WO2016078397A1 - 新型胞苷衍生物及其应用 - Google Patents

新型胞苷衍生物及其应用 Download PDF

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WO2016078397A1
WO2016078397A1 PCT/CN2015/081047 CN2015081047W WO2016078397A1 WO 2016078397 A1 WO2016078397 A1 WO 2016078397A1 CN 2015081047 W CN2015081047 W CN 2015081047W WO 2016078397 A1 WO2016078397 A1 WO 2016078397A1
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substituted
group
compound
nitro
cyano
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PCT/CN2015/081047
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English (en)
French (fr)
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杨达丽亚
王海东
刘昕
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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Priority claimed from CN201510167477.3A external-priority patent/CN106146583B/zh
Application filed by 常州方圆制药有限公司, 常州优谱生物医药有限公司 filed Critical 常州方圆制药有限公司
Priority to EP15860978.4A priority Critical patent/EP3210992B1/en
Priority to JP2017526507A priority patent/JP2017534657A/ja
Priority to RU2017120030A priority patent/RU2684402C2/ru
Priority to US15/527,313 priority patent/US10174067B2/en
Priority to CA2967058A priority patent/CA2967058C/en
Priority to KR1020177016332A priority patent/KR101982951B1/ko
Priority to AU2015349390A priority patent/AU2015349390B2/en
Publication of WO2016078397A1 publication Critical patent/WO2016078397A1/zh

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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

Definitions

  • the present invention relates to the use of a novel cytidine derivative and a derivative for the preparation of an antitumor drug.
  • 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.
  • Cytosine derivatives having antitumor effects are cytarabine and gemcitabine. Cytarabine is converted into active cytarabine cytarabine in vivo to exert an anticancer effect. Cytarabine triphosphate inhibits the synthesis of DNA and inhibits the growth of cells by inhibiting NDA polymerase and a small amount of DNA, and is mainly used for the treatment of acute myeloid leukemia. However, the cytotoxic side effects of cytarabine are also large.
  • the hematopoietic system is mainly myelosuppression, white blood cells and thrombocytopenia, severe aplastic anemia or megaloblastic anemia can occur; it can occur in the early stage of treatment for leukemia and lymphoma patients. Hyperuricemia, severe cases can occur uric acid nephropathy.
  • Gemcitabine is a derivative of deoxycytidine that is similar in structure and metabolism to cytarabine. Gemcitabine is catalyzed by the action of nucleotide kinases in cells to form active difluorocytidine diphosphate (dFdCDP) and difluorocytidine triphosphate (dFdCTP), which inhibit DNA polymerase and impede DNA synthesis. Due to incorporation into the DNA, the continued extension of the DNA strand is terminated, thereby inhibiting the growth of tumor cells.
  • dFdCDP active difluorocytidine diphosphate
  • dFdCTP difluorocytidine triphosphate
  • Gemcitabine is indicated for pancreatic cancer (first- and second-line treatment), non-small cell lung cancer, breast cancer, ovarian cancer, and head and neck squamous cell carcinoma. However, gemcitabine is also more toxic. Adverse reactions include myelosuppression, leukopenia, thrombocytopenia, anemia; digestive tract reactions such as mild nausea, vomiting, and abnormal liver function; fever, flu-like symptoms, fatigue, mucositis, and the like.
  • the tumor cell When the above cytidine derivative enters the human body, the tumor cell will produce a multi-drug resistance gene, and the amino group on the ring is easily acetylated to cause the compound to lose anticancer activity, and other drug resistance factors, the cytosine derivative has a large side effect and It is easy to produce drug resistance.
  • a gemcitabine prodrug, a pharmaceutical composition and use thereof are disclosed in U.
  • the hydrogen atom of the methylol group on the ribofuranose is substituted by H, an acyl group, a substituted acyl group, an acyloxycarbonyl group, a substituted acyloxycarbonyl group, an oxycarbonyl group, a substituted oxycarbonyl group or the like
  • the hydrogen atom of the hydroxyl group on the ribofuranose is a substituent of H, an acyl group, a substituted acyl group, an acyloxycarbonyl group, a substituted acyloxycarbonyl group, an oxycarbonyl group, a substituted oxycarbonyl group or the like
  • the compound prepared by the patent is a prodrug, and has antitumor activity after being transformed into the body; in addition, clinical studies have found that the gemcitabine prodrug is highly toxic and the antitumor activity is not strong enough, and no drug has been developed yet.
  • the technical problem to be solved by the present invention is to provide a novel cytidine derivative and the use of the above derivative in the preparation of an antitumor drug.
  • a technical solution for achieving the object of the present invention is: a novel cytidine derivative having the following general formula (I):
  • R2 is H, halogen or X1 is a C 1 to C 10 alkyl group, a C 1 to C 10 substituted alkyl group, a C 1 to C 10 alkoxy group, a C 1 to C 10 substituted alkoxy group, a C 1 to C 6 alkyl group.
  • R3 is H or Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three a substituted heterocyclic ring substituted with a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group, or a substituted heterocyclic ring independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups a ring; the heterocyclic ring is imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine or piperidine; the fused heterocyclic ring is quinoline or hydrazine; X2 is -(CH 2 ) n-, wherein
  • R2 is H.
  • R2 is not H; and R3 is not H.
  • R2 is not H, R2 is halogen or X1 is -(CH 2 )n-Ph or is substituted by -(CH 2 )n-Ph.
  • Rl is a C 1 to C 4 alkyl group, a substituted a C 1 to C 4 alkyl group, a benzyl group, or a substituted benzyl group; R3 X3, independently by one or two or three halogen, a substituted cyano, nitro, amino, hydroxy or carboxy substituted imidazole, substituted pyridine independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, or independently Or a substituted benzene ring substituted with two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups.
  • the tumor is a hematological tumor or a malignant solid tumor.
  • the salts are hydrochloride, phosphate, sulfate, carbonate, nitrate, citrate, tartrate, maleate, succinate, sulfonate, p-toluenesulfonate, methanesulfonate.
  • An acid salt a benzoate or a fumarate.
  • a pharmaceutical composition comprising, as an active ingredient, a cytidine derivative represented by the formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.
  • the dosage form of the above composition is an injection preparation or an oral dosage form, wherein the injection preparation is a solution injection, a suspension injection, an emulsion injection, or a sterile powder for injection, and the oral dosage form is a tablet, a powder, a granule, a capsule, a micro. Pill preparations, solutions, suspensions, emulsions, syrups or elixirs.
  • the present invention has a positive effect: the growth inhibition test of the compound of the present invention on colon cancer HCT-116 tumor-bearing nude mouse xenografts confirms that the compound of the present invention has high antitumor activity and simultaneously colonizes human colon cancer HCT-116. The effect of mouse body weight was small, demonstrating that the toxicity of the compound is low.
  • R2 is H, halogen or X1 is a C 1 to C 10 alkyl group, a C 1 to C 10 substituted alkyl group, a C 1 to C 10 alkoxy group, a C 1 to C 10 substituted alkoxy group, a C 1 to C 6 alkyl group.
  • R3 is H or Wherein X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene independently substituted with one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups, independently of one or two or three a substituted heterocyclic ring substituted with a halogen, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group, or a substituted heterocyclic ring independently substituted by one or two or three halogen, cyano, nitro, amino, hydroxy or carboxy groups a ring; the heterocyclic ring is imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine or piperidine; the fused heterocyclic ring is quinoline or hydrazine; X2 is -(CH 2 ) n-, wherein
  • cytidine derivative of the present invention the following compounds are given in Table 1, but the cytidine derivatives of the present invention are 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 of the present example is 4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 4, code G2), which is synthesized by a three-step reaction.
  • the reaction formula is as follows (in the reaction formula, HMDS is hexamethyldisilazane, reflux is reflux, chloridate is chloride, rt is room temperature, TEA is triethylamine, the same applies hereinafter).
  • the carbon chain of the substituted alkyl group is independently One or two or three halogen, cyano, nitro, amino, hydroxy or carboxy substituted; substituted -(CH 2 ) n-Ph on the carbon chain or on the phenyl ring independently from one or two or three halogens Substituted with cyano, nitro, amino, hydroxy or carboxy.
  • the cytidine derivative of the present example is 4-N-(tert-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 6, code G3), which is synthesized by a three-step reaction.
  • the reaction formula is as follows.
  • the cytidine derivative of the present example is 4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 8, code G4), which is synthesized by a three-step reaction.
  • the formula is as follows.
  • the cytidine derivative of the present example is 4-N-(4-nitrobenzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 10, code G5), after three steps
  • the reaction was synthesized, and the reaction formula was as follows.
  • the cytidine derivative of the present example is 5-bromo-4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 11, code G6), reaction formula as follows.
  • the cytidine derivative of the present example is 5-bromo-4-N-(benzyloxycarbonyl)-2'-deoxy-2',2'-difluorocytidine (Structure 12, code G7), and the reaction formula is as follows (DMF in the reaction formula is N,N-dimethylformamide, the same applies hereinafter).
  • the cytidine derivative of this example is 5'-O-[3,5-dinitrosalicylate]-4-N-(n-butoxycarbonyl)-2'-deoxy-2', 2'- Difluorocytidine (Structure 14, code G8).
