WO2018090979A1 - 一种具有抗癌作用的化合物及其制备方法和应用 - Google Patents

一种具有抗癌作用的化合物及其制备方法和应用 Download PDF

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WO2018090979A1
WO2018090979A1 PCT/CN2017/111666 CN2017111666W WO2018090979A1 WO 2018090979 A1 WO2018090979 A1 WO 2018090979A1 CN 2017111666 W CN2017111666 W CN 2017111666W WO 2018090979 A1 WO2018090979 A1 WO 2018090979A1
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cancer
group
compound
linifanib
alkyl
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PCT/CN2017/111666
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French (fr)
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龚兆龙
林毅晖
李风庆
唐方强
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思路迪（北京）医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a compound and a preparation method and application thereof, in particular to a compound which can be selectively converted into a stronger anticancer activity in vivo, a preparation method and application thereof.
  • Linifanib is a multi-target anti-cancer compound whose target is mostly angiogenesis-related kinase, which has good inhibitory effects on VEGFRs, PDGFRs, CSF-1R and Flt-1/3.
  • THP time to progression, time to response rate
  • ORR overall response rate
  • the present application binds Linifanib or a derivative thereof to a polypeptide through a multi-carbon chain to form a compound Linifanib-Cx-AAy (ie, a compound of the formula I), using PSMA (Prostate-Specific Membrane Antigen, prostate specific) High expression of membrane antigen in tumor endothelial cells and part of tumor cells of solid tumors, specifically degrading Linifanib-Cx-AAy at the tumor site to form active anticancer compound Linifanib or its derivatives, thereby specificity at the tumor site Enrichment of anticancer compounds while reducing systemic toxicity.
  • PSMA Prostate-Specific Membrane Antigen, prostate specific
  • the present application provides a compound having the structure of Formula I, a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof:
  • A is selected from (CH 2 ) e N(R 7 )C(O)N(R 8 )(CH 2 ) f and CH 2 C(O)NR 7 , wherein e and f are independently 0 or 1, wherein each group a ring in which the group is replaced by R 3 and R 4 at its left end;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkoxy, alkoxyalkoxy, alkoxyalkyl, alkyl, aryloxy, aryloxyalkyl, halo, haloalkoxy, haloalkyl, Heterocyclyl, heterocyclylalkenyl, heterocyclylalkoxy, heterocyclylalkyl, heterocyclyloxyalkyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, (NR a R b ) alkoxy a group, (NR a R b )alkenyl, (NR a R b )alkyl, (NR a R b )carbonylalkenyl, and (NR a R b )carbonylalkyl;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen, alkoxy, alkyl, halo, haloalkoxy, haloalkyl and hydroxy;
  • R 5 and R 6 are independently selected from the group consisting of hydrogen, alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, aryloxy, arylalkyl, carboxyl, cyano, halo, haloalkoxy, haloalkane Base, hydroxyl, hydroxyalkyl, nitro and -NR c R d ;
  • R 7 and R 8 are independently selected from the group consisting of hydrogen and alkyl
  • R 9 is selected from the group consisting of hydrogen, hydroxy, amino, alkenyl, alkynyl, alkoxy, alkylamino, alkoxyalkyl, alkyl, alkoxycarbonyl, aryl, heterocycloalkyl;
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkoxy, aryloxy, alkenyloxy, nitro, halo, primary, secondary and tertiary amine;
  • R a and R b are independently selected from the group consisting of hydrogen, alkyl, alkylcarbonyl, alkylsulfonyl, arylsulfonyl, haloalkylsulfonyl and heterocyclylsulfonyl;
  • R c and R d are independently selected from the group consisting of hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl.
  • each compound is as follows:
  • the polypeptide (reactant 1) and the benzyl protected L (reactant 2) are reacted in the presence of a catalyst and a condensing agent to obtain a protected group-containing intermediate compound 1, which is an intermediate Compound 1 is further subjected to catalytic hydrogenation in a polar solvent to remove the protecting group to obtain intermediate compound 2;
  • the intermediate compound 2 is reacted with Linifanib or a derivative thereof in the presence of a catalyst and a condensing agent to obtain a protecting group-containing intermediate compound 3, which further removes the protecting group under acidic conditions.
  • a compound of formula I a compound of formula I.
  • the reaction temperature is carried out at -20 ° C to 125 ° C;
  • the organic solvent is selected from the group consisting of ethers, alcohols, alkanes, and aromatics having 1 to 20 carbon atoms.
  • the catalyst is 1-hydroxybenzotriazole (HOBT);
  • the condensing agent is 1-ethyl-3-(3-dimethylamine C Any one or more of carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) or 4-dimethylaminopyridine (DMAP).
  • reaction molar ratio of the reactants 1 and 2 in the reaction is 1:1 to 1:10, and the molar ratio of the reactant 1 to the condensing agent is 1:0.1 to 1:10; the molar ratio of the reactant 1 to the catalyst is 1:0.1 to 1:10.
  • the reaction temperature is carried out at -20 ° C to 250 ° C;
  • the organic solvent is selected from the group consisting of ethers, alcohols, alkyl halides, amides, nitriles having 1 to 20 carbon atoms or a mixture thereof, or a mixture with water in various ratios;
  • the catalyst is palladium carbon, palladium hydroxide is dry or wet.
  • the molar ratio of the intermediate compound 2 to the catalyst is from 1:0.1 to 1:10.
  • the above reaction temperature is carried out at -20 ° C to 125 ° C;
  • the organic solvent is selected from the group consisting of ethers, alcohols, alkanes, aromatic hydrocarbons, ketones having 1 to 20 carbon atoms.
  • the catalyst is 1-hydroxybenzotriazole (HOBT);
  • the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Any one or more of amine hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) or 4-dimethylaminopyridine (DMAP).
  • EDCI amine hydrochloride
  • DCC 1,3-dicyclohexylcarbodiimide
  • DMAP 4-dimethylaminopyridine
  • the reaction temperature is carried out at -20 ° C to 125 ° C;
  • the organic solvent is an ether, an alcohol, an alkane, an aromatic hydrocarbon, a ketone or an alkyl halide having 1 to 20 carbon atoms. , an amide, a nitrile, an ester or a mixture thereof in various ratios;
  • the acidic reagent is formic acid, acetic acid, trifluoroacetic acid.
  • the molar ratio of the intermediate compound 3 to the acidic reagent is from 1:1 to 1:10.
  • the present application provides a pharmaceutical composition comprising a compound of the above formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph thereof, and pharmaceutically acceptable according to the present application.
  • the pharmaceutical compositions include, but are not limited to, oral dosage forms, parenteral dosage forms, topical dosage forms, and rectal administration dosage forms.
  • the pharmaceutical composition may be an oral tablet, a capsule, a pill, a powder, a sustained release preparation, a solution, and a suspension. Sterile solutions, suspensions or emulsions for parenteral injection, ointments or creams for topical use, or suppositories for rectal administration.
  • the pharmaceutical composition and the at least one therapeutic agent are each combined in a separate dosage form into a combined product, such as a kit.
  • the application provides the use of a compound of formula I, a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph thereof, for the manufacture of a medicament having anticancer effects.
  • the cancer includes esophageal cancer, endometrial cancer, malignant lymphoma, multiple myeloma, gastrointestinal stromal tumor, colon cancer, rectal cancer, breast cancer, liver cancer, stomach cancer, ovarian cancer, uterine cancer, cervical cancer, Vaginal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer, melanoma, etc.
  • the effect is optimal for liver cancer.
  • the present application provides a method of treating cancer comprising administering a therapeutically effective amount of a compound of formula I, a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph thereof
  • a method of treating cancer comprising administering a therapeutically effective amount of a compound of formula I, a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph thereof
  • the cancer comprises esophageal cancer, endometrial cancer, malignant lymphoma, multiple myeloma, gastrointestinal stromal tumor, colon cancer, rectal cancer, breast cancer, liver cancer, gastric cancer, ovarian cancer, Uterine cancer, cervical cancer, vaginal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer, melanoma, etc.
  • the effect is optimal for liver cancer.
  • solvate refers to a combination of a compound of the present application and a solvent molecule formed by solvation. Such as hydrates, ethanol solvates, methanol solvates, and the like.
  • polymorph or “polymorph” refers to a compound of the present application that exists in a different lattice form.
