WO2019096095A1 - 整合素受体靶向的抗癌偶联物 - Google Patents

整合素受体靶向的抗癌偶联物 Download PDF

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WO2019096095A1
WO2019096095A1 PCT/CN2018/115058 CN2018115058W WO2019096095A1 WO 2019096095 A1 WO2019096095 A1 WO 2019096095A1 CN 2018115058 W CN2018115058 W CN 2018115058W WO 2019096095 A1 WO2019096095 A1 WO 2019096095A1
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cancer
compound
tumor
conjugate
irinotecan
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PCT/CN2018/115058
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袁建栋
黄仰青
宋云松
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博瑞生物医药（苏州）股份有限公司
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines

Definitions

  • the present invention relates to an integrin receptor-targeted anti-cancer conjugate, and more particularly to a multi-arm polymer modified integrin receptor-targeted anti-cancer conjugate which is an integrin receptor
  • the ligand is linked to the anticancer drug by a multi-arm polymer to form a conjugate.
  • WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a clinical phase III drug nktr 102, which is mainly used for metastatic breast cancer, developed by Nektar Therapeutics.
  • the drug is a water-soluble multi-branched polymer prodrug to increase the drug loading, and the structure is as follows:
  • the compound is linked to irinotecan using a multi-arm PEG to increase water solubility, increase drug loading, and reduce side effects without changing the anticancer effect.
  • the drug still has disadvantages, for example, poor targeting, can not act on specific cancer cells, while killing cancer cells, it also affects the performance of normal cells, and the incidence of adverse reactions is still relatively high.
  • Integrins are a class of cell adhesion receptor molecules that are widely expressed on the surface of nucleated cells. Among them, integrin ⁇ ⁇ ⁇ 3 is involved in various tumor cells and tumors such as glioma, melanoma and ovarian cancer. The high expression of endothelial cells is closely related to tumor angiogenesis, tumor metastasis and anti-radiation therapy of tumors, so integrin ⁇ ⁇ ⁇ 3 is often used as a specific target for tumor targeting.
  • arginine-glycine-aspartate (Arg-Gly-Asp, RGD) tripeptide sequence can specifically recognize the integrin family containing ⁇ ⁇ subunits, with high affinity, can carry effects Molecules specifically bind to it, thereby inhibiting tumor growth and neovascularization.
  • the therapeutic effector molecules may be introduced into the targeted tumor sites, effectively reducing the tumor treatment of damage to normal tissue cells.
  • the present invention introduces the compound a referred to in Chinese Patent Application No. 201710263114.9 into the present invention.
  • the structure of the compound a is as shown in the formula (I):
  • the conjugate of the invention is a heptane sulfonate of compound a, which typically comprises from one to eight molecules of heptane sulfonate.
  • each branch is bound to a conjugate of two molecules of heptane sulfonate, respectively.
  • the preferred conjugate is eight molecules of heptane sulfonate, referred to herein as compound H:
  • R is the organic center, ie in the structure of the conjugate Represents the junction of atoms.
  • R is the organic center, ie in the structure of the conjugate Represents the junction of atoms.
  • branch consists of a polymer POLY, a multivalent linker L, a targeting molecule T, and an active agent D.
  • the polymer POLY is polyethylene glycol, and in the present invention, it is specifically:
  • n 113, Representing the junction of an atom, the oxygen atom labeled "&” is an atom attached to the organic center "R".
  • n represents the degree of polymerization of the polymer, that is, the average number of repeating units contained in the polymer macromolecular chain, depending on the molecular weight of the polymer. For example, when n is 113, it means that the average value is 113.
  • Multi-valent joint L is:
  • the targeting molecule T is the RGD peptide cRGD, which is a series of compounds, preferably cRGD is as follows:
  • the active agent D is irinotecan and has the following structure:
  • Conjugates of the invention using targeting cRGD peptide, binding specifically with ⁇ ⁇ ⁇ 3 integrin, targeted therapeutic agent may be introduced into the tumor site, effectively reduced the tumor treatment of damage to normal tissue cells.
  • the conjugates of the invention also exhibit high loading capacity so that the total dose can be lowered to treat a particular condition, such as cancer. That is, the conjugate active agent carrier of the present invention is capable of effectively covalently bonding to a plurality of active agent molecules, allowing a greater amount of therapeutic dosage form (i.e., active agent portion) to be administered per certain amount of conjugate.
  • the modification of the conjugate of the present invention by the water-soluble polymer is essentially that the conjugate is also hydrophilic, and in particular, when the active agent is a water-insoluble drug, the bioavailability of the conjugate is improved.
  • the conjugates of the invention are capable of exhibiting a stronger effect than tissues that are not coupled, and are more enriched in the human or other animal tissues.
  • Suitable solid tumor types for the conjugates of the invention include colon cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, glioma, and malignant sarcoma, carcinoma, and lymph of the breast, ovary, colon, kidney, bile duct, lung, and brain. tumor.
  • 4arm-PEG20K-SCM The structure of 4arm-PEG20K-SCM is as follows:
  • the molecular weight of the MALDI-TOF of Compound H was 25,506.34.
  • Examples 5-9 are pharmacological data of Compound a. It should be noted here that since Compound H is a salt of Compound a, Compound H enters human or animal body, and compound A is actually used for anticancer action. The pharmacological data of Compound a was used to demonstrate the anticancer effect of Compound H.
  • Irinotecan raw drug
  • nktr-102 The preparation method of nktr-102 is as follows:
  • MDA-MB-231 was cultured in DMEM medium (GIBCO, USA) containing 10% fetal calf serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Matrigel (Mat Matelel) Matrigel was purchased from BD Corporation of the United States.