  • reaction formula is as follows (in the reaction formula, (Boc) 2 O is di-tert-butyl dicarbonate, dioxane is 1,4-dioxane, DMAP is 4-dimethylaminopyridine, and EDCL is 1-(3-dimethylamino).
  • (Boc) 2 O is di-tert-butyl dicarbonate
  • dioxane is 1,4-dioxane
  • DMAP 4-dimethylaminopyridine
  • EDCL 1-(3-dimethylamino).
  • Propyl)-3-ethylcarbodiimide hydrochloride DCM is dichloromethane
  • TFA trifluoroacetic acid, the same below):
  • Compound 13 was first prepared. 60 mg (0.16 mmol) of G2 and 106 mg (1 mmol) of sodium carbonate prepared in Example 1 were mixed, and added to 5 mL of a mixed solution of 1,4-dioxane and water (4:1 by volume). 44 mg (0.2 mmol) of di-tert-butyl dicarbonate (Boc) 2 O was added to the solution, and then the reaction was stirred at 24 ° C, and TLC was used to detect whether G 2 was completely reacted during the reaction. After the reaction was completed, 2 mL of water was added to the system after the reaction, and then extracted twice with ethyl acetate for 30 mL each time.
  • Boc di-tert-butyl dicarbonate
  • R3 By changing 3,5-dinitrosalicylic acid to another acid, R3 can be prepared.
  • X 3 is a benzene ring, a heterocyclic ring, a fused heterocyclic ring, a substituted benzene, a substituted heterocyclic ring or a substituted fused heterocyclic ring independently substituted by one or two or three halogens, a cyano group, a nitro group, an amino group, a hydroxyl group or a carboxyl group;
  • the heterocyclic ring includes imidazole, pyridine, furan, thiophene, thiazole, pyrimidine, piperazine and piperidine; fused heterocyclic ring includes quinoline and hydrazine; and X2 is C 1 to C 3 -(CH 2 )n- or C a compound of 0 to C 3 -O-(CH 2 )n-.
  • the cytidine derivative of this example is 5'-O-[2-(4-nitro-1H-imidazolium) acetate]-4-N-(tert-butoxycarbonyl)-2'-deoxy-2' , 2'-difluorocytidine (Structure 16, code G9).
  • Compound 15 was first prepared. 60 mg (0.16 mmol) of the compound 6 (G3) prepared in Example 2 and 106 mg (1 mmol) of sodium carbonate were mixed, and then added to a mixed solution of 5 mL of 1,4-dioxane and water (4:1 by volume). 44 mg (0.2 mmol) of di-tert-butyl dicarbonate (Boc) 2 O was added to the solution, and then the reaction was stirred at 24 ° C, and TLC was used to detect whether G 2 was completely reacted during the reaction. After the reaction was completed, 2 mL of water was added to the system after the reaction, and then extracted twice with ethyl acetate for 30 mL each time.
  • Boc di-tert-butyl dicarbonate
  • the cytidine derivative of this example has the code G10, and the reaction formula is as follows (DCC in the reaction formula is N,N'-dicyclohexylcarbodiimide):
  • the code of the cytidine derivative of this example is G11.
  • Compound 24 is first prepared and has the following reaction formula:
  • the reaction flask was charged with iodine (2.8 g, 11 mmol), iodic acid (0.83 g, 4.7 mmol), acetic acid (37.5 mL), carbon tetrachloride (25.5 mL), water (25.5 ml) and compound 23, and stirred at 40 ° C. Reaction for 24h.
  • the solvent was dried and dichloromethane and water were added.
  • the pH was adjusted to 6-7, and the organic phase was washed with sodium thiosulfate and washed with water.
  • the organic phases were combined and dried over anhydrous sodium sulfate. Filter and the filtrate was dried.
  • the compound 24, i.e., the product 5 g was obtained (yield 55% in two steps).
  • the code of the cytidine derivative of this example is G13.
  • 1,4-dioxane is 1,4-dioxane
  • HMDS is hexamethyldisilazane
  • C 1 to C 10 substituted alkoxy, C alkylsulfonyl group of 1 to C 6, C 1 to C 6 alkylthio of, - (CH 2) n- Ph or substituted -(CH 2 )n-Ph; wherein the substituted alkyl, substituted alkoxy carbon chain is independently substituted by one or two or three halogens, cyano, nitro, amino, hydroxy or carboxy; -(CH 2 ) n-Ph and substituted -(CH 2 )n-Ph n 0, 1, 2, 3 - 10; substituted -(CH 2 ) n-Ph on the carbon chain or on the benzene ring by one or two Or three H, halogen, cyano, nitro, amino, hydroxy or carboxy substituted.
  • the code of the cytidine derivative of this example is G14.
  • the code of the cytidine derivative of this example is G15.
  • the code of the cytidine derivative of this example is G16.
  • Boc-protected compound 45 (355 mg, 0.65 mmol) was combined with compound 22 (499 mg, 1.95 mmol), DCC (401 mg, 1.95 mmol), and then added to 45 mL of dichloromethane, and DMAP (2 mg, 0.016 mmol) was added. Stir at room temperature overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted with 2 ⁇ 20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of brine, dried over anhydrous sodium sulfate and then added to TFA to give the target compound G16 ( 110 mg, 2 steps yield 24%).
  • the code of the cytidine derivative of this example is G17.
  • the Boc-protected compound 48 (540 mg, 0.869 mmol) was combined with compound 22 (667 mg, 2.60 mmol), DCC (537 mg, 2.60 mmol), then added to 45 mL of dichloromethane and DMAP (2 mg, 0.016 mmol) Stir at room temperature overnight. After TLC detection, after the reaction was completed, it was diluted with 5 mL of water, extracted with 2 ⁇ 20 mL of dichloromethane, and the organic phase was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous sodium sulfate and then added to TFA to obtain the target compound G17 ( 115mg, 2 step yield 17%)
  • hydrochloride salt of 4-N-(n-butoxycarbonyl)-2'-deoxy-2',2'-difluorocytidine of the compound of Example 1 was prepared.
  • hydrochloride salt it is also possible to prepare a phosphate, a sulfate, a carbonate, a nitrate, a citrate, a tartrate, a maleate, a succinate, a sulfonate, a p-toluene of a cytidine derivative. Sulfonate, methanesulfonate, benzoate or fumarate.
  • This example prepared a lyophilized powder injection of the compound G14 of Example 13.
  • the lyophilized powder injection of G14 comprises 30 g of compound G14, 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 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 of the present embodiment is composed of an active ingredient and an adjuvant, wherein the pharmaceutically active component is the cytidine derivative 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, Sorbic 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 maximum tolerated dose (MTD) is the dose at which the animal does not die, the animal's weight loss does not exceed 10% (compared to Day 0), or does not produce significant toxic side effects.
  • test object is configured as follows.
  • the solvent used to dissolve the test substance is as follows:
  • ICR mice ICR mice; grade: SPF; gender: female.
  • Feeding method six cages.
  • the animal room has an ambient temperature of 18-26 ° C, a relative humidity of 30-70%, and 12 hours of light.
  • the experimental animals were acclimated for 5-7 days before the experiment.
  • the SPF large mouse growth and propagation feed Co60 was sterilized and purchased from Beijing Keao Xieli Co., Ltd.
  • the experimental animals were filtered and sterilized with water, and the animals were free to eat and drink.
  • Mode of administration ip. If the animal dies, the dose is reduced until the animal survives, and if there is no animal death, the dose is increased; if the animal is normally alive at a given high dose, the experiment is over. Finally, the MTD of the test subject was determined according to the experimental results; the animals were continuously observed for 7 days after acute administration.
  • All animals in the experiment were subjected to detailed clinical observations of all the tested animals. Two times a day (10:00, 16:00 each), continuous observation for 14 days, including but not limited to: skin, Hair, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs and feet, respiratory and circulatory systems, autonomic effects (such as salivation), nervous system (such as tremors, convulsions, stress reactions, and abnormal behavior).
  • 14 days including but not limited to: skin, Hair, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs and feet, respiratory and circulatory systems, autonomic effects (such as salivation), nervous system (such as tremors, convulsions, stress reactions, and abnormal behavior).
  • the body weight of the animals was weighed before administration, and then the body weight of the animals was weighed and recorded at the same time every day.
  • G3 and G4 can tolerate at 350mg/kg
  • G5 can tolerate at 300mg/kg
  • G6 can tolerate at 200mg/kg
  • G7 can tolerate at 200mg/kg
  • G8 can tolerate at 300mg/kg
  • G10 at 400mg /kg can be tolerated
  • G11 can tolerate at 400mg/kg
  • G12 can tolerate at 400mg/kg
  • G13 can tolerate at 400mg/kg
  • G15 can tolerate at 400mg/kg
  • G16 can tolerate at 400mg/kg .
  • the growth inhibitory effect and toxicity of the cytidine derivative sample of the present invention on 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 100, 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 3.
  • the weight change rate of G10 to G16 is shown in Table 4-2.