  • stereoisomer refers to isomers resulting from the different arrangement of atoms in a molecule in space.
  • pharmaceutical composition refers to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical component including, but not limited to, carriers, stabilizers, dilutions. Agents, dispersing agents, suspending agents, thickening agents and/or excipients.
  • carrier refers to a relatively non-toxic chemical agent that facilitates the introduction of a compound into a cell or tissue.
  • alkyl refers to a straight or branched saturated hydrocarbon chain containing from 1 to 10 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, Sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethyl Amyl, n-heptyl, n-octyl, n-decyl and n-decyl.
  • the "aryl group” means an aromatic carbocyclic group having 6 to 14 carbon ring atoms.
  • the aryl group can be monocyclic or polycyclic. In the case of a polycyclic aromatic ring, only one of the polycyclic systems needs to be unsaturated, while the remaining one or more rings may be saturated, partially saturated or unsaturated.
  • Examples of the aryl group include a phenyl group, a naphthyl group, an anthracenyl group, an indanyl group, and a tetrahydronaphthyl group.
  • heteroaryl refers to a five- or six-membered aromatic ring having at least one carbon atom and one or more independently selected nitrogen, oxygen or sulfur atoms.
  • the “heteroaryl group” means an aromatic heterocyclic group having 5 to 14 ring atoms.
  • the heteroaryl group can be a single ring or two or three fused rings.
  • heteroaryl substituents include: 6-membered ring substituents such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and 1,3,5-, 1,2,4- or 1,2,3- Triazinyl; 5-membered ring substituent such as imidazolyl, furyl, thienyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1,3,4-oxadiazolyl and isothiazolyl; 6/5-membered fused ring substituents such as benzothienyl, benzisoxazolyl, benzoxazole Base, imidazolyl, fluorenyl, benzimidazolyl, pyrrolo[2,3-b]pyridinyl, fluorenyl; and 6/6-membered fused ring, such as benzopyranyl
  • cycloalkenyl refers to a monocyclic or bridged hydrocarbon ring system.
  • the monocyclic cycloalkenyl group has 4, 5, 6, 7 or 8 carbon atoms and 0 heteroatoms.
  • the four-membered ring system has one double bond, and the five- or six-membered ring system has one or two double bonds, and the seven- or eight-membered ring system has one, two or three double bonds.
  • Representative examples of monocyclic cycloalkenyl include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • heterocycloalkyl group means a saturated ring structure containing a total of 3 to 14 ring atoms. At least one of the ring atoms is a hetero atom (i.e., oxygen, nitrogen or sulfur), and the remaining ring atoms are independently selected from the group consisting of carbon, oxygen, nitrogen and sulfur.
  • an “effective amount” of an ingredient or formulation unit refers to an amount of the ingredient that is effective to provide the desired effect when used in combination with other ingredients.
  • the “effective amount” will vary from subject to subject, depending on the age and general condition of the individual, the particular active drug, and the like. Thus, it is not always possible to refer to an accurate “effective amount”, however, a suitable “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimental methods.
  • subject can refer to a patient or other animal, particularly a mammal, such as a human, a dog, a monkey, that receives a compound or pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or alleviate the disease of the invention. Cows, horses, etc.
  • Linifanib or its derivative is reacted with Boc-protected L (Reactant 3) under the conditions of a condensing agent and a catalyst to form an intermediate compound Ma, which is removed from the Boc protection intermediate under the action of trifluoroacetic acid.
  • Compound Mb the intermediate compound Mb is further condensed with a protecting group of aspartic acid to obtain an intermediate compound Mc, and the intermediate compound Mc is removed under the trifluoroacetic acid condition to remove the Boc protection to form an intermediate compound Md, Md in a noble metal catalyst condition
  • the lower catalytic hydrogenation of the benzyl group gives the metabolite of the compound of formula 2.
  • A is selected from (CH 2 ) e N(R 7 )C(O)N(R 8 )(CH 2 ) f and CH 2 C(O)NR 7 , wherein e and f are independently 0 or 1, wherein each group a ring in which the group is replaced by R 3 and R 4 at its left end;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkoxy, alkoxyalkoxy, alkoxyalkyl, alkyl, aryloxy, aryloxyalkyl, halo, haloalkoxy, haloalkyl, Heterocyclyl, heterocyclylalkenyl, heterocyclylalkoxy, heterocyclylalkyl, heterocyclyloxyalkyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, (NR a R b ) alkoxy a group, (NR a R b )alkenyl, (NR a R b )alkyl, (NR a R b )carbonylalkenyl, and (NR a R b )carbonylalkyl;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen, alkoxy, alkyl, halo, haloalkoxy, haloalkyl and hydroxy;
  • R 5 and R 6 are independently selected from the group consisting of hydrogen, alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, aryloxy, arylalkyl, carboxyl, cyano, halo, haloalkoxy, haloalkane Base, hydroxyl, hydroxyalkyl, nitro and -NR c R d ;
  • R 7 and R 8 are independently selected from the group consisting of hydrogen and alkyl
  • R 10 is selected from the group consisting of hydrogen, alkyl, alkoxy, aryloxy, alkenyloxy, nitro, halo, primary, secondary and tertiary amine;
  • R a and R b are independently selected from the group consisting of hydrogen, alkyl, alkylcarbonyl, alkylsulfonyl, arylsulfonyl, haloalkylsulfonyl and heterocyclylsulfonyl;
  • R c and R d are independently selected from the group consisting of hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl.
  • each metabolite is as follows:
  • the present invention attempts to structurally link carbon chains and amino acids at multiple sites of Linifanib, but the results are not satisfactory, most of the compounds are not synthesized successfully, the yield is very low, and some compounds are successfully synthesized, It does not act to block the activity of Linifanib, or it cannot be stably present in plasma.
  • only one product of the ligation is not only high in yield, but also can successfully block the activity of Linifanib, and can be biologically converted into the active drug Linifanib in vitro (in liver homogenate and spleen homogenate) to inhibit tumor cells at a lower dose, especially It is the proliferation of liver cancer cells.
  • the reaction solution was diluted with 100 ml of dichloromethane, and washed twice with 250 ml of deionized water, and the organic phase was separated.
  • the intermediate compound prepared in Example 5 was weighed in 595 mg (0.63 mmol) dissolved in 20 ml of dichloromethane, and the reaction temperature was -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added thereto, and the reaction was stirred at the reaction temperature.
  • the reaction solution was diluted with 40 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • the intermediate compound 3 prepared in Example 9 was weighed in 585 mg (0.63 mmol), dissolved in 20 ml of dichloromethane, and the reaction temperature was -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added thereto, and the reaction temperature was maintained at 20 ° C.
  • the reaction solution was diluted with 40 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • Benzyl-(12-amino)dodecanoate hydrochloride 404 mg (1.18 mmol), HOBT 238 mg (1.76 mmol), and EDCI 192 mg (1.76 mmol) were weighed and dissolved in dichloromethane (250 ml).
  • Control reaction temperature 20 ⁇ 40 ° C slowly add Asp (Boc)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)- (OtBu) 1267mg (1.23mmol), add, complete the reaction temperature
  • the intermediate compound 3 prepared in Example 13 was weighed and dissolved in 60 ml of dichloromethane, and the reaction temperature was -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added thereto, and the reaction temperature was maintained at 20 ° C.
  • the reaction solution was diluted with 40 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • the organic phase was separated and the organic phase dried over anhydrous sodium sulfate.
  • reaction mixture was diluted with 50 ml of methylene chloride and washed twice with 100 ml of deionized water, and the organic phase was dried over anhydrous sodium sulfate.
  • the reaction solution was diluted with 50 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • the organic phase was separated and the organic phase dried over anhydrous sodium sulfate.
  • the desiccant was filtered off and the filtrate was concentrated to a yellow oil.
  • the oil was chromatographed to give a yellow oil (yield: 71.
  • the reaction mixture was diluted with 50 ml of methylene chloride and washed twice with 100 ml of deionized water, and the organic phase was dried over anhydrous sodium sulfate. The desiccant was filtered off and the filtrate was concentrated at low temperature to give a brown oil. The oil was subjected to silica gel column chromatography (DCM:MeOH:EtOAc:
  • the intermediate Ma 194 mg (0.33 mmol) prepared in Example 18 was weighed and dissolved in 20 ml of dichloromethane, and the reaction temperature was -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added thereto, and the reaction temperature was maintained at 1.5 to maintain the reaction temperature.
