WO2016078163A1 - 新型胞苷衍生物二聚体及其应用 - Google Patents

新型胞苷衍生物二聚体及其应用 Download PDF

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WO2016078163A1
WO2016078163A1 PCT/CN2014/093220 CN2014093220W WO2016078163A1 WO 2016078163 A1 WO2016078163 A1 WO 2016078163A1 CN 2014093220 W CN2014093220 W CN 2014093220W WO 2016078163 A1 WO2016078163 A1 WO 2016078163A1
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group
substituted
compound
tumor
cytidine derivative
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French (fr)
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杨达丽亚
王海东
王慧娟
廖雄登
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常州方圆制药有限公司
常州优谱生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/09—Pyrimidine radicals with arabinosyl as the saccharide radical
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical

Definitions

  • the present invention relates to an antitumor compound, and in particular to a novel cytidine derivative dimer and use thereof.
  • Malignant tumors are one of the common diseases that threaten human health, and tumor mortality ranks first among various diseases.
  • the anti-tumor drugs used in clinical practice are a prominent problem that plagues tumor chemotherapy. Improving the therapeutic effect of tumors while reducing the toxicity of drugs is an important research topic in the current treatment of oncology drugs.
  • the existing cytidine compounds are mainly used for the treatment of hematological tumors, and also have cytidine compounds for solid tumors, but have problems of high toxicity, narrow application range, and poor effect.
  • the existing cytidine compounds are susceptible to drug resistance, treatment failure, and tumor recurrence.
  • the technical problem to be solved by the present invention is to provide a novel cytidine derivative dimer with high efficiency, high activity and low toxic and side effects and application thereof.
  • a technical solution for achieving the object of the present invention is a novel cytidine derivative dimer having the following general formula (I):
  • R1 is the same as R2.
  • R3 is H, alkoxycarbonyl, substituted alkoxycarbonyl, and the substituent of the substituted alkoxycarbonyl group is halogen, cyano, nitro, amino, hydroxy or carboxy; preferably H or alkoxycarbonyl; further preferably H Or n-butoxycarbonyl.
  • R4 is H, an alkoxycarbonyl group or a substituted alkoxycarbonyl group, and the substituent of the substituted alkoxycarbonyl group is a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group; preferably an H or an alkoxycarbonyl group; further preferably H or n-butoxycarbonyl.
  • both R3 and R4 are H.
  • the tumor is a hematological tumor or a malignant solid tumor.
  • a pharmaceutical composition comprising, as an active ingredient, the cytidine derivative dimer of the formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more drugs Use a carrier or excipient.
  • the dosage form of the composition is an injection or an oral dosage form, wherein the oral dosage form includes a tablet, a powder, a granule, a capsule, a pellet preparation, a solution, a suspension, an emulsion, a syrup or an expector; the injection includes A solution injection, a suspension injection, an emulsion injection, or a sterile powder for injection.
  • Example 1 is a synthetic route diagram of the cytidine derivative dimer of Example 1;
  • Example 3 is a synthetic route diagram of the cytidine derivative dimer of Example 3;
  • Example 4 is a synthetic route diagram of the cytidine derivative dimer of Example 4.
  • Figure 5 is a bar graph showing the inhibition rate of colony formation of human colon cancer cell line HCT-116 cells by using four compounds of application example 1 at a concentration of 50 nM, 150 nM and 450 nM;
  • Fig. 6 is a graph showing the relationship between the inhibition rate of the four compounds of Application Example 1 on human colon cancer cell line HCT-116 cells and the concentration of the compound.
  • R3 is H, an alkoxycarbonyl group, a substituted alkoxycarbonyl group, and the substituent of the substituted alkoxycarbonyl group is a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group.
  • R4 is H, an alkoxycarbonyl group or a substituted alkoxycarbonyl group, and the substituent of the substituted alkoxycarbonyl group is a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group.
  • cytidine derivative dimer of the present invention the following compounds are given in Table 1, but the cytidine derivative dimer of the present invention is not limited to these compounds.
  • the compound in the above table was prepared, and the solid reagent used in the synthesis was directly used without further treatment, and the liquid reagent was used after being re-distilled and dried.
  • the cytidine derivative dimer of this example is 1.5-bis-[4-N-n-butoxycarbonyl-3'-O-n-butoxycarbonyl-2'-deoxy-2',2'-difluoro Cytidine] glutarate (code D1, number 101 in Table 1), the structural formula is as follows:
  • the compounds 102 to 105 in Table 1 were obtained by changing glutaric anhydride to other corresponding dianhydrides.
  • Compound 106 in Table 1 was prepared by substituting tert-butyl chloroformate for butyl chloroformate.
  • the cytidine derivative dimer of the present example is 1.5-bis-[4-N-n-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine]glutarate (code D2) , number 107 in Table 1, the structural formula is as follows:
  • intermediate 8 was prepared.
  • the reaction process is shown in Figure 2.
  • the preparation process is as follows:
  • the synthetic route of D2 is shown in Figure 2.
  • the preparation process is as follows:
  • the compound 108 is reacted with another dianhydride such as glutaric anhydride to obtain the compounds 108 to 110 in Table 1.
  • another dianhydride such as glutaric anhydride
  • Compound 111 in Table 1 can be obtained by substituting tert-butyl chloroformate for butyl chloroformate.
  • the cytidine derivative dimer of the present example is 1.5-bis-[4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine]glutarate (code number) D3),
  • the synthetic route of D3 is shown in Figure 3.
  • the preparation process is as follows:
  • compound 13 was prepared: 300 mg (1 mmol) of 2'-deoxy-2',2'-difluorocytidine hydrochloride, 5 mL (0.023 mmol) of hexamethyldisilazane, and a catalytic amount of ammonium sulfate 5 mg was dissolved. 5 mL of 1,4-dioxane was heated and refluxed for 2 hours; after the refluxing reaction was completed, the reaction liquid was concentrated, toluene was added thereto, and the mixture was concentrated to dryness twice.
  • compound 14 can be reacted with other dianhydrides, such as COOHCHBr(CH 2 ) 2 COOH, COOH CHPh(CH 2 ) 2 COOH, COOH CHCNCH 2 COOH, etc. to obtain compounds 113 to 116 in Table 1. .
  • other dianhydrides such as COOHCHBr(CH 2 ) 2 COOH, COOH CHPh(CH 2 ) 2 COOH, COOH CHCNCH 2 COOH, etc.
  • the cytidine derivative dimer of this example is 1-O-(4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine)-5-O-( 4-N-n-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine-succinate (1-O-(4-N-(Benzyloxycarbonyl)-gemcitabine)-4-O -(4-N-(n-Butoxycarbonyl)- Gemcitabine)-succinate, code D4), the structure is as follows:
  • hydrochloride of other cytidine derivative dimers is prepared as above.
  • hydrochloride it is also possible to prepare phosphate, sulfate, carbonate, nitrate, citrate, tartrate, maleate, succinate, sulfonate of the cytidine derivative dimer. , p-toluenesulfonate, methanesulfonate, benzoate or fumarate.
  • This example prepared a lyophilized powder injection of the compound D3 of Example 3.
  • the lyophilized powder injection of D3 comprises 30 g of compound D3, mannitol (20% w/v) 300 g, buffer buffer 7 g of sodium dihydrogen phosphate dihydrate, and surfactant poloxamer 188 (F68) 4.0 g.