  • Biosafety cabinet (model: AC2-6E1), purchased from ESCO; CO 2 water-tight cell incubator (model: 3111), purchased from Thermo Scientific Forma; inverted microscope (model: CKX41SF), purchased from Olympus;
  • tumor-related parameters refers to the Chinese CFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs", according to the Chinese SFDA "Guidelines for Non-Clinical Research Techniques of Cytotoxic Antitumor Drugs” (November 2006), %T/ C ⁇ 40% and statistically analyzed P ⁇ 0.05 was effective.
  • TV initial is the tumor volume measured at the time of group administration
  • TV t is the tumor volume at each measurement during administration.
  • RTV T represents the treatment group RTV
  • RTV C represents the solvent control group RTV.
  • TGI 100% ⁇ [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]]
  • TV t(T) represents the tumor volume measured by the treatment group at each time
  • TV initial (T) represents the tumor volume of the treatment group at the time of group administration
  • TV t (C) represents the tumor volume measured by the solvent control group at each time
  • TV Initial (C) indicates the tumor volume of the solvent control group at the time of group administration.
  • animal weight loss rate 100% ⁇ (BW initial - BW t ) / BW initial
  • BW t represents the body weight of the animal measured each time during administration
  • BW initial represents the body weight of the animal at the time of group administration.
  • IR (%) 100% ⁇ (W C - W T ) / W C
  • W C represents the tumor weight of the control group
  • W T represents the tumor weight of the treatment group
  • test data was calculated using Microsoft Office Excel 2007 software and related statistical processing. Data were expressed as mean ⁇ standard error (Mean ⁇ SE) unless otherwise specified. T-test was used for comparison between groups, P ⁇ 0.05 was considered as significant difference.
  • Test samples irinotecan, nktr-102, compound a.
  • McCoy's 5A medium McCoy's 5A medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, water for injection, physiological saline, lactic acid, sorbitol.
  • FBS fetal bovine serum
  • trypsin trypsin
  • cyan-chain double antibody water for injection, physiological saline, lactic acid, sorbitol.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-7 weeks) were purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., and were raised in SPF animal room at a temperature of 20-25 °C. Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human colon cancer cell line HT-29, derived from the cell bank of the Institute of Culture Collection of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • HT-29 cell culture HT-29 cells were cultured in 10% fetal bovine serum McCoy's 5A culture medium under 5% CO 2 at 37 ° C; digested with 0.25% trypsin; cell growth In the case, it is passed 2 to 3 times a week, and the passage ratio is 1:4 to 1:6.
  • HT-29 cells in logarithmic growth phase were collected, cell counted and resuspended in serum-free McCoy's 5A medium, adjusted to a cell concentration of 4 ⁇ 10 7 cells/mL; pipetted cells to make them evenly dispersed Then, the tube was placed in a 50 mL centrifuge tube, and the tube was placed in an ice box; the cell suspension was pipetted with a 1 mL syringe, and injected into the right armpit of the nude mouse, and each animal was inoculated with 100 ⁇ L (4 ⁇ 10 6 cells/cell). An HT-29 nude mouse xenograft model was established.
  • Solvent preparation Weigh 0.5g of sorbitol into a 50mL centrifuge tube, add 50mL of water for injection in the centrifuge tube, vortex to completely dissolve the solid substance, and prepare a 1% concentration of sorbitol aqueous solution (w/v) Store in a refrigerator at 4 °C.
  • Formulation of irinotecan preparation Weigh 12.0 mg of irinotecan, add 0.15 mL of 1% lactic acid, vortex to completely dissolve the drug, and then add 2.85 mL of 1% sorbitol aqueous solution separately, vortex and mix. Evenly, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The effective concentration of irinotecan in the solution was 4.0 mg ⁇ mL -1 .
  • nktr-102 preparation Before each administration, accurately weigh 101.5mg of nktr-102, add 2.5mL of normal saline, vortex to completely dissolve the drug, and the effective concentration of irinotecan in the solution is 4.0mg ⁇ mL -1 .
  • Preparation of the compound of the present invention preparation preparation Before each administration, accurately weigh 120.3 mg of compound a, add 2.5 mL of physiological saline, vortex, and ultrasonically (if necessary) to completely dissolve the drug, and irinotecan is effective in the solution. The concentration was 4.0 mg ⁇ mL -1 .
  • Animal grouping and dosing schedule The first administration was started on the day of grouping, and the experiment was terminated after about 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered intravenously to the test samples of irinotecan, nktr-102, and compound a, all administered once every 4 days, Q4D ⁇ 3.
  • Test samples irinotecan, nktr-102, compound a.
  • mice Female BALB/c nude mice, 150 inoculated, 66 experimental, age 6-8 weeks, body weight 20-22g ⁇ 20% body weight mean, animal source is Shanghai Xipuer - Bikai Experimental Animal Co., Ltd. ( BK), license number SCXK (Shanghai) 2008-0016. All experimental animals were housed in SPF-level laboratories. The experimenter is responsible for daily care and experimental research. Each rat cage is attached with an identity card containing information such as the experiment number, experimental group, experimenter's name, mouse breed and gender, and the mouse is marked with earrings.
  • Randomization When the tumor volume reached 150-200 mm 3 , it was divided into 11 groups by random block method, and each group of 6 mice ensured that the tumor volume and mouse body weight were uniform among the groups. The mean value of tumor volume of each group differed from the mean of tumor volume of all experimental animals by no more than ⁇ 10%.
  • breeding conditions living conditions: IVC system, 6 per cage; temperature: 20 ° C - 26 ° C; humidity: 40% ⁇ 70%; light: 12 hours alternate day and night.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat. Drinking water is urban tap water, which is filtered and autoclaved for drinking.
  • the litter is corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving. Change the litter twice a week. The mice were given the shortest environmental adaptation period before the experiment.
  • Human breast cancer MDA-MB-231 was purchased from the Institute of Cell Biology, Shanghai Academy of Sciences.
  • a subcutaneous xenograft model of human breast cancer MDA-MB-231 nude mice was established, each inoculated with 1 ⁇ 10 6 cells.
