WO2022193898A1 - 一种多肽偶联硼携带剂及其制备方法和药物制剂 - Google Patents

一种多肽偶联硼携带剂及其制备方法和药物制剂 Download PDF

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WO2022193898A1
WO2022193898A1 PCT/CN2022/076659 CN2022076659W WO2022193898A1 WO 2022193898 A1 WO2022193898 A1 WO 2022193898A1 CN 2022076659 W CN2022076659 W CN 2022076659W WO 2022193898 A1 WO2022193898 A1 WO 2022193898A1
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boron
dmf
arginine
glycan
compound
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向婧
任秋实
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北京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/009—Neutron capture therapy, e.g. using uranium or non-boron material
    • A61K41/0095—Boron neutron capture therapy, i.e. BNCT, e.g. using boronated porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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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  • the present invention relates to a polypeptide-coupled boron carrier and its preparation method and pharmaceutical preparation, in particular to an N-4-carboxyphenylboronic acid amide-threo-benzene-benzene-phenol-glycan-glycan-silk-essence-glycan- Lai-Jing-Aspart-Aspart-Benzene-Lys-Sue-Glu-Glu-Tyro-Cysteine and its preparation method and pharmaceutical preparation.
  • Tumors especially malignant tumors, are diseases that seriously endanger human health in the world today, and their mortality rate is second only to cardiovascular diseases, ranking second in the mortality rate of various diseases. According to the current trend of cancer incidence, the number of new cancer patients worldwide will reach 15 million every year.
  • Glioma is a tumor that occurs in the neuroectoderm, so it is also called neuroectodermal tumor or neuroepithelial tumor.
  • Tumors originate from interstitial cells, ie glia, ependymal, choroid plexus epithelium, and neural parenchyma cells, ie neurons. Most tumors originate from different types of glia, but according to their histological origin and similar biological characteristics, various tumors that occur in the neuroectoderm are generally referred to as gliomas.
  • gliomas There are many classification methods for gliomas, and clinicians often use the Kernohan classification, which is relatively simple. Among all types of gliomas, astrocytoma is the most common, followed by glioblastoma, followed by medulloblastoma, ependymoma, oligodendroglioma, pineal tumor, and mixed glioma. stromal tumor, choroid plexus papilloma, unclassified glioma, and neuronal tumor. Different types of gliomas have different predilection sites.
  • astrocytomas are more common in the cerebral hemisphere in adults, while children are more common in the cerebellum; almost all glioblastomas occur in the cerebral hemisphere; medulloblastomas occur in the cerebellar vermis; Ependymomas are more common in the fourth ventricle.
  • gliomas grow aggressively and have unclear boundaries with surrounding tissues, despite the use of microscopes during surgery, a small amount of tumor cells inevitably remains, leading to tumor recurrence.
  • new progress has been made in chemotherapy, it is difficult for systemic chemotherapy to pass through the blood-brain barrier, and it is difficult to achieve effective drug concentration in the local tumor. reduction, pulmonary fibrosis, etc.
  • significant progress has been made in each treatment modality, and the combined treatment of the three approaches can prolong patient survival, gliomas are still difficult to cure.
  • new treatments such as boron neutron capture therapy have moved from the laboratory to the clinic.
  • Boron neutron capture therapy is a new method for the treatment of tumors. It was originally proposed by Locher in 1936. It is to inject a boron ( 10 B) carrier with a specific affinity to the tumor into the human body. Aggregate in tumor cells, and then irradiated with ultra-thermal low-energy neutrons (1eV ⁇ 10keV), 10 B nuclei and neutrons undergo nuclear reaction, and the resulting ⁇ particles and 7 Li 3+ kill tumor cells: 10 B+ 1 n ⁇ [ 11 B] ⁇ 4 He 2+ ( ⁇ )+ 7 Li 3+ +2.31MeV.