  • the cytidine derivative dimer of the present invention can be prepared into other forms of injections such as a solution injection, a suspension injection, and an emulsion injection.
  • the pharmaceutical composition of the cytidine derivative dimer of the present embodiment is composed of an active ingredient and an adjuvant, wherein the pharmaceutically active component is the cytidine derivative dimer prepared in the above examples or a corresponding salt thereof.
  • the proportion of the pharmaceutically active component in the composition is from 1% to 95% (30% in this embodiment).
  • the excipient consists of water, lactose, corn starch, hydroxypropyl methylcellulose and magnesium stearate.
  • the pharmaceutical composition of the present embodiment is in the form of a tablet.
  • the pharmaceutically active component can be formulated into oral powders, granules, capsules, pellets, solutions, suspensions, emulsions, syrups or An expectorant, or a sustained release and controlled release preparation in oral form, or a pharmaceutical composition in other oral form, which contains common corresponding excipients (additives, addenda, etc. depending on the effect), such as additives
  • mannitol lactose
  • starch magnesium stearate
  • saccharin salts cellulose or magnesium sulfate.
  • a pharmaceutically acceptable addenda may be selected as a carrier for the pharmaceutically active ingredient, including materials mature in the prior art, such as inert solid diluents, aqueous solvents, liposomes, microspheres or/and none.
  • Toxic organic solvents, etc. preferred additions are: moisturizer, emulsifier, pH buffer, human serum albumin, antioxidants, preservatives, bacteriostatic agents, glucose, sucrose, trehalose, maltose, lecithin, glycine, Yamanashi Acid, propylene alcohol, polyethylene, protamine, boric acid, sodium chloride, or potassium chloride, mineral oil, vegetable oil, etc.; one or several combinations may be selected as a pharmaceutical carrier.
  • the target tumor of the pharmaceutical composition of the present invention includes a hematological tumor or a malignant solid tumor; specifically, the target tumor includes lung cancer, prostate cancer, breast cancer, colon cancer, gastric cancer, pancreatic cancer, liver cancer, esophageal cancer, brain tumor, ovarian cancer , uterine cancer, kidney cancer, head and neck cancer, skin cancer, bladder cancer, vulvar cancer, testicular tumor, rectal cancer, villus cancer, germ cell tumor, malignant lymphoma, leukemia and multiple myeloma, and even more preferred target tumor Pancreatic cancer (first- and second-line treatment), non-small cell lung cancer, breast cancer, ovarian cancer, and head and neck squamous cell carcinoma, colon cancer may be included, but the present invention is not limited thereto.
  • the target tumor includes lung cancer, prostate cancer, breast cancer, colon cancer, gastric cancer, pancreatic cancer, liver cancer, esophageal cancer, brain tumor, ovarian cancer , uterine cancer, kidney cancer, head and
  • the proliferation inhibition effect of four candidate compounds (D1, D2, D3, D4) on human colon cancer cell line HCT-116 cells was evaluated by cell colony formation inhibition assay at 50nM, 150nM and 50nM.
  • Test materials Cell line: HCT-116 human colon cancer cell line was ordered from Shanghai Cell Resource Center of Chinese Academy of Sciences, Cat#TCHu 99.
  • HCT-116 human colon cancer cell culture medium DMEM + 10% FBS.
  • Cell culture collect the cells in logarithmic growth phase, count, resuspend the cells with complete medium; adjust the cell concentration to the appropriate concentration, inoculate a 6-well plate culture dish, inoculate about 300 cells per well, 1.8 ml medium; Incubate for 5 hours at 37 ° C in a 100% relative humidity, 5% CO 2 incubator.
  • a s number of cell clones sampled (cell + test compound).
  • a c number of cell clones (cell + 1% DMSO) of the negative control (no sample treatment).
  • the number of clones of the four compounds against human colon cancer cell line HCT-116 cells is shown in Table 2 below.
  • the inhibition rate of colonization of human colon cancer cell line HCT-116 cells by the four compounds at 50 nM, 150 nM and 450 nM is shown in Fig. 5.
  • Fig. 6 The relationship between the inhibition rate of four compounds on human colon cancer cell line HCT-116 cells and inhibitor concentration is shown in Fig. 6. As can be seen from Fig. 6, the IC50 value of D1 is 245.3 nM, and the IC50 value of D2 is 226.6 nM. D3 has an IC50 value of 99.80 nM and D1 has an IC50 value of 111.7 nM.
  • the growth inhibitory effect and toxicity of the cytidine derivative dimer sample of the present invention on the transplanted tumor of colon cancer HCT-116 tumor-bearing nude mice were determined.
  • the solvent used to dissolve the test substance is as follows:
  • Number of animals Order 40, choose the ones that are in good health for the experiment.
  • Animal numbering method tail number.
  • the animal room environment maintained a temperature of 23 ⁇ 2 ° C, humidity of 40-70%, alternating 12 hours of light and dark.
  • Animal feed (SLAC-M01) was purchased from Beijing Keao Xieli Co., Ltd. The experimental animals were filtered and sterilized with water. Animals were free to eat and drink during the experiment.
  • Colon cancer HCT-116 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in a carbon dioxide incubator at 37 ° C, saturated humidity, and containing a volume fraction of 5% CO 2 and 95% air using F-12 medium (containing 10% FBS). Logarithmic growth phase cells were taken before inoculation, digested with 0.25% trypsin, washed once with PBS, resuspended in PBS, resuspended in serum-free medium, and adjusted to a cell concentration of about 3 x 10 ⁇ 7 cells/mL.
  • Each nude mouse was subcutaneously inoculated with 0.1 mL of cell suspension (3x10 ⁇ 6 cells/mouse) under sterile conditions. When the tumor grows to a volume of about 60-150 mm 3 , nude mice with similar tumor volume and good shape are selected (the shape is as single spherical as possible, no irregular shape or multiple tumors are gathered together), grouped, each group 6 Only, the grouping situation is as follows:
  • IP intraperitoneal injection
  • QD ⁇ 1 injection once.
  • the control control group that is, the model control group, was injected with a mixed solution of 5:5:90 ethanol, Cremophor EL, and physiological saline.
  • the formation of tumors at the inoculation site of each group of nude mice was observed.
  • the evaluation index of antitumor activity is the tumor growth inhibition rate TGI (%), and the relative tumor growth rate T/C (%).
  • TGI (%) (V control - V Treatment ) / V control ) ⁇ 100.
  • T/C (%) T RTV / C RTV ⁇ 100.
  • mice The body weight of the mice was weighed 3 times a week.
  • the weight loss is >20% after administration of the test substance, the sudden death of the animal or the tumor volume exceeds 2800 mm ⁇ 3, the CO 2 is sacrificed, the tumor is isolated and weighed, autopsy is performed, and the diseased organ is visually observed and recorded.
  • the average body weight of each group of animals is shown in Table 4.
  • TGI growth inhibition rate
  • the maximum tumor inhibition rate in the compound D1400mg/kg group was 91.49% on Day 7, and 72.62% in 16 days; the maximum tumor inhibition rate in the compound D2400mg/kg group was 94.79% in Day 9, and 90.63% in 16 days;
  • the maximum tumor inhibition rate of Compound D3350mg/kg was 98.54% on Day 11, and 95.58% on 16 days;
  • the maximum tumor inhibition rate of Compound D4300mg/kg was 97.32% on Day 11, and 92.12% on 16 days.