  • the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered intravenously to the test samples of irinotecan, nktr-102, and compound a, all administered once every 4 days, Q4D ⁇ 3.
  • Example 5 For the preparation of the test article, see Example 5.
  • the volume required for a single administration is 3 mL.
  • MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA). The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 1 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 1 x 10 6 / 0.1 ml / mouse.
  • the animals were randomly divided into 11 groups according to the random block method, so that the tumor difference of each group was less than 10% of the mean value, 6 groups in each group, and the group was used as Day1. medicine.
  • the experimental period was carried out for 3 weeks, and the animal body weight and tumor size were measured twice a week during the experiment. Daily observations record clinical symptoms.
  • the animals were sacrificed, the body weight was weighed, the tumor was removed, weighed and photographed. The results are shown in Table 2.
  • Test samples irinotecan, nktr-102, compound a.
  • mice Female BALB/c nude mice, 150 inoculated, 66 experimental, age 6-8 weeks, body weight 20-22g ⁇ 20% body weight mean, animal source is Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. ( BK), license number SCXK (Shanghai) 2008-0016. All experimental animals were housed in SPF-level laboratories. The experimenter is responsible for daily care and experimental research. Each rat cage is attached with an identity card containing information such as the experiment number, experimental group, experimenter's name, mouse breed and gender, and the mouse is marked with earrings. When the tumor volume reached 150-200 mm 3 , they were divided into 11 groups by random block method, and 6 mice in each group ensured that the tumor volume and mouse body weight were uniform among the groups. The mean value of tumor volume of each group differed from the mean of tumor volume of all experimental animals by no more than ⁇ 10%.
  • breeding conditions living conditions are IVC system, 6 per cage; temperature: 20 ° C - 26 ° C; humidity: 40% ⁇ 70%; light: 12 hours day and night alternate.
  • the irradiated large mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Free to eat. Drinking water is urban tap water, which is filtered and autoclaved for drinking.
  • the litter is corn cob, Shanghai Maosheng Derivative Technology Co., Ltd., used after autoclaving. Change the litter twice a week. The mice were given the shortest environmental adaptation period before the experiment.
  • a subcutaneous xenograft model of human pancreatic cancer MIA Paca-2 nude mice was established, each inoculated with 3 ⁇ 10 6 cells.
  • the administration volume was 10 mL ⁇ kg -1 .
  • the effective dose equivalent to irinotecan is 40mg ⁇ kg -1 .
  • the first group was a solvent control group, and normal saline was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered intravenously to the test samples of irinotecan, nktr-102, and compound a, all administered once every 4 days, Q4D ⁇ 3.
  • Example 5 For the preparation of the test article, see Example 5.
  • the volume required for a single administration is 3 mL.
  • MIA Paca-2 cells were cultured in DMEM containing 10% fetal bovine serum FBS (GIBCO, USA) and 2.5% HS. The cells were cultured in a 5% CO 2 incubator at 37 °C.
  • the subcutaneous transplantation model of tumor nude mice was established by cell inoculation method: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 3 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 3 x 10 6 /0.1 ml/mouse.
  • the animals were randomly divided into 4 groups according to the random block method, so that the tumor difference of each group was less than 10% of the mean value, 6 groups in each group, and the day of the group was used as Day1.
  • the experimental period was carried out for 3 weeks, and the animal body weight and tumor size were measured twice a week during the experiment. Daily observations record clinical symptoms.
  • the animals were sacrificed, the body weight was weighed, the tumor was removed, weighed and photographed. The results are shown in Table 3.
  • Example 8 Inhibition of tumor growth in nude mice xenograft model of human gastric cancer NCI-N87 cell line.
  • Test samples irinotecan, nktr-102, compound a.
  • Reagents RPMI-1640 medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, physiological saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ⁇ 25 ° C, relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human gastric cancer cell NCI-N87, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • NCI-N87 cell culture NCI-N87 cells were cultured in 10% fetal bovine serum RPMI-1640 medium in 5% CO 2 at 37 ° C; digested with 0.25% trypsin; The growth condition was 1 to 2 times per week, and the passage ratio was 1:2 to 1:6.
  • NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 5 ⁇ 10 7 cells/mL; pipette the cells with a pipette. Disperse evenly, put into a 50mL centrifuge tube, place the centrifuge tube in an ice box; use a 1mL syringe to aspirate the cell suspension, and inject it into the right armpit of the nude mouse before inoculation, each animal is inoculated with 100 ⁇ L (5 ⁇ 10 6 cells / ), establish a NCI-N87 nude mouse xenograft model.
  • Example 5 For the preparation of the test article, see Example 5.
  • the volume required for a single administration is 3 mL.
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan), Q4D ⁇ 3, respectively.
  • Example 9 Effect on the survival rate of brain in situ model of U87MG nude mice.
  • Test samples irinotecan, nktr-102, compound a.
  • Reagents RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., raised in SPF animal room, temperature 20 ⁇ 25 ° C, Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain glioma cell line U87MG, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • NCI-N87 cell culture NCI-N87 cells were cultured in RPMI-1640 medium in 5% CO 2 at 37 ° C; digested with 0.25% trypsin; passaged weekly according to cell growth 1 to 2 times, the passage ratio is 1:2 to 1:6.
  • NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 1 ⁇ 10 8 cells/mL; pipette the cells with a pipette. After dispersing evenly, it was placed in a 50 mL centrifuge tube, and the centrifuge tube was placed in an ice box; the cell suspension was aspirated with a 1 mL syringe, and the human glioma cell line U87MG was cultured in vitro by microinjection using the guidance of an animal stereotactic instrument.
  • Formulation of the administration preparation The preparation of the test sample is shown in Example 5. The volume required for a single administration is 3 mL.
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan), Q4D ⁇ 3, respectively.

