结果发现聚乙二醇修饰不仅可以增加蛋白质的水溶性，还能减少肾脏的清除，改变了蛋白质的药代动力学和药效学性质。经过近几十年的发展，PEG化技术不仅在蛋白质类药物的开发中得到普遍的应用，而且已扩展到新型药物载体、控释制剂等各个领域。PEGylation technology (PEGylation PEGylation), also known as chemical modification, is one of the most important technologies in molecular altering structure chemistry (MASC). The research on PEGylation technology began in the 1980s. For the first time, Abuchowski et al. covalently bound polyethylene glycol to proteins to protect them from damage. They found that polyethylene glycol modification can not only increase the water solubility of proteins , can also reduce renal clearance and change the pharmacokinetic and pharmacodynamic properties of the protein. After decades of development, PEGylation technology has not only been widely used in the development of protein drugs, but also has been extended to various fields such as new drug carriers and controlled release preparations.

在聚乙二醇化技术中，需要对聚乙二醇的端基进行活化，引入适当的活性基团，此活性基团对要结合的药物分子中的至少一个官能团具有活性，能与之形成稳定的化学键。In PEGylation technology, it is necessary to activate the end group of polyethylene glycol and introduce an appropriate active group, which is active to at least one functional group in the drug molecule to be combined and can form a stable compound with it. chemical bond.

许多制备聚乙二醇活性衍生物的方法已被报道，Davis等人在美国专利4,179,337种公开了将聚乙二醇结合到蛋白质上，从而得到一种免疫原性较低，且能保持大部分生理活性的结合物。Nakagawa等人公开了将PEG结合到小岛活化蛋白质上以减少其副作用和免疫原性。Veronese等人在Applied Biochem.And Biotech，11：141-152(1985)上公开了用氯甲酸苯酯类活化聚乙二醇以修饰核糖核酸酶和超氧化物歧化酶。Katre等人在美国专利4,766,106和4,917,888上也公开了通过聚合物结合使蛋白质增溶，将聚乙二醇同重组蛋白质结合以减弱蛋白质的免疫原性和提高他们的半衰期。Many methods for preparing active derivatives of polyethylene glycol have been reported. Davis et al. disclosed in US Patent No. 4,179,337 that polyethylene glycol is bound to proteins, thereby obtaining a low immunogenicity and keeping most of the protein. Physiologically active compounds. Nakagawa et al. disclose the conjugation of PEG to islet-activating proteins to reduce their side effects and immunogenicity. Veronese et al. in Applied Biochem. And Biotech, 11:141-152 (1985) disclose the activation of polyethylene glycol with phenyl chloroformates to modify ribonucleases and superoxide dismutases. Katre et al. in US Patents 4,766,106 and 4,917,888 also disclose protein solubilization by polymer conjugation, combining polyethylene glycol with recombinant proteins to attenuate the immunogenicity of the proteins and increase their half-life.

目前，聚乙二醇衍生物广泛的用于与蛋白质、多肽以及其他治疗药物的结合以延长所述药物的半衰期，降低其免疫原性和毒性。在临床使用中，PEG及其衍生物作为药物制剂的载体已经在很多商业药品中得到了广泛的应用，而将PEG结合到药物分子上的技术也在许多批准药品中被广泛的使用。如PEG-intron，一种干扰素和聚乙二醇的结合物就表现出了更长的半衰期和更好的治疗效果。紫杉醇与聚乙二醇的结合物也相应的降低了毒性和延长了生物活性。At present, polyethylene glycol derivatives are widely used in combination with proteins, polypeptides and other therapeutic drugs to prolong the half-life of the drugs and reduce their immunogenicity and toxicity. In clinical use, PEG and its derivatives have been widely used as carriers of pharmaceutical preparations in many commercial drugs, and the technology of combining PEG to drug molecules is also widely used in many approved drugs. For example, PEG-intron, a combination of interferon and polyethylene glycol, has shown a longer half-life and better therapeutic effect. The combination of paclitaxel and polyethylene glycol also correspondingly reduces toxicity and prolongs biological activity.

上述形成PEG-蛋白质结合物的方法以及由所述方法得到的结合物存在几个问题，其一是形成这些结合物的方法会使蛋白质失活。另外，在形成这些PEG-蛋白质结合物时所用的某些连接基团易在体内水解，断裂，当给药以后发生这样的断裂时，这些结合物便失去了由PEG带来的有利性能。There are several problems with the above methods of forming PEG-protein conjugates and the conjugates resulting from said methods, one being that the methods of forming these conjugates inactivate the protein. In addition, certain linking groups used in the formation of these PEG-protein conjugates are prone to hydrolysis and cleavage in vivo, and when such cleavage occurs after administration, the conjugates lose the beneficial properties imparted by PEG.