  • BNCT neutron reaction cross-section of 10 B in tumor cells
  • 3800b barn
  • normal cells around the tumor can also undergo neutron capture reactions with hydrogen ( 1 H) and nitrogen ( 14 N), but in 1 H and 14 N
  • the sub-reaction cross-sections are only 0.332b and 1.82b, respectively, so compared with the surrounding tissue, the reaction cross-section of 10 B to neutrons in tumor cells is large, and the toxic and side effects are small.
  • BNCT is mainly used for the treatment of brain tumors, gliomas, and melanomas.
  • the boron-carrying agents used in BNCT are a series of passively or actively targeted boron-containing compounds whose molecules can be selectively taken up by tumor cells.
  • An ideal boron carrier should have the following characteristics: 1) The boron dose should meet the requirements, at least ensure that each gram of tumor tissue absorbs about 10-30 ⁇ g 10 B, that is, each tumor cell contains 10 9 10 B atoms, and during irradiation, The radiation dose of boron in tumor cells should be kept above 1Gy. 2) The ratio of the boron concentration of tumor cells to normal tissues and the ratio of the boron concentration of tumor cells to blood should be guaranteed to be more than 3, so as to ensure the targeting of the boron-carrying agent. 3) In addition to being taken up by tumor cells in the dividing phase, it can also be taken up by tumor cells. 4) Non-toxic, good water solubility.
  • the second-generation BNCT reagents are a series of passively targeted boron-containing organic small molecule compounds whose molecules can be selectively taken up by tumor cells.
  • boron-containing drugs approved by the FDA in clinical treatment:
  • BSH Sulfanyl dodecaborane disodium salt
  • BSH Na 2 B 12 H 11 SH
  • BSH is a water-soluble compound containing 12 B atoms in the molecule.
  • BSH has the best aggregation effect in the treatment of brain tumors. It was originally synthesized by Miller et al. in 1964. curative effect.
  • Goeun Choi et al. combined layered double hydroxide (LDH) and BSH. After U87 cells absorbed BSH-LDH nanoparticles, the 10 B content was 42.4 ⁇ g/10 6 cells, which was larger than the minimum boron requirement of BNCT. About 2000 times.
  • the T/B value of 10 B in the BSH-LDH group in mice was also 4.4 times higher than that in the BSH group within 2 hours. Excellent neutron capture efficiency was also finally produced at 30 ⁇ g 10 B mL -1 .
  • BPA 10 B-p-dicarboxyboronylphenylalanine
  • BPA-F fructose complex of BPA
  • boron-carrying agents Due to the defects of molecular properties, the tumor cell specificity of BPA and BSH is insufficient and cannot meet the ideal standard of the aforementioned boron-carrying agents.
  • a major challenge in the development of boron-carrying agents is the selective targeting of tumor cells and the delivery of therapeutic concentrations of boron with minimal normal tissue uptake and retention.
  • Directions for improving the selectivity of boron-carrying agents include integration with tumor targeting groups, such as unnatural amino acids, polyamines, polypeptides, proteins, antibodies, nucleosides, carbohydrates, porphyrins, liposomes, and nanoparticles, etc. .
  • boron neutron capture therapy is brain glioma and head and neck tumors.
  • the drug needs to penetrate the blood-brain barrier to reach brain glioma, so the follow-up research on boron-carrying agents needs to meet the needs of sufficient doses.
  • 10 B molecules are delivered to tumor cells and maintain a high T/N value. Among them, effective killing of glioma cells without destroying normal brain tissue is more challenging than the treatment of malignant tumors in other sites.
  • the blood-brain barrier excludes drugs with molecular weights greater than 200 Da, and treatment of gliomas is made more difficult by the highly infiltrating nature of glioma cells and their genomic heterogeneity.
  • An object of the present invention is to provide a polypeptide boron-carrying agent and a pharmaceutically acceptable salt thereof.
  • the boron-carrying agent of the present invention is an N-4-carboxyphenylboronamide type polypeptide compound, which has the advantages of stable quality and excellent properties, and is more suitable for preparing various forms of pharmaceutical preparations as raw materials.