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Abstract

本发明公开了一种多臂的、由水溶性聚合物修饰的靶向药物偶联物的庚烷磺酸盐。该偶联物典型的庚烷磺酸盐包括一至八分子的庚烷磺酸盐。优选每个支链分别结合两分子庚烷磺酸盐的偶联物，该优选的偶联物为八分子庚烷磺酸盐。本发明为一个多臂聚合物修饰的靶向抗癌偶联物，其中，水溶性的聚合物修饰可增强该偶联物的水溶性，提高载药量；靶向分子增加靶向性，使其在目标组织的浓度更高。

Description

整合素受体靶向的抗癌偶联物 技术领域
本发明涉及整合素受体靶向的抗癌偶联物，更具体地说，本发明涉及多臂聚合物修饰的整合素受体靶向的抗癌偶联物，其为整合素受体的配体通过多臂聚合物与抗癌药物连接成偶联物。
背景技术
WO2005028539、WO2010019233、WO2011063156、WO2011063158公开了一种处于临床三期的药物nktr 102，该药物主要用于转移性乳腺癌，由Nektar Therapeutics研发。该药物是一种水溶性的多支链聚合物药物前体，以提高药物的负载，结构如下：
Figure PCTCN2018115058-appb-000001
该化合物是使用多臂PEG与伊立替康连接，以提高水溶性，增加载药量，在抗癌作用不变的情况下，降低副作用。但该药物仍具有缺点，比如，靶向性较差，不能作用于特定的癌细胞，在杀死癌细胞的同时，也会影响正常细胞的性能，而使不良反应发生率仍然比较高。
整合素(Integrins)是一类细胞黏附受体分子，在有核细胞表面均广泛表达，其中整合素α γβ 3在神经胶质瘤、黑色素瘤、卵巢癌等多种肿瘤细胞及肿瘤相关的内皮细胞表面高度表达，与肿瘤的血管发生、肿瘤转移及肿瘤的抗放射治疗密 切相关，因此整合素α γβ 3常被用作肿瘤靶向的特异性靶点。已有研究表明，精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp，RGD)的三肽序列能够特异性地识别含α γ亚基的整合素家族，具有高度亲和力，可以携带效应分子特异地与其结合，从而抑制肿瘤生长和新生血管的形成。
利用RGD肽与整合素的α γβ 3特异结合，可以将治疗效应分子靶向性地导入肿瘤部位，有效地减少了肿瘤治疗中对正常组织细胞的损害。
发明内容
本发明将中国专利申请201710263114.9所涉及的化合物a引入本发明。化合物a的结构如式(I)所示：
Figure PCTCN2018115058-appb-000002
本发明偶联物为化合物a的庚烷磺酸盐，典型的庚烷磺酸盐包括一至八分子的庚烷磺酸盐。优选每个支链分别结合两分子庚烷磺酸盐的偶联物，该优选的偶联物为八分子庚烷磺酸盐，在本发明称之为化合物H：
Figure PCTCN2018115058-appb-000003
为进一步说明本发明的发明构思，上述偶联物可表述为式(II)：
Figure PCTCN2018115058-appb-000004
其中，R为有机中心，即偶联物结构中的
Figure PCTCN2018115058-appb-000005
代表原子的连接处。从有机中心的中心碳原子出发，发出四条支链，每一条支链都是相同的。每一条支链由聚合物POLY、多价接头L、靶向分子T、活性剂D构成。
聚合物POLY为聚乙二醇，在本发明中，其具体为：
Figure PCTCN2018115058-appb-000006
n为113，
Figure PCTCN2018115058-appb-000007
代表原子的连接处，标“&”号的氧原子为与有机中心“R”连接的原子。
本领域技术人员应该知晓，在高分子领域，n代表所述的聚合物的聚合度，即聚合物大分子链上所含重复单元数目的平均值，其取决于所述的聚合物的分子量，例如，当n为113时，是指平均值为113。
多价接头L为：
Figure PCTCN2018115058-appb-000008
符号“*”代表多价接头L通过半胱氨酸与靶向分子T的连接点，“#”代表多价接头L与活性剂D的连接点，“％”代表多价接头L与POLY的连接点。
靶向分子T为RGD肽cRGD，cRGD是一系列化合物，优选的cRGD如下：
Figure PCTCN2018115058-appb-000009
活性剂D为伊立替康，结构如下：
Figure PCTCN2018115058-appb-000010
本发明偶联物利用cRGD肽的靶向作用，与整合素的α γβ 3特异结合，可以将治疗药物靶向性地导入肿瘤部位，有效地减少了肿瘤治疗中对正常组织细胞的损害。
本发明偶联物还呈现出高载荷能力，这样就可以降低总剂量来治疗一种特殊的疾病，例如癌症等。也就是说，本发明偶联物活性剂载体能够有效地和多种活性剂分子以共价键结合，允许每一定的偶联物量可以服用更多量的治疗剂型(也就是活性剂部分)。本发明偶联物通过水溶性聚合物的修饰，本质上是偶联物也是亲水的，特别是活性剂为水难溶药物时，提高偶联物的生物利用度。相比未偶联的药物，本发明偶联物能够表现出更强的作用，在人体或其他动物体内组织更加富集。
本发明偶联物适合的实体肿瘤类型包括结肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤。
具体实施例
在下面将对本发明进行详细描述。然而，本发明可能具体体现为许多不同的形式，而且它不应该被局限于此处所描述的实施例中，提供这些实施例中的目的是使所披露内容更完整与全面。所用试剂和原料，除了提供制备方法的除外，其余均为市售，其中4arm-PEG20K-SCM购自北京键凯科技有限公司，分子量约为20kDa左右。除非另有定义，否则本文中所有科技术语具有的含义与权利要求主题所属技术领域人员通常理解的含义相同。
4arm-PEG20K-SCM结构如下：
Figure PCTCN2018115058-appb-000011
名词解释
Figure PCTCN2018115058-appb-000012
Figure PCTCN2018115058-appb-000013
实施例1
Figure PCTCN2018115058-appb-000014
化合物2的制备