                                 发明内容Contents of Invention

本发明提供一种新型的，以硫辛酸作为连接体，具有双链结构的聚乙二醇活性衍生物，具有以下结构通式：The present invention provides a novel active derivative of polyethylene glycol with lipoic acid as a linker and a double-chain structure, which has the following general structural formula:

其中R1，R2为PEG本身的羟基或一个官能团，选自由C1~C12烷氧基、环烷氧基和芳烷基组成的基团。F为活性功能基团，选自由琥珀酰亚胺基、马来酰亚胺基或醛基组成的基团，可以与药物或基体上的氨基或巯基形成共价键连接。Wherein R1 and R2 are the hydroxyl group of PEG itself or a functional group, selected from groups consisting of C1~C12 alkoxy, cycloalkoxy and aralkyl groups. F is an active functional group, selected from a group consisting of succinimide, maleimide or aldehyde, which can form a covalent bond with an amino group or a sulfhydryl group on the drug or the substrate.

根据本发明，这种活性衍生物优选分子具有以下结构，但不仅限于以下结构：According to the present invention, such active derivative preferably has the following structure, but not limited to the following structure:

                                结构1Structure 1

                                结构2Structure 2

                                结构3Structure 3

                                结构4Structure 4

                                结构5Structure 5

                                结构6Structure 6

本发明还提供了制备上述活性衍生物的方法，包括以下步骤：The present invention also provides a method for preparing the above-mentioned active derivative, comprising the following steps:

取单甲氧基聚乙二醇酸溶于二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物溶于二氯甲烷中，再加入硫辛酸(还原型)和吡啶，室温下搅拌回流16-24小时。过滤并将所得滤液减压蒸干，固体物溶于二氯甲烷中，再加入N-羟基琥珀酰亚胺(N-羟基马来酰亚胺)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫代S-酸酯硫辛酸琥珀酰亚胺酯(或马来酰亚胺酯)(结构1和2)，置-20℃保存。Dissolve monomethoxy polyethylene glycol acid in dichloromethane, add appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, remove solvent and remaining thionyl chloride by rotary evaporation, and dissolve the obtained solid in Add lipoic acid (reduced form) and pyridine to dichloromethane, stir and reflux at room temperature for 16-24 hours. Filtrate and evaporate the resulting filtrate to dryness under reduced pressure, dissolve the solid in dichloromethane, then add N-hydroxysuccinimide (N-hydroxymaleimide), in dimethylaminopyridine (DMAP) and bicyclic In the presence of hexylcarbodiimide (DCCI), react for 4 hours at a temperature of -20~10°C. Separate the water with a water separator, continue to reflux overnight, and then remove the dichloromethane in a vacuum. The mixture is precipitated with ether, recrystallized with ethanol, and further purified with a Sephadex G-25 column to obtain pure double polyethylene glycol sulfide. Substitute S-ester lipoic acid succinimidyl ester (or maleimide ester) (structures 1 and 2), store at -20°C.

取单甲氧基聚乙二醇溶于二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于乙醇中，再加入硫辛酸(还原型)和NaOH，室温下搅拌回流18-24小时后用盐酸调节pH值至5.0。将混合物溶剂减压蒸干，固体物溶于二氯甲烷中，再加入N-羟基琥珀酰亚胺(N-羟基马来酰亚胺)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时或过夜。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经SephadexG-25柱进一步纯化，可得纯品双聚乙二醇硫醚硫辛酸琥珀酰亚胺酯(马来酰亚胺酯)(结构3和4)，置-20℃保存。Dissolve monomethoxypolyethylene glycol in dichloromethane, add an appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, remove the solvent and the remaining thionyl chloride by rotary evaporation, and dissolve the obtained solid substance in ethanol Add lipoic acid (reduced form) and NaOH, stir and reflux at room temperature for 18-24 hours, then adjust the pH value to 5.0 with hydrochloric acid. The solvent of the mixture was evaporated to dryness under reduced pressure, and the solid was dissolved in dichloromethane, and then N-hydroxysuccinimide (N-hydroxymaleimide) was added, and in dimethylaminopyridine (DMAP) and dicyclohexyl carbon In the presence of diimine (DCCI), react at -20~10°C for 4 hours or overnight. Use a water separator to remove water, continue to reflux overnight, and then remove the dichloromethane in a vacuum. The mixture is precipitated with ether, recrystallized with ethanol, and further purified through a Sephadex G-25 column to obtain pure bis-polyethylene glycol thioether lipoic acid Succinimidyl esters (maleimide esters) (structures 3 and 4), store at -20°C.