  • the reaction solution was diluted with 50 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • reaction mixture was diluted with 50 ml of methylene chloride and washed twice with 100 ml of deionized water, and the organic phase was dried over anhydrous sodium sulfate.
  • the intermediate Md prepared in Example 21 was weighed 425 mg (0.61 mmol), dissolved in 30 ml of anhydrous methanol, and added with 10% Pd/C 25 mg under nitrogen atmosphere, replaced with hydrogen for 3 times, and controlled under a hydrogen atmosphere at 2 MPa.
  • the reaction solution was filtered under nitrogen atmosphere to recover palladium carbon.
  • the filtrate was concentrated at low temperature to give a pale yellow oil.
  • the oil was subjected to preparative chromatography to give a white solid powder (yield: 234 mg, 63.4%).
  • reaction mixture was diluted with 50 ml of methylene chloride and washed twice with 100 ml of deionized water, and the organic phase was dried over anhydrous sodium sulfate.
  • the desiccant was filtered off and the filtrate was concentrated at low temperature to give a brown oil. This oil was subjected to silica gel column chromatography (DCM:MeOH:EtOAc:EtOAc:
  • the intermediate Ma 225 mg (0.33 mmol) prepared in Example 23 was weighed and dissolved in 20 ml of dichloromethane, and the reaction temperature was -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added thereto, and the reaction temperature was maintained at 1.5 to maintain the reaction temperature.
  • Into the reaction solution It was diluted with 50 ml of dichloromethane, washed twice with 120 ml of deionized water, twice with 60 ml of 5% sodium hydrogen carbonate solution, and twice with 120 ml of deionized water.
  • reaction mixture was diluted with 50 ml of methylene chloride and washed twice with 100 ml of deionized water, and the organic phase was dried over anhydrous sodium sulfate.
  • the present application determines the half-inhibition of six compounds (compounds 1, 4, 7, 8, 12, and Linifanib) on 54 commercial tumor cell lines (including 26 liver cancer cell lines) by cell proliferation assay (Alamar Blue assay platform). Concentration (IC 50 value), the difference between the activity of the five compounds and the active drug Linifanib was examined.
  • Cell test 54 cell lines in logarithmic growth phase were seeded in 96-well culture plates at 500 to 1 ⁇ 10 4 /well (the optimal seeding density of each cell line in the pre-experiment). After incubating at 37 ° C for 4 h in a humidified incubator of % CO 2 , 10 ⁇ L of compound 1, 4, 7, 8, 12 and Linifanib were added to each well, and 9 drug concentration gradients were tested for each compound (from the highest test concentration 3.16 times) Gradient dilution), starting concentrations of 30 or 100 ⁇ M, respectively, depending on the solubility of each compound.
  • the QC reference compound Doxorubicin was added simultaneously to each cell line test, and the final drug concentrations were 10, 3.16, 1, 0.31, 0.1, 0.03, 0.01, 0.003, and 0.001 ⁇ M, respectively.
  • a positive control group (100% inhibition) and a negative control group (0% inhibition) were set at the same time.
  • the drug group was repeated for 2 wells per concentration, and the positive control group and the negative control group were repeated for 6 wells. After the culture was continued for 6 days in the incubator, follow-up AlamarBlue test operation;
  • Cell line 29-54 in the table is the response of liver cancer cell lines to each compound.
  • liver cancer cell lines were liver cancer cell lines, and nearly half of the liver cancer cell lines (12/26, 46%) were moderately sensitive to Linifanib, IC 50 ⁇ 5 ⁇ M, and most of the liver cancer cell lines (15) /26,58%)
  • the IC 50 difference for the precursors Linifanib-C 12 -AA 5 and for Linifanib was more than 8 times, and almost all liver cancer cells did not respond to the intermediates, see Table 1.
  • the purpose of this example was to investigate the stability of the precursor Linifanib-C 12 -AA 5 (compound 8), intermediate Linifanib-C 12 -Asp (compound 12) in plasma, liver homogenate, and spleen homogenate (precursor Linifanib-C 12 -AA 5 metabolism intermediates Linifanib-C 12 -Asp and Linifanib; intermediate Linifanib-C 12 -Asp further metabolizes Linifanib), quantitative analysis of metabolites, and verification of incubation system with positive drugs Stability, for the reference of compound drug evaluation.
  • MATERIALS Male SD rats (200-250g), Beijing Vitallihua
  • Test sample positive drug and its metabolites M1 and M2, Linifanib-C 12 -AA 5 and its metabolites Linifanib-C 12 -Asp and Linifanib.
  • Sex male; weight: 200-250g;
  • Bioanalytical method establishing a biological analysis method of the compound Linifanib-C 12 -Asp and Linifanib;
  • Rat liver/spleen was taken on the day of the experiment, 2 pieces were chopped and mixed, and homogenized with 4 volumes (1 g: 4 mL) of phosphate buffer (pH 7.4), the temperature of the homogenization process was controlled to not exceed 10 ° C; protein quantitative detection was performed. Control liver and spleen protein concentrations.
  • Positive drugs are metabolized in plasma to their metabolites as expected, indicating that the plasma system is stable and the subsequent test results are reliable.
  • Linifanib-C 12 -AA 5 was stable in plasma and did not produce intermediate LC 12 -ASP and Linifanib; the intermediate Linifanib-C 12 -Asp was stably incubated in plasma, and the level of Linifanib produced was close to 0. .
  • the positive drug was metabolized to its metabolite in the liver homogenate as expected, and the liver homogenate system was stable, and the subsequent detection results were reliable.
  • liver homogenate stability of precursors and intermediates are shown in Table 3-4 and Figure 1-2.
  • the intermediate Linifanib-C 12 -Asp produced by the precursor Linifanib-C 12 -AA 5 can be rapidly converted into Linifanib in liver homogenate, so the intermediate in the liver homogenate The accumulation concentration is low.
  • the positive drug was metabolized into its metabolite in the spleen homogenate as expected, and the spleen homogenate system was stable, and the subsequent detection results were reliable.
  • the metabolism of the precursor in the spleen homogenate is also the same as in the liver homogenate, and both are intermediates, and the intermediate is metabolized to Linifanib.
  • the accumulation concentration of the intermediate in the spleen homogenate is higher, and the amount of the produced Linifanib is less than that in the liver homogenate.