  • a boron carrying agent and a pharmaceutically acceptable salt thereof according to the present invention are N-4-carboxyphenylboronic acid amide-threo-benzene-benzene-phenol-glycan-glycan-silk-essence-glycan-lysine-essence- Aspartame-aspartate-benzene-lys-threo-gluten-glutamate-tyro-cysteine and pharmaceutically acceptable salts thereof, the specific molecular formula is:
  • N-4-carboxyphenylboronic acid amide of the present invention -threo-benzene-benzene-phenol-glycan-glycan-silk-essence-glycan-lysine-essence-aspart-aspart-benzene-lys-threo-gu- Gluten-tyrosine-cysteine compound (ANG)
  • the molecular formula is C 114 H 158 BN 30 O 35 S 1
  • the molecular weight is 2552.4
  • compound 2 N-4-carboxyphenylboronic acid amide-arginine-lys-lys-lysine-arginine-arginine-glutyl-arginine-arginine-arginine (TAT).
  • compound 3 N-4-carboxyphenylboronic acid amide-methyl-propane-silk-methyl-threo-glycan-glycan-glutyl-glutamyl-methyl-glycine (T7).
  • Another object of the present invention is to provide a pharmaceutical preparation, which comprises: (1), the boron carrier of the present invention and/or a pharmaceutically acceptable salt thereof; and (2), one or more A pharmaceutically acceptable carrier or excipient.
  • the compounds or pharmaceutical preparations of the present invention can prepare a targeted boron neutron capture therapeutic drug for the treatment of gliomas and head and neck tumors.
  • Another object of the present invention is to provide the preparation method of above-mentioned compound, comprises the steps:
  • connection method of the remaining amino acids follows the conventional Fmoc synthesis method.
  • the present invention is used as a targeted boron neutron capture therapeutic drug, and one of its main indications is brain glioma and head and neck tumor. Because gliomas grow aggressively and have unclear boundaries with surrounding tissues, surgery cannot avoid a small amount of tumor cells remaining, leading to tumor recurrence. Although new progress has been made in chemotherapy, it is difficult for systemic chemotherapy to pass through the blood-brain barrier, and it is difficult to achieve effective drug concentration in the local area of the tumor, and high-dose chemotherapy makes the systemic toxicity and side effects obvious.
  • the present invention is expected to effectively kill glioma cells without destroying normal brain tissue, reduce damage to the brain, and has a wide range of applications.
  • Fig. 1 is the mass spectrum of N-4-carboxyphenylboronamide type polypeptide compound prepared in Example 1 of the present invention
  • Fig. 2 is the mass spectrum of N-4-carboxyphenylboronic acid amide-essence-lys-lys-lys-lys-sing-sing-glutyl-sing-sing-arginine,
  • Figure 3 is a mass spectrum of N-4-carboxyphenylboronic acid amide-methyl-propane-silk-methyl-threo-glycan-glycan-glutyl-glutyl-methyl-glycine,
  • Figure 5 shows the absorption of polypeptides in different tissues.
  • the first amino acid is attached to 4-carboxyphenylboronic acid.
  • the MBHA resin was pre-swollen in DCM for 30 min, filtered with suction and rinsed with DMF. A solution of threonine and DIPEA in dichloromethane was added to the resin. The resin mixture was reacted with stirring for 2 hours. After suction filtration, the resin was treated with a mixture of DCM/DIPEA/MeOH (17:1:2, volume ratio), followed by DMF (3 ⁇ 30 sec), methanol (3 ⁇ 30 sec) and DCM (3 ⁇ 30 sec) rinse.
  • the synthesized polypeptide was purified and separated by HPLC high performance liquid chromatography, conditions: C18 column ODS-34.6 ⁇ 250mm, mobile phase A: H 2 O (0.065% trifluoroacetic acid), mobile phase B: acetonitrile (0.05% trifluoroacetic acid) Fluoroacetic acid), flow rate 1 mL/min, gradient conditions: A: 0-25 min, 5%-65%; 25-27 min, 65%-95%; 27-35 min, 95%-5%.