取硫辛酸(还原型)和N，N-羰基二咪唑混悬于二氯甲烷中，室温搅拌反应12小时或过夜，将滤液旋转蒸干，所得固体溶于四氢呋喃中并在四氢铝锂存在下，室温反应过夜。将所得混合物过滤后旋转蒸干。所得固体溶于二氯甲烷中，加入已制备的单甲氧基聚乙二醇氯(mPEG-Cl)或单甲氧基聚乙二醇酰氯(mPEG-COCl)和适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于二氯甲烷中，硫酸钠干燥，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫代S-酸酯硫辛酸醛(或双聚乙二醇硫醚硫辛酸醛)(结构5和6)，置-20℃保存。Suspend lipoic acid (reduced form) and N, N-carbonyldiimidazole in dichloromethane, stir at room temperature for 12 hours or overnight, spin the filtrate to dryness, and dissolve the resulting solid in tetrahydrofuran in the presence of lithium aluminum hydride reaction overnight at room temperature. The resulting mixture was filtered and spun to dryness. The resulting solid was dissolved in dichloromethane, and the prepared monomethoxypolyethylene glycol chloride (mPEG-Cl) or monomethoxypolyethylene glycol acid chloride (mPEG-COCl) and an appropriate amount of thionyl chloride were added, at room temperature After stirring for 5-7 hours, the solvent and the remaining thionyl chloride were removed by rotary evaporation, and the obtained solid matter was dissolved in dichloromethane, dried over sodium sulfate, the mixture was precipitated by ether, recrystallized by ethanol, and passed through a Sephadex G-25 column After further purification, the pure bis-polyethylene glycol thioether lipoic acid aldehyde (or bis-polyethylene glycol thioether lipoic acid aldehyde) (structures 5 and 6) can be obtained and stored at -20°C.

根据本发明的另一个方面，提供上述活性衍生物通过F基团与药物分子形成的结合物以及包含这种结合物的药物组合物。上述聚乙二醇活性衍生物可以在温和的生理条件下(反应pH值在4~9之间，反应温度0~20℃)与具有自由氨基或巯基的药物分子结合，形成大分子结合物。这种结合物具有以下结构通式：According to another aspect of the present invention, there is provided a conjugate formed by the above-mentioned active derivative and a drug molecule through the F group and a pharmaceutical composition comprising the conjugate. The above active derivatives of polyethylene glycol can be combined with drug molecules with free amino groups or sulfhydryl groups under mild physiological conditions (reaction pH value between 4-9, reaction temperature 0-20°C) to form macromolecular conjugates. This conjugate has the general structural formula:

以上所述药物分子可以是蛋白质或多肽，优选但不限于干扰素、红细胞生成素、G-CSF、胸腺肽，胸腺五肽(TP-5)、胸腺肽α1、肌肽、亮丙瑞林、戈那瑞林、阿拉瑞林、舍莫瑞林、奥曲肽、生长抑素、血管紧张素、阿基瑞林等。The above-mentioned drug molecules can be proteins or polypeptides, preferably but not limited to interferon, erythropoietin, G-CSF, thymosin, thymopentin (TP-5), thymosin α1, carnosine, leuprolide, gonaurel Lin, alarelin, sermorelin, octreotide, somatostatin, angiotensin, ajirelin, etc.

除了蛋白质或多肽以外，药物分子还可以是选自氨基酸、糖类、有机酸、生物碱、黄酮类、甙类、醌类、萜类等药物分子中具有自由氨基或巯基的分子。In addition to proteins or polypeptides, drug molecules can also be selected from amino acids, sugars, organic acids, alkaloids, flavonoids, glycosides, quinones, terpenes and other molecules with free amino groups or sulfhydryl groups.

