CN117362478A - 一种去硫酸肝素载体及其制备方法和应用 - Google Patents
一种去硫酸肝素载体及其制备方法和应用 Download PDFInfo
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- CN117362478A CN117362478A CN202311300901.8A CN202311300901A CN117362478A CN 117362478 A CN117362478 A CN 117362478A CN 202311300901 A CN202311300901 A CN 202311300901A CN 117362478 A CN117362478 A CN 117362478A
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明提供了一种去硫酸肝素载体及其制备方法和应用,属于生物医药技术领域。本发明为解决肝素作为载体在全身给药治疗可能出现的潜在出血风险,进行肝素完全脱硫酸基,并利用脱硫酸肝素作为药物载体构建三组分型药物递送系统,制备得到抗肿瘤药物,其中,脱硫酸肝素作为药物载体,半乳糖作为靶向肝脏肿瘤细胞基团,紫杉醇为药物。所述的抗肿瘤药物的溶解度得到改善,并且在体外实验中该系统表现出了优异的抗肿瘤活性。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种去硫酸肝素载体及其制备方法和应用。
背景技术
药物载体的种类众多,较为成熟的有微囊与微球、纳米粒、脂质体。从20 世纪60年代以来,药物控制释放体系的研究引起人们广泛的重视。随着科技的发展,人们对疾病的治疗效果和治疗手段的要求也日益提高。提高药物的生物利用度、延长药物作用时间、实现药物体内靶向定位,降低毒副作用一直是药物载体领域研究的重点。就目前情况来看,许多药物载体仍存在研制成本高、包封率低、适用范围窄、临床应用率不高、安全性低等问题。
肝素是一种天然存在的糖胺聚糖,1916年首先由Mclean发现,由嗜碱性粒细胞和肥大细胞在体内产生,可与抗凝血酶(AT)结合,形成LMWH-AT复合物,该复合物能抑制凝血酶(Fa)和Xa因子(FXa)等凝血因子的活性,从而发挥抗凝血作用,其作为抗凝剂在临床上应用已超过百年历史。近年来,许多科学家探索了肝素在药物载体方面的应用,其优点为:可修饰位点多(每个二糖重复单元上都有可修饰的羧基),水溶性好(改善药物分子水溶性),安全性好(近百年的抗凝血应用证明了其安全性)。例如,专利CN201710374727.X公开了一种低分子量肝素以及在制备肺纤维化中的用途,该肝素的数均分子量(Mn)的范围为1500-10000Da,重均分子量(Mw)的范围为3500-11500Da。专利CN200910136329.X则公开了一种含有吡格列酮和肝素或低分子肝素的药物组合物在预防或治疗脂肪肝中的应用。
肝素作为一种众所周知的抗凝剂,由于其与多种蛋白质和生长因子结合的能力以及生物相容性,已被研究用作靶向治疗癌症的药物载体。然而,它的使用也存在挑战,特别是因为它的抗凝作用会导致出血。这种风险在癌症患者中更高,因为癌症本身或其治疗可能已经增加了出血的风险。目前大部分药物载体均选用肝素,或选择性脱除N-位,C2、C3或C6位硫酸基,在全身给药治疗过程中仍然存在出血等不良反应和潜在风险。因此,亟需提供一种制备工艺简单,成本低,安全性高,毒性小的肝素载体。
发明内容
针对上述不足,本发明提供了一种去硫酸肝素载体及其制备方法和应用。本发明以去硫酸基肝素作为载体,经靶向基团和药物分子化学修饰后,实现药物水溶性的改善,并可靶向递送药物。肝素作为抗凝血药物,也是人体内本来就有的分子,该分子作为药物载体不会被人体产生排斥作用,脱除硫酸基后,载体无抗凝血活性。本发明所述的去硫酸肝素载体研制成本低,安全性高,毒性小,具有较好的应用前景。
为了实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种去硫酸肝素载体,所述的去硫酸肝素载体如下式所示:
。
另一方面,本发明提供了上述去硫酸肝素载体的制备方法,所述的制备方法如下所示:
。
具体地,所述的制备方法包括以下步骤:
(1)将肝素钠溶解在水溶液中,通过H离子交换树脂转换成吡啶盐的形式,将洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌,蒸干;