Abstract

本发明提供一种式I结构的化合物及其制备方法和治疗癌症的用途。本发明化合物对多种癌细胞具有抑制作用，可在体外(肝匀浆和脾匀浆中)经生物学转化为活性药物Linifanib，以更低剂量抑制肿瘤细胞尤其是肝癌细胞的增殖。

Description

一种具有抗癌作用的化合物及其制备方法和应用 技术领域
本发明涉及一种化合物及其制备方法和应用，具体涉及一种在体内选择性转化为更强抗癌活性的化合物及其制备方法和应用。
背景技术
利用抗肿瘤药物选择性地杀伤肿瘤细胞而对正常细胞毒性较小，一直是肿瘤治疗中的难题。近年来兴起的靶向治疗主要针对肿瘤细胞中特定靶点的变异，为肿瘤患者带来了福音。但是靶向治疗也存在受益患者群体小以及用药后迅速耐药等诸多限制。这就迫使生物医药研发必需另辟蹊径，来为更多的患者提供新的治疗方案。
Linifanib是一个多靶点的抗癌化合物，其靶点多为血管新生相关的激酶，对VEGFRs，PDGFRs，CSF-1R和Flt-1/3均有较好的抑制作用。在针对肝癌的大型随机III期临床试验中发现，Linifanib对肝癌患者的TTP(time to progression，疾病进展时间)及ORR(overall response rate，总体应答率)均明显优于肝癌唯一批准的靶向药Sorafenib(TTP 5.4月vs 4.0月，ORR 13.0％vs 6.9％)，但是其毒副作用也大于Sorafenib，因此其总体药效并不强于Sorafenib，因而没有通过FDA的审批(J Clin Oncol，2014，33，172-179)。
发明内容
为解决上述问题，本申请将Linifanib或其衍生物通过多碳链与多肽连接在一起，形成化合物Linifanib-Cx-AAy(即本申请式I化合物)，利用PSMA(Prostate-Specific Membrane Antigen，前列腺特异膜抗原)在实体瘤的肿瘤内皮细胞及部分肿瘤细胞中的高表达，在肿瘤部位特异性地降解Linifanib-Cx-AAy形成有活性的抗癌化合物Linifanib或其衍生物，从而在肿瘤部位特异性地富集抗癌化合物同时降低其全身毒性。
作为本申请的一个方面，本申请提供一种具有式I结构的化合物、其药学上可接受的盐、立体异构体、溶剂化物或多晶形物：
Figure PCTCN2017111666-appb-000001
其中，
A选自(CH2)eN(R7)C(O)N(R8)(CH2)f和CH2C(O)NR7，其中e和f独立为0或1，其中各个基团由其左端连接R3和R4取代的环；
L为-[Cm(O)(Z)n(NH)q]-，其中m,q分别为0或1，n为0～11，p为0～8；Z选自-CR10-，-CR10-O-CR10-，-S-S-，-CR10＝CR10-，-CR10≡CR10-，-Ar，-CO-NH-和-N＝CR10-中的任意一种基团或几种基团以常规方式相连接；
R1和R2独立选自氢、烷氧基、烷氧基烷氧基、烷氧基烷基、烷基、芳氧基、芳氧基烷基、卤基、卤代烷氧基、卤代烷基、杂环基、杂环基烯基、杂环基烷氧基、杂环基烷基、杂环基氧基烷基、羟基、羟基烷氧基、羟基烷基、(NRaRb)烷氧基、(NRaRb)烯基、(NRaRb)烷基、(NRaRb)羰基烯基和(NRaRb)羰基烷基；
R3和R4独立选自氢、烷氧基、烷基、卤基、卤代烷氧基、卤代烷基和羟基；
R5和R6独立选自氢、烷氧基、烷氧基烷基、烷氧基羰基、烷基、芳氧基、芳基烷基、羧基、氰基、卤基、卤代烷氧基、卤代烷基、羟基、羟基烷基、硝基和-NRcRd；
R7和R8独立选自氢和烷基；
R9选自氢、羟基，氨基，烯基、炔基，烷氧基，烷胺基，烷氧基烷基、烷基、烷氧基羰基、芳基、杂环烷基；
R10选自氢，烷基、烷氧基、芳氧基、链烯氧基、硝基、卤基、伯胺基、仲胺基和叔胺基；
Ra和Rb独立选自氢、烷基、烷基羰基、烷基磺酰基、芳基磺酰基、卤代烷基磺酰基和杂环基磺酰基；
Rc和Rd独立选自氢、烷基、烷基羰基、芳基、芳基烷基、环烷基、环烷基烷基、杂环基和杂环基烷基。
具体的，各化合物结构如下：
Figure PCTCN2017111666-appb-000002
Figure PCTCN2017111666-appb-000003
反应路线：
首先将多肽(反应物1)与带苄基保护的L(反应物2)在催化剂和缩合剂存在的条件下进行反应，得到带保护基团的中间体化合物1，该中间体 化合物1进一步在极性溶剂中进行催化氢化脱除保护基团得到中间体化合物2；
将中间体化合物2与Linifanib或其衍生物在催化剂和缩合剂存在的条件下进行反应，得到带保护基团的中间体化合物3，该中间体化合物3进一步在酸性条件下脱除保护基团得到式I化合物。
反应路线图：
Figure PCTCN2017111666-appb-000004
进一步，在上述制备中间体化合物1方法中，所述反应温度在-20℃至125℃下进行；所述有机溶剂选自含有1-20个碳原子的醚、醇、烷烃、芳 香烃、酮、卤代烷、酰胺、腈、酯或者其混合物；所述催化剂为1-羟基苯并三唑(HOBT)；所述缩合剂为1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐(EDCI)、1,3-二环己基碳二亚胺(DCC)或4-二甲氨基吡啶(DMAP)中的任意一种或几种。该步骤中，反应中反应物1、2反应摩尔比为1:1～1:10，反应物1与缩合剂的摩尔比为1:0.1～1:10；反应物1与催化剂的摩尔比为1:0.1～1:10。
进一步，在上述制备中间体化合物2方法中，所述反应温度在-20℃至250℃下进行；所述有机溶剂选自含有1-20个碳原子的醚、醇、卤代烷、酰胺、腈或者其混合物，或与水各种比例的混合物；所述催化剂为钯碳，氢氧化钯干性或湿性。上述制备方法中，中间体化合物2与催化剂反应摩尔比为1:0.1～1:10。
进一步，在上述制备中间体化合物3方法中，上述反应温度在-20℃至125℃下进行；所述有机溶剂选自含有1-20个碳原子的醚、醇、烷烃、芳香烃、酮、卤代烷、酰胺、腈、酯或其混合物；所述催化剂为1-羟基苯并三唑(HOBT)；所述缩合剂为1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐(EDCI)、1,3-二环己基碳二亚胺(DCC)或4-二甲氨基吡啶(DMAP)中的任意一种或几种。该步骤中，Linifanib或其衍生物与中间体化合物2摩尔比为1:1～1:10，Linifanib或其衍生物与缩合剂的摩尔比为1:0.1～1:10；与催化剂的摩尔比为1:0.1～1:10。
进一步，在上述制备式1化合物方法中，所述反应温度在-20℃至125℃下进行；所述有机溶剂是含有1-20个碳原子的醚、醇、烷烃、芳香烃、酮、卤代烷、酰胺、腈、酯或者它们各种比例的混合物；所述酸性试剂为甲酸，乙酸，三氟乙酸。上述制备方法中，中间体化合物3与酸性试剂反应摩尔比为1:1～1:10。
作为本申请的另一个方面，本申请提供了一种药物组合物，其包含本申请上述的式I化合物或其药学上可接受的盐、立体异构体、溶剂化物、多晶形物，和药学上可接受的载体。所述药物组合物包括但不限于口服剂型、胃肠外给药剂型、外用剂型和直肠给药剂型。在一些实施方式中，所述药物组合物可以是口服的片剂、胶囊剂、丸剂、散剂、缓释制剂、溶液剂和悬浮液， 用于胃肠外注射的无菌溶液、悬浮液或乳液，用于外用的软膏或乳膏，或者用于直肠给药的栓剂。在一些实施方式中，所述药物组合物和至少一种治疗剂分别以独立的剂型组合成组合产品，如药剂盒。
作为本申请的另一个方面，本申请提供所述式I化合物、其药学上可接受的盐、立体异构体、溶剂化物、多晶形物在制备具有抗癌作用的药物中的应用。所述癌症包括食管癌、子宫内膜癌、恶性淋巴瘤、多发性骨髓瘤、胃肠道间质瘤、结肠癌、直肠癌、乳腺癌、肝癌、胃癌、卵巢癌、子宫癌、宫颈癌、阴道癌、肺癌、肾癌、前列腺癌、膀胱癌、胰腺癌、脑癌、黑色素瘤等。优选针对肝癌效果最佳。
作为本申请的另一个方面，本申请提供一种治疗癌症的方法，该方法包括将治疗有效量的所述式I化合物、其药学上可接受的盐、立体异构体、溶剂化物、多晶形物施用于由此需求的个体。在一些实施方式中，所述癌症包括食管癌、子宫内膜癌、恶性淋巴瘤、多发性骨髓瘤、胃肠道间质瘤、结肠癌、直肠癌、乳腺癌、肝癌、胃癌、卵巢癌、子宫癌、宫颈癌、阴道癌、肺癌、肾癌、前列腺癌、膀胱癌、胰腺癌、脑癌、黑色素瘤等。优选针对肝癌效果最佳。
本申请所述“药学上可接受的盐”是指保留了指定化合物的游离酸和游离碱的生物效力，并且在生物学或其他方面没有不良作用的盐。本申请中的盐指用有机酸/无机酸形成的酸式盐，以及用有机碱/无机碱形成的碱式盐。
本申请所述“溶剂化物”是指通过溶剂化作用形成的本申请化合物与溶剂分子的组合。如水合物、乙醇溶剂化物、甲醇溶剂化物等。
本申请所述“多晶形物”或“多晶形”是指以不同的晶格形式存在的本申请化合物。