  • the body weight of G4 350mg/kg was significantly decreased on the 4th day of administration (p ⁇ 0.05), and the average weight loss rate was average. After 10.91 ⁇ 3.45%, the body weight increased steadily, and the body weight was significantly increased on the 18th to 20th day compared with the model control group (p ⁇ 0.05).
  • the body weight of G5 325mg/kg was significantly decreased (p ⁇ 0.05), and the body weight loss rate was ⁇ 10%. After that, the body weight increased steadily, and the body weight increased significantly on the 13th to 20th day compared with the model control group (p ⁇ 0.05 to 0.01).
  • G7 250mg/kg showed a significant decrease in body weight (p ⁇ 0.05) on days 4 and 6 of administration, and the body weight loss rates were 12.28 ⁇ 4.78% and 4.39 ⁇ 3.6%, respectively, and then the body weight increased steadily. There was no significant difference in body weight between the other drug-administered groups and the model control.
  • TGI growth inhibition rate
  • the maximum tumor inhibition rate of the compound G3 350 mg/kg group was 58.10% in Day8 and 46.82% in Day22.
  • the tumor inhibition rate of the compound G4 350 mg/kg group was better at Day 11 and reached a maximum of 92.58%, and remained at 70% or more for Day 22 days.
  • the tumor inhibition rate of the compound G5 325mg/kg group was better at Day11, reaching a maximum of 94.46%, and remained above 70% for Day24.
  • the maximum tumor inhibition rate in the G9 325 mg/kg group was 80.77% in Day4 and about 40% in Day22.
  • the maximum tumor inhibition rate in the G7 250 mg/kg group was 82.62% in Day8, and the inhibition rate in Day22 was 44.07%.
  • test compound G3-G9 against human colon cancer HCT-116 tumor-bearing mice is shown in Table 7-1 below:
  • the relative tumor volume of the compound G4 350 mg/kg group was significantly lower from Day 4 to Day 18 (p ⁇ 0.05 to 0.01) compared with the model control group.
  • the G5 325 mg/kg group had a significant decrease in tumor relative volume from Day 4 to Day 18 (p ⁇ 0.05 to 0.01).
  • the G7 250 mg/kg group had a significant decrease in tumor relative volume from Day 4 to Day 15 (p ⁇ 0.05 to 0.01).
  • the relative volume of tumor in the G9 325 mg/kg group was only significantly decreased in Day 4 (p ⁇ 0.05). There was no significant difference in tumor relative volume between the other drug-administered groups and the model control group.
  • the relative tumor proliferation rate of the compound G4 350 mg/kg group reached a minimum of 17.62% in Day 13, and the tumor proliferation rate in Day 22 was 75.38%.
  • the relative tumor proliferation rate of the compound G5 325 mg/kg group reached a minimum of 10.01% on Day 11, and the tumor proliferation rate on Day 22 was 68.77%.
  • Compound G7 250mg/kg group relative tumor proliferation The rate reached a minimum of 17.67% in Day 8 and a tumor proliferation rate of 58.62% in Day 22 days.
  • the compound G4, G5, G7 had a good tumor inhibition rate on colon cancer HCT-116 tumor-bearing nude mice xenografts.
  • Day 8 to Day 13 had good tumor inhibition after intraperitoneal administration.
  • the relative growth rate of G5 in Day 11 reached a minimum of 10.01%, and the effect on animal body weight was reduced, and the average weight loss rate was less than 10%.

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Abstract

本发明公开了一种新型胞苷衍生物及其应用，具有下述通式(Ⅰ) 本发明的新型胞苷衍生物对结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制试验证实，本发明的化合物的抗肿瘤活性高，同时对人结肠癌HCT-116荷瘤小鼠体重的影响以及死亡率数据证明化合物的毒性较低。

Description

新型胞苷衍生物及其应用 技术领域
本发明涉及一种新型的胞苷衍生物以及衍生物在制备抗肿瘤药物中的应用。
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。
具有抗肿瘤作用的胞苷衍生物有阿糖胞苷和吉西他滨。阿糖胞苷在体内转化为活性的三磷酸阿糖胞苷而发挥抗癌作用。三磷酸阿糖胞苷通过抑制NDA多聚酶及少量掺入DNA，阻止DNA的合成，抑制细胞的增长，主要用于治疗急性粒细胞白血病。但是阿糖胞苷的毒副作用也较大，对造血系统主要是骨髓抑制，白细胞及血小板减少，严重者可发生再生障碍性贫血或巨幼细胞性贫血；对白血病、淋巴癌患者治疗初期可发生高尿酸血症，严重者可发生尿酸性肾病。
吉西他滨(Gemcitabine)是去氧胞苷的衍生物，结构和代谢均与阿糖胞苷相似。吉西他滨在细胞内通过核苷酸激酶作用，催化成有活性的二磷酸双氟胞苷(dFdCDP)和三磷酸双氟胞苷(dFdCTP)，后者抑制DNA多聚酶而阻碍DNA的合成。由于掺合到DNA上，终止了DNA链的继续延长，从而抑制肿瘤细胞的生长。
吉西他滨适用于胰腺癌(一、二线治疗)、非小细胞性肺癌、乳腺癌、卵巢癌和头颈部鳞癌等。但是吉西他滨的毒性也较大，不良反应有骨髓抑制即白细胞、血小板减少，贫血；消化道反应如轻度恶心、呕吐和肝功能异常；发热、流感样症状、乏力、黏膜炎等。
上述胞苷衍生物进入人体后，肿瘤细胞会产生多耐药基因，并且环上的氨基易被乙酰化而致化合物失去抗癌活性，以及其它耐药因素，上述胞苷衍生物毒副作用大并且容易产生耐药性。
为了降低阿糖胞苷和吉西他滨的毒性，提高或维持抗肿瘤药效，研究人员对胞苷衍生物的化学结构进行修饰。
例如《Synthesis and Biological Activity of a Gemcitabine Phosphoramidate Prodrug》(J.Med.Chem 2007,50,3743-3746；Weidong Wu)报道了一种吉西他滨磷酸酯前体药物。
美国专利US 7265096 B2(申请号10/701965)公开了一种吉西他滨前体药物、 药物组合物及应用，该文献对吉西他滨的氨基、呋喃核糖上的羟基的氢原子和羟甲基的氢原子进行了取代，呋喃核糖上的羟甲基的氢原子由H、酰基、取代酰基、酰氧基羰基、取代酰氧基羰基、氧羰基、取代氧羰基等取代；呋喃核糖上的羟基的氢原子由H、酰基、取代酰基、酰氧基羰基、取代酰氧基羰基、氧羰基、取代氧羰基等取代；氨基由-N＝C(R10)(R11)或-NHR12取代，其中R12为C5-C9的酰基或者是C5-C9的取代酰基。该专利所制备的化合物是前药，进入体内转化后才具有抗肿瘤活性；另外，临床研究发现该吉西他滨前体药物毒性大，抗肿瘤活性不够强，目前还未开发成药。
发明内容
本发明所要解决的技术问题是提供一种新型的胞苷衍生物以及上述衍生物在制备抗肿瘤药物中的应用。
实现本发明目的的技术方案是：一种新型胞苷衍生物，具有下述通式(Ⅰ)：
Figure PCTCN2015081047-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；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H、卤素或
Figure PCTCN2015081047-appb-000002
X1是C1至C10的烷基、C1至C10的取代烷基、C1至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph、或者是取代-(CH2)n-Ph；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；所述的取代烷氧基，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-(CH2)n-Ph，其中n＝0、1、2、3～10；所述的取代-(CH2)n-Ph，其中n＝0、1、2、 3～10，其碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。
R3是H或
Figure PCTCN2015081047-appb-000003
其中X3是苯环，杂环，稠杂环，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代杂环，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代稠杂环；所述杂环是咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪或哌啶；所述稠杂环是喹啉或吲哚；X2是-(CH2)n-，其中n＝1、2、3，或者X2是-O-(CH2)n-，其中n＝0、1、2、3。