(2)将步骤(1)的产物溶解在DMSO和MEOH的混合溶液中,加热回流,反应液纯化后收集产物,溶解到水中透析,得到未修饰肝素脱硫酸基产物。
进一步具体地,所述的DMSO:MEOH=9:1。
又一方面,本发明提供了上述去硫酸肝素载体在制备抗肿瘤药物中的应用。
又一方面,本发明提供了一种抗肿瘤药物,所述的抗肿瘤药物为利用上述脱硫酸肝素作为药物载体构建的三组分型药物递送系统:脱硫酸肝素作为药物载体,半乳糖作为靶向肝脏肿瘤细胞基团,紫杉醇为药物。
具体地,所述的抗肿瘤药物如下式所示:
。
进一步具体地,所述的抗肿瘤药物还包含任选的药学上可接受的载体。
进一步具体地,所述的药学上可接受的载体包括但不限于:稀释剂、赋形剂、填充剂、润湿剂、崩解剂、矫味剂和粘合剂。
又一方面,本发明提供了上述抗肿瘤药物的制备方法,所述的制备方法如下所示:
。
具体地,所述的制备方法包括以下步骤:
(1)肝素全乙酰化:compound 5的合成
a.将肝素钠溶解在水中,加入乙酸酐,用TEA调节PH=8,室温搅拌,蒸干溶剂,混合物用G-10纯化,洗脱液收集蒸干得到乙酰化肝素;
b.将乙酰化肝素通过H离子交换树脂,收集洗脱液在0℃下搅拌,并在冰浴下用TEA调节PH至8-9,冰浴下继续搅拌,冻干,得到compound 5,合成三组分/二组分化合物备用;
(2)缩合反应:化合物6的合成
将compound 5溶解在DMSO和水的混合溶液中,依次加入Ethanamine、2-[2-(2-azidoethoxy)ethoxy]、compound 3、EDC、HOBt和TEA,室温搅拌过夜,混合物用G-10纯化,洗脱液收集蒸干后再次溶解在少量水中,用去离子水透析,冻干后得到compound 6;
(3)脱硫酸基:化合物7的合成
c.将compound 6溶解在水中,通过H离子交换树脂,洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌,蒸干。
d.将上步产物溶解在DMSO:MEOH=9:1混合溶液中,加热回流,反应液用G-10纯化,得到compound 7;
(4)点击反应:三组分产物1的合成
将Compound 7溶解在DMSO: H2O = 1.5:1混合溶液中,加入 compound 4。加热搅拌过夜,蒸干溶剂,粗产物用DCM:MeOH=1:1溶解,超声后离心,更换有机溶液反复离心多次,直到上清液TLC检测没有残留的compound 4,固体加水冻干,得到终产物compound 1。
与现有技术相比,本发明的积极和有益效果在于:
(1)本发明为解决肝素作为载体在全身给药治疗可能出现的潜在出血风险,进行肝素完全脱硫酸基,表现出无抗凝血活性,可作为药物载体经化学修饰药物偶联,实现了高效药物安全递送。可解决肝素在体内治疗过程中引发出血副作用的问题,制备工艺简单,成本低,安全性高,毒性小,具有较好的应用前景。
(2)本发明利用脱硫酸肝素作为药物载体构建三组分型药物递送系统,制备得到抗肿瘤药物,其中,脱硫酸肝素作为药物载体,半乳糖作为靶向肝脏肿瘤细胞基团,紫杉醇为药物。所述的抗肿瘤药物的溶解度得到改善,并且在体外实验中该系统表现出了优异的抗肿瘤活性。
附图说明
图1为凝血酶时间(TT)实验结果。
图2为脱硫酸肝素、化合物7和9的HepG2细胞毒性实验结果。
图3为PTX、化合物1,2和4的HepG2细胞毒性实验结果。
图4为细胞抑制实验结果。
图5为未经修饰脱硫酸基肝素(Desulfated Heparin)核磁数据。
图6为Compound 5核磁数据。
图7为三组分(compound 7)核磁数据。
图8为Compound 1核磁数据。
图9为二组分(compound 9)核磁数据。
图10为Compound 2核磁数据。
图11为Compound 3核磁数据。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1.未经修饰脱硫酸基肝素(Desulfated Heparin)的合成
(1)将肝素钠(500mg)溶解在20mL水溶液中,通过H离子交换树脂转换成吡啶盐的形式,将洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌30min,蒸干,直接投下一步。
(2)将步骤(1)的产物溶解在DMSO:MEOH=9:1(10mL)混合溶液中,100℃加热回流12h,反应液用G-10纯化,产物收集,溶解到2-3mL水中透析12h。得到未修饰肝素脱硫酸基产物(Desulfated Heparin)。(两步产率98%)