向250mL圆底烧瓶中加入3.50g化合物1(1.0eq)，52.5ml DMF，加热至60℃溶解，5-10min后减压蒸去DMF，加入300ml正庚烷减压蒸馏，重复三次，旋干后加入105ml DCM，1.08g Boc-Gly-OH(1.2eq)，63mg DMAP(0.1eq)，滴加1.59g DCC(1.5eq)溶于10ml DCM的溶液，20℃反应4小时，TLC监控反应完毕后，过滤，浓缩至剩余25％体积时加入120ml IPA，蒸去75％的溶剂，加入150ml正庚烷，室温搅拌1小时，过滤，正庚烷洗涤2次，干燥得淡黄色固体4.02g化合物2。
化合物3的制备
向100mL三口瓶中加入4.02g化合物2，50ml DCM，搅拌溶解后滴加11.6ml  TFA，室温反应2h，TLC监控反应完毕后加入150ml乙腈，减压蒸馏120ml溶剂后倒入320ml TBME溶液中，搅拌30min，过滤，滤饼用TBME洗涤得4.00g淡黄色固体化合物3。
实施例2
Figure PCTCN2018115058-appb-000015
化合物5的制备
向250mL三口瓶中加入6.9g化合物4，30ml EA，搅拌溶解后降温至0℃，加40ml 0.3M的HCl/EA，保温反应2h，TLC监控反应完毕浓缩至干，得到化合物5，直接进行下一步反应。
化合物6的制备
将化合物5(1.0eq)用50ml纯化水溶解，加入3.96g碳酸氢钠(2.0eq)，用50ml DME将5.30g Fmoc-OSU(1.0eq)溶解，加入到化合物5的反应瓶中，补加25ml THF，室温搅拌2小时，TLC监控反应完毕后，蒸去有机溶剂，EA萃取杂质，水相用稀盐酸调节pH至3-4，EA萃取2次，合并有机相，水洗一次，饱和食盐水洗涤后无水硫酸钠干燥，浓缩得8.4g淡黄色油状物化合物6。
化合物7的制备
在100ml反应瓶中加入4.00g化合物6(1.0eq)、2.92g H-Lys(Boc)-OBzl·HCl、40ml DCM溶解，加入2.76g DIEA(3.0eq)，1.74g DEPC(1.5eq)，室温搅拌2小时，TLC监控反应完毕后，醋酸水溶液洗涤，碳酸氢钠溶液洗涤，水洗一次，饱和食盐水洗涤一次后无水硫酸钠干燥，浓缩得7.0g淡黄色油状物化合物7，不纯化直接进行下一步反应。(利用同样的方法制备化合物16)
化合物8的制备
用140ml 25％的DEA/DCM将7.0g化合物7溶解，室温搅拌6小时，TLC监控反应完毕后，浓缩至干，加入100ml，50ml EA，用稀盐酸将pH调节至3-4，分液，水相用EA萃取2次后浓缩至干，得3.5g淡黄色固体化合物8。(利用同样的方法制备化合物17)
实施例3
连有保护基的靶向分子cRGD(化合物11)的制备
Figure PCTCN2018115058-appb-000016
化合物9的制备
利用2Cl-Trt Resin，Fmoc保护法，偶联试剂采用HOBT/DIC，DMF为反应溶剂，反应监控采用茚三酮检测，依次将下列保护氨基酸连接到树脂上：Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Glu(OBzl)-OH、Fmoc-D-Phe-OH、 Fmoc-Asp(OtBu)OH、脱除Fmoc，DMF洗涤、DCM洗涤、甲醇洗涤后干燥，加入裂解试剂：醋酸/TFE/DCM＝1/2/7，反应2小时，冰的MTBE沉淀、洗涤，干燥得类白色固体化合物9。
化合物10的制备
2L的三口烧瓶中加入14.0g化合物9(1.0eq)，加入1L DMF，降温至0℃，加入9.2g碳酸氢钠(8.0eq)，溶清后加入15.1g DPPA(4.0eq)，保温过夜，TLC反应完毕后倒入5L水中，EA萃取2次，水洗，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得类白色固体化合物10 11.5g
化合物11的制备
1L的氢化釜中加入11.5g化合物10，1L甲醇，2.5g Pd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物11 11.0g
实施例4 化合物a和化合物H的制备
Figure PCTCN2018115058-appb-000017
化合物12的制备
5ml反应瓶中加入480mg化合物11(1.0eq)，380mg化合物8(1.1eq)，1ml DMF，203mg DIEA(3.0eq)，128mg DEPC(1.5eq)，室温反应2h，TLC反应完毕后倒入10mL水中，EA萃取2次，稀盐酸洗涤，碳酸氢钠溶液洗涤，饱和氯化钠洗涤后无水硫酸钠干燥，浓缩得果冻状固体化合物12 0.8g，直接进行下一步反应。
化合物13的制备
200ml的氢化釜中加入0.8g化合物10，30ml甲醇，0.28g Pd/C，加氢过夜，TLC反应完毕后过滤，浓缩得灰色固体化合物13 0.66g
化合物14的制备
100ml反应瓶中加入6.60g化合物13(1.0eq)，3.59g化合物3(1.05eq)，66ml DMF，1.16g DIEA(3.0eq)，1.10g DEPC(1.5eq)，室温反应2h，TLC反应完 毕后倒入700ml TBME中，打浆后抽滤，固体用150DCM溶解后倒入1.5L TBME中，打浆抽滤，干燥灰色粉末化合物14 9.0g，直接进行下一步反应。
化合物15的制备
250ml反应瓶中，加入9.0g化合物14，裂解试剂92.5％TFA/2.5％水/2.5％TIS，室温搅拌2h，用冰的MTBE沉淀、离心、洗涤，粗品经反相HPLC纯化、冻干，得淡黄色絮状物5.0g化合物15。
化合物a的制备
反应瓶中加入2.3g化合物15(4.5eq)，6.0g 4arm-PEG20K-SCM(1.0eq)，60ml DMF，0.27g TEA(9.0eq)，室温反应，HPLC监控反应无明显进展后，倒入1000ml TBME中，打浆、抽滤，干燥得类色粉末粗品化合物a 7.6g
化合物H的制备
化合物a经反向HPLC纯化(硅胶：C18，300A；流动相：庚烷磺酸钠/水，乙腈)后，收集纯品后调节pH＝4～5，再经反向HPLC脱盐(硅胶：C18，300A；流动相：醋酸/水，乙腈)，收集纯品后浓缩除去有机溶剂，冻干得类白色粉末化合物H。
化合物H的MALDI-TOF检测分子量为25506.34。
以下实施例5-9为化合物a的药理数据，在这里需要说明的是，由于化合物H是化合物a的盐，在化合物H进入人体或动物体内，实际起抗癌作用的是化合物a，故可以使用化合物a的药理数据来证明化合物H的抗癌作用。