  • test compound D1-D4 against human colon cancer HCT-116 tumor-bearing mice is shown in Table 8 below:
  • the relative tumor proliferation rate of the compound D1400mg/kg group reached 28.36% in Day 7 and the tumor proliferation rate in Day 16 was 89.47%.
  • the relative tumor proliferation rate of the compound D2400 mg/kg group reached a minimum of 14.41% in Day 9 and a tumor proliferation rate of 21.64% in Day 16 days.
  • the relative tumor proliferation rate of the compound D3350 mg/kg group reached a minimum of 3.41% in Day 11 and a tumor proliferation rate of 10.49% in Day 16 days.
  • the relative tumor proliferation rate of the compound D4300 mg/kg group reached a minimum of 25.94% in Day 11 and a tumor proliferation rate of 37.96% in Day 16 days.
  • the invention has a positive effect: the novel cytidine derivative dimer prepared by the molecular optimization design of the cytidine compound has obvious inhibitory effect on human colon cancer HCT-116 tumor cells, and the tumor-bearing nude
  • the mouse human colon cancer HCT-116 xenograft has a strong growth inhibitory effect;
  • the novel cytidine derivative dimer compound of the present invention has a very high antitumor activity, and the toxicity of the compound is low.

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Abstract

本发明提供通式（I）的胞苷衍生物二聚体及其应用。所述胞苷衍生物二聚体对人结肠癌HCT-116肿瘤细胞有明显的抑制作用，对荷瘤裸小鼠人结肠癌HCT-116移植瘤具有强烈的抑制作用。本发明的胞苷衍生物二聚体具有强抗肿瘤活性和低毒性。

Description

新型胞苷衍生物二聚体及其应用 技术领域
本发明涉及一种抗肿瘤化合物，具体涉及一种新型胞苷衍生物二聚体及其应用。
背景技术
恶性肿瘤是威胁人类健康的常见疾病之一，肿瘤死亡率居于各种疾病之首。目前临床使用的抗肿瘤药物，其毒性是困扰肿瘤化疗的突出问题。提高肿瘤治疗效果同时降低药物毒性，是当前治疗肿瘤药物的重要研究课题。
现有的胞苷化合物主要用于治疗血液肿瘤，也有用于实体肿瘤的胞苷化合物，但是存在毒性大、适用范围窄、效果差的问题。另外，对于现有的胞苷化合物人体易产生抗药性，治疗失败，导致肿瘤复发。
发明内容
本发明所要解决的技术问题是提供一种高效能、高活性同时毒副作用低的新型胞苷衍生物二聚体及其应用。
实现本发明目的的技术方案是一种新型胞苷衍生物二聚体，具有下述通式(Ⅰ)：
Figure PCTCN2014093220-appb-000001
其中，R1是C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph、或取代-(CH2)n-Ph；所述的-(CH2)n-Ph，其中n＝0、1、2、3～10，Ph为苯环；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代。优选为C1至C10的烷基或者-(CH2)n-Ph，n＝0、1、2、3～10；进一步优选为C1至C4的烷基或者-(CH2)n-Ph，n＝0、1、2、3；更进一步优选为正丁基或者苄基。
R2是C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph、或取代-(CH2)n-Ph；所述的-(CH2)n-Ph，其中n＝0、1、2、3～10；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代。优选为C1至C10的烷基或者-(CH2)n-Ph，n＝0、1、2、3～10；进一步优选为C1至C4的烷基或者-(CH2)n-Ph，n＝0、1、2、3；更进一步优选为正丁基或者苄基。
作为再进一步的优选，R1与R2相同。
R3是H、烷氧羰基、取代烷氧羰基，所述的取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基；优选为H或者烷氧羰基；进一步优选为H或者正丁氧羰基。
R4是H、烷氧羰基、或取代烷氧羰基，所述的取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基；优选为H或者烷氧羰基；进一步优选为H或者正丁氧羰基。
作为再进一步的优选，R3和R4均为H。
R5是-(CH2)n-，其中n＝1至15，或者是碳链上有取代基的-(CH2)n-，所述的取代基是苯基、取代苯基、卤素、氰基、硝基、氨基、羟基、羧基，或者是-(CH2)n-X1-X2-；所述的-(CH2)n-X1-X2-，其中n＝0、1、2或3，X1是O、S，X2是-(CH2)n–Ph，其中n＝0、1、2或3，或者X2是嘧啶基、吡喃基、咪唑基、吡嗪基或吡啶基；优选为-(CH2)n-，n＝1至15或者-(CH2)n-X1-X2-，n＝0、1、2或3，X1为O、S，X2为Ph；进一步优选为-(CH2)n-，n＝1至5或者-(CH2)-O-Ph-；更进一步优选为-(CH2)2-或者-(CH2)3-。
上述的新型胞苷衍生物二聚体或其盐在制备抑制肿瘤的药物中的应用。
所述肿瘤为血液肿瘤或恶性实体性肿瘤。
一种药物组合物，其中含有作为活性成分的的权利要求1所述的通式(Ⅰ)所示的胞苷衍生物二聚体或其药学上可接受的盐，以及一种或多种药用载体或赋形剂。
所述组合物的剂型是注射剂，或者是口服剂型，其中口服剂型包括片剂、散剂、颗粒剂、胶囊剂、微丸制剂、溶液剂、混悬剂、乳剂、糖浆剂或酏剂；注射剂包括溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉末。
一种如上所述的新型胞苷衍生物二聚体的制备方法，包括以下步骤：
①制备通式(Ⅱ)的化合物，
Figure PCTCN2014093220-appb-000002