实施例2.经修饰脱硫酸基肝素(脱硫酸肝素偶联靶向分子和药物)的合成
1、肝素全乙酰化:compound 5的合成
将1g 肝素钠溶解在12mL水中,加入800µL乙酸酐,用TEA调节PH=8,室温搅拌4h,蒸干溶剂,混合物用G-10纯化,洗脱液收集蒸干得到乙酰化肝素。
将乙酰化肝素通过H离子交换树脂,收集洗脱液在0℃下搅拌,并在冰浴下用TEA调节PH至8-9,冰浴下继续搅拌30min,冻干,得到compound 5,合成三组分/二组分化合物备用。
2、缩合反应:化合物6的合成
将compound 5(177mg, 188µmol, 1eq.)溶解在DMSO和水的混合溶液中,依次加入Ethanamine、2-[2-(2-azidoethoxy)ethoxy](32.8mg,188µmol,1eq.)、compound3(89.2mg,188µmol,1eq.)、EDC(54.1mg,282µmol,1.5eq.)、HOBT(43.2mg,282µmol,1.5eq.)和TEA(130µL,940µmol,5eq.)。室温搅拌过夜,混合物用G-10纯化,洗脱液收集蒸干后再次溶解在少量水中,用去离子水透析12h,冻干后得到compound 6。直接投下一步。
3、脱硫酸基:化合物7的合成
将compound 6(261 mg)溶解在6mL H2O中,通过H离子交换树脂,洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌30 min,蒸干,直接投下一步。
将上步产物溶解在DMSO:MEOH=9:1(10mL)混合溶液中,100℃回流12h,反应液用G-10纯化,得到compound 7。(三步产率 125.3mg, 48%)
4、点击反应:三组分产物1的合成
将Compound 7 (84.5 mg, 60.8 µmol) 溶解在DMSO: H2O = 1.5:1 (5mL)混合溶液中,加入 compound 4 (84.3 mg, 66.9 µmol)。70℃加热搅拌过夜。蒸干溶剂,粗产物用DCM:MeOH=1:1 超声5min,3000rpm离心15min,更换有机溶液反复离心多次,直到上清液TLC检测没有残留的compound 4 为止。固体加水冻干。得到终产物compound 1。(92.7 mg, 59.4%)
实施例3.经修饰脱硫酸基肝素(脱硫酸肝素偶联药物)的合成
1、缩合反应:8的合成
将compound 5 (200mg,213µmol,1eq.)溶解在DMSO:H2O=2.5:1(7mL)的混合溶液中,依次加入Ethanamine、2-[2-(2-azidoethoxy)ethoxy](18.5mg, 106µmol, 0.5eq.)、EDC(30.6mg, 159µmol, 0.75eq.)、HOBt(24.4mg, 159µmol, 0.75eq.)和TEA(74µL, 532.5µmol, 2.5eq.)。室温搅拌过夜,混合物用G-10纯化,洗脱液收集蒸干后溶解在少量水中,用去离子水透析12h,冻干后得到compound 8。直接投下一步。
2、脱硫酸基:化合物9的合成
将compound 8(200 mg)溶解在6mL H2O中,通过H离子交换树脂,洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌30 min,蒸干,直接投下一步。
将上步产物溶解在DMSO:MEOH=9:1(10mL)混合溶液中,100℃回流12h,反应液用G-10纯化,得到compound 9。(三步产率118.3 mg,59.7%)
3、点击反应:三组分产物2的合成
将Compound9 (50 mg, 53.6 µmol)溶解在DMSO: H2O = 1.5:1 (5 mL)的混合溶液中,加入 compound 4 (74.3 mg, 59 µmol) 。70℃加热搅拌过夜。蒸干溶剂,粗产物用DCM:MeOH=1:1 超声5min,7000rpm离心15min,更换有机溶液反复离心多次,直到上清液TLC检测没有残留的compound 4为止。固体加水冻干。得到终产物 compound 2。(44.2 mg,37.6%)
实验例1.凝血时间(TT)测试