本申请所述“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体。
本申请所述“药物组合物”是指任选的混合有至少一种药学上可接受的化学成分的生物活性化合物，所述药学上可接受的化学成分包括但不限于载体、稳定剂、稀释剂、分散剂、悬浮剂、增稠剂和/或赋形剂。所述“载体”是指相对无毒的化学试剂，其有助于将化合物引入到细胞或组织中。
本申请所述“烷基”是指含1至10个碳原子的直链或支链饱和烃链，包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、3-甲基己基、2,2-二甲基戊基、2,3-二甲基戊基、正庚基、正辛基、正壬基和正癸基。
所述“芳基”是指含有6至14个碳环原子的芳族碳环基。芳基可以是单环的或多环的。在多环芳族环的情况下，所述多环系中的仅一个环需要是不饱和的，而剩余的一或多个环可以是饱和的、部分饱和的或不饱和的。芳基的例子包括苯基、萘基、茚基、茚满基和四氢萘基。
所述“杂芳基”是指具有至少一个碳原子和一个或多个的独立地选择的氮、氧或硫原子的五元或六元芳族环。具体地，所述“杂芳基”是指含有5至14个环原子的芳族杂环基。杂芳基可以是单环或2个或3个稠合环。杂芳基取代基的例子包括：6元环取代基，诸如吡啶基、吡嗪基、嘧啶基、哒嗪基和1,3,5-、1,2,4-或1,2,3-三嗪基；5元环取代基，诸如咪唑基、呋喃基、噻吩基、吡唑基、噁唑基、异噁唑基、噻唑基、1,2,3-、1,2,4-、1,2,5-、或1,3,4-噁二唑基和异噻唑基；6/5元稠合环取代基，诸如苯并噻吩基、苯并异噁唑基、苯并噁唑基、咪唑基、吲哚基、苯并咪唑基、吡咯并[2,3-b]吡啶基、嘌呤基；和6/6元稠合环，诸如苯并吡喃基、喹啉基、异喹啉基、噌啉基、喹唑啉基和苯并噁嗪基。
所述“环烯基”是指单环或桥连烃环系。单环环烯基具有4、5、6、7或8个碳原子和0个杂原子。四元环系具有一个双键，五元或六元环系具有一个或两个双键，七元或八元环系具有一个、两个或三个双键。单环环烯基的代表性例子包括、但不限于：环丁烯基、环戊烯基、环己烯基、环庚烯基和环辛烯基。
所述“杂环烷基”是指含有共计3至14个环原子的饱和环结构。环原子中的至少一个是杂原子(即氧、氮或硫)，其余环原子独立地选自碳、氧、氮和硫。如四氢呋喃基、四氢吡喃基、四氢噻吩基、吡咯基、吡咯啉基、吡咯烷基、咪唑基、咪唑啉基、咪唑烷基、吡唑基、吡唑啉基、吡唑烷基、三唑基、四唑基。
所述的“有效量”是指无毒性，但足够量的提供所需的作用的药物或药 剂。在本发明的药物组合物或药剂盒中，一种成分或制剂单元的“有效量”是指该成分在和其他成分联合应用时有效提供所需效应的量。“有效量”会因受试者的不同而不同，依据年龄和个体的一般情况，特定的活性药物等等。因此，不可能总是指精确的“有效量”，然而，任何个体病例中合适的“有效量”可以由本领域普通技术人员应用常规的实验方法来测定。
所述“受试者”可以指患者或者其它接受本发明化合物或药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
为进行体外实验，本申请还合成了式I化合物的代谢产物式II化合物，其反应路线为：
首先Linifanib或其衍生物与带Boc保护的L(反应物3)在缩合剂和催化剂条件下反应生成中间体化合物Ma，该中间体化合物Ma在三氟乙酸作用条件下脱除Boc保护生成中间体化合物Mb，该中间体化合物Mb进一步与带保护基的天门冬氨酸缩合得到中间体化合物Mc，中间体化合物Mc在三氟乙酸条件下脱除Boc保护生成中间体化合物Md，Md在贵金属催化剂条件下催化氢化脱除苄基得到代谢产物式2化合物。
反应路线图：
Figure PCTCN2017111666-appb-000005
其中，
A选自(CH2)eN(R7)C(O)N(R8)(CH2)f和CH2C(O)NR7，其中e和f独立为0或1，其中各个基团由其左端连接R3和R4取代的环；
L为-[Cm(O)(Z)n(NH)q]-，其中m,q分别为0或1，n为0～11，p为0～8；Z选自-CR10-，-CR10-O-CR10-，-S-S-，-CR10＝CR10-，-CR10≡CR10-，-Ar，-CO-NH-和-N＝CR10-中的任意一种基团或几种基团以常规方式相连接；
R1和R2独立选自氢、烷氧基、烷氧基烷氧基、烷氧基烷基、烷基、芳氧基、芳氧基烷基、卤基、卤代烷氧基、卤代烷基、杂环基、杂环基烯基、 杂环基烷氧基、杂环基烷基、杂环基氧基烷基、羟基、羟基烷氧基、羟基烷基、(NRaRb)烷氧基、(NRaRb)烯基、(NRaRb)烷基、(NRaRb)羰基烯基和(NRaRb)羰基烷基；
R3和R4独立选自氢、烷氧基、烷基、卤基、卤代烷氧基、卤代烷基和羟基；
R5和R6独立选自氢、烷氧基、烷氧基烷基、烷氧基羰基、烷基、芳氧基、芳基烷基、羧基、氰基、卤基、卤代烷氧基、卤代烷基、羟基、羟基烷基、硝基和-NRcRd；
R7和R8独立选自氢和烷基；
R10选自氢，烷基、烷氧基、芳氧基、链烯氧基、硝基、卤基、伯胺基、仲胺基和叔胺基；
Ra和Rb独立选自氢、烷基、烷基羰基、烷基磺酰基、芳基磺酰基、卤代烷基磺酰基和杂环基磺酰基；
Rc和Rd独立选自氢、烷基、烷基羰基、芳基、芳基烷基、环烷基、环烷基烷基、杂环基和杂环基烷基。
具体的，各代谢产物结构见如下：
Figure PCTCN2017111666-appb-000006
Figure PCTCN2017111666-appb-000007
本发明在合成阶段曾尝试在Linifanib多个位点进行结构修饰连接碳链和氨基酸，但结果均不理想，大部分化合物均未合成成功，产率很低，还有一部分化合物虽然合成成功，但没有起到封闭Linifanib活性的作用，或是在血浆中不能稳定存在。最终仅1位连接的产物不仅得率高，且能成功封闭Linifanib活性，并能在体外(肝匀浆和脾匀浆中)经生物学转化为活性药物Linifanib，以更低剂量抑制肿瘤细胞尤其是肝癌细胞的增殖。
附图说明
图1前体和中间体肝匀浆稳定性第1次实验
图2前体和中间体肝匀浆稳定性第2次实验
图3前体和中间体脾匀浆稳定性第1次实验
图4前体和中间体脾匀浆稳定性第2次实验
图5肝脾匀浆对比
具体实施方式
实施例1～2：制备目标化合物1
实施例1 中间体化合物3的制备
称取Linifanib 600mg(1.6mmol)，HOBT 324mg(2.4mmol)、EDCI 460mg(2.4mmol)溶于50ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入市售中间体化合物2Asp(Boc)-Glu(OtBu)-(OtBu)912mg(1.92mmol)，最后再加入DIPEA 516mg(4.0mmol)加毕，维持该反应温度搅拌反应12h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150 ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝0:1～100:1)，得类白色固体粉末716mg，收率53.8％。
实施例2 目标化合物1的制备
称取实施例1制备的中间体化合物3 500mg(0.38mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应20～24h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入40ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得类白色固体粉末106mg，收率44.9％。1H NMR(400MHz,DMSO-d6)δ:9.37(s,1H),8.67(s,1H),8.32-8.27(m,2H),7.99-7.96(m,1H),7.65-7.59(m,3H),7.42-7.39(m,2H),7.27-7.18(m,2H),7.13-7.08(m,1H),6.81-6.78(m,2H),5.25(s,2H),4.34-4.11(m,2H),3.71-3.60(m,1H),2.91-2.81(m,1H),2.37-2.31(m,2H),2.28(s,3H),2.24-2.20(m,1H),1.89-1.86(m,1H).HPLC purity:96.1％(214nm),95.5％(254nm).MS(ESI):m/z 620.0[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000008
实施例3～6：制备目标化合物4
实施例3 中间体化合物1的制备