②制备通式(Ⅲ)的化合物，
Figure PCTCN2014093220-appb-000003
③将通式(Ⅱ)的化合物与碳酸钠混合加入到1,4-二氧六环和水的体系中，然后加入(Boc)2O，TLC检测，待反应结束后萃取洗涤，干燥后减压浓缩至干；将得到的化合物加入到三氯甲烷中，加入吡啶和二酸酐R5(CO)2O，反应过夜，浓缩得黏稠油，柱层析得到通式(Ⅳ)化合物待用；
Figure PCTCN2014093220-appb-000004
④将得到的通式(Ⅳ)化合物与通式(Ⅲ)化合物、DCC混合后加入到二氯甲烷中，加入DMAP，TLC检测，待反应结束后洗涤干燥，浓缩至干，再加入TFA和DCM，室温搅拌，冰浴冷却后滤去白色固体，浓缩得黏稠油，柱层析得到产物。
附图说明
图1为实施例1的胞苷衍生物二聚体的合成路线图；
图2为实施例2的胞苷衍生物二聚体的合成路线图；
图3为实施例3的胞苷衍生物二聚体的合成路线图；
图4为实施例4的胞苷衍生物二聚体的合成路线图；
图5为应用例1的4种化合物在作用浓度为50nM,150nM和450nM对人结肠癌细胞株HCT-116细胞的克隆形成抑制率柱形图；
图6为应用例1的4种化合物对人结肠癌细胞株HCT-116细胞的抑制率与化合物浓度的关系曲线图。
具体实施方式
本发明的胞苷衍生物二聚体的结构式如式(Ⅰ)：
Figure PCTCN2014093220-appb-000005
其中R1为C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph，n＝0、1、2、3～10或取代-(CH2)n-Ph，n＝0、1、2、3～10，Ph为苯；取代烷基的碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；取代-(CH2)n-Ph的碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代。
R2为C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph，n＝0、1、2、3～10或取代-(CH2)n-Ph，n＝0、1、2、3～10，Ph为苯；取代烷基的碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；取代-(CH2)n-Ph的碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代。
R3为H、烷氧羰基、取代烷氧羰基，取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基。
R4为H、烷氧羰基、取代烷氧羰基，取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基。
R5为-(CH2)n-，n＝1至15。或者是碳链上有取代基的-(CH2)n-，取代基为卤素、氰基、硝基、氨基、羟基或羧基。或者是-(CH2)n-X1-X2-，n＝0、1、2或3； X1为O、S；X2为-(CH2)n–Ph，n＝0、1、2或3，或者X2为嘧啶基、吡喃基、吡啶基。
对于本发明的胞苷衍生物二聚体，在表1中给出如下化合物，但本发明的胞苷衍生物二聚体不限于这些化合物。
表1
Figure PCTCN2014093220-appb-000006
对上表中的化合物进行制备，合成过程中所用到的固体试剂没有经过进一步处理直接使用，液体试剂经过重蒸干燥后使用。
(实施例1)
本实施例的胞苷衍生物二聚体为1.5–二–[4–N–正丁氧羰基–3’–O–正丁氧羰基-2’-脱氧-2’，2’-二氟代胞苷]戊二酸酯(代号D1，表1中编号101)，结构式如下：
Figure PCTCN2014093220-appb-000007
D1的合成路线见图1，具体制备过程如下：
将2’-脱氧-2’，2’-二氟代胞苷盐酸盐(3g，10mmol)、咪唑(0.875g，12.8mmol)加入到10mL的无水吡啶中，冰浴降温，在0℃下加入TBDMsCl(3.3g，21mmol)，搅拌0.5h，升至室温，继续反应12h，用甲醇(8.0mL)处理，搅拌60min后，减压除去溶剂，得到化合物2。反应体系继续加入50mLDCM和10ml吡啶，在冰浴和氮气保护条件下加入氯甲酸丁酯(5.46g，40mmol)，室温下搅拌反应12h，旋干，残留物溶于乙酸乙酯(100mL)，用冷饱和碳酸氢钠溶液(30mLX2)、浓盐水(30mL)洗涤；溶液使用无水硫酸钠干燥3h并过滤，滤液经柱层析(二氯甲烷/甲醇，40∶1)得到中间体3(3.6g，两步产率62％)。
将化合物3(3.6g,6.23mmol)加入到40mL THF，冰浴降温至0℃，慢慢加入三乙胺三氢氟酸盐4mL，反应24h，将溶剂真空旋干得到橙色固体，直接过柱(二氯甲烷/甲醇，20∶1)得到化合物4(1.68g，产率58％)。
将得到的中间体4(1.68g，3.62mmol)加入到30mL的三氯甲烷中，加入吡啶30mL，再加入戊二酸酐(620mg，5.44mmol)，在反应搅拌过夜，在加入DMAP(7mg,0.057mmol)后，搅拌3h，然后浓缩得黏稠油状物，柱层析得到化合物5(1.11g，产率53％)。
将化合物5(58mg，0.1mmol)与化合物4(92mg，0.2mmol)、DCC(42mg，0.2mmol)混合后加入到15mL二氯甲烷中，加入DMAP(6mg,0.049mmol)，反应在24℃搅拌24h。TLC检测，待反应结束后加入50mL二氯甲烷，使用10mL水和20mL饱和食盐水洗涤，使用无水硫酸钠干燥，浓缩至干。柱层析(二氯甲烷/甲醇，20∶1) 得到化合物6(49mg，产率48％)。
1H-NMR(MeOD-d4,400MHz)δ：7.97(d,2H,J＝7.68Hz,H6-1,H6-2),7.40(d,2H,J＝7.68Hz,H5-1,H5-2),6.35(t,2H,J＝7.24Hz,H1'-1,H1'-2),4.47(m,6H,H5a'-1,H5a'-2,H5b'-1,H5b'-2,H4'-1,H4'-2),4.21(m,8H,O-CH2×4),2.53(t,4H,J＝7.16Hz,CH2-CH2-CH2),1.97(m,2H,CH2-CH2-CH2),1.64(m,8H,O-CH2-CH2×4),1.42(m,8H,O-CH2-CH2-CH2×4),0.98(m,12H,CH2-CH3×4)。
13C NMR(MeOD-d4,100MHz)δ：172.83,164.51,153.87,144.53,96.26,77.52,69.13,65.89,61.94,32.42,30.66,30.47,18.83,18.67,12.79,12.74,8.48。
ESIMS：calcd for C43H58F4N6O18m/z 1023.37(M+H)+,found 1023.66。
按照上述合成路线，将戊二酸酐改为其他对应的二酸酐，即可制备获得表1中的化合物102至105。将氯甲酸叔丁酯替代氯甲酸丁酯，即可制得表1中化合物106。
(实施例2)
本实施例的胞苷衍生物二聚体为1.5-二-[4-N-正丁氧羰基-2’-脱氧-2’，2’-二氟代胞苷]戊二酸酯(代号D2，表1中编号107)，结构式如下：
Figure PCTCN2014093220-appb-000008
首先制备中间体8，反应过程见图2，制备过程如下：