作为可选择的，R2为H。
进一步优选的，R2不为H；并且R3不为H。
R2不为H时，R2为卤素或
Figure PCTCN2015081047-appb-000004
X1是-(CH2)n-Ph或者是取代-(CH2)n-Ph。
作为优选的，R1是C1至C4的烷基、C1至C4的取代烷基、苄基，或者是取代苄基；R3的X3是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代咪唑，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代吡啶，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯环。
上述化合物或其盐在制备治疗肿瘤药物中的应用。
所述肿瘤为血液肿瘤或恶性实体肿瘤。
所述的盐为盐酸盐、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。
一种药物组合物，其中含有作为活性成分的通式(Ⅰ)所示的胞苷衍生物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。
上述组合物的剂型为注射剂或者是口服剂型，其中注射剂为溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉末，口服剂型为片剂、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。
本发明具有积极的效果：本发明的化合物对结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制试验证实，本发明的化合物的抗肿瘤活性高，同时对人结肠癌HCT-116荷瘤小鼠体重的影响小，证明化合物的毒性较低。
具体实施方式
本发明的新型胞苷衍生物的结构式如式(Ⅰ)：
Figure PCTCN2015081047-appb-000005
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；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H、卤素或
Figure PCTCN2015081047-appb-000006
X1是C1至C10的烷基、C1至C10的取代烷基、C1至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph或者是取代-(CH2)n-Ph；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；所述的取代烷氧基，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-(CH2)n-Ph，其中n＝0、1、2、3～10；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。
R3是H或
Figure PCTCN2015081047-appb-000007
其中X3是苯环，杂环，稠杂环，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代杂环，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代稠杂环；所述杂环是咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪或哌啶；所述稠杂环是喹啉或吲哚；X2是-(CH2)n-，其中n＝1、2、3，或者X2是-O-(CH2)n-，其中n＝0、1、2、3。
对于本发明的胞苷衍生物，在表1中给出如下化合物，但本发明的胞苷衍生物不限于这些化合物。
表1
Figure PCTCN2015081047-appb-000008
Figure PCTCN2015081047-appb-000009
Figure PCTCN2015081047-appb-000010
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为4-N-(正丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式4，代号G2)，经三步反应合成，反应式如下(反应式中HMDS为六甲基二硅氮烷，reflux为回流，chloridate为氯化物，rt为室温，TEA为三乙胺，下同)。
Figure PCTCN2015081047-appb-000011
将300mg(1mmol)2’-脱氧-2’，2’-二氟代胞苷盐酸盐(结构式1，代号G1)、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，反应产物化学结构式3，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230mg的G2，三步反应产率55.5％。
G2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。
按照上述制备方法，改变反应原料，R1除正丁基外，可为其他基团，如：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。
(实施例2)
本实施例的胞苷衍生物为4-N-(叔丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式6，代号G3)，经三步反应合成，反应式如下。
Figure PCTCN2015081047-appb-000012
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、416mg(3mmol)二碳酸二叔丁酯，室温搅拌反应4h，反应产物化学结构式5，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199mg的产物G3，，三步反应产率54％。
G3核磁共振表征：
1H-NMR(MeOD-d4,400MHz)δ：8.27(d,1H,J＝7.68Hz,H6),7.32(d,1H,J＝7.68Hz,H5),6.28(t,1H,J＝7.08Hz,H1'),4.25(m,1H,H5a'),3.93(m,2H,H5b',H4'),3.78(m,1H,H3'),1.52(s,9H,t-Bu)。
13C-NMR(MeOD-d4,100MHz)δ：164.36,152.17,144.24,125.29,122.71,120.13,109.98,95.78,82.17,81.60,70.45,69.30,68.90,65.57,55.90,27.12,23.57。
(实施例3)
本实施例的胞苷衍生物为4-N-(苄氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式8，代号G4)，经三步反应合成，反应式如下。
Figure PCTCN2015081047-appb-000013
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162mg的产物G4，，三步反应产率41％。
G4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。
(实施例4)
本实施例的胞苷衍生物为4-N-(4-硝基苄氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式10，代号G5)，经三步反应合成，反应式如下。
Figure PCTCN2015081047-appb-000014
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、430mg(3mmol)氯甲酸对硝基苄酯，室温搅拌反应4h，反应产物化学结构式9，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160mg的产物G5，三步反应产率36％。
G5核磁共振表征：
1H-NMR(DMSO-d6,400MHz)δ：11.11(s,1H),8.25(m,3H,Ph),7.68(d,2H,J＝8.64Hz,Ph),7.08(d,1H,J＝7.44Hz,H6),6.29(d,1H,J＝7.44Hz,H5),6.17(t,1H,J＝7.4Hz,H1'),5.33(s,2H,CH2-Ph),5.29(t,1H,J＝5.44Hz),4.19(m,1H,H5a'),3.66(m,1H,H5b'),3.61(m,1H,H4'),3.29(m,1H,H3')。
13C-NMR(DMSO-d6,100MHz)δ：163.93,153.47,147.86,144.38,128.97,126.72,126.19,124.26,123.62,95.57,84.82,83.99,81.77,71.91,69.13,68.42,66.06,62.34,59.52。
按照上述合成方法，能够合成各种具有不同取代基团的衍生物，例如G5-1和G5-2。
(实施例5)
本实施例的胞苷衍生物为5-溴-4-N-(正丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式11，代号G6)，反应式如下。
Figure PCTCN2015081047-appb-000015
按照实施例1的方法制得G2后，取1g(2.75mmol)G2溶于150mL二甲基甲酰胺中，搅拌下加入500mg(1.75mmol)二溴海因，得到的淡黄色溶液室温下搅拌反应1h，经LCMS检测反应完全，G2完全转化为G6。减压旋蒸除去溶剂，用乙氰浓缩，浓缩后粗产品用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到263mg的产物G6，自G1开始反应至获得G6，反应总产率51％。
G6核磁共振表征：
1H-NMR(MeOD-d4,400MHz)δ：8.62(s,1H,H5),6.18(t,1H,J＝6.52Hz,H1'),4.19(m,1H,H5a'),4.15(m,2H,H5b',H4'),3.95(m,2H,O-CH2-CH3),3.17(m,1H,H3'),1.36(m,2H,-O-CH2-CH2-CH2-CH3),1.31(m,2H,-O-CH2-CH2-CH2-CH3),0.98(m,2H,-O-CH2-CH2-CH2-CH3)。
13C-NMR(MeOD-d4,100MHz)δ：143.05,124.56,84.59,81.91,75.45,66.17,58.74,58.42,32.96,30.64,28.32,18.84,12.79,8.17.
ESIMS：calcd for C14H18BrF2N3O6m/z 442.03(M+H)+,found 442.02。
(实施例6)
本实施例的胞苷衍生物为5-溴-4-N-(苄氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式12，代号G7)，反应式如下(反应式中DMF为N，N-二甲基甲酰胺，下同)。
Figure PCTCN2015081047-appb-000016
按照实施例3的方法制得G4后，取1g(2.51mmol)G4溶于150mLN，N-二甲基甲酰胺中，搅拌下加入500mg(1.75mmol)二溴海因，得到的淡黄色溶液室温下搅拌反应1h，经LCMS检测反应完全，G4完全转化为G7。减压旋蒸除去溶剂，用乙氰浓缩，浓缩后粗产品用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到283mg的产物G7，自G1开始反应至获得G7，反应总产率32％。
G7核磁共振表征：
1H-NMR(MeOD-d4,400MHz)δ：8.60(s,1H,H5),7.45(m,2H,Ph),7.36(m,3H,Ph),6.18(t,1H,J＝6.52Hz,H1'),5.24(s,2H,CH2-Ph),4.33(m,1H,H5a'),4.0(m,2H,H5b',H4'),3.81(m,1H,H3')。
13C-NMR(MeOD-d4,100MHz)δ：143.23,135.93,128.37,128.27,125.22,122.63,120.05,85.41,85.08,84.76,81.85,68.77,68.53,68.31,67.93,58.76.