  • BPA was prepared into 0.1mg/g, 0.2mg/g, 0.4mg/g, 0.6mg/g, 0.8mg/g aqueous solutions, and the U87MG, U251, A375, A549, HS683 and Hep cell lines were cultured in a constant temperature incubator for 12 hours. After taking out, the cells and medium were separated, and the number of cells was detected. After the cells were digested with concentrated nitric acid, the content of B in the cells was detected by ICP-AES.
  • the detection line is 249.772nm. After detection, the B content in every 10 6 cells can be calculated, and Figure 4 is drawn. It can be seen that in 12 hours, the boron concentration in the cells and the added product concentration are positively correlated, and increase The trend is gradually slowing down. The product was best absorbed in the glioma cell line U251 within 12 hours.
  • mice with an average weight of about 21 g were selected and inoculated with HS683 glioma to establish a mouse glioma model. After 21 days, the mouse brain glioma model was generated, and 500 mg/kg of BPA, N-4-carboxyphenylboronic acid amide-threo-benzene-benzene-phenol-glycan-glycan-silk-jing- Glycine-Lysin-Jing-Aspartame-Aspartic-Benzene-Lys-Sue-Glu-Glu-Tyro-Cysteine Acyl-arginine-arginine-arginine, N-4-carboxyphenylboronic acid amide-methyl-propane-silk-methyl-threo-glycan-glycine-glutamyl-glutamyl-methyl-glycine solution.
  • mice After administration, the main tissues and organs (heart, liver, spleen, lung, kidney and tumor site) of mice were excised at selected time periods (1 hour, 2 hours, 4 hours), washed and weighed, and then digested with concentrated nitric acid. . After the digested tissue samples were diluted, the concentration in the samples was determined by ICP-MS.

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Abstract