将300mg(1mmol)2’-脱氧-2’，2’-二氟代胞苷盐酸盐(结构式1)、5mL(0.023mmol)六甲基二硅氮烷HMDS，催化量硫酸铵5mg溶于5mL 1,4-二氧六环中，加热回流反应2h，反应产物化学结构式为2。回流反应结束后反应液浓缩，向其中加入甲苯，浓缩至干2次，浓缩所得产物溶于10mL二氯甲烷中。
向上述二氯甲烷溶液中加入0.24mL(3mmol)N-甲基咪唑、0.32mL(3mmol)氯甲酸丁酯，室温搅拌反应4h，反应液浓缩得粘稠油状物。
将上述粘稠油状物溶于3mL三乙胺和20mL甲醇组成的混合溶液中，室温搅拌4h。减压蒸馏除去溶剂，粗产品用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到17230mg的化合物7，三步反应产率55.5％。
化合物7核磁共振表征：
1H-NMR(MeOD-d4，400MHz)δ：8.30(d，1H，J＝7.68Hz，H6)，7.34(d，1H，J＝7.68Hz，H5)，6.28(t，1H，J＝7.08Hz，H1')，4.33(m，1H，H5a')，4.0(m，2H，O-CH2-CH2-)，3.81(m，1H，H5b')，3.79(m，1H，H4')，1.68(m，2H，O-CH2-CH2-)，1.45(m，2H，O-CH2-CH2-CH2)，0.98(t，3H，J＝7.4Hz，-CH2-CH3)。
13C-NMR(MeOD-d4，100MHz)δ：164.28，156.27，153.50，144.39，128.33，122.72，95.81，84.90，81.71，74.87，68.88，63.69，59.15，30.66，32.40，18.81，11.23，8.06。
取60mg(0.16mmol)化合物7和106mg(1mmol)碳酸钠，混合后加入到5mL1,4-二氧六环和水(体积比4∶1)的混合溶液中。向溶液中加入44mg(0.2mmol)二碳酸二叔丁酯(Boc)2O，然后在24℃下搅拌反应，反应过程中TLC检测化合物7是否完全反应完毕。待反应结束后向反应后的体系中加入2mL水稀释，然后用乙酸乙酯萃取2次，每次用量30mL。萃取得到的有机相用5mL水和5mL饱和食盐水依次洗涤，洗涤完毕无水硫酸钠干燥，接着减压浓缩至干；浓缩后用硅胶层析柱纯化，用二氯甲烷/丙酮/甲醇(1∶1∶0.02)洗脱得到51mg的化合物8，上述反应产率76％。
D2的合成路线见图2，制备过程如下：
将得到的化合物8(223mg，0.25mmol)溶解到6mL的三氯甲烷中，加入吡啶5mL，再加入丁二酸酐(100mg，1mmol)，在45℃下反应过夜，浓缩得黏稠油，柱层析(DCM-MeOH 20∶1至10∶1)得到化合物9(211mg，产率75％)。
将化合物9(56mg，0.1mmol)与化合物8(92mg，0.2mmol)、DCC(42mg，0.2mmol)混合后加入到15mL二氯甲烷中，加入DMAP(6mg,0.049mmol)，反应在24℃搅拌24h。TLC检测，待反应结束后加入50mL二氯甲烷，使用10mL水和20mL饱和食盐水洗涤，使用无水硫酸钠干燥，浓缩至干。再加入TFA5mL和DCM10mL，室温搅拌0.5h；冰浴冷却后滤去白色固体。浓缩得黏稠油，柱层析(DCM-MeOH 20∶1至10∶1)得到产物D2(30mg，产率35％)。
1H-NMR(MeOD-d4,400MHz)δ7.85(d,2H,J＝7.68Hz,H6-1,H6-2),7.37(d,2H,J＝7.68Hz,H5-1,H5-2),6.26(t,2H,J＝7.24Hz,H1'-1,H1'-2),4.53(m,2H,H5a'-1,H5a'-2),4.40(m,4H,H5b'-1,H5b'-2,H4'-1,H4'-2),4.20(m,2H,H3-1,H3-2),2.73(m,4H, -CH2-CH2-),1.64(m,4H,O-CH2-CH2),1.37(m,4H,O-CH2-CH2-CH2),0.93(m,6H,CH2-CH3).
13C-NMR(MeOD-d4,100MHz)δ172.41,164.01,155.95,153.52,144.36,96.56,70.53,66.29,62.49,30.74,28.76,19.01,13.49.
ESIMS：calcd for C32H40F4N6O14m/z 809.25(M+H)+,found 809.34。
按照上述制备方法，将化合物8与其他二酸酐反应，如戊二酸酐等反应即可制得表1中的化合物108至110。将氯甲酸叔丁酯替代氯甲酸丁酯，即可制得表1中化合物111。
(实施例3)
本实施例的胞苷衍生物二聚体为1.5-二-[4-N-(苄氧羰基)-2’-脱氧-2’，2’-二氟代胞苷]戊二酸酯(代号D3)，
Figure PCTCN2014093220-appb-000009
D3的合成路线见图3，制备过程如下：
首先制备化合物13：将300mg(1mmol)2’-脱氧-2’，2’-二氟代胞苷盐酸盐、5mL(0.023mmol)六甲基二硅氮烷，催化量硫酸铵5mg溶于5mL 1,4-二氧六环中，加热回流反应2h；回流反应结束后反应液浓缩，向其中加入甲苯，浓缩至干2次，浓缩所得产物溶于10mL二氯甲烷中。
向上述二氯甲烷溶液中加入0.24mL(3mmol)N-甲基咪唑、340mg(3mmol)氯甲酸卞酯，室温搅拌反应4h，反应产物化学结构式7，反应液浓缩得粘稠油状物。
将上述粘稠油状物溶于3mL三乙胺和20mL甲醇组成的混合溶液中，室温搅拌下过夜。然后减压蒸馏除去溶剂，粗产品用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到21162mg的化合物12，三步反应产率41％。
化合物12核磁共振表征：
1H-NMR(MeOD-d4,400MHz)δ：8.31(d,1H,J＝7.64Hz,H6),7.39(m,5H,J＝7.68Hz,Ph),6.25(t,1H,J＝7.12Hz,H1'),5.21(s,2H.CH2-Ph),4.31(m,1H,H5a'),3.82(m,2H,H5b,H4'),3.79(m,1H,H3')。
13C-NMR(MeOD-d4,100MHz)δ：164.22,156.22,153.27,144.48,135.87,128.42, 128.10,125.31,122.74,120.16,95.89,85.35,84.91,81.7,81.66,68.87,67.54,58.31。
将化合物12(80mg，0.2mmol)与碳酸钠(106mg,1mmol)混合加入到1,4-二氧六环和水的体系中(体积比4∶1,5mL)。后加入(Boc)2O(44mg,0.2mmol)，反应在24℃搅拌48h，TLC检测，待反应结束后加入2mL水稀释，使用2*30mL乙酸乙酯萃取，有机相使用5mL水和5mL饱和食盐水洗涤，使用无水硫酸钠干燥后减压浓缩至干，柱层析(二氯甲烷-丙酮-乙醇1∶1∶0.02)得到化合物13(64mg，产率64％)。
将得到的化合物13(248mg，0.5mmol)加入到15mL三氯甲烷中，加入吡啶5mL，再加入戊二酸酐(100mg，1mmol)，在45℃下反应过夜，浓缩得黏稠油，柱层析(二氯甲烷/甲醇，20∶1至10∶1)得到化合物14(223mg，产率73％)。