ESIMS：calcd for C17H16BrF2N3O6m/z 476.02(M+H)+,found 477.09。
同样的，按照实施例1的合成路线在氨基上进行取代后，按照实施例6的方法在5 位上实现Br取代，即可制得G7-1和G7-2。
按照上述制备方法，可制得R2为其他基团的衍生物。
(实施例7)
本实施例的胞苷衍生物为5'-O-[3,5-二硝基水杨酸酯]-4-N-(正丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式14，代号G8)。
反应式如下(反应式中(Boc)2O为二碳酸二叔丁酯，dioxane为1,4-二氧六环，DMAP为4-二甲氨基吡啶，EDCL为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，DCM为二氯甲烷，TFA为三氟乙酸，下同)：
Figure PCTCN2015081047-appb-000017
首先制备化合物13。取实施例1制备的60mg(0.16mmol)G2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G2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13，上述反应产率76％。
取上述制备的51mg(0.11mmol)的化合物13，与98mg(0.42mmol)3,5-二硝 基水杨酸、60mg(0.31mmol)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCL)混合后加入到15mL二氯甲烷中，再向二氯甲烷中加入2mg 4-二甲氨基吡啶(DMAP)，在24℃下搅拌反应24h，反应过程中薄层色谱TLC检测化合物13是否完全反应完毕。
反应结束后向反应后物料中加入50mL二氯甲烷，接着使用10mL水和20mL饱和食盐水依次洗涤，洗涤完毕使用无水硫酸钠干燥，浓缩至干。向浓缩后得到的物料中加入5mL三氟乙酸(TFA)，室温搅拌12h后浓缩至干。浓缩后用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到18mg的产物G8，由化合物13制得化合物14的反应产率为28％。
将3,5-二硝基水杨酸改变为其他酸，即可制备R3为
Figure PCTCN2015081047-appb-000018
其中X3为苯环、杂环、稠杂环、独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯、取代杂环、取代稠杂环；所述杂环包括咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪和哌啶；稠杂环包括喹啉和吲哚；X2为C1至C3的-(CH2)n-或C0至C3的-O-(CH2)n-的化合物。
(实施例8)
本实施例的胞苷衍生物为5'-O-[2-(4-硝基-1H-咪唑)乙酸酯]-4-N-(叔丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷(结构式16，代号G9)。
反应式如下：
Figure PCTCN2015081047-appb-000019
首先制备化合物15。取60mg(0.16mmol)实施例2制备的化合物6(G3)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G2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/乙醇(1∶1∶0.02)洗脱得到48mg的化合物15，上述反应产率64％。
取上述制备的51mg(0.11mmol)的化合物15，与98mg(0.57mmol)的2-(4-硝基-1H-咪唑)乙酸、60mg(0.31mmol)EDCL混合后加入到15mL二氯甲烷中，再向二氯甲烷中加入2mg DMAP，在24℃下搅拌反应24h，反应过程中薄层色谱TLC检测化合物15是否完全反应完毕。
反应结束后向反应后物料中加入50mL二氯甲烷，接着使用10mL水和20mL饱和食盐水依次洗涤，洗涤完毕使用无水硫酸钠干燥，浓缩至干。向浓缩后得到的物料中加入5mL三氟乙酸(TFA)，室温搅拌12h后浓缩至干。浓缩后用硅胶层析柱纯化，用二氯甲烷/甲醇(20∶1)洗脱得到18mg的产物G9，由化合物15制得化合物16的反应产率为31％。
(实施例9)
本实施例的胞苷衍生物的代号G10，反应式如下(反应式中DCC为N,N'-二环己基碳二亚胺)：
Figure PCTCN2015081047-appb-000020
首先准备化合物22，反应式如下(反应式中DMF为N,N-二甲基甲酰胺)：
Figure PCTCN2015081047-appb-000021
向反应瓶中加入化合物20即3,5,6-三氯-2-吡啶酚(5g,25.2mmol)，碳酸钾(7g,50.6mmol)和N,N-二甲基甲酰胺DMF(30ml)，搅拌15min。冷却到0℃，滴加溴乙酸乙酯(2.78ml,25.2mmol)。室温反应4h。加入水(15ml)，二氯甲烷提取(15ml×3)。有机相旋干，得到化合物21直接进行下一步反应。
将化合物21(5.4g,18.8mmol)加入54mL水中，加入氢氧化钠(0.864g,21.6mmol)，在80℃下搅拌4h。然后滴加浓盐酸调pH至1，过滤得产物化合物22待用，3.3g(70％)。
将化合物12即5-溴-4-N-(苄氧羰基)--2’-脱氧-2’，2’-二氟代胞苷(2g，4.21mmol)与碳酸钠(3.3g,31.1mmol)混合后加入到1,4-二氧六环和水的体系中(体积比4∶1,200mL)。加入(Boc)2O(1.8g,8.25mmol,Di-t-butyldicarbonate)，在25℃下搅拌反应48h，反应过程中TLC检测，待反应结束后加入20mL水稀释，使用2×100mL乙酸乙酯2次萃取，有机相使用50mL水和50mL饱和食盐水洗涤，使用无水硫酸钠干燥，柱层析(二氯甲烷/丙酮/甲醇，1∶1∶0.02)得到化合物17，产量690mg，产率29％。
ESIMS：calcd for C22H24BrF2N3O8m/z 576.07(M+H)+,found 576.16。
将化合物17(350mg，0.61mmol)与化合物22(186mg，0.73mmol)、DCC(250mg，1.21mmol，Dicydohexylcarbodiimide，N,N'-二环己基碳二亚胺)混合后加入到7mL二氯甲烷中，加入DMAP(15mg,0.12mmol，4-dimethylaminopyridine，4-二甲氨基吡啶)，反应在常温下搅拌过夜。TLC检测，待反应结束后加入5mL水稀释，使用2×20mL二氯甲烷萃取，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，柱层析(二氯甲烷/甲醇，45∶1)得到中间体18(260mg，产率70％)。该中间体直 接使用TFA(三氟乙酸，trifluoroacetic acid)的二氯甲烷溶液处理，直接得到产物G10(175mg，产率77％)。
G10核磁共振表征：
1H-NMR(CDCl3,400MHz)δ7.81(s,1H),7.75(s,1H),7.35(m,5H),6.24(m,1H),5.27(s,2H),5.22(s,2H),5.03(m,2H),4.67(m,1H),4.47(m,1H),4.30(s,2H)。
13C NMR(CDCl3,100MHz)δ167.80,162.56,157.21,155.57,143.28,140.91,128.90,128.78,124.55,123.44,118.57,117.47,72.61,68.68,64.94,63.83,63.37,62.84。
ESIMS：calcd for C24H18BrCl3F2N4O8m/z 712.93(M+H)+,found 712.99。
(实施例10)
本实施例的胞苷衍生物的代号G11。
首先制备化合物24，反应式如下：
Figure PCTCN2015081047-appb-000022
将2’-脱氧-2’，2’-二氟代胞苷(5g，16mmol)溶于乙酸(56mL)，35℃下搅拌2h；加入三氯甲烷(17mL)，0℃下搅拌15min。滴加三氯甲烷/乙酰氯(28ml/33ml)混合液，50℃下搅拌反应24h。旋干溶剂，加入甲醇(35ml)，旋干，得到产物23，直接进行下一步反应。
反应瓶加入碘单质(2.8g，11mmol)、碘酸(0.83g，4.7mmol)、乙酸(37.5mL)、四氯化碳(25.5mL)、水(25.5ml)和化合物23，40℃下搅拌反应24h。旋干溶剂，加入二氯甲烷和水。调节pH至6～7，有机相用硫代硫酸钠洗涤，水洗。合并有机相，无水硫酸钠干燥。过滤，滤液旋干。得到化合物24即产物5g待用(两步产率55％)。
ESIMS：calcd for C13H14F2N3O6m/z 473.99(M+H)+,found 474.14。
制备化合物G11的反应式如下：
Figure PCTCN2015081047-appb-000023
Figure PCTCN2015081047-appb-000024
将化合物24(2.5g,5.3mmol)、吡啶(1.24g,15.8mmol)溶于二氯甲烷(35mL)；0℃下滴加氯甲酸丁酯(2.15g,15.8mmol)，10℃下反应过夜。旋干溶剂，柱分离(二氯甲烷∶甲醇＝60∶1)得到1.9g(产率63％)化合物25。
ESIMS：calcd for C18H22F2N3O8m/z 574.04(M+H)+,found 574.14。
将化合物25(2.5g,4.3mmol)溶于甲醇(40mL)，搅拌5min，加入碳酸钾(2.1g,15.2mmol)，室温搅拌过夜，过滤，滤液旋干得1.5g(产率70.4％)化合物26。
ESIMS：calcd for C14H18F2N3O6m/z 490.02(M+H)+,found 490.07。
将化合物26(3.9g,8mmol)、碳酸钠(4.24g,40mmol)溶于1,4-二氧六环(22.5mL)和水(4.5mL)的混合液中，搅拌10min。加入二碳酸二叔丁酯(2.1g,9.6mmol)。室温反应至少24h。旋干溶剂，加入二氯甲烷(70mL)和水(100mL)，二氯甲烷提取(70mL×3)。有机相旋干。柱分离(二氯甲烷∶甲醇＝80∶1)得到2.8g(产率60％)化合物27。
ESIMS：calcd for C19H26F2IN3O8m/z 590.07(M+H)+,found 590.02.