称取苄基-(6-氨基)己烷酸酯盐酸盐304mg(1.18mmol)、HOBT 238mg(1.76mmol)、EDCI 192mg(1.76mmol)溶于50ml二氯甲烷中，室温搅拌溶解。控制反应温度20～40℃缓慢加入Asp(Boc)-Glu(OtBu)-(OtBu)584mg (1.23mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(石油醚/丙酮＝10:1～2:1)，得黄色固体粉末338mg，收率42.3％。
实施例4 中间体化合物2的制备
称取实施例3制备的中间体化合物1 285mg(0.42mmol)，溶于60ml无水甲醇中，氮气保护条件下加入10％Pd/C 25mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得黄褐色油状物。将该油状物进行制备色谱分离，得浅黄色固体粉末115mg，收率46.6％。
实施例5 中间体化合物3的制备
称取Linifanib 600mg(1.6mmol)，HOBT 324mg(2.4mmol)、EDCI 460mg(2.4mmol)溶于50ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入实施例4制备的中间体化合物2 1129mg(1.92mmol)，最后再加入DIPEA 516mg(4.0mmol)加毕，维持该反应温度搅拌反应12h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝0:1～100:1)，得类白色固体粉末776mg，收率51.3％。
实施例6 目标化合物4的制备
称取实施例5制备的中间体化合物3 595mg(0.63mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应20～24h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入40ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相， 有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得类白色固体粉末193mg，收率41.9％。1H NMR(400MHz,DMSO-d6)δ:9.30(s,1H),8.67(d,J＝7.6Hz,1H),8.58(d,J＝2.0Hz,1H)8.31(d,J＝8.0Hz,1H)8.25-8.22(m,1H),8.09(s,3H),7.99(d,J＝6.4Hz,1H),7.64-7.58(m,3H),7.41(d,J＝8.4Hz,1H),7.19-7.09(m,2H),6.83(d,J＝5.6Hz,1H),5.17(s,2H),4.26-4.14(m,2H),3.15-2.97(m,4H),2.70-2.54(m,2H),2.33-2.32(m,2H),2.28(s,3H),2.00-1.69(m,4H),1.50-1.36(m,4H).HPLC purity:98.23％(214nm),98.45％(254nm).MS(ESI):m/z 733.0[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000009
实施例7～10：制备目标化合物7
实施例7 中间体化合物1的制备
称取苄基-(12-氨基)十二烷酸酯盐酸盐404mg(1.18mmol)、HOBT 238mg(1.76mmol)、EDCI 192mg(1.76mmol)溶于50ml二氯甲烷中，室温搅拌溶解。控制反应温度20～40℃缓慢加入Asp(Boc)-Glu(OtBu)-(OtBu)584mg(1.23mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(石油醚/丙酮＝10:1～2:1)，得黄色固体粉末278mg，收率35.6％。
实施例8 中间体化合物2的制备
称取实施例7制备的中间体化合物1 2270mg(3.43mmol)，溶于100ml无水甲醇中，氮气保护条件下加入10％Pd/C 50mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得黄褐色油状物。将该油状物进行制备色谱分离，得浅黄色固体粉末1101mg，收率56.1％。
实施例9 中间体化合物3的制备
称取Linifanib 600mg(1.6mmol)，HOBT 324mg(2.4mmol)、EDCI 460mg(2.4mmol)溶于50ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入实施例8制备的中间体化合物2 1098mg(1.92mmol)，最后再加入DIPEA 516mg(4.0mmol)加毕，维持该反应温度搅拌反应12h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝0:1～100:1)，得类白色固体粉末589mg，收率39.6％。
实施例10 目标化合物7的制备
称取实施例9制备的中间体化合物3 585mg(0.63mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应20～24h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入40ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得类白色固体粉末223mg，收率43.3％。1H NMR(400MHz,DMSO-d6)δ:9.31(s,1H),8.67(d,J＝7.6Hz,1H)8.59(s,1H),8.31(d,J＝8.4Hz,1H),8.21(s,1H),8.19-7.98(m,4H),7.64-7.57(m,3H),7.42(d,J＝8.0Hz,2H),7.19-7.09(m,2H),6.83-6.82(m,1H),5.18(s,2H),4.29-4.13(m,2H),3.12-2.96(m,4H),2.73-2.68(m,2H),2.67-2.63(m,2H),2.28(s,3H),2.12-1.67(m,4H),1.37-1.23(m,18H).HPLC purity:99.04％(214nm), 99.01％(254nm).MS(ESI):m/z 817.1[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000010
实施例11～14：制备目标化合物8(Linifanib-C12-AA5)
实施例11 中间体化合物1的制备
称取苄基-(12-氨基)十二烷酸酯盐酸盐404mg(1.18mmol)、HOBT 238mg(1.76mmol)、EDCI 192mg(1.76mmol)溶于250ml二氯甲烷中，室温搅拌溶解。控制反应温度20～40℃缓慢加入Asp(Boc)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-(OtBu)1267mg(1.23mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(石油醚/丙酮＝10:1～2:1)，得黄色固体粉末553mg，收率35.6％。
实施例12 中间体化合物2的制备
称取实施例11制备的中间体化合物1 4000mg(3.0mmol)，溶于100ml无水甲醇中，氮气保护条件下加入10％Pd/C 50mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得黄褐色油状物。将该油状物进行制备色谱分离，得浅黄色固体粉末1595 mg，收率42.8％。1H NMR(CDCl3)δ1.27(brs,14H),1.46～1.47(m,54H),1.65～1.85(m,8H),2.34～2.35(brs,16H),,3.06～3.36(brs,2H),4.46-4.52(m,5H),6.31(brs,1H,-NH-C＝O),6.68(brs,1H,-NH-C＝O),6.91(brs,2H,-NH-C＝O),7.19(brs,1H,-NH-C＝O),7.54(brs,1H,-NH-C＝O).13C NMR(CDCl3)δ192.97,190.34,173.02,172.22,172.00,171.81,171.22,171.08,170.76,82.42,82.27,82.08,82.02,80.64,80.53,52.35,51.83,51.44,39.84,33.79,32.52,32.15,31.61,31.11,29.26,29.11,28.97,28.92,28.86,28.78,28.71,28.48,28.33,28.10,28.01,27.98,27.76,27.65,26.68,24.61,12.10.