将得到的化合物14(61mg，0.1mmol)与化合物13(99mg，0.2mmol)、DCC(42mg，0.2mmol)混合后加入到15mL二氯甲烷中，加入DMAP(6mg,0.049mmol)，反应在24℃搅拌24h。TLC检测，待反应结束后加入50mL二氯甲烷，使用10mL水和20mL饱和食盐水洗涤，使用无水硫酸钠干燥，浓缩至干。再加入TFA(5ml)和DCM(10ml)，室温搅拌0.5h。冰浴冷却后滤去白色固体。浓缩得黏稠油，柱层析(DCM-MeOH20∶1至10∶1)得到产物D3(30mg，产率35％)。
1H-NMR(MeOD-d4,400MHz)δ：8.31(d,1H,J＝7.64Hz,H6),7.39(m,5H,J＝7.68Hz,Ph),6.27(t,2H,J＝7.8Hz,H1'-1,H1'-2),5.17(s,4H,CH2-Ph×2),4.46(m,4H,H5a'-1,H5a'-2,H5b'-1,H5b'-2),4.21(m,2H,H4'-1,H4'-2),4.10(m,2H,H3'-1,H3'-2),,2.53(t,4H,J＝7.16Hz,-CH2-CH2-CH2-),1.99(q,2H,J＝7.2Hz,-CH2-CH2-CH2-)。
13C NMR(MeOD-d4,100MHz)δ172.90,164.21,155.90,153.31,144.25,141.03,135.86,128.41,128.28,128.10,124.85,123.25,122.26,96.14,79.17,74.97,67.59,62.06,33.19,32.51,19.96。
ESIMS：calcd for C39H38F4N6O14m/z 891.24(M+H)+,found 891.31。
按照上述制备方法，将化合物14与其他二酸酐反应，如COOHCHBr(CH2)2COOH、COOH CHPh(CH2)2COOH、COOH CHCNCH2COOH等反应即可制得表1中的化合物113至116。
(实施例4)
本实施例的胞苷衍生物二聚体为1-O-(4-N-(苄氧羰基)–2’-脱氧-2’，2’-二氟代胞苷)-5-O-(4-N-正丁氧羰基-2’-脱氧-2’，2’-二氟代胞苷)-琥珀酸酯(1-O-(4-N-(Benzyloxycarbonyl)–gemcitabine)-4-O-(4-N-(n-Butoxycarbonyl)- gemcitabine)-succinate，代号D4)，结构式如下：
Figure PCTCN2014093220-appb-000010
D4的合成路线见图4，具体制备过程如下：
将化合物9(56mg，0.1mmol)与化合物13(99mg，0.2mmol)、DCC(42mg，0.2mmol)混合后加入到15mL二氯甲烷中，加入DMAP(6mg,0.049mmol)，反应在24℃搅拌24h。TLC检测，待反应结束后加入50mL二氯甲烷，使用10mL水和20mL饱和食盐水洗涤，使用无水硫酸钠干燥，浓缩至干。再加入TFA(5ml)和DCM(10ml)，室温搅拌0.5h。冰浴冷却后滤去白色固体。浓缩得黏稠油，柱层析(DCM-MeOH20∶1至10∶1)得到产物D4(30mg，产率36％)。
1H-NMR(MeOD-d4,400MHz)δ7.98(m,2H,H6-1,H6-2),7.40(d,2H,J＝7.68Hz,H5-1,H5-2),7.38(m,6H,Ph),6.26(t,2H,J＝8Hz,H1'-1,H1'-2),5.21(s,2H,CH2-Ph),4.43(m,2H,H5a'-1,H5a'-2),4.29(m,2H,H5b'-1,H5b'-2),4.21(m,6H,H4'-1,H4'-2H3'-1,H3'-2),2.74(m,4H,-CH2-CH2-),1.43(m,2H,O-CH2-CH2-),1.28(m,2H,O-CH2-CH2-CH2-),0.97(t,3H,J＝7.4Hz,-CH2-CH3)。
13C NMR(MeOD-d4,100MHz)δ172.56,164.19,155.89,153.52,144.61,135.86,128.09,122.22,96.21,79.38,78.91,70.83,67.60,65.92,62.11,56.72,30.64,28.66,28.51,25.93,18.82,14.26,12.77。
ESIMS：calcd for C35H38F4N6O14m/z 843.24(M+H)+,found 843.33。
按照上述制备方法，将其他二酸酐代替丁二酸酐与化合物8反应，得到的中间体与化合物13反应即可制得表1中的化合物118至120。
(实施例5、胞苷衍生物二聚体的盐酸盐)
本实施例制备实施例1的胞苷衍生物二聚体的盐酸盐。
取1.5–二–[4–N–正丁氧羰基–3’–O–正丁氧羰基-2’-脱氧-2’，2’-二氟代胞苷]戊二酸酯0.50g溶解于60mL乙酸乙酯中，冰浴下通入干燥的盐酸气，搅拌15分钟后去除溶剂得到白色固体产物。
其他胞苷衍生物二聚体的盐酸盐的制备方法同上。
除了上述盐酸盐外，还可以制备胞苷衍生物二聚体的磷酸盐、硫酸盐、碳酸盐、硝酸盐、柠檬酸盐、酒石酸盐、马来酸盐、琥珀酸盐、磺酸盐、对甲苯磺酸盐、甲磺酸盐、苯甲酸盐或富马酸盐。
(实施例6、胞苷衍生物二聚体的注射用冻干粉针剂)
本实施例制备实施例3的化合物D3的冻干粉针剂。
D3的冻干粉针剂包括30g的化合物D3、甘露醇(20％w/v)300g，缓冲剂二水合磷酸二氢钠7克、表面活性剂泊洛沙姆188(F68)4.0g。
将按照上述处方量准确称取的二水合磷酸二氢钠、泊洛沙姆188(F68)(CAS号：9003-11-6)、甘露醇(20％w/v)加入300g预冷至10℃以下的注射用水中溶解后，用0.1mol/L的NaOH调节溶液pH值为7.3～7.5；再向上述溶液中加入处方量的D3混合均匀，用0.1mol/L的NaOH溶液或0.1mol/L的HCl调节pH值为7.3±0.2(本实施例为7.5)；加水至2000g，溶液用0.22μm微孔滤膜过滤除菌；按每瓶2.0g将滤液分装于管制瓶中，半加塞后置于冷冻干燥机中冻干，待干燥后真空压塞，轧盖，贴标签，即得冻干粉针剂1000支，保存在2～8℃温度下。
除了上述冻干粉针剂即注射用无菌粉末外，本发明的胞苷衍生物二聚体还可制备成其他形式的注射剂，如溶液型注射剂、混悬型注射剂、乳剂型注射剂。
(实施例7、胞苷衍生物二聚体的药物组合物)
本实施例的胞苷衍生物二聚体的药物组合物由活性组分和辅料组成，其中的药物活性组分为上述实施例制备的胞苷衍生物二聚体或其对应的盐。药物活性组分的重量在组合物中所占比例为1％～95％(本实施例为30％)。辅料由水、乳糖、玉米淀粉、羟丙基甲基纤维素和硬脂酸镁组成。本实施例的药物组合物的剂型为片剂。
药物组合物的适用剂型除除上涉及的片剂形式外，药物活性组分可以被制成口服的散剂、颗粒剂、胶囊剂、微丸制剂、溶液剂、混悬剂、乳剂、糖浆剂或酏剂，或者是口服形式的缓释及控释制剂，或者是其他口服形式的药物组合物，这些口服剂型含有常见的相应的辅料(根据不同的作用分为添加剂、附加物等)，如添加剂有药物等级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精盐、纤维素或硫酸镁等。
在实现上述口服剂型中，可以选择药学上的附加物作为药物活性组分的载体，包括已有技术成熟的物质，例如：惰性固体稀释液、水溶剂、脂质体、微球体或/和无毒有机溶剂等；优选的附加物有：加湿剂、乳化剂、pH缓冲液、人血清白蛋白、抗氧化剂、防腐剂、抑菌剂、葡萄糖、蔗糖、海藻糖、麦芽糖、卵磷脂、甘氨酸、山梨 酸、丙烯醇、聚乙烯、鱼精蛋白、硼酸、氯化钠、或者氯化钾、矿物油、植物油等；可从中选择一种或几种组合作为药物载体。