所使用到的供试品及试剂、仪器等来源如下：
伊立替康(原料药)系购买所得。nktr-102的制备方法如下：
将实施例1中的化合物3(829mg，4.5eq)添加到250mL的反应瓶中，加入DCM(50mL)，三乙胺(221mg，9.0eq)，溶解后加入4ARM-PEG20K-SCM(5.00g，1.0eq)添加到该反应瓶中。HPLC监控反应没有明显进展后，减压蒸馏出去约20mL DCM，将溶液倒入300mL TBME中搅拌沉淀，过滤，得到5.4g粗品，粗品经HPLC制备纯化，脱盐，用稀盐酸调节pH至5-6，冻干得到2.71g淡绿色粉末nktr-102。
MDA-MB-231培养于DMEM培养基(GIBCO，美国)，含10％胎牛血清FBS(GIBCO，美国)，培养于含5％CO 2的37℃培养箱。基质胶(BD Matrigel) Matrigel购自美国BD公司。
生物安全柜(型号：AC2-6E1)，购自ESCO；CO 2隔水细胞培养箱(型号：3111)，购自Thermo Scientific Forma；倒置显微镜(型号：CKX41SF)，购自Olympus；。
在本发明实施例中，所有动物实验操作严格遵守动物使用和管理规范。肿瘤相关参数的计算参考中国CFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》，根据中国SFDA《细胞毒类抗肿瘤药物非临床研究技术指导原则》(2006年11月)，％T/C≤40％并经统计学分析P＜0.05为有效。相对肿瘤增殖率越低，说明抑制肿瘤效果越良好
肿瘤体积(TV)计算公式如下：TV(mm 3)＝l×w 2/2
其中，l表示肿瘤长径(mm)；w表示肿瘤短径(mm)。
相对肿瘤体积(RTV)的计算公式为：RTV＝TV t/TV initial
其中，TV initial为分组给药时测量到的肿瘤体积；TV t为给药期间每一次测量时的肿瘤体积。
相对肿瘤增殖率(％T/C)的计算公式为：％T/C＝100％×(RTV T/RTV C)
其中，RTV T表示治疗组RTV；RTV C表示溶剂对照组RTV。
肿瘤生长抑制率TGI(％)的计算公式为：TGI＝100％×[1-(TV t(T)-TV initial(T))/(TV t(C)-TV initial(C))]
其中，TV t(T)表示治疗组每次测量的肿瘤体积；TV initial(T)表示分组给药时治疗组的肿瘤体积；TV t(C)表示溶剂对照组每次测量的肿瘤体积；TV initial(C)表示分组给药时溶剂对照组的肿瘤体积。
动物体重下降率的计算公式为：动物体重下降率＝100％×(BW initial-BW t)/BW initial
其中，BW t表示给药期间每次测量的动物体重；BW initial表示分组给药时的动物体重。
瘤重抑瘤率IR(％)的计算公式为：IR(％)＝100％×(W C-W T)/W C
其中，W C表示对照组瘤重；W T表示治疗组瘤重。
试验数据用Microsoft Office Excel 2007软件进行计算和相关统计学处理。数据除特别说明外，用均数±标准误(Mean±SE)表示，组间比较采用t-检验，P＜0.05 为显著性差异。
实施例5 化合物a在HT-29裸鼠移植瘤模型体内药效评价
供试品：伊立替康、nktr-102、化合物a。
试剂：McCoy’s 5A培养液，胎牛血清(FBS)，胰蛋白酶，青-链双抗，注射用水，生理盐水，乳酸，山梨糖醇。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～7周)从北京维通利华实验动物技术有限公司购买，饲养于SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：人结肠癌细胞HT-29，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
HT-29细胞培养：在5％CO 2、37℃培养条件下，HT-29细胞在含10％胎牛血清McCoy’s 5A培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代2到3次，传代比例为1∶4到1∶6。
动物模型制备：收取对数生长期HT-29细胞，细胞计数后重悬于无血清McCoy’s 5A培养基中，调整细胞浓度至4×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(4×10 6细胞/只)，建立HT-29裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物66只，采用随机区组法分为11组(n＝6)。实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
溶剂配制：称取0.5g山梨糖醇装入50mL离心管中，在离心管中加入50mL注射用水，涡旋振荡使固体物质完全溶解，配制成浓度1％的山梨糖醇水溶液(w/v)，保存于4℃冰箱备用。
伊立替康给药制剂配制：称取12.0mg的伊立替康，加入0.15mL的1％乳酸，涡旋振荡使药物完全溶解，再分别加入2.85mL的1％山梨糖醇水溶液，涡 旋振荡混合均匀，溶液中1％乳酸、1％山梨糖醇水溶液的比例约为5∶95(v/v)。溶液中伊立替康有效浓度为4.0mg·mL -1。
nktr-102给药制剂配制：每次给药前，准确称量101.5mg的nktr-102，加入2.5mL的生理盐水，涡旋振荡使药物完全溶解，溶液中伊立替康有效浓度为4.0mg·mL -1。
本发明化合物给药制剂配制：每次给药前，准确称量120.3mg的化合物a，加入2.5mL的生理盐水，涡旋振荡，超声(如需要)使药物完全溶解，溶液中伊立替康有效浓度为4.0mg·mL -1。
动物分组及给药方案：于分组当天开始第一次给药，21天左右后结束实验，给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-4组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a，均为每4天给药一次，Q4D×3。
实验最后一天，称量体重、测量瘤径后动物安乐死(CO 2)。剥取肿瘤组织并称重，结果见表1。