                             具体实施方案Specific implementation plan

                                实施例1Example 1

双聚乙二醇硫代S-酸酯硫辛酸琥珀酰亚胺酯的制备Preparation of Dipolyethylene Glycol Thio S-ester Lipoic Acid Succinimide Ester

反应式：Reaction formula:

取10g单甲氧基聚乙二醇酸5000(0.002mol)溶于50ml二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物溶于75ml二氯甲烷中，再加入0.21g硫辛酸(还原型)(0.001mol)和0.40g吡啶(0.005mol)，搅拌回流16-24小时。过滤并将所得滤液减压蒸干，固体物溶于50ml二氯甲烷中，再加入0.58g N-羟基琥珀酰亚胺(0.005mol)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时或过夜。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫代S-酸酯硫辛酸琥珀酰亚胺酯8.2g，置-20℃保存。Take 10g of monomethoxy polyethylene glycol acid 5000 (0.002mol) and dissolve it in 50ml of dichloromethane, add an appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, and remove the solvent and the remaining thionyl chloride by rotary evaporation , Dissolve the obtained solid in 75ml of dichloromethane, then add 0.21g lipoic acid (reduced form) (0.001mol) and 0.40g pyridine (0.005mol), stir and reflux for 16-24 hours. Filter and evaporate the resulting filtrate to dryness under reduced pressure, dissolve the solid in 50ml of dichloromethane, then add 0.58g of N-hydroxysuccinimide (0.005mol), in dimethylaminopyridine (DMAP) and dicyclohexyl carbon di In the presence of imine (DCCI), react at -20~10°C for 4 hours or overnight. Separate the water with a water separator, continue to reflux overnight, and then remove the dichloromethane in a vacuum. The mixture is precipitated with ether, recrystallized with ethanol, and further purified with a Sephadex G-25 column to obtain pure double polyethylene glycol sulfide. Substitute S-ester lipoic acid succinimidyl ester 8.2g, store at -20°C.

                                实施例2Example 2

双聚乙二醇硫代S-酸酯硫辛酸马来酰亚胺酯的制备Preparation of Dipolyethylene Glycol Thio S-ester Lipoic Acid Maleimide Ester

反应式：Reaction formula:

取16g单甲氧基聚乙二醇酸8000(0.002mol)溶于50ml二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于75ml二氯甲烷中，再加入0.21g硫辛酸(还原型)(0.001mol)和0.40g吡啶(0.005mol)，搅拌回流16小时。过滤并将所得滤液减压蒸干，固体物溶于50ml二氯甲烷中，再加入0.57g N-羟基马来酰亚胺(0.005mol)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时或过夜。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫代S-酸酯硫辛酸马来酰亚胺酯14.1g，置-20℃保存。Take 16g of monomethoxy polyethylene glycol acid 8000 (0.002mol) and dissolve it in 50ml of dichloromethane, add an appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, and remove the solvent and the remaining thionyl chloride by rotary evaporation , The resulting solid matter was dissolved in 75ml of dichloromethane, then added 0.21g of lipoic acid (reduced form) (0.001mol) and 0.40g of pyridine (0.005mol), and stirred and refluxed for 16 hours. Filter and evaporate the obtained filtrate to dryness under reduced pressure, the solid is dissolved in 50ml of dichloromethane, then add 0.57g N-hydroxyl maleimide (0.005mol), in dimethylaminopyridine (DMAP) and dicyclohexyl carbon In the presence of diimine (DCCI), react at -20~10°C for 4 hours or overnight. Separate the water with a water separator, continue to reflux overnight, and then remove the dichloromethane in a vacuum. The mixture is precipitated with ether, recrystallized with ethanol, and further purified with a Sephadex G-25 column to obtain pure double polyethylene glycol sulfide. Substitute S-ester lipoic acid maleimide ester 14.1g, store at -20°C.

                                实施例3Example 3

双聚乙二醇硫醚硫辛酸琥珀酰亚胺酯的制备Preparation of Dipolyethylene glycol thioether lipoic acid succinimidyl ester

反应式：Reaction formula:

取20g单甲氧基聚乙二醇10000(0.002mol)溶于50ml二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于75ml二氯甲烷中，再加入0.21g硫辛酸(还原型)(0.001mol)和0.20g NaOH(0.005mol)的乙醇溶液20ml，搅拌回流18-24小时后用盐酸调节pH值至5.0。将混合物溶剂减压蒸干，固体物溶于50ml二氯甲烷中，再加入0.58g N-羟基琥珀酰亚胺(0.005mol)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时或过夜。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经SephadexG-25柱进一步纯化，可得纯品双聚乙二醇硫醚硫辛酸琥珀酰亚胺酯18.3g，置-20℃保存。Take 20g of monomethoxypolyethylene glycol 10000 (0.002mol) and dissolve it in 50ml of dichloromethane, add an appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, remove the solvent and the remaining thionyl chloride by rotary evaporation, The obtained solid matter was dissolved in 75ml of dichloromethane, then 20ml of ethanol solution of 0.21g lipoic acid (reduced form) (0.001mol) and 0.20g NaOH (0.005mol) was added, and the pH was adjusted with hydrochloric acid after stirring and reflux for 18-24 hours value to 5.0. The mixture solvent was evaporated to dryness under reduced pressure, and the solid was dissolved in 50ml of dichloromethane, then 0.58g of N-hydroxysuccinimide (0.005mol) was added, and in dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide In the presence of (DCCI), react at -20~10°C for 4 hours or overnight. Use a water separator to remove water, continue to reflux overnight, and then remove the dichloromethane in a vacuum. The mixture is precipitated with ether, recrystallized with ethanol, and further purified through a Sephadex G-25 column to obtain pure bis-polyethylene glycol thioether lipoic acid Succinimide ester 18.3g, stored at -20°C.

                                实施例4Example 4

双聚乙二醇硫醚硫辛酸马来酰亚胺酯的制备Preparation of Bis-polyethylene glycol thioether lipoic acid maleimide ester

反应式：Reaction formula:

取40g单甲氧基聚乙二醇20000(0.002mol)溶于100ml二氯甲烷中，加入适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物溶于150ml二氯甲烷中，再加入0.21g硫辛酸(还原型)(0.001mol)和0.2g NaOH(0.005mol)的乙醇溶液30ml，室温下搅拌回流18-24小时后用盐酸调节pH值至5.0。将混合物溶剂减压蒸干，固体物溶于100ml二氯甲烷中，再加入0.57g N-羟基马来酰亚胺(0.005mol)，在二甲氨基吡啶(DMAP)与二环己基碳二亚胺(DCCI)存在下，温度-20~10℃，反应4小时或过夜。用水分离器分去水分，继续回流过夜，然后真空抽去二氯甲烷，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得纯品双聚乙二醇硫醚硫辛酸马来酰亚胺酯35.4g，置-20℃保存。Take 40g of monomethoxypolyethylene glycol 20000 (0.002mol) and dissolve it in 100ml of dichloromethane, add an appropriate amount of thionyl chloride, stir at room temperature for 5-7 hours, remove the solvent and the remaining thionyl chloride by rotary evaporation, Dissolve the resulting solid in 150ml of dichloromethane, then add 0.21g of lipoic acid (reduced form) (0.001mol) and 30ml of ethanol solution of 0.2g NaOH (0.005mol), stir and reflux at room temperature for 18-24 hours, then wash with hydrochloric acid Adjust the pH to 5.0. The mixture solvent was evaporated to dryness under reduced pressure, and the solid was dissolved in 100ml of dichloromethane, and then 0.57g of N-hydroxyl maleimide (0.005mol) was added, and in dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide In the presence of amine (DCCI), the temperature is -20~10°C, react for 4 hours or overnight. Separate the water with a water separator, continue to reflux overnight, and then vacuum the dichloromethane. The mixture is precipitated with ether, recrystallized with ethanol, and further purified with a Sephadex G-25 column to obtain pure double polyethylene glycol sulfide sulfide. Caprylic acid maleimide ester 35.4g, stored at -20°C.

                                实施例5Example 5

双聚乙二醇硫代S-酸酯硫辛酸醛的制备Preparation of Dipolyethylene Glycol Thio S-ester Lipoic Acid Aldehyde

反应式：Reaction formula:

取0.21g硫辛酸(还原型)(0.001mol)和0.24g N，N-羰基二咪唑(0.0015mol)混悬于25ml二氯甲烷中，室温搅拌反应12小时或过夜，将滤液旋转蒸干，所得固体溶于20ml四氢呋喃中并在四氢铝锂存在下，室温反应过夜。将所得混合物过滤后旋转蒸干。所得固体溶于50ml二氯甲烷中，加入16g已制备的单甲氧基聚乙二醇氯8000(mPEG-Cl)(0.002mol)和适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于50ml二氯甲烷中，硫酸钠干燥，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫代S-酸酯硫辛酸醛15.0g，置-20℃保存。Get 0.21g lipoic acid (reduced form) (0.001mol) and 0.24g N, N-carbonyldiimidazole (0.0015mol) are suspended in 25ml dichloromethane, stir reaction at room temperature for 12 hours or overnight, the filtrate is spun to dryness, The resulting solid was dissolved in 20 ml of tetrahydrofuran and reacted overnight at room temperature in the presence of lithium aluminum hydride. The resulting mixture was filtered and spun to dryness. The obtained solid was dissolved in 50ml of dichloromethane, 16g of prepared monomethoxypolyethylene glycol chloride 8000 (mPEG-Cl) (0.002mol) and an appropriate amount of thionyl chloride were added, stirred at room temperature for 5-7 hours, and then rotated The solvent and the remaining thionyl chloride were removed by evaporation, the resulting solid matter was dissolved in 50ml of dichloromethane, dried over sodium sulfate, the mixture was precipitated with ether, recrystallized with ethanol, and then further purified by a Sephadex G-25 column to obtain Pure bis-polyethylene glycol thio S-ester lipoic acid aldehyde 15.0g, stored at -20°C.