结构式为：
Figure PCTCN2017111666-appb-000011
实施例13 中间体化合物3的制备
称取Linifanib 600mg(1.6mmol)，HOBT 324mg(2.4mmol)、EDCI 460mg(2.4mmol)溶于250ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入实施例12制备的中间体化合物2 2340mg(1.9mmol)，最后再加入DIPEA 516mg(4.0mmol)加毕，维持该反应温度搅拌反应12h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入100ml二氯甲烷稀释，再用250ml去离子水洗涤两次，分离有机相。有机相再用150ml饱和食盐水洗涤，分液，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝0:1～100:1)，得类白色固体粉末1132mg，收率44.7％。
实施例14 目标化合物8(Linifanib-C12-AA5)的制备
称取实施例13制备的中间体化合物3 1000mg(0.63mmol)溶于60ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应20～24h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入40ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用 60ml 5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得类白色固体粉末293mg，收率38.6％。1H NMR(400MHz,DMSO-d6)δ:9.51(s,1H),8.97(s,1H),8.56(s,1H),8.32(d,J＝8.4Hz,1H),8.18(s,1H),8.02-7.96(m,4H),7.66-7.57(m,3H),7.42(d,J＝8.8Hz,2H),7.19-7.08(m,2H),6.82-6.81(m,1H),5.18(s,2H),4.16-3.98(m,6H),3.07-2.96(m,4H),2.67-2.63(m,2H),2.28(s,3H),2.24-2.14(m,8H),2.03-1.87(m,5H),1.77-1.67(m,4H),1.37-1.23(m,18H).HPLC purity:99.3％(214nm),99.1％(254nm).MS(ESI):m/z 1204.5[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000012
实施例15～17：制备目标化合物10
实施例15 代谢产物中间体Mc的制备
称取Boc-L-天冬氨酸1-苄酯137mg(0.42mmol)，HOBT 77.8mg(0.58mmol)、EDCI 110mg(0.58mmol)溶于10ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入Linifanib 143mg(0.38mmol)，最后再加入DIPEA 124mg(0.96mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用100ml去离子水洗涤两次，分离有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝1:0～30:1)，得黄色油状物221mg，收率77.3％。
实施例16 代谢产物中间体Md的制备
称取实施例15制备的中间体化合物Mc 198mg(0.29mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应1.5～2h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得黄色油状物。将该油状物进行制备色谱分离，得黄色油状物131mg，收率77.8％。
实施例17 目标化合物10的制备
称取实施例16制备的中间体Md 336mg(0.58mmol)，溶于30ml无水甲醇中，氮气保护条件下加入10％Pd/C 25mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得淡黄色油状物。将该油状物进行制备色谱分离，得白固体粉末178mg收率62.6％。HPLC purity:97.2％(214nm),98.6％(254nm).MS(ESI):m/z 491.0[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000013
实施例18～22：制备目标化合物11
实施例18 代谢产物中间体Ma的制备
称取6-(BOC-氨基)己烷酸136mg(0.59mmol)，HOBT 107mg(0.8mmol)、EDCI 152mg(0.8mmol)溶于10ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入Linifanib 200mg(0.53mmol)，最后再加入 DIPEA 171mg(1.3mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用100ml去离子水洗涤两次，分离有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝1:0～30:1)，得黄色油状物207mg，收率59.8％。
实施例19 代谢产物中间体Mb的制备
称取实施例18制备的中间体Ma 194mg(0.33mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应1.5～2h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml 5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得黄色油状物147mg，收率91.1％。
实施例20 代谢产物中间体Mc的制备
称取Boc-L-天冬氨酸1-苄酯137mg(0.42mmol)，HOBT 77.8mg(0.58mmol)、EDCI 110mg(0.58mmol)溶于10ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入实施例19制备的中间体Mb 185mg(0.38mmol)，最后再加入DIPEA 124mg(0.96mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用100ml去离子水洗涤两次，分离有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝1:0～30:1)，得黄色油状物200mg，收率66.3％。
实施例21 代谢产物中间体Md的制备
称取实施例20制备的中间体化合物Mc 230mg(0.29mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应1.5～2h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得黄色油状物。将该 油状物进行制备色谱分离，得黄色油状物158mg，收率78.4％。
实施例22 目标化合物11的制备
称取实施例21制备的中间体Md 425mg(0.61mmol)，溶于30ml无水甲醇中，氮气保护条件下加入10％Pd/C 25mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得淡黄色油状物。将该油状物进行制备色谱分离，得白固体粉末234mg收率63.4％。HPLC purity:96.2％(214nm),98.1％(254nm).MS(ESI):m/z 604.3[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000014
实施例23～27：制备目标化合物12(Linifanib-C12-Asp)
实施例23 代谢产物中间体Ma的制备
称取12-(BOC-氨基)十二烷酸186mg(0.59mmol)，HOBT 107mg(0.8mmol)、EDCI 152mg(0.8mmol)溶于10ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入Linifanib 200mg(0.53mmol)，最后再加入DIPEA 171mg(1.3mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用100ml去离子水洗涤两次，分离有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝1:0～30:1)，得黄色油状物225mg，收率63.2％。
实施例24 代谢产物中间体Mb的制备
称取实施例23制备的中间体Ma 225mg(0.33mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应1.5～2h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中 加入50ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml 5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得红褐色油状物。将该油状物进行制备色谱分离，得黄色油状物220mg，收率95％。
实施例25 代谢产物中间体Mc的制备
称取Boc-L-天冬氨酸1-苄酯137mg(0.42mmol)，HOBT 77.8mg(0.58mmol)、EDCI 110mg(0.58mmol)溶于10ml二氯甲烷中，搅拌反应0.5h，控制反应温度20～40℃缓慢加入实施例24制备的中间体Mb 220mg(0.38mmol)，最后再加入DIPEA 124mg(0.96mmol)，加毕，维持该反应温度搅拌反应4h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用100ml去离子水洗涤两次，分离有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得褐色油状物。将该油状物进行硅胶柱层析(DCM:MeOH＝1:0～30:1)，得黄色油状物250mg，收率74.2％。
实施例26 代谢产物中间体Md的制备
称取实施例25制备的中间体化合物Mc 250mg(0.29mmol)溶于20ml二氯甲烷中，控制反应温度-5～5℃缓慢加入三氟乙酸3ml(0.04mmol)，维持该反应温度搅拌反应1.5～2h，TLC(DCM/MeOH＝40:1)检测反应完全。向反应液中加入50ml二氯甲烷稀释，再用120ml去离子水洗涤两次，再用60ml5％碳酸氢钠溶液洗涤两次，再用120ml去离子水洗涤两次。分离有机相，有机相用无水硫酸钠干燥。滤除干燥剂，滤液低温浓缩得黄色油状物。将该油状物进行制备色谱分离，得黄色油状物158mg，收率71.3％。
实施例27 目标化合物12(Linifanib-C12-Asp)的制备