本发明的药物组合物的靶肿瘤包括血液肿瘤或恶性实体性肿瘤；具体的，靶肿瘤包括肺癌、前列腺癌、乳腺癌、结肠癌、胃癌、胰腺癌、肝癌、食道癌、脑肿瘤、卵巢癌、子宫癌、肾癌、头颈癌、皮肤癌、膀胱癌、外阴癌、睾丸瘤、直肠癌、绒毛癌、生殖细胞瘤、恶性淋巴瘤、白血病和多发性骨髓瘤，并且甚至更优选的靶肿瘤可包括胰腺癌(一、二线治疗)、非小细胞性肺癌、乳腺癌、卵巢癌和头颈部鳞癌、结肠癌，但本发明不限于此。
(应用例1、系列化合物对HCT-116细胞集落形成的抑制实验)
1、通过细胞集落形成抑制试验评估4个候选化合物(D1,D2,D3,D4)在作用浓度为50nM,150nM和50nM对人结肠癌细胞株HCT-116细胞的增殖抑制作用。
2、试验材料：细胞株：HCT-116人结肠癌细胞株订购于中科院上海细胞资源中心，Cat#TCHu 99。
3、试剂配制
HCT-116人结肠癌细胞培养基：DMEM+10％FBS。
化合物的准备：用DMSO稀释化合物使终浓度为100μM。
细胞染色液的配制：用无水乙醇配制0.5％结晶紫溶液避光保存；染色前与PBS按体积比1：4的比例配成细胞染色液。
4、细胞培养：收集对数生长期细胞，计数，用完全培养基重新悬浮细胞；调整细胞浓度至合适浓度，接种6孔板培养皿，每孔接种约300个细胞，1.8ml培养基；细胞在37℃，100％相对湿度，5％CO2培养箱中孵育5小时。
5、细胞克隆形成抑制实验和数据处理
①收集对数生长期细胞，计数，用含5％FBS的培养基重新悬浮细胞，计数，按300个细胞/孔接种6孔板培养皿；细胞在37℃，100％相对湿度，5％CO2培养箱中孵育5小时。
②用培养基(含5％FBS)将化合物稀释至0.5μM,1.5μM和4.5μM。按200
μl/孔加入细胞，使化合物终浓度为50nM,150nM和450nM，每个浓度点进行3
重复测试。
③细胞在37℃，100％相对湿度，5％CO2培养箱中孵育72小时。
④吸弃皿中培养基(含化合物的培养基)，用Hank’s Balance Salt Solution (HBSS))溶液润洗两遍，更换新鲜培养基(15％FBS的DMEM培养基)。
⑤细胞在37℃，100％相对湿度，5％CO2培养箱中孵育7至10天，直到形成肉眼可见的克隆斑。
⑥吸弃皿中培养基，用PBS溶液润洗两遍。
⑦吸净残余PBS，加入无水乙醇1ml/皿，固定30分钟。
⑧吸净乙醇，加入细胞染色液，染色3分钟。
⑨吸弃染色液，用PBS润洗三遍后计数。
数据处理：
克隆形成率＝[As/Ac]×100％；克隆形成抑制率＝1-克隆形成率。
As：样品处理的细胞克隆数目(细胞+待测化合物)。
Ac：阴性对照(不加样品处理)的细胞克隆数目(细胞+1％DMSO)。
6、结果和讨论
四种化合物对人结肠癌细胞株HCT-116细胞的克隆数目见下表2。
表2
Figure PCTCN2014093220-appb-000011
％inhibition：抑制率。
4种化合物在作用浓度为50nM,150nM和450nM对人结肠癌细胞株HCT-116细胞的克隆形成抑制率见图5。
由表2及图5可见本发明化合物对肿瘤细胞有明显的抑制作用。
4种化合物对人结肠癌细胞株HCT-116细胞的抑制率与抑制剂浓度的关系曲线见图6，由图6可看到，D1的IC50值为245.3nM，D2的IC50值为226.6nM，D3的IC50值为99.80nM，D1的IC50值为111.7nM。
(应用例2、系列化合物对肿瘤的生长抑制作用)
本应用例通过观察接种部位肿瘤的形成情况和受试动物的体重变化来评价单次腹腔注射化合物D1至D4对结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制作用及其毒性。
1、试验目的
测定本发明的胞苷衍生物二聚体样品对结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制作用及其毒性。
2、受试物的配制
受试物溶解所用溶剂来源如下：
溶剂 批号 厂商
无水乙醇 10009218 国药集团化学试剂有限公司
Cremophor EL 27963 Sigma
0.9％生理盐水 13083004 华裕制药有限公司
称取定量对应的受试物于5mL玻璃试管中，在5mm的磁力搅拌子搅拌下溶解于乙醇中，全部溶解后加入Cremophor EL，保持搅拌，临用前加入标示量的生理盐水搅拌均匀，配置时乙醇、Cremophor EL、生理盐水的体积比为5∶5∶90。
3、实验动物
品种和品系：Balb/c Nude小鼠；级别：SPF；性别：雌性。
来源：上海西普尔-毕凯实验动物有限公司。
动物数量：订购40只，选择其中健康状况良好的用于实验。
动物合格证号：0123627。
实验开始时动物年龄：7-9周龄。
实验开始时动物体重：18-22克。
适应环境时间：5-7天。
动物编号方式：尾号。
动物房环境保持温度23±2℃,湿度40-70％，12小时明暗交替。
动物饲料(SLAC-M01)购自北京科澳协力有限公司。实验动物用水采用过滤灭菌水。实验过程中动物自由饮食和饮水。
4、实验方法
4.1肿瘤细胞：结肠癌HCT-116细胞，购于中科院细胞生物研究所。用F-12培养基，(含10％的FBS)培养在37℃，饱和湿度，含体积分数为5％CO2、95％空气的二氧化碳培养箱内。接种前取对数生长期细胞，以0.25％胰蛋白酶消化后，PBS洗涤1次，PBS重新悬浮计数，用不含血清的培养基重新悬浮细胞，调整细胞浓度至约3x10^7cell/mL。
4.2动物接种及分组：每个裸鼠在无菌状态下，右侧后肢皮下接种0.1mL细胞悬液(3x10^6cell/mouse)。待肿瘤长至体积60-150mm3左右时，选出肿瘤体积相近、形状较好的裸鼠(形状尽量为单一圆球形，无不规则的形状或多个肿瘤聚在一起)，分组，每组6只，分组情况如下：
表3
Figure PCTCN2014093220-appb-000012
IP：腹腔注射；QD×1：注射一次。
Control控制组即模型对照组的小鼠注射5:5:90的乙醇、Cremophor EL、生理盐水组成的混合溶液。
4.3动物给药和观察
观察各组裸鼠接种部位肿瘤的形成状况，每周3次用圆洞尺测量肿瘤结节的直径(D),并按如下公式计算肿瘤结节的体积(V)：V＝3/4π(D/2)3。
抗肿瘤活性的评价指标为肿瘤生长抑制率TGI(％)，相对肿瘤增殖率T/C(％)。
肿瘤生长抑制率TGI(％)的计算公式为：TGI(％)＝(Vcontrol-VTreatment)/Vcontrol)×100。
相对肿瘤体积(relative tumor volume，RTV)计算公式为：RTV＝Vt/V0。其中V0为分组给药时的肿瘤体积，Vt为测量时的肿瘤体积。
相对肿瘤增殖率T/C(％)，计算公式为：T/C(％)＝TRTV/CRTV×100。
TRTV：治疗组RTV；CRTV：阴性对照组RTV。
每周3次称量小鼠体重。
4.4临床症状
在实验开始和实验过程中每个动物所有的临床症状都应记录。观察应在每天的同一时间进行。
给予受试物后如出现体重减低>20％，濒死动物或肿瘤体积超过2800mm^3，则CO2处死，分离肿瘤并称重，尸检，肉眼观察是否有病变器官并记录。
4.5数据统计
实验数据除特别指出外，均以Mean±SEM表示；两组间数据采用非配对T检验，P<0.05认为有显著性差异。
5试验结果
(1)受试化合物对人结肠癌HCT-116荷瘤小鼠体重的影响。
各组动物平均体重见表4。