用生理盐水制备10mg/mL的肝素、脱硫酸基肝素(Desulfated Heparin)、compound7和compound 9标准溶液。使用3.8%(w/v)枸橼酸钠抗凝管采取空腹12h的患者静脉血,以1500rpm离心收集的血液样品15分钟分离血浆,并在不干扰细胞成分的情况采取100µL上层血浆。向血浆样品中依次加入上述药物标准液,使用STA自动凝血仪检测凝血酶时间(TT)。确保血浆采集后2h内完成测量,避免血浆成分的降解或者变化。每个样品重复三次,确保准确定和再现性。对于空白组,则用血清和生理盐水代替药物。
凝血酶时间(TT)测试是衡量在凝血酶存在的情况下形成血栓所需的时间。肝素通过增强抗凝血酶III的活性,抑制凝血酶和因子Xa的作用,从而防止血栓形成。当使用肝素时,它可以延长TT测试结果。如图1所示,其他化合物均未表现出任何抗凝血作用。这表明,与肝素对比,本发明所述的药物载体无抗凝血活性。
实验例2.细胞毒性实验
(1)脱硫酸肝素、化合物7和9的QSG-7701细胞毒性实验
将QSG-7701细胞接种在96孔板(2×103 /孔)中。孵育24h后,弃去培养基,用不同浓度的脱硫酸肝素、化合物7和9溶液分别处理细胞72h。孵育72h后弃去培养基。随后,将含有10%CCK-8的RPMI 1640溶液加入到96孔板的每个孔中,再孵育2小时。在SpectraMax-M2板读数器上读取450nm处的吸光度读数。检测结果如图2A所示。
(2)脱硫酸肝素、化合物7和9的HepG2细胞毒性实验
将HepG2细胞接种在96孔板(5×103 /孔)中。孵育24h后,弃去培养基,用不同浓度的脱硫酸肝素、化合物7和9溶液分别处理细胞72h。孵育72h后弃去培养基。随后,将含有10%CCK-8的DMEM溶液加入到96孔板的每个孔中,再孵育2小时。在SpectraMax-M2板读数器上读取450nm处的吸光度读数。检测结果如图2B所示。
(3)PTX、化合物1,2和4的HepG2细胞毒性实验
将HepG2细胞接种在96孔板(2×103 /孔)中。孵育24h后,弃去培养基,用不同浓度的PTX、化合物1、2和4溶液分别处理细胞72h。孵育72h后弃去培养基。随后,将含有10%CCK-8的DMEM溶液加入每个孔中并再孵育2小时。在SpectraMax-M2板读数器上读取450nm处的吸光度读数。检测结果如图3A所示。
(4)PTX、化合物1,2和4的QSG-7701细胞毒性实验
将QSG-7701细胞接种在96孔板(5×103 /孔)中。孵育24h后,弃去培养基,用不同浓度的PTX、化合物1、2和4溶液分别处理细胞72h。孵育72h后弃去培养基。随后,将含有10%CCK-8的RPMI 1640溶液加入每个孔中并再孵育2小时。在SpectraMax-M2板读数器上读取450nm处的吸光度读数。检测结果如图3B所示。
细胞毒性实验均通过以下公式计算细胞活力:
细胞活力(%)=[(As-Ab)/(Ac-Ab)] ×100%。
其中As是用不同药物制剂孵育的细胞的OD值,Ac是用DMEM或RPMI 1640和CCK8孵育的细胞的OD值,Ab是仅用DMEM孵育的细胞的OD值。使用GraphPad软件计算药物的IC50。
由图2和图3可知,在测试浓度范围内(1µg/mL至16µg/mL)未观察到细胞毒性。
从图3A可以看出,天然PTX显示出高效的抗HepG2细胞系,IC50值为0.653nM。BCN修饰的PTX-化合物4,具有类似的抗HepG2活性,它含有可水解的酯键以释放天然PTX药物。与PTX相比,化合物1的IC50值降至1.055nM。而对于不含半乳糖的化合物2,IC50值进一步下降至10 nM。这是因为化合物1上的半乳糖可以帮助靶向药物递送到HepG2,HepG2具有大量的半乳糖受体。另一方面,如图3B所示,对于正常人肝细胞系QSG-7701,PTX表示毒性高,与BCN衍生化化合物4具有妥协的毒性(~100倍)。另一方面,化合物1和2的IC50值比天然PTX高得多(~高1000倍)。
实验例3.Gla-Heparin-FITC(化合物11)靶向抑制实验
1、三组分点击:化合物10的合成
将Compound 7 (30 mg, 22.5 µmol)溶解在DMSO: H2O = 2:1 (3 mL)的混合溶液中,加入BCN-FITC (17.7 mg, 24.8 µmol)。37℃搅拌过夜。蒸干溶剂,粗产物用150µL饱和碳酸氢钠溶液搅拌半天,蒸干溶剂,得到终产物 compound 10。(26 mg, 56.4%)
2、FITC标记载体的Confocol成像:
将HepG2细胞(1×105个细胞/孔)接种在玻璃底培养皿(D29-14-1.5-N,Sunnyvale, California, USA)中并孵育24小时。