一种硼携带剂及其制备方法和药物制剂，该硼携带剂包含化合物1 N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸，分子式为C 114H 158BN 30O 35S 1，分子量为2552.4，还包含化合物2 N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸，和/或化合物3 N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸。化合物1-3可以作为一种靶向性的硼中子俘获治疗药物，用于治疗脑胶质瘤和头颈部肿瘤。

Description

一种多肽偶联硼携带剂及其制备方法和药物制剂 技术领域：
本发涉及一种多肽偶联硼携带剂及其制备方法和药物制剂，尤其涉及一种N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸及其制备方法和药物制剂。
背景技术：
肿瘤尤其是恶性肿瘤是当今世界严重危害人类健康的疾病，其死亡率仅次于心血管疾病，居各类疾病死亡率的第二位，而且近年来，其发病率呈明显上升趋势。根据目前癌症的发病趋势，全球每年新增癌症患者人数将达到1500万人。
胶质瘤是发生于神经外胚层的肿瘤，故亦称神经外胚层肿瘤或神经上皮肿瘤。肿瘤起源于神经间质细胞，即神经胶质、室管膜、脉络丛上皮和神经实质细胞，即神经元。大多数肿瘤起源于不同类型的神经胶质，但根据组织发生学来源及生物学特征类似，对发生于神经外胚层的各种肿瘤，一般都称为神经胶质瘤。
胶质瘤的分类方法很多，临床工作者往往采用的是分类比较简单的Kernohan分类法。各型胶质瘤中，以星形细胞瘤最多，其次为胶质母细胞瘤，其后依次为髓母细胞瘤、室管膜瘤、少枝胶质瘤、松果体瘤、混合性胶质瘤、脉络丛乳头状瘤、未分类胶质瘤及神经元性肿瘤。各型胶质瘤的好发部位不同，如星形细胞瘤成人多见于大脑半球，儿童则多发在小脑；胶质母细胞瘤几乎均发生于大脑半球；髓母细胞瘤发生于小脑蚓部；室管膜瘤多见于第4脑室。
目前，胶质瘤的治疗按效果分依次为外科手术、放射治疗、化学治疗。由于胶质瘤呈侵袭性生长，与周围的组织界限不清，尽管在手术中应用了显微镜，也不可避免少量肿瘤细胞残留，导致肿瘤复发。虽然在化疗方面不断取得新的进展，但由于全身化疗很难通过血脑屏障，在肿瘤的局部很难达到有效的药物浓度，而且大剂量化疗使得全身毒副作用明显，如骨髓抑制、白细胞血小板的减少、肺纤维化等。虽然每一种治疗方式都取得了明显进步，并且3种方法的综合治疗能延长病人的生存期，但胶质瘤仍难治愈。近年来，硼中子俘获疗法等新疗法已从实验室走向临床。
硼中子俘获疗法(BNCT)是一种新型的治疗肿瘤的方法，最初是由美国Locher于1936年提出的设想，它是把与肿瘤有特异性亲和力的硼( 10B)携带剂注入人体，聚集于肿瘤细胞之内，随后用超热低能中子(1eV～10keV)进行照射， 10B原子核与中子发生核反应，产生的α粒子和 7Li 3+杀死肿瘤细胞： 10B+ 1n→[ 11B]→ 4He 2+(α)+ 7Li 3++2.31MeV。研究表明肿瘤细胞中 10B的中子反应截面为3800b(barn)，而肿瘤周围的正常细胞氢( 1H)和氮( 14N)也可发生中子俘获反应，但是 1H和 14N中子反应截面仅分别为0.332b和1.82b，所以相对于肿瘤周围的组织，肿瘤细胞内 10B对中子的反应截面很大，毒副作用较小。但直到上世纪80年代后，硼化合物载体的制备有了重大突破，同时解决了热中子源的问题，才使得BNCT进入临床实用阶段。临床上BNCT主要用于脑部肿瘤、神经胶质瘤和黑色素瘤等癌症的治疗。
BNCT中使用的硼携带剂是一系列被动或主动靶向的含硼化合物，其分子能够选择性地被肿瘤细胞吸收。理想的硼携带剂应该具备以下特点：1)硼剂量要达到要求，至少保证每克肿瘤组织吸收约10～30μg 10B，即每个肿瘤细胞含有10 9个 10B原子，并且在照射期间，肿瘤细胞内硼的辐射剂量要保持在1Gy以上。2)肿瘤细胞对正常组织的硼浓度之比以及肿瘤细胞对血液的硼浓度之比均要保证达到3以上，保证硼携带剂的靶向性。3)除了被处于分裂期的肿瘤细胞摄取之外，还可以被肿瘤细胞吸收。4)无毒，水溶性好。
第二代BNCT试剂是一系列被动靶向的含硼有机小分子化合物，其分子能够选择性地被肿瘤细胞吸收。在临床治疗方面已有两种被美国FDA批准的含硼药物：