表4
Figure PCTCN2014093220-appb-000013
表格中Day：天。
表5体重改变率
Figure PCTCN2014093220-appb-000014
由上表中数据可看到，对结肠癌HCT-116荷瘤裸小鼠腹腔注射各化合物之后，D1组400mg/kg在给药第4天动物体重显著降低，其后体重稳定增长，在第14-16天与模型对照组比较体重显著升高。其他给药组动物体重与模型对照比较无显著性差异。
(2)受试化合物对人结肠癌HCT-116荷瘤小鼠肿瘤体积的影响
各组肿瘤体积具体数据见表6。
Figure PCTCN2014093220-appb-000015
Figure PCTCN2014093220-appb-000016
由上述各组肿瘤体积的数据可见，本发明的胞苷衍生物对肿瘤生长具有明显的抑制作用。
(3)受试化合物对人结肠癌HCT-116荷瘤小鼠肿瘤的生长抑制率(TGI％)
受试化合物对人结肠癌HCT-116荷瘤小鼠肿瘤的生长抑制率(TGI)见如下表7：
表7 D1-D4对人结肠癌HCT-116荷瘤小鼠肿瘤的生长抑制率
Figure PCTCN2014093220-appb-000017
化合物D1400mg/kg组肿瘤抑制率最大值在Day 7，为91.49％，到16天为72.62％；化合物D2400mg/kg组肿瘤抑制率最大值在Day 9，为94.79％，到16天为90.63％；化合物D3350mg/kg组肿瘤抑制率最大值在Day 11，为98.54％，到16天为95.58％；化合物D4300mg/kg组肿瘤抑制率最大值在Day 11，为97.32％，到16天为92.12％。
(4)受试化合物对人结肠癌HCT-116荷瘤小鼠的肿瘤相对体积(RTV)
受试化合物D1-D4对人结肠癌HCT-116荷瘤小鼠的肿瘤相对体积见如下表8：
表8
Figure PCTCN2014093220-appb-000018
(5)受试化合物对人结肠癌HCT-116荷瘤小鼠的相对肿瘤增值率(T/C％)
受试化合物对人结肠癌HCT-116荷瘤小鼠的相对肿瘤增值率数据见如下表9：
表9
Figure PCTCN2014093220-appb-000019
Figure PCTCN2014093220-appb-000020
化合物D1400mg/kg组相对肿瘤增殖率在Day 7达到最小值28.36％，至Day 16天肿瘤增殖率为89.47％。化合物D2400mg/kg组相对肿瘤增殖率在Day 9达到最小值14.41％，至Day 16天肿瘤增殖率为21.64％。化合物D3350mg/kg组相对肿瘤增殖率在Day 11达到最小值3.41％，至Day 16天肿瘤增殖率为10.49％。化合物D4300mg/kg组相对肿瘤增殖率在Day 11达到最小值25.94％，至Day 16天肿瘤增殖率为37.96％。
在系列化合物对人结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制实验中，化合物D2、D3、D4对结肠癌HCT-116荷瘤裸小鼠移植瘤的肿瘤抑制率较好，一次性腹腔给药后至16天有显著的肿瘤抑制作用，对动物体重无明显影响，说明本发明的胞苷衍生物二聚体抗肿瘤活性高同时毒副作用小。
工业应用性
本发明具有积极的效果：本发明通过对胞苷化合物进行分子优化设计，制备的新型的胞苷衍生物二聚体对人结肠癌HCT-116肿瘤细胞有明显的抑制作用，同时对荷瘤裸小鼠人结肠癌HCT-116移植瘤具有强烈的生长抑制作用；本发明的新型胞苷衍生物二聚体化合物的抗肿瘤活性非常高，同时化合物的毒性很低。

Claims (8)

  1. 一种新型胞苷衍生物二聚体，具有下述通式(Ⅰ)：
    Figure PCTCN2014093220-appb-100001
    其中，R1是C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph、或取代-(CH2)n-Ph；所述的-(CH2)n-Ph，其中n＝0、1、2、3～10，Ph为苯环；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；
    R2是C1至C10的烷基、C1至C10的取代烷基、-(CH2)n-Ph、或取代-(CH2)n-Ph；所述的-(CH2)n-Ph，其中n＝0、1、2、3～10；所述的取代烷基，其碳链上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代；
    R3是H、烷氧羰基、取代烷氧羰基，所述的取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基；
    R4是H、烷氧羰基、或取代烷氧羰基，所述的取代烷氧羰基的取代基为卤素、氰基、硝基、氨基、羟基或羧基；
    R5是-(CH2)n-，其中n＝1至15，或者是碳链上有取代基的-(CH2)n-，所述的取代基是苯基、取代苯基、卤素、氰基、硝基、氨基、羟基、羧基，或者是-(CH2)n-X1-X2-；所述的-(CH2)n-X1-X2-，其中n＝0、1、2或3，X1是O、S，X2是-(CH2)n–Ph，其中n＝0、1、2或3，或者X2是嘧啶基、吡喃基、咪唑基、吡嗪基或吡啶基。
  2. 根据权利要求1所述的新型胞苷衍生物二聚体，其特征在于：R3为H，并且R4为H。
  3. 根据权利要求2所述的新型胞苷衍生物二聚体，其特征在于：R1与R2相同。
  4. 权利要求1所述的新型胞苷衍生物二聚体或其盐在制备抑制肿瘤的药物中的应用。
  5. 根据权利要求4所述的新型胞苷衍生物二聚体或其盐在制备抑制肿瘤的药物中的应用，其特征在于所述肿瘤为血液肿瘤或恶性实体性肿瘤。
  6. 一种药物组合物，其特征在于：其中含有作为活性成分的的权利要求1所述的通式(Ⅰ)所示的胞苷衍生物二聚体或其药学上可接受的盐，以及一种或多种药用载体或赋形剂。
  7. 权利要求6所述的药物组合物，其特征在于：组合物的剂型是注射剂，或者是口服剂型，其中口服剂型包括片剂、散剂、颗粒剂、胶囊剂、微丸制剂、溶液剂、混悬剂、乳剂、糖浆剂或酏剂；注射剂包括溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉末。
  8. 一种如权利要求1所述的新型胞苷衍生物二聚体的制备方法，其特征在于包括以下步骤：
    ①制备通式(Ⅱ)的化合物，
    Figure PCTCN2014093220-appb-100002
    ②制备通式(Ⅲ)的化合物，
    Figure PCTCN2014093220-appb-100003
    ③将通式(Ⅱ)的化合物与碳酸钠混合加入到1,4-二氧六环和水的体系中，然后加入(Boc)2O，TLC检测，待反应结束后萃取洗涤，干燥后减压浓缩至干；将得到的化合物加入到三氯甲烷中，加入吡啶和二酸酐R5(CO)2O，反应过夜，浓缩得黏稠油，柱层析得到通式(Ⅳ)化合物待用；
    Figure PCTCN2014093220-appb-100004
    ④将得到的通式(Ⅳ)化合物与通式(Ⅲ)化合物、DCC混合后加入到二氯甲烷中，加入DMAP，TLC检测，待反应结束后洗涤干燥，浓缩至干，再加入TFA和DCM，室温搅拌，冰浴冷却后滤去白色固体，浓缩得黏稠油，柱层析得到产物。
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