将化合物27(50mg，0.08mmol)与化合物22(32.4mg，0.13mmol)、DCC(35mg，0.17mmol)混合后加入到2mL二氯甲烷中，加入DMAP(2mg,0.016mmol)，反应在常温下搅拌过夜。TLC检测，待反应结束后加入5mL水稀释，使用2×20mL二氯甲烷2次萃取，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，柱层析(二氯甲烷/甲醇，150∶1)得到中间体(50mg，产率71％)。该中间体直接使用TFA的二氯甲烷溶液处理，直接得到G11(35mg，产率80％)。
G11的表征如下：
ESIMS：calcd for C21H20Cl3F2IN4O8m/z 726.94(M+H)+,found 727.12。
1H-NMR(MeOD-d4,400MHz)δ8.06(s,1H),5.49(s,1H),5.07(m,3H),4.51(s,1H),4.48(m,1H),4.21(s,5H),1.69(m,3H),1.44(m,3H),0.97(m,3H)。
13C-NMR(MeOD-d4,100MHz)δ168.06,141.08,140.91,122.67,81.73,66.15,63.85,63.54,59.38,58.64,33.27,30.68,19.73,18.89,15.75,12.84,8.78。
(实施例11)
本实施例的胞苷衍生物的代号G12，反应式如下：
Figure PCTCN2015081047-appb-000025
将化合物27(300mg，0.51mmol)与2-(4-硝基-1H-咪唑)乙酸(105mg，0.61mmol)、DCC(210mg，1.02mmol)混合后加入到10mL二氯甲烷中，加入DMAP(9mg,0.073mmol)，反应在常温下搅拌过夜。TLC检测，待反应结束后加入20mL水稀释，使用3×30mL二氯甲烷3次萃取，有机相使用20mL水和20mL饱和食盐水洗涤，使用无水硫酸钠干燥，柱层析(二氯甲烷/甲醇，66∶1)得到中间体200mg(53％)，该中间体直接使用TFA的二氯甲烷溶液处理，直接得到70mg(40％)产物G12。
(实施例12)
本实施例的胞苷衍生物的代号G13。
首先制备化合物35。
Figure PCTCN2015081047-appb-000026
将化合物24(5g，0.01mol)、(Ph3)PdCl2(1.5g，2.14mmol)、CuI(1.0g，5.27mmol)加入预干燥的反应瓶中，N2保护下注入干燥的THF(100ml)，注入Me3SiC≡CH(5.2g，0.053mol)，注入TEA(15ml)。室温反应过夜。反应结束后减压蒸馏除去溶剂。使用硅胶层析柱(乙酸乙酯/石油醚：1∶1)纯化得到2.67g(产率57％)化合物30。
将化合物30(10g，1.1mmol)加入到MeOH(200ml)中，搅至全溶。在0℃ 下，冷却15分钟。加入K2CO3(10.92g，3.95mmol)，0℃下搅拌2小时。反应结束后先过滤除去碳酸钾，再减压蒸馏除去溶剂。使用硅胶层析柱(二氯甲烷/甲醇：10∶1)纯化得到5.46g(产率84.27％)化合物31。
将化合物61(5.46g,0.019mol)，CuSO4.5H2O(475mg,1.9mmol)，Vc.Na(1.13g,5.7mmol)，加入到THF(55mL)和水(49ml)中，N2保护下常温搅拌10分钟。然后注入
Figure PCTCN2015081047-appb-000027
N2保护下常温搅拌36小时。反应结束后减压蒸馏除去溶剂。使用硅胶层析柱(二氯甲烷/甲醇：15∶1)纯化得到4.88g(产率61.0％)化合物32。
化合物32：ESIMS：calcd for C18H18F2N6O4m/z 421.18(M+H)+,found 421.36。
Figure PCTCN2015081047-appb-000028
上述反应式中1,4-dioxane为1,4-二氧六环，HMDS为六甲基二硅氮烷。
将化合物32(2.0g，4.76mmol)、六甲基二硅氮烷HMDS(20mL)、(NH4)2SO4(50.3mg，0.38mmol)加入到1,4-二氧六环(20mL)中，在80℃下搅拌回流4小时；反应结束后减压蒸馏除去溶剂，加入甲苯5ml×2旋干带溶剂，油泵抽干。加入二氯甲烷(40ml)、吡啶(1.13g,14.3mmol)，降温至0℃，缓慢加入氯甲酸对硝基卞酯(3.08g,14.3mmol)，10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂。加入甲醇(40mL)，0℃冷却15分钟，滴加三乙胺(6.7ml)。10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：40∶1)纯化得到780mg(三步产率27.3％)化合物33。
化合物33：ESIMS：calcd for C26H23F2N7O8m/z 600.22(M+H)+,found 600.25。
Figure PCTCN2015081047-appb-000029
将化合物33(780mg,1.3mmol)、Na2CO3(690mg,6.5mmol)加入至1,4-二氧六环(20ml)和水(5ml)中，搅拌至溶解。常温下加入(Boc)2O(340.6mg,1.56mmol)。常温搅拌过夜；反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：80∶1)纯化得到366mg(产率40.2％)化合物34。
化合物34：ESIMS：calcd for C31H31F2N7O10m/z 700.28(M+H)+,found 700.08。
Figure PCTCN2015081047-appb-000030
将化合物34(250mg,0.36mmol)、化合物22(109mg,0.43mmol)、DCC(148mg,0.72mmol),DMAP(5mg)加入至二氯甲烷(12ml)中，常温搅拌过夜。反应结束后抽滤除去DCC，减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：100∶1)纯化得到210mg(产率60.0％)化合物35。
化合物35：ESIMS：calcd for C38H33Cl3F2N8O12m/z 937.28(M+H)+,found 937.28。
将化合物35(210mg,0.224mmol)加入至10ml的DCM/TFA＝5∶1体系中，常温搅拌4小时；反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：60∶1)纯化得到产物140mg(产率75.0％)G13。
ESIMS：calcd for C33H25Cl3F2N8O10m/z 837.17(M+H)+,found 837.27。
1H-NMR(CDCl3,400MHz)δ8.34(s,1H),8.23(m,3H),7.57(m,3H),7.33(m,3H),7.23(m,2H),6.38(m,1H),5.49(m,3H),5.30(s,3H),5.13(m,2H),4.73(m,1H),4.33(m,3H)。
Figure PCTCN2015081047-appb-000031
将现在市场上能够购买到的或者是已经合成的叠氮化合物代替
Figure PCTCN2015081047-appb-000032
进行反
Figure PCTCN2015081047-appb-000033
烷基、C1
至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph或取代-(CH2)n-Ph；其中取代烷基、取代烷氧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；-(CH2)n-Ph和取代-(CH2)n-Ph的n＝0、1、2、3～10；取代-(CH2)n-Ph的碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。
(实施例13)
本实施例的胞苷衍生物的代号G14。
首先制备化合物39。
Figure PCTCN2015081047-appb-000034
将化合物32(1.5g，3.57mmol)，HMDS(15mL),(NH4)2SO4(37.7mg，0.285mmol)加入到1,4-二氧六环(15mL)中，在80℃下搅拌回流4小时。反应结束后减压蒸馏除去溶剂，加入甲苯5ml×2旋干带溶剂，油泵抽干。加入二氯甲烷(30mL)、吡啶(0.846g,0.01mol)，降温至0℃慢加氯甲酸卞酯(1.83g,0.01mol)，10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂。加入甲醇40mL，0℃冷却15分钟，滴加三乙胺6mL。10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：40∶1)纯化得到570mg(产率28.8％)化合物37。
ESIMS：calcd for C25H24F2N6O6 m/z 555.24 (M+H)+, found 555.04。
将化合物37(520mg，0.939mmol)，Na2CO3(497.5mg,4.69mmol)加入至12 mL1,4-二氧六环和3mL水中，搅拌至溶解；常温下加入(Boc)2O(245.5mg，1.126mmol)，常温搅拌过夜。反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：80∶1)纯化得到278mg(产率45.3％)化合物38。
ESIMS：calcd for C31H32F2N6O8m/z 655.28(M+H)+,found 655.08。
Figure PCTCN2015081047-appb-000035
将化合物38(200mg,0.3mmol),化合物22(93.6mg,0.367mmol),DCC(126mg,0.612mmol),DMAP(10mg)加入至二氯甲烷(10ml)中，常温搅拌过夜。反应结束后抽滤除去DCC，减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：125∶1)纯化得到170mg(产率62.5％)化合物39。
ESIMS：calcd for C38H34Cl3F2N7O10m/z 914.14(M+Na)+,found 914.36。
Figure PCTCN2015081047-appb-000036
将化合物39(140mg,0.157mmol)加入至10mL的DCM/TFA＝5∶1体系中，常温搅拌4小时；反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：50∶1)纯化得到产物G14 95mg(产率76.43％)。
ESIMS：calcd for C33H26Cl3F2N7O8m/z 814.26(M+Na)+,found 814.61。
1H-NMR(CDCl3,400MHz)δ8.50(s,1H),8.36(s,1H),7.63(s,1H),7.33(m,9H),7.23(m,2H),6.38(m,1H),5.49(m,2H),5.30(s,4H),5.13(m,1H),4.80(m,1H),4.50(m,2H),4.30(m,1H).