1)硫基十二硼烷二钠盐(BSH：Na 2B 12H 11SH)：BSH是一种水溶性化合物，分子中含有12个B原子。经验证明在脑肿瘤的治疗中BSH聚集效果最为良好，最初是由Miller等人在1964年合成的，1968年被首次用于脑肿瘤的治疗，在开颅术中使用BNCT照射，取得了良好的疗效。2018年Goeun Choi等将层状双氢氧化物(LDH)和BSH结合，在U87细胞吸收BSH-LDH纳米颗粒后， 10B含量为42.4μg/10 6个细胞，比BNCT的最小硼需求大了约2000倍。小鼠体内BSH-LDH组 10B的T/B值在2小时内也比使用BSH的组高4.4倍。在30μg 10B mL -1条件下也最终产生优异的中子捕获效率。
2) 10B-对二羧硼酰苯丙氨酸(BPA： 10BC 9H 12NO 4)。BPA是一种脂溶性化合物，其结构类似于苯丙氨酸，因为肿瘤细胞代谢旺盛，需要的苯丙氨酸比正常细胞多，因此肿瘤细胞可主动地大量摄取BPA，但是因为其结构中含有硼，所以其并不参与蛋白质合成。基于BPA优先被黑色素合成细胞摄取的假设，它最初通过静脉注射治疗皮肤黑色素瘤患者。Coderre等人在1990年的实验表明，BPA也被其他类型的肿瘤细胞所吸收，包括大鼠脑9L胶质肉瘤等。Mallesh等人发现BPA的果糖复合物(BPA-F)能显著提高其水溶性，增加在血液内的溶解度，可以通过静脉注射，故BPA很快进入临床阶段，用于治疗恶性黑色素瘤和脑胶质瘤患者。
受制于分子特性层面的缺陷，BPA和BSH的肿瘤细胞特异性不足，无法满足前述硼携带剂的理想标准。硼携带剂的发展面临的主要挑战是需满足对肿瘤细胞的选择性靶向，以及在最小正常组织摄取和滞留情况下递送治疗浓度的硼。提高硼携带剂选择性的方向包括与肿瘤靶向基团的整合，如非天然氨基酸、多胺、多肽、蛋白质、抗体、核苷、糖类、卟啉类化合物、脂质体和纳米颗粒等。
硼中子俘获治疗针对的主要适应症之一是脑胶质瘤和头颈部肿瘤，药物到达脑胶质瘤需要穿透血脑屏障，所以后续对硼携带剂的研究需要满足将足够剂量的 10B分子输送到肿瘤细胞中，并要保持较高的T/N值。其中，在不破坏正常脑组织的条件下有效杀死胶质瘤细胞，这比其他部位的恶性肿瘤的治疗更具挑战性。血脑屏障会排除分子量大于200Da的药物，同时还因为胶质瘤细胞的高度浸润特性及其基因组异质性导致治疗脑胶质瘤变得更加困难。
发明内容：
本发明的一个目的是提供一种多肽硼携带剂及其药学上可接受的盐。
本发明所述硼携带剂是一种N-4-羧基苯硼酰胺型多肽化合物，该化合物具有质量稳定的优点，并且性质优良，更适于作为原料药制备各种形式的药物制剂。
本发明实现上述目的的技术方案是：
本发明所述的一种硼携带剂及其药学上可接受的盐为N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸及其药学上可接受的盐，具体分子式为：
本发明所述的N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸类化合物(ANG)，确定分子式为C 114H 158BN 30O 35S 1，分子量为2552.4的化合物，此为化合物1
化合物2化学名称为N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸(TAT)。
化合物3化学名称为N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸(T7)。
本发明的另一个目的是提供一种药物制剂，其包含：(1)、本发明所述的硼携带剂和/或及其药学上可接受的盐；和(2)、一种或多种药学上可接受的载体或赋形剂。
本发明所述化合物或药物制剂可以制备一种靶向性的硼中子俘获治疗药物，用于治疗脑胶质瘤和头颈部肿瘤。
本发明的另一目的是提供上述化合物的制备方法，包括如下步骤：
1.向4-羧基苯硼酸上连接第一个氨基酸。将MBHA树脂预先在DCM中溶胀30min,抽滤并用DMF冲洗干净。将含苏氨酸和DIPEA的二氯甲烷溶液加入到树脂中。树脂混合物在搅拌下反应2小时。
2.抽滤,用DCM/DIPEA/MeOH(17:l:2)的混合液处理树脂,然后用DMF(3×30秒),甲醇(3×30秒)和DCM(3×30秒)冲洗。
3.其余氨基酸的连接方法按照常规的Fmoc合成法。