                                实施例6Example 6

双聚乙二醇硫醚硫辛酸醛的制备Preparation of Dipolyethylene Glycol Thioether Lipoic Acid Aldehyde

反应式：Reaction formula:

取0.21g硫辛酸(还原型)(0.001mol)和0.24g N，N-羰基二咪唑(0.0015mol)混悬于25ml二氯甲烷中，室温搅拌反应12小时或过夜，将滤液旋转蒸干，所得固体溶于20ml四氢呋喃中并在四氢铝锂存在下，室温反应过夜。将所得混合物过滤后旋转蒸干。所得固体溶于100ml二氯甲烷中，加入40g已制备的单甲氧基聚乙二醇酰氯20000(mPEG-COCl)(0.002mol)和适量氯化亚砜，室温搅拌5-7小时后，旋转蒸发除去溶剂及剩余的氯化亚砜，将所得固体物质溶于75ml二氯甲烷中，硫酸钠干燥，混合物经乙醚沉淀，乙醇重结晶，再经Sephadex G-25柱进一步纯化，可得可得纯品双聚乙二醇硫醚硫辛酸醛35.1g，置-20℃保存。Get 0.21g lipoic acid (reduced form) (0.001mol) and 0.24g N, N-carbonyldiimidazole (0.0015mol) are suspended in 25ml dichloromethane, stir reaction at room temperature for 12 hours or overnight, the filtrate is spun to dryness, The resulting solid was dissolved in 20 ml of tetrahydrofuran and reacted overnight at room temperature in the presence of lithium aluminum hydride. The resulting mixture was filtered and spun to dryness. The obtained solid was dissolved in 100ml of dichloromethane, and 40g of prepared monomethoxypolyethylene glycol acid chloride 20000 (mPEG-COCl) (0.002mol) and an appropriate amount of thionyl chloride were added, stirred at room temperature for 5-7 hours, and then rotated The solvent and the remaining thionyl chloride were removed by evaporation, and the resulting solid matter was dissolved in 75ml of dichloromethane, dried over sodium sulfate, the mixture was precipitated with ether, recrystallized with ethanol, and further purified through a Sephadex G-25 column to obtain Pure bis-polyethylene glycol thioether lipoic acid aldehyde 35.1g, stored at -20°C.

                                实施例7Example 7

双链聚乙二醇-干扰素的制备Preparation of double-stranded polyethylene glycol-interferon

取10mg/ml的干扰素硼酸缓冲液，pH＝9.0，加入双聚乙二醇硫代S-酸酯硫辛酸琥珀酰亚胺酯5000干粉500mg，4℃条件下反应8小时，经阴离子交换柱纯化，可得PEG-IFN样品。Take 10mg/ml interferon borate buffer solution, pH = 9.0, add 500mg of bis-polyethylene glycol thio S-ester lipoic acid succinimide ester 5000 dry powder, react at 4°C for 8 hours, pass through an anion exchange column After purification, PEG-IFN samples can be obtained.

Claims (3)

1. the polyethylene active derivatives that has following general structure

R wherein¹And R²Be selected from methoxyl group, oxyethyl group or benzyl group.

2. polyethylene active derivatives as claimed in claim 1 is characterized in that: the molecular weight of polyoxyethylene glycol is 1000～40,000.

3. a polyethylene active derivatives as claimed in claim 1 is by its active end group and the formed mixture of drug molecule, and this mixture has following general structure:

Y is a linking group, is selected from the group of being made up of alkyl, amide group; Described drug molecule is selected from protein, polypeptide or contains free amino group or the organic acid of sulfydryl, carbohydrate, alkaloid, flavonoid, glucoside, quinones, terpene.