13C-NMR(CDCl3,100MHz)δ168.61157.65155.68146.20143.22140.59128.87128.02123.07117.2468.1964.1154.4012.69。
Figure PCTCN2015081047-appb-000037
将现在市场上能够购买到的或者是已经合成的叠氮化合物代替
Figure PCTCN2015081047-appb-000038
进行反
Figure PCTCN2015081047-appb-000039
C1至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph或取代-(CH2)n-Ph；其中取代烷基、取代烷氧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；-(CH2)n-Ph和取代-(CH2)n-Ph的n＝0、1、2、3～10；取代-(CH2)n-Ph的碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。
(实施例14)
本实施例的胞苷衍生物的代号G15。
首先制备化合物43。
Figure PCTCN2015081047-appb-000040
将化合物32(1.3g，3.0mmol)、HMDS(13mL)、(NH4)2SO4(32.7mg，0.247mmol)加入到1,4-二氧六环(13mL)中，在80℃下搅拌回流4小时。反应结束后减压蒸馏除 去溶剂，加入甲苯5ml×2旋干带溶剂，油泵抽干。加入二氯甲烷(26ml)、吡啶(0.733g,9.3mmol)，降温至0℃慢加氯甲酸丁酯(1.268g,9.3mmmol)，10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂。加入甲醇(30ml)，0℃冷却15分钟，滴加三乙胺(4.5ml)。10℃下搅拌过夜。反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：40∶1)纯化得到850mg(产率52.8％)化合物41。
将化合物41(800mg,1.538mmol)、Na2CO3(815mg,7.69mmol)加入至25mL1,4-二氧六环和6mL水中，搅拌至溶解；常温下加入(Boc)2O(403mg,1.85mmol)，常温搅拌过夜。反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：80∶1)纯化得到458mg(产率48.1％)化合物42。
ESIMS：calcd for C28H34F2N6O8m/z 637.76(M+NH4)+,found 637.46。
Figure PCTCN2015081047-appb-000041
将化合物42(380mg,0.613mmol)、化合物22(187.5mg,0.736mmol)、DCC(252.5mg,1.226mmol)、DMAP(20mg)加入至二氯甲烷(20ml)中，常温搅拌过夜。反应结束后抽滤除去DCC，减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：125∶1)纯化得到333mg(产率63.5％)化合物43。
ESIMS：calcd for C35H36Cl3F2N7O10m/z 858.18(M+H)+,found 858.28。
Figure PCTCN2015081047-appb-000042
Figure PCTCN2015081047-appb-000043
将化合物43(310mg,0.362mmol)加入至12ml的DCM/TFA＝5∶1体系中，常温搅拌4小时；反应结束后减压蒸馏除去溶剂，使用硅胶层析柱(二氯甲烷/甲醇：50∶1)纯化得到235mg(产率85.8％)产物G15。
ESIMS：calcd for C30H28Cl3F2N7O8m/z 758.18(M+H)+,found 758.18。
1HNMR(CDCl3,400MHz)δ8.43(s,1H),8.33(s,1H),8.11(m,1H),7.70(m,1H),7.63(m,1H),7.34(m,4H),6.34(m,1H),5.53(m,3H),5.21(m,2H),4.76(m,1H),4.54(m,1H),4.46(m,1H),4.43(m,1H),4.20(m,2H),1.70(m,2H),1.41(m,2H),0.95(m,3H)。
将现在市场上能够购买到的或者是已经合成的叠氮化合物代替
Figure PCTCN2015081047-appb-000044
进行反
Figure PCTCN2015081047-appb-000045
C1至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph或取代-(CH2)n-Ph；其中取代烷基、取代烷氧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；-(CH2)n-Ph和取代-(CH2)n-Ph的n＝0、1、2、3～10；取代-(CH2)n-Ph的碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代。
(实施例15)
本实施例的胞苷衍生物的代号G16。
Figure PCTCN2015081047-appb-000046
将化合物11(2g,4.52mmol)、碳酸钠(3.4g)溶于133mL1,4-二氧六环和34mL水的混合液中，搅拌10min；加入二碳酸二叔丁酯(1.47g,6.78mmol)，室温反应至少48h。旋干溶剂，加入二氯甲烷(70mL)和水(100mL)，二氯甲烷提取(70ml×3)。有机相旋干。柱分离(二氯甲烷∶甲醇＝80∶1)得到1.2g(48.9％)化合物45。
ESIMS：calcd for C19H26BrF2N3O8m/z 542.09(M+H)+,found 542.11
Figure PCTCN2015081047-appb-000047
将Boc保护的化合物45(355mg，0.65mmol)与化合物22(499mg，1.95mmol)、DCC(401mg，1.95mmol)混合后加入到45mL二氯甲烷中，加入DMAP(2mg,0.016mmol)，反应在常温下搅拌过夜。TLC检测，待反应结束后加入5mL水稀释，使用2×20mL二氯甲烷萃取，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加入TFA处理，直接得到目标化合物G16(110mg，2步产率24％)。
1H-NMR(MeOD-d4,400MHz)δ：8.05(s,2H,Ar),6.22(t,1H,J＝7.6Hz,H1'),5.15(m,2H),4.67(m,1H,H5a'),4.48(m,4H,H5b',H4',O-CH2-CH3),1.70(m,2H,-O-CH2-CH2-CH2-CH3),1.28(m,2H,-O-CH2-CH2-CH2-CH3),0.97(m,2H,-O-CH2-CH2-CH2-CH3)。
13C-NMR(MeOD-d4,100MHz)δ：168.12,155.98,142.88,141.03,124.15,122.75, 117.32,82.73,78.37,77.97,63.76,63.55,62.67,41.82,31.04,30.63,29.98,18.88,18.84,12.81,8.29。
ESIMS：calcd for C21H20BrCl3F2N4O8m/z 681.94(M+2H)+,found 681.15。
(实施例16)
本实施例的胞苷衍生物的代号G17。
Figure PCTCN2015081047-appb-000048
将化合物10(911mg,1.75mmol)溶于50mL二甲基甲酰胺中，加入二溴海因(500mg,1.75mmol)，淡黄色溶液室温搅拌1h，LCMS检测反应完全。减压旋蒸除去溶剂，柱层析(二氯甲烷/甲醇，20∶1)，获得化合物47(483mg，总产率52.9％，白色固体)。
1H-NMR(CDCl3,400MHz)δ8.40(s,1H,Ar),8.23(d,J＝4Hz,2H,Ar),7.60(d,J＝4Hz,2H,Ar),6.15(m,1H,H1'),5.30(s,2H,Ar-CH2),4.50(m,1H,H5a'),4.48(m,4H,H5b',H4',O-CH2-CH3)。
将化合物47(2.33g,5.72mmol)，碳酸钠(4.12g)溶于133mL1,4-二氧六环和34mL水的混合液中，搅拌10min。加入二碳酸二叔丁酯(1.72g,7.9mmol)；室温反应至少48h。旋干溶剂，加入二氯甲烷(70ml)和水(100ml)，二氯甲烷提取(70ml×3)。有机相旋干。柱分离(二氯甲烷∶甲醇＝80∶1)得到1.4g(49％)化合物48。
Figure PCTCN2015081047-appb-000049
将Boc保护的化合物48(540mg，0.869mmol)与化合物22(667mg，2.60mmol)、DCC(537mg，2.60mmol)混合后加入到45mL二氯甲烷中，加DMAP(2mg,0.016mmol)，反应在常温下搅拌过夜。TLC检测，待反应结束后加入5mL水稀释，使用2×20mL二氯甲烷萃取，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加入TFA处理，直接得到目标化合物G17(115mg，2步产率17％)
1H-NMR(MeOD-d4,400MHz)δ：.05(s,2H,Ar),6.22(t,1H,J＝7.6Hz,H1'),5.15(m,2H),4.67(m,1H,H5a'),4.48(m,4H,H5b',H4',O-CH2-CH3),1.70(m,2H,-O-CH2-CH2-CH2-CH3),1.28(m,2H,-O-CH2-CH2-CH2-CH3),0.97(m,2H,-O-CH2-CH2-CH2-CH3)。
13C-NMR(MeOD-d4,100MHz)δ：67.81,157.47,155.55,142.93,141.00,128.98,128.42,123.99,117.54,79.92,76.89,76.55,66.69,65.80,63.78,62.54。
ESIMS：calcd for C24H17BrCl3F2N5O10m/z 760.92(M+2H)+,found 760.10。
(实施例17胞苷衍生物的盐酸盐)
本实施例制备实施例1的化合物4-N-(正丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷的盐酸盐。
取4-N-(正丁氧羰基)-2’-脱氧-2’，2’-二氟代胞苷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18、胞苷衍生物的注射用冻干粉针剂)
本实施例制备实施例13的化合物G14的冻干粉针剂。
G14的冻干粉针剂包括30g的化合物G14、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G14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19、胞苷衍生物的口服药物组合物)
本实施例的胞苷衍生物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、单次腹腔给予系列化合物在ICR小鼠的最大耐受量试验)
本实验是研究受试物单次腹腔给药对ICR小鼠的毒性反应，确定各受试物的最大耐受量(MTD)。最大耐受量(MTD)是指动物不会发生死亡，动物的体重降低不会超过10％(与Day 0相比)，或者不产生明显毒副作用的剂量。
1、受试物配置如下。
受试物溶解所用溶剂来源如下：
Figure PCTCN2015081047-appb-000050
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。
2、试验动物
品种和品系：ICR小鼠；级别：SPF；性别：雌性。
来源：上海斯莱克实验动物有限公司。
合格证号：0130749。
实验开始动物体重：18-20g。
数量和性别：41只，雌性。
饲养方式：六只一笼。
适应期：5～7天，与实验时相同饲养条件。
动物房环境温度18-26℃，相对湿度30-70％，12小时光照。实验动物在实验前适应5-7天。SPF大小鼠生长繁殖饲料Co60灭菌，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，动物自由饮食及饮水。
3、实验方法
给药方式：ip。如动物死亡则减低剂量，直至动物存活，如无动物死亡，则增加剂量；如在给给定高剂量下动物正常存活则实验结束。最终根据实验结果确定小鼠对受试物的MTD；急性给药后连续观察动物7天。
实验过程中所有动物，对所有的受试动物进行详细临床观察，给药后每天两次(上10：00、下午16：00各一次)，连续观察14天，观察包括但不仅限于：皮肤，毛，眼，耳，鼻，口腔，胸腔，腹部，外生殖器，四肢和脚，呼吸道及循环系统，自主效应(如流涎)，神经系统(如震颤，抽搐，应激反应以及反常行为)。
给药前称重动物的体重，随后每天在同一时间称量动物的体重并记录。
每天详细记录观察结果、动物体重以及给药一周后动物存活情况。
4试验结果