4.用含20％哌淀的DMF溶液处理10分钟,依次用DMF、CH 2Cl 2和DMF冲洗、耦合、向Fmoc氨基酸溶液5ml中依次加入4mLHBTU、2mLHOBT和206.58μL DIPEA,然后加入700μL DMF溶解,将以上混合液加入树脂混合物中,反应1小时后,依次用DMF、CH 2Cl 2(2x)和DMF清洗；
5.使用HPLC高效液相色谱仪对合成的多肽进行纯化分离，条件：C18色谱柱ODS-34.6×250mm，流动相A：H 2O(0.065％三氟乙酸)，流动相B：乙腈(0.05％三氟乙酸)，流速1mL/min，梯度条件：A:0-25min，5％-65％；25-27min,65％-95％；27-35min，95％-5％。
本发明的优点是：
本发明是作为一种靶向性的硼中子俘获治疗药物，其对的主要适应症之一是脑胶质瘤和头颈部肿瘤。由于胶质瘤呈侵袭性生长，与周围的组织界限不清，外科手术无法避免少量肿瘤细胞残留，导致肿瘤复发。虽然在化疗方面不断取得新的进展，但由于全身化疗很难通过血脑屏障，在肿瘤的局部很难达到有效的药物浓度，而且大剂量化疗使得全身毒副作用明显。本发明有望在不破坏正常脑组织的条件下有效杀死胶质瘤细胞，减少对大脑的损伤，有广泛的应用。
附图说明：
图1为本发明实施例1制得的N-4-羧基苯硼酰胺型多肽化合物质谱图，
图2为N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸质谱图，
图3为N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸质谱图，
图4为本产品和BPA12小时内在不同细胞中的吸收，
图5为多肽在不同组织中的吸收。
具体实施方式：
下文结合具体实施例对本发明的内容做进一步详细地说明，所述实施例的目的仅用于说明和描述本发明当前的最佳模式。本发明的保护范围不以任何方式受此处所述实施例的限制。
实施例1
N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸的制备
向4-羧基苯硼酸上连接第一个氨基酸。将MBHA树脂预先在DCM中溶胀30min,抽滤并用DMF冲洗干净。将含苏氨酸和DIPEA的二氯甲烷溶液加入到树脂中。树脂混合物在搅拌下反应2小时。抽滤,用DCM/DIPEA/MeOH(17:l:2，体积比)的混合液处理树脂,然后用DMF(3×30秒),甲醇(3×30秒)和DCM(3×30秒)冲洗。
实施例2 N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸的纯化
用含20％哌淀的DMF溶液处理10分钟,依次用DMF、CH 2Cl 2和DMF冲洗、耦合、向Fmoc氨基酸溶液5ml中(中依次加入4mLHBTU(189.62mg)、2mLHOBT(33.78mg)和206.58μLDIPEA,然后加入700μLDMF溶解,将以上混合液加入树脂混合物中,反应1小时后,依次用DMF、CH 2Cl 2(2x)和DMF清洗；
使用HPLC高效液相色谱仪对合成的多肽进行纯化分离，条件：C18色谱柱ODS-34.6×250mm，流动相A：H 2O(0.065％三氟乙酸)，流动相B：乙腈(0.05％三氟乙酸)，流速1mL/min，梯度条件：A:0-25min，5％-65％；25-27min,65％-95％；27-35min，95％-5％。
实施例3 根据本发明的化合物的体外研究
将N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸、N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸、N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸配置成0.1mg/g、0.2mg/g、0.4mg/g、0.6mg/g、0.8mg/g的水溶液，分别加入25T培养皿培养的U87MG、U251、A375、A549、HS683和Hep细胞系中，在恒温培养箱中培养12小时。取出后分离细胞和培养基，检测细胞数量，用浓硝酸消化细胞后，通过ICP-AES检测细胞中B的含量。