表1 对肿瘤增殖率T/C(％)
Figure PCTCN2018115058-appb-000018
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，化合物a对人结肠癌HT-29裸鼠移植瘤模型肿瘤体内生长有良好抑制作用，且优于伊立替康和nktr-102。
实施例6 人乳腺癌MDA-MB-231裸鼠异种移植模型的抑制作用
供试品：伊立替康、nktr-102、化合物a。
实验动物：雌性BALB/c裸鼠，接种150只，实验用66只，年龄为6-8周， 体重20-22g±20％体重均值，动物来源为上海西普尔-必凯实验动物有限公司(BK)，许可证号SCXK(沪)2008-0016。所有实验动物均饲养在SPF级别实验室。实验人员负责日常护理和实验研究。每个鼠笼均佩挂有实验编号、实验组别、实验人员姓名、小鼠品种和性别等信息的身份卡片，小鼠用耳钉标记。
随机分组：当肿瘤体积达到150-200mm 3后用随机区组法分为11组，每组6只小鼠，保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10％。
饲养条件：居住条件：IVC系统，每笼6只；温度：20℃-26℃；湿度：40％±70％；光照：12小时昼夜交替。辐照大小鼠饲料，购自北京科澳协力饲料有限公司。自由进食。饮水为城市自来水，经过滤高压灭菌后饮用。垫料为玉米芯，上海茂生衍生物科技有限公司，高压灭菌后使用。每周换两次垫料。实验前给予小鼠最短一周环境适应期。
其他化学试剂和材料：人乳腺癌MDA-MB-231购于上海中科院细胞生物研究所。
建立人乳腺癌MDA-MB-231裸鼠皮下移植瘤模型，每只接种1×10 6个细胞。
给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-4组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a，均为每4天给药一次，Q4D×3。
供试品的配制见实施例5。单次给药需要的体积3mL。
实验方法：MDA-MB-231细胞培养于DMEM，含10％胎牛血清FBS(GIBCO，美国)。细胞放置于5％CO 2培养箱37℃培养。细胞接种法建立肿瘤裸鼠皮下移植模型：收集对数生长期的肿瘤细胞，计数后重悬于1×PBS，调整细胞悬液浓度至1×10 7/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞，1×10 6/0.1ml/鼠。在肿瘤体积达到100-200mm 3时，将动物按随机区组法进行随机分组，分为11组，使各组肿瘤差异小于均值的10％，每组6只，分组当日作为Day1，分组当天给药。实验周期进行3周，实验期间每周测定两次动物体重和肿瘤大小。每日观察记录临床症状。实验最后一天处死动物，称量体重，剥离肿瘤，称重并拍照记录，结果见表2。
表2 对肿瘤增殖率T/C(％)
Figure PCTCN2018115058-appb-000019
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，化合物a对人乳腺癌MDA-MB-231裸鼠移植瘤有良好抑制作用，且优于伊立替康和nktr-102。
实施例7 对人胰腺癌MIA Paca-2裸鼠异种移植模型的抑制作用
供试品：伊立替康、nktr-102、化合物a。
实验动物：雌性BALB/c裸鼠，接种150只，实验用66只，年龄为6-8周，体重20-22g±20％体重均值，动物来源为上海西普尔-必凯实验动物有限公司(BK)，许可证号SCXK(沪)2008-0016。所有实验动物均饲养在SPF级别实验室。实验人员负责日常护理和实验研究。每个鼠笼均佩挂有实验编号、实验组别、实验人员姓名、小鼠品种和性别等信息的身份卡片，小鼠用耳钉标记。当肿瘤体积达到150-200mm 3后用随机区组法分为11组，每组6只小鼠，保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10％。
饲养条件：居住条件为IVC系统，每笼6只；温度：20℃-26℃；湿度：40％±70％；光照：12小时昼夜交替。辐照大小鼠饲料，购自北京科澳协力饲料有限公司。自由进食。饮水为城市自来水，经过滤高压灭菌后饮用。垫料为玉米芯，上海茂生衍生物科技有限公司，高压灭菌后使用。每周换两次垫料。实验前给予小鼠最短一周环境适应期。
其他化学试剂和材料：人胰腺癌MIA Paca-2购于上海中科院细胞生物研究所。
建立人胰腺癌MIA Paca-2裸鼠皮下移植瘤模型，每只接种3×10 6个细胞。 给药体积均为10mL·kg -1。折合伊立替康的有效剂量均为40mg·kg -1。第一组为溶剂对照组，尾静脉注射给予生理盐水，每4天1次，共给药3次(Q4D×3)。第2-4组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a，均为每4天给药一次，Q4D×3。
供试品的配制见实施例5。单次给药需要的体积3mL。
实验方法：MIA Paca-2细胞培养于DMEM，含10％胎牛血清FBS(GIBCO，美国)和2.5％HS。细胞放置于5％CO 2培养箱37℃培养。
细胞接种法建立肿瘤裸鼠皮下移植模型：收集对数生长期的肿瘤细胞，计数后重悬于1×PBS，调整细胞悬液浓度至3×10 7/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞，3×10 6/0.1ml/鼠。
在肿瘤体积达到100-200mm 3时，将动物按随机区组法进行随机分组，分为4组，使各组肿瘤差异小于均值的10％，每组6只，分组当日作为Day1，分组当天给药。实验周期进行3周，实验期间每周测定两次动物体重和肿瘤大小。每日观察记录临床症状。实验最后一天处死动物，称量体重，剥离肿瘤，称重并拍照记录。结果见表3。
表3 对肿瘤增殖率T/C(％)
Figure PCTCN2018115058-appb-000020
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，化合物a对人胰腺癌MIA Paca-2裸鼠移植瘤有良好抑制作用，且优于伊立替康和nktr-102。