G3和G4在350mg/kg可以耐受，G5在300mg/kg可以耐受，G6在200mg/kg可以耐受，G7在200mg/kg可以耐受，G8在300mg/kg可以耐受，G10在400mg/kg可以耐受，G11在400mg/kg可以耐受，G12在400mg/kg可以耐受，G13在400mg/kg可以耐受，G15在400mg/kg可以耐受，G16在400mg/kg可以耐受。
(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G2至G17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100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：
Figure PCTCN2015081047-appb-000051
Figure PCTCN2015081047-appb-000052
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)临床观察和死亡率
G4 350mg/kg组在给药后第4天1只动物死亡，3只动物出现活动减少、体重降低、体表温度低于正常等临床症状。G8 350mg/kg组动物在给药后第4天全部死亡。G6 300mg/kg组在给药后第4天5只动物死亡。模型对照组和其余各化合物组(G3，G5，G7，G9，G10，G11，G12，G13，G15，G16)动物临床症状未出现明显异常。各组动物死亡率见表2(表中QD*1：单次腹腔注射)。
表2
Figure PCTCN2015081047-appb-000053
(2)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3。
表3-1G3至G9不同日期小鼠体重
Figure PCTCN2015081047-appb-000054
Figure PCTCN2015081047-appb-000055
*p<0.05,**p<0.01vs vehicle group
表3-2G10至G16不同日期小鼠体重
Figure PCTCN2015081047-appb-000056
Figure PCTCN2015081047-appb-000057
*p<0.05,**p<0.01vs vehicle group
表4-1G3至G9体重改变率
Figure PCTCN2015081047-appb-000058
*p<0.05,**p<0.01vs vehicle group
G10至G16体重改变率见表4-2
表4-2
Figure PCTCN2015081047-appb-000060
Figure PCTCN2015081047-appb-000061
*p<0.05,**p<0.01vs vehicle group
由上述图表数据，在化合物对结肠癌HCT-116荷瘤裸小鼠移植瘤的生长抑制作用中，G4 350mg/kg在给药第4天动物体重显著降低(p<0.05)，体重降低率平均为10.91±3.45％，其后体重稳定增长，在第18～20天与模型对照组比较体重显著升高(p<0.05)。G5 325mg/kg在给药4天动物体重显著降低(p<0.05)，体重降低率<10％，其后体重稳定增长，在第13～20天与模型对照组比较体重显著升高(p<0.05～0.01)。G7 250mg/kg在给药第4，6天动物体重显著降低(p<0.05)，体重降低率分别为12.28±4.78％和4.39±3.6％，其后体重稳定增长。其他给药组动物体重与模型对照比较无显著性差异。
(3)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5。
表5-1 G3-G9肿瘤体积
Figure PCTCN2015081047-appb-000062
Figure PCTCN2015081047-appb-000063
*p<0.05,**p<0.01vs vehicle group
表5-2 G10-G16肿瘤体积
Figure PCTCN2015081047-appb-000064
Figure PCTCN2015081047-appb-000065
*p<0.05,**p<0.01vs vehicle group
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具有明显的抑制作用。
(4)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6：
表6-1 G3-G9对人结肠癌HCT-116荷瘤小鼠肿瘤的生长抑制率
Figure PCTCN2015081047-appb-000066
Figure PCTCN2015081047-appb-000067
表6-2 G10-G16对人结肠癌HCT-116荷瘤小鼠肿瘤的生长抑制率
Figure PCTCN2015081047-appb-000068
Figure PCTCN2015081047-appb-000069
化合物G3 350mg/kg组肿瘤抑制率最大值在Day8，为58.10％，至Day22天约为46.82％。化合物G4 350mg/kg组肿瘤抑制率较好在Day11达到最大值92.58％，至Day22天仍维持在70％以上。化合物G5 325mg/kg组肿瘤抑制率较好在Day11达到最大值94.46％，至Day24天仍维持在70％以上。G9 325mg/kg组肿瘤抑制率最大值在Day4为80.77％，至Day22天约为40％。G7 250mg/kg组肿瘤抑制率最大值在Day8为82.62％，至Day22天抑制率为44.07％。
(5)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G3-G9对人结肠癌HCT-116荷瘤小鼠的肿瘤相对体积见如下表7-1：
表7-1
Figure PCTCN2015081047-appb-000070
Figure PCTCN2015081047-appb-000071
化合物G4 350mg/kg组与模型对照组比较肿瘤相对体积在Day 4到Day 18有显著性降低(p<0.05～0.01)。G5 325mg/kg组与模型对照组比较肿瘤相对体积在Day 4到Day 18有显著性降低(p<0.05～0.01)。G7 250mg/kg组与模型对照组比较肿瘤相对体积在Day 4到Day 15有显著性降低(p<0.05～0.01)。G9 325mg/kg组与模型对照组比较肿瘤相对体积仅在Day 4有显著性降低(p<0.05)。其他给药组与模型对照组比较肿瘤相对体积无显著性差异。
G10-G16对人结肠癌HCT-116荷瘤小鼠的肿瘤相对体积见如下表7-2：
表7-2
Figure PCTCN2015081047-appb-000072
Figure PCTCN2015081047-appb-000073
(6)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8：
表8-1G3-G9对人结肠癌HCT-116荷瘤小鼠的相对肿瘤增值率
Figure PCTCN2015081047-appb-000074
化合物G4 350mg/kg组相对肿瘤增殖率在Day 13达到最小值17.62％，至Day 22天肿瘤增殖率为75.38％。化合物G5 325mg/kg组相对肿瘤增殖率在Day 11达到最小值10.01％，至Day 22天肿瘤增殖率为68.77％。化合物G7 250mg/kg组相对肿瘤增殖 率在Day 8达到最小值17.67％，至Day 22天肿瘤增殖率为58.62％。
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G4，G5，G7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Day 8到Day 13有较好的肿瘤抑制作用，其中G5在Day 11相对肿瘤增殖率达到最小值10.01％，对动物体重的降低作用较小，平均体重降低率小于10％。
表8-2G10-G16对人结肠癌HCT-116荷瘤小鼠的相对肿瘤增值率
Figure PCTCN2015081047-appb-000075
基于上述实验的结果，能够确定本发明的新型胞苷衍生物提供了作为抗肿瘤剂的优异作用。
上述实施例对本发明进行示例性的说明，本领域技术人员能够理解可能存在各种变型且这些变型在本发明的范围内。

Claims (10)

  1. 一种新型胞苷衍生物，具有下述通式(Ⅰ)：
    Figure PCTCN2015081047-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；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H、卤素、或
    Figure PCTCN2015081047-appb-100002
    X1是C1至C10的烷基、C1至C10的取代烷基、C1至C10的烷氧基、C1至C10的取代烷氧基、C1至C6的烷基磺酰基、C1至C6的烷硫基、-(CH2)n-Ph、或者是取代-(CH2)n-Ph；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；所述的取代烷氧基，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-(CH2)n-Ph，其中n＝0、1、2、3～10；所述的取代-(CH2)n-Ph，其中n＝0、1、2、3～10，其碳链上或苯环上由一个或两个或三个H、卤素、氰基、硝基、氨基、羟基或羧基取代；
    R3是H或
    Figure PCTCN2015081047-appb-100003
    其中X3是苯环，杂环，稠杂环，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代杂环，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代稠杂环；所述杂环是咪唑、吡啶、呋喃、噻吩、噻唑、嘧啶、哌嗪或哌啶；所述稠杂环是喹啉或吲哚；X2是-(CH2)n-，其中n＝1、2、3，或者X2是-O-(CH2) n-，其中n＝0、1、2、3。
  2. 根据权利要求1所述的新型胞苷衍生物，其特征在于：R2为H。
  3. 根据权利要求1所述的新型胞苷衍生物，其特征在于：R2不为H，并且R3不为H。
  4. 根据权利要求3所述的新型胞苷衍生物，其特征在于：R2为卤素或
    Figure PCTCN2015081047-appb-100004
    X1是-(CH2)n-Ph，或者是取代-(CH2)n-Ph。
  5. 根据权利要求4所述的新型胞苷衍生物，其特征在于：R1是C1至C4的烷基、C1至C4的取代烷基、苄基，或者是取代苄基；R3的X3是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代咪唑，独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代吡啶，或者是独立地由一个或两个或三个卤素、氰基、硝基、氨基、羟基或羧基取代的取代苯环。
  6. 权利要求1所述的化合物或其盐在制备治疗肿瘤药物中的应用。
  7. 根据权利要求6所述的化合物在制备治疗肿瘤药物中的应用，其特征在于：所述肿瘤为血液肿瘤或恶性实体肿瘤。
  8. 根据权利要求6所述的化合物在药学上可接受的盐，其特征在于：所述的盐为盐酸盐、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。
  9. 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。
  10. 根据权利要求9所述的药物组合物，其特征在于：组合物的剂型为注射剂或者是口服剂型，其中注射剂为溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉末，口服剂型为片剂、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。
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CN109824746A (zh) * 2019-03-15 2019-05-31 杭州科兴生物化工有限公司 一种友霉素类化合物及其制备方法和应用
JP2020518657A (ja) * 2017-04-26 2020-06-25 カールマン，トーマス，アイ． 多標的ヌクレオシド誘導体

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JP2020518657A (ja) * 2017-04-26 2020-06-25 カールマン，トーマス，アイ． 多標的ヌクレオシド誘導体
JP7173613B2 (ja) 2017-04-26 2022-11-16 カールマン，トーマス，アイ． 多標的ヌクレオシド誘導体
CN109824746A (zh) * 2019-03-15 2019-05-31 杭州科兴生物化工有限公司 一种友霉素类化合物及其制备方法和应用

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