同时将BPA配置成0.1mg/g、0.2mg/g、0.4mg/g、0.6mg/g、0.8mg/g的水溶液，分别加入25T培养皿培养的的U87MG、U251、A375、A549、HS683和Hep细胞系中，在恒温培养箱中培养12小时。取出后分离细胞和培养基，检测细胞数量，用浓硝酸消化细胞后，通过ICP-AES检测细胞中B的含量。
检测线为249.772nm，检测后计算可得每10 6个细胞中所含的B含量，绘制图4，由此可见，在12小时中，细胞中硼浓度和加入的产品浓度呈正相关，且增长趋势逐渐减缓。12小时内，本产品在胶质瘤细胞系U251中吸收最佳。
对比可得在脑胶质瘤HS683、A375细胞吸收中，化合物1与化合物2均显著高于BPA。
实施例4 根据本发明的化合物的体内研究
选取健康的BALB/c小鼠，平均体重约为21g，接种HS683脑胶质瘤，建立小鼠脑胶质瘤模型。待21天后，小鼠脑胶质瘤模型生成，通过小鼠尾静脉注射500mg/kg的BPA、N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸、N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸、N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸溶液。给药后，于选定时间段(1小时、2小时、4小时)切除小鼠主要组织器官(心，肝，脾，肺、肾及肿瘤部位)，清洗称重后用浓硝酸消解组织器官。消解后的组织样品经稀释后，通过ICP-MS测定样品中的浓度。
如图5所示，给药后两小时，可以看出，小鼠脑胶质瘤部位，三种化合物吸收量均高于BPA，且化合物1最高，表示本产品能够提高硼携带剂的靶向效果，增加BNCT照射时的有效中子吸收量。
以上对本发明的具体实施例进行了描述，但需要理解的是，本发明并不局限于上述特定的实施方式，本领域技术人员可以在不脱离本发明构思的前提下，做出各种变形和修改，这些都属于本发明的保护范围。

Claims (6)

  1. 一种硼携带剂，其特征在于选自：化合物1，所述化合物1为N-4-羧基苯硼酸酰胺-苏-苯-苯-酪-甘-甘-丝-精-甘-赖-精-天冬-天冬-苯-赖-苏-谷-谷-酪-半胱氨酸；化合物2N-4-羧基苯硼酸酰胺精-赖-赖-赖-精-精-谷酰-精-精-精氨酸；化合物3：N-4-羧基苯硼酸酰胺-甲-丙-丝-甲-苏-甘-甘-谷酰-谷酰-甲-甘氨酸。
  2. 如权利要求1所述的硼携带剂，其中，所述化合物1的确定分子式为C 114H 158BN 30O 35S 1，分子量为2552.4。
  3. 一种药物制剂，其特征在于包含：(1)、权利要求1所述的硼携带剂和/或及其药学上可接受的盐；和(2)、一种或多种药学上可接受的载体或赋形剂。
  4. 如权利要求1-3所述任一项在制备一种靶向性硼中子俘获治疗药物中的应用。
  5. 根据权利要求4所述的应用，其特征在于所述靶向性硼中子俘获治疗药物用于治疗脑胶质瘤和头颈部肿瘤。
  6. 一种制备权利要求1所述的硼携带剂的方法，包括如下步骤：
    1)向4-羧基苯硼酸上连接第一个氨基酸，将MBHA树脂预先在DCM中溶胀30min,抽滤并用DMF冲洗干净，将含苏氨酸和DIPEA的二氯甲烷溶液加入到树脂中，树脂混合物在搅拌下反应2小时；
    2)抽滤,用DCM/DIPEA/MeOH，体积比为17:l:2的混合液处理树脂,然后用DMF 3×30秒,甲醇3×30秒和DCM 3×30秒冲洗；
    3)其余氨基酸的连接方法按照常规的Fmoc合成法；
    4)用含20％哌淀的DMF溶液处理10分钟,依次用DMF、CH 2Cl 2和DMF冲洗、耦合、向Fmoc氨基酸溶液5ml中依次加入4mLHBTU、2mLHOBT和206.58μL DIPEA,然后加入700μL DMF溶解,将以上混合液加入树脂混合物中,反应1小时后,依次用DMF、CH 2Cl 2(2x)和DMF清洗；
    5)使用HPLC高效液相色谱仪对合成的多肽进行纯化分离，条件：C18色谱柱ODS-34.6×250mm，流动相A：H 2O 0.065％三氟乙酸，流动相B：乙腈0.05％三氟乙酸，流速1mL/min，梯度条件：A:0-25min，5％-65％；25-27min,65％-95％；27-35min，95％-5％。
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