实施例8 对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤体内生长的抑制作用。
供试品：伊立替康、nktr-102、化合物a。
试剂：RPMI-1640培养液，胎牛血清(FBS)，胰蛋白酶，青-链双抗，生理 盐水。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～8周)从北京维通利华实验动物技术有限公司购买，饲养于苏州圣苏新药开发有限公司SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：人胃癌细胞NCI-N87，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
实验方法
NCI-N87细胞培养：在5％CO 2、37℃培养条件下，NCI-N87细胞在含10％胎牛血清RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代1到2次，传代比例为1∶2到1∶6。
动物模型制备：收取对数生长期NCI-N87细胞，细胞计数后重悬于无血清的RPMI-1640培养基中，调整细胞浓度至5×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(5×10 6细胞/只)，建立NCI-N87裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物66只，采用随机区组法分为11组(n＝6)。实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
供试品的配制见实施例5。单次给药需要的体积3mL。
动物分组及给药：于分组当天开始第一次给药，21天后结束实验，给药体积均为10mL·kg -1。第1组为溶剂对照组，尾静脉注射给予空白溶剂，每4天1次，共给药3次(Q4D×3)。第2-4组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a，给药剂量均为40mg·kg -1(以伊立替康含量计算)，Q4D×3。
实验结束后，称量体重、测量瘤径后动物安乐死(CO 2)。剥取肿瘤组织并称重。结果见表4。
表4 对肿瘤增殖率T/C(％)
Figure PCTCN2018115058-appb-000021
*与空白溶剂、伊立替康和nktr-102组的RTV相比，P＜0.05
#与空白溶剂、伊立替康和nktr-102组的％T/C相比，P＜0.05
实验结果显示，化合物a对人胃癌NCI-N87细胞株裸鼠移植瘤模型肿瘤生长有良好抑制作用，且优于伊立替康和nktr-102。
实施例9 对U87MG裸鼠脑原位模型生存率的影响。
供试品：伊立替康、nktr-102、化合物a。
试剂：RPMI-1640培养液，胰蛋白酶，青-链双抗，生理盐水。
实验动物：雌性BALB/c裸小鼠(只数：150只；周龄：6～8周)从北京维通利华实验动物技术有限公司购买，饲养于SPF动物房，温度20～25℃，相对湿度40％～70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
移植性肿瘤瘤株：脑胶质瘤细胞U87MG，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验室液氮冻存)。
实验方法：
NCI-N87细胞培养：在5％CO 2、37℃培养条件下，NCI-N87细胞在RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代1到2次，传代比例为1∶2到1∶6。
动物模型制备：收取对数生长期NCI-N87细胞，细胞计数后重悬于无血清的RPMI-1640培养基中，调整细胞浓度至1×10 8细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，借助动物立体定向仪的引导，采用微注射方法将体外培养人脑胶质瘤细胞U87MG细胞1μL(1×10 5细胞/只)，建立U87MG脑胶质瘤原位模型，接种后定期 观察动物状态。接种后第12天，挑选动物66只，采用随机区组法分为11组(n＝6)。
给药制剂配制：供试品的配制见实施例5。单次给药需要的体积3mL。
动物分组及给药：于分组当天开始第一次给药，21天后结束实验，给药体积均为10mL·kg -1。第1组为溶剂对照组，尾静脉注射给予空白溶剂，每4天1次，共给药3次(Q4D×3)。第2-4组分别尾静脉注射给予受试样品伊立替康、nktr-102、化合物a，给药剂量均为40mg·kg -1(以伊立替康含量计算)，Q4D×3。
结果见表5。
表5 动物生存时间(天)
Figure PCTCN2018115058-appb-000022
*与空白溶剂、伊立替康和nktr-102组的中位生存时间相比，P＜0.05
实验结果显示，化合物a对脑胶质瘤有良好抑制作用，且优于伊立替康和nktr-102。

Claims (7)

  1. 一种化合物，其为具有式(I)结构的多支链药物偶联物的庚烷磺酸盐，
    Figure PCTCN2018115058-appb-100001
  2. 如权利要求1所述的化合物，其为每个支链分别结合两分子庚烷磺酸盐的八分子庚烷磺酸盐，如式(II)所示：
    Figure PCTCN2018115058-appb-100002
  3. 如权利要求1-2任一所述的化合物在制备用于治疗癌症的药物中的应用。
  4. 如权利要求1-2任一所述的化合物在制备用于治疗选自结肠癌、肺癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤的疾病的药物中的应用。
  5. 一种药学上可接受的组合物，其包含权利要求1-2任一所述的化合物，以及药学上可接受的赋形剂。
  6. 如权利要求5所述的组合物在制备用于治疗癌症的药物中的应用。
  7. 如权利要求6所述的应用，其在制备用于治疗选自结肠癌、肺癌、乳腺癌、卵巢癌、胰腺癌、胃癌、脑胶质瘤及乳房、卵巢、结肠、肾、胆管、肺和脑的恶性肉瘤、癌和淋巴瘤的疾病的药物中的应用。
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