第1组:然后将细胞用化合物10(2µM或10 µM FITC)的DMEM溶液处理4小时。
第2组:将细胞用GalNAc(30mM)的DMEM溶液预处理1小时,然后加入化合物10(2µM或10µM FITC)的DMEM溶液共孵育4小时。
空白组:仅用培养基处理细胞。
孵育后,用PBS洗涤细胞3次,并用4%多聚甲醛处理15分钟进行固定。为了标记细胞核,将细胞在室温下用1µg/mL DAPI进一步染色10分钟。轻轻吸出DAPI染色溶液(1µg/mL)。用无菌PBS或生理盐水洗涤2-3次,每次3-5分钟。在超高分辨率激光共聚焦显微镜(A1RHD25+N-SIM+N-STORM, Nikon, Japan)下观察细胞。检测结果如图4所示。
HepG2的半乳糖受体水平很高。为了可视化靶向效应,合成了BCN-FITC,代替BCN-PTX(化合物4),BCN-FITC通过SPAAC与化合物7偶联。然后将该化合物与HepG2以2µM/10µM的浓度孵育。如图4所示,HepG2细胞在与游离FITC孵育时几乎没有标记。然而,当HepG2细胞与FITC标记的化合物10一起孵育4h/10h时,在细胞周围观察到绿色荧光。通过与30 mM半乳糖共孵育来抑制荧光。这些数据表明化合物10上的半乳糖可以靶向HepG2细胞。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种去硫酸肝素载体,其特征在于:所述的去硫酸肝素载体如下式所示:
。
2.一种权利要求1所述的去硫酸肝素载体的制备方法,其特征在于:所述的制备方法如下所示:
。
3.根据权利要求2所述的制备方法,其特征在于:所述的制备方法包括以下步骤:
(1)将肝素钠溶解在水溶液中,通过H离子交换树脂转换成吡啶盐的形式,将洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌,蒸干;
(2)将步骤(1)的产物溶解在DMSO和MEOH的混合溶液中,加热回流,反应液纯化后收集产物,溶解到水中透析,得到未修饰肝素脱硫酸基产物。
4.根据权利要求3所述的制备方法,其特征在于:所述的DMSO:MEOH=9:1。
5.一种权利要求1所述的去硫酸肝素载体在制备抗肿瘤药物中的应用。
6.一种抗肿瘤药物,其特征在于:所述的抗肿瘤药物为利用上述脱硫酸肝素作为药物载体构建的三组分型药物递送系统:脱硫酸肝素作为药物载体,半乳糖作为靶向肝脏肿瘤细胞基团,紫杉醇为药物。
7.根据权利要求6所述的抗肿瘤药物,其特征在于:所述的抗肿瘤药物如下式所示:
。
8.根据权利要求6所述的抗肿瘤药物,其特征在于:所述的抗肿瘤药物还包含任选的药学上可接受的载体。
9.一种权利要求6-8任一项所述的抗肿瘤药物的制备方法,其特征在于:所述的制备方法如下所示:
。
10.根据权利要求9所述的制备方法,其特征在于:所述的制备方法包括以下步骤:
(1)肝素全乙酰化:compound 5的合成
a.将肝素钠溶解在水中,加入乙酸酐,用TEA调节PH=8,室温搅拌,蒸干溶剂,混合物用G-10纯化,洗脱液收集蒸干得到乙酰化肝素;
b.将乙酰化肝素通过H离子交换树脂,收集洗脱液在0℃下搅拌,并在冰浴下用TEA调节PH至8-9,冰浴下继续搅拌,冻干,得到compound 5,合成三组分/二组分化合物备用;
(2)缩合反应:化合物6的合成
将compound 5溶解在DMSO和水的混合溶液中,依次加入Ethanamine、2-[2-(2-azidoethoxy)ethoxy]、compound 3、EDC、HOBt和TEA,室温搅拌过夜,混合物用G-10纯化,洗脱液收集蒸干后再次溶解在少量水中,用去离子水透析,冻干后得到compound 6;
(3)脱硫酸基:化合物7的合成
c.将compound 6溶解在水中,通过H离子交换树脂,洗脱液置于冰浴中,用吡啶调节PH=9,冰浴下搅拌,蒸干;
d.将上步产物溶解在DMSO:MEOH=9:1混合溶液中,加热回流,反应液用G-10纯化,得到compound 7;
(4)点击反应:三组分产物1的合成
将Compound 7溶解在DMSO: H2O = 1.5:1混合溶液中,加入 compound 4。加热搅拌过夜,蒸干溶剂,粗产物用DCM:MeOH=1:1溶解,超声后离心,更换有机溶液反复离心多次,直到上清液TLC检测没有残留的compound 4,固体加水冻干,得到终产物compound 1。
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