称取实施例26制备的中间体Md 210mg，溶于30ml无水甲醇中，氮气保护条件下加入10％Pd/C 25mg，通入氢气进行3次置换，在氢气氛下控制2MPa，20～65℃反应6～12h，TLC(DCM/MeOH＝40:1)检测反应完全。在氮气保护条件下，将反应液过滤，回收钯碳。滤液低温浓缩得淡黄色油状物。将该油状物进行制备色谱分离，得白固体粉末102mg收率54.8％。1H-NMR(DMSO)δ:1.22(m,12H),1.35(m,4H),1.71(s,2H),2.41(s,3H,-CH3),2.64(m, 1H),2.72(m,1H),3.34(m,6H),3.50(m,2H),5.20(s,2H),6.83(brs,1H),7.10(m,1H),7.18(m,1H),7.40(d,J＝7.5Hz,2H),7.58(m,1H),7.67(d,J＝7.5Hz,2H),7.91(m,1H),8.23(m,1H),8.32(m,1H),9.01(m,1H),9.83(m,1H)1.HPLC purity:98.5％(214nm),99.3％(254nm).MS(ESI):m/z 688.4[M+1]+
结构式为：
Figure PCTCN2017111666-appb-000015
实施例28 Linifanib相关化合物对肿瘤细胞株增殖的影响
本申请通过细胞增殖实验(Alamar Blue检测平台)测定6个化合物(化合物1、4、7、8、12以及Linifanib)在54株商业化肿瘤细胞株(含26株肝癌细胞株)上的半数抑制浓度(IC50值)，考察5个化合物与活性药Linifanib活性的差别。
1.仪器与材料
Thermo 311型CO2培养箱；Haier生物安全柜；Molecular Devices酶标仪；湘仪牌L530型台式低速离心机；Olympus IX51倒置荧光显微镜,DMEM、RPMI 1640、MEM、DMEM/F12 1：1培养基、胎牛血清、0.25％胰蛋白酶溶液、磷酸盐缓冲液(赛默飞世尔上海有限公司)；sigma二甲基亚砜(DMSO)、刃天青；
54株商业化肿瘤细胞株(含26株肝癌细胞株)。
实验药物：化合物1、4、7、8、12及Linifanib；化疗药阿霉素(Doxorubicin)(HY-15142；上海皓元生物医药科技有限公司)。
2.实验方法
2.1不同细胞株的培养
54株细胞株培养在含胎牛血清的培养液中，放置于37℃、5％的CO2 培养箱中温育。细胞均呈贴壁状态生长，在倒置显微镜下观察生长状况，待细胞汇合率达到80％-90％时进行传代培养。传代比例和数量以实验需求为准，此次细胞株传代培养比例一般为1：2～1：3。
2.2对不同肿瘤细胞株的增殖抑制作用
细胞测试：取对数生长期的54株细胞株，以500～1×104/孔(预实验中确定各细胞株的最佳接种密度)的数量接种于96孔培养板中，在含5％CO2的湿化培养箱中37℃培养4h后，每孔分别加入10μL化合物1、4、7、8、12及Linifanib，每个化合物测试9个药物浓度梯度(从最高测试浓度按3.16倍梯度稀释)，依据各化合物的溶解度不同起始浓度分别为30或100μM。并在每株细胞株测试时同步加入QC参考化合物Doxorubicin，其最终药物浓度依次分别为10、3.16、1、0.31、0.1、0.03、0.01、0.003和0.001μM。此外同时设置阳性对照组(100％抑制)及阴性对照组(0％抑制)，药物组每浓度重复2孔，阳性对照组和阴性对照组重复6孔，培养箱中继续培养6天后，接后续AlamarBlue测试操作；
AlamarBlue测试操作：每孔加10μL AlamarBlue试剂孵育1-4h，振荡1-2min，MD酶标仪EX:560nm,EM:590nm波长测得荧光值，记录结果，通过计算本发明化合物对细胞抑制率(％)＝(A0％抑制－A给药)/(A0％抑制－A100％抑 制)×100％，再利用GraphPad Prism 5.0或MATILAB软件采用非线性回归的方法(常采用四参数拟合曲线方程)作图得到药物剂量反应曲线，从而获得本发明化合物作用于癌细胞株的IC50值。
3.结果与分析
3.1 6个待测样品(化合物1、4、7、8、12及Linifanib)在54株商业化肿瘤细胞株上的IC50汇总结果如表1所示。
表1 六个化合物对54个商业化肿瘤细胞株的IC50(μM)值汇总
Figure PCTCN2017111666-appb-000016
Figure PCTCN2017111666-appb-000017
注：表中细胞株29-54是肝癌细胞株对各化合物的应答
由表1结果可见，几乎所有肿瘤细胞株对化合物1的IC50与对Linifanib的IC50值很接近，这个化合物的改构几乎没有起到封闭Linifanib活性的作用；而其他的4个化合物(化合物4，7，8和12)的改构都成功地封闭了Linifanib抑制肿瘤细胞增殖的活性，绝大部分肿瘤细胞对这4个化合物和对Linifanib的IC50差5倍以上。对Linifanib敏感的细胞有3株，分别为KASUMI-1(白血病细胞)，NCI-H1703(肺癌细胞)和AN3-CA(子宫内膜瘤细胞)，其IC50分别为0.01，0.02和0.19μM，其对前体Linifanib-C12-AA5的IC50分别为0.10，0.08和3.14μM，相差分别为10，4和16.7倍。
54株商业化肿瘤细胞株中有26株为肝癌细胞株，近一半肝癌细胞株(12/26，46％)对Linifanib都是中度敏感，IC50<5μM，同时大部分肝癌 细胞株(15/26，58％)对前体Linifanib-C12-AA5和对Linifanib的IC50差8倍以上，而几乎所有肝癌细胞对中间体都无应答，见表1。
实施例29 体外(血浆/肝匀浆/脾匀浆)稳定性研究
本实施例的目的是考察前体Linifanib-C12-AA5(化合物8)、中间体Linifanib-C12-Asp(化合物12)在血浆、肝匀浆、脾匀浆中孵育稳定性(前体Linifanib-C12-AA5代谢生成中间体Linifanib-C12-Asp和Linifanib；中间体Linifanib-C12-Asp进一步代谢生成Linifanib)，并对代谢产物进行定量分析，同时以阳性药验证孵育体系的稳定性，为化合物成药性评价等提供参考。
1仪器与材料
仪器：API3000液质联用仪，ABI
材料：雄性SD大鼠(200-250g)，北京维通利华
供试品：阳性药及其代谢产物M1和M2，Linifanib-C12-AA5及其代谢产物Linifanib-C12-Asp和Linifanib。
2Linifanib-C12-AA5和Linifanib-C12-Asp稳定性研究
2.1Linifanib-C12-AA5和Linifanib-C12-Asp血浆孵育稳定性研究方案
试验动物
种类：SD大鼠；数量：2
性别：雄；体重：200-250g；
实验步骤
1动物采血，血样置于EDTA抗凝管中，在4℃条件下，3000g下离心15min，分离血浆，2份血样等体积混合；
2称取一定量Linifanib-C12-AA5/Linifanib-C12-Asp溶于DMSO:MeOH(2:8)，经纯度折算配制成200μM母液，在血浆中加入化合物使其终浓度为2μg/mL，体系中有机相比例不超过0.5％。
3 37℃水浴温孵，设置取样点0,0.5,1,2,4,6,8h。每个取样点取100μL样品加入300μL乙腈(含内标)进行沉淀处理，12000rpm离心5min，取上清200μL待LC-MS/MS进行分析。
4配置标准曲线，定量检测Linifanib,Linifanib-C12-Asp。
2.2Linifanib-C12-AA5和Linifanib-C12-Asp肝/脾匀浆孵育稳定性研究方案
试验动物
种类：SD大鼠；数量：2
性别：雄；体重：200-250g
实验步骤
1生物分析方法：建立化合物Linifanib-C12-Asp和Linifanib生物分析方法；
2实验当天取大鼠肝脏/脾脏，2份剪碎混合，用4倍体积(1g:4mL)磷酸盐缓冲液(pH 7.4)匀浆，匀浆过程控制温度不超过10℃；进行蛋白定量检测，控制肝、脾脏蛋白浓度。
3往肝/脾脏匀浆中分别加入药物Linifanib-C12-AA5，Linifanib-C12-Asp，使其浓度分别为1000ng/mL，孵育体积为1mL，并控制孵育体系中有机溶剂浓度不超过0.5％。
4 37℃水浴孵育，分别在0，0.5，1，2，4，6，8h各取样100μL，用确定的方法对样品进行预处理后进行LC-MS/MS分析。
5以空白肝/脾脏匀浆液配制标准曲线，定量检测Linifanib-C12-Asp，Linifanib。
3稳定性研究结果
3.1血浆稳定性实验结果
阳性药按照预期随时间延长在血浆中代谢为其代谢产物，说明血浆体系稳定，后续检测结果可靠。
结果见表2。
由表2可知，Linifanib-C12-AA5在血浆中孵育稳定，不产生中间体L-C12-ASP和Linifanib；中间体Linifanib-C12-Asp在血浆中孵育稳定，产生的Linifanib水平接近于0。
表2 前体与中间体化合物在大鼠血浆中稳定性实验结果
Figure PCTCN2017111666-appb-000018
3.2肝匀浆稳定性结果
阳性药按照预期随时间延长在肝匀浆中代谢为其代谢产物，说明肝匀浆体系稳定，后续检测结果可靠。
前体和中间体的肝匀浆稳定性结果见表3-4及图1-2。
表3 肝匀浆稳定性第1次实验
Figure PCTCN2017111666-appb-000019
表4 肝匀浆稳定性第2次实验
Figure PCTCN2017111666-appb-000020
由表3-4及图1-2可知，前体Linifanib-C12-AA5生成的中间体Linifanib-C12-Asp在肝匀浆中能迅速转化为Linifanib，所以肝匀浆中中间体的蓄积浓度较低。
同时，当加入相同摩尔浓度的前体和中间体时，在肝匀浆中各个时间点上生成的Linifanib的量基本相当，说明Linifanib的生成基本上都是通过中间体生成的，由前体直接生成的Linifanib非常少。
3.3脾匀浆稳定性结果
阳性药按照预期随时间延长在脾匀浆中代谢为其代谢产物，说明脾匀浆体系稳定，后续检测结果可靠。
前体和中间体的脾匀浆稳定性结果见表5-6及图3-4。
表5 脾匀浆稳定性第1次实验
Figure PCTCN2017111666-appb-000021
表6 脾匀浆稳定性第2次实验
Figure PCTCN2017111666-appb-000022
由表5-6及图3-4可知，在脾匀浆中前体的代谢也与肝匀浆中相同，都是先生成中间体，而后中间体代谢为Linifanib。
另外，脾匀浆中中间体的蓄积浓度较高，而生成的Linifanib的量比肝匀浆中少。
从以上体外稳定性研究研究结果可以得出以下结论：
(1)前体Linifanib-C12-AA5和中间体Linifanib-C12-Asp在血浆中稳定。
(2)前体Linifanib-C12-AA5在体外的代谢途径基本清楚，即通过PSMA代谢为中间体Linifanib-C12-Asp，而后中间体Linifanib-C12-Asp通过某种酰胺酯酶代谢为活性药Linifanib。
(3)肝匀浆中产生的活性药物Linifanib更多，如图5，表示药物前体Linifanib-C12-AA5在肝中更特异性的转化为活性药物。

Claims (10)

  1. 一种具有式I结构的化合物、其药学上可接受的盐、立体异构体、溶剂化物或多晶形物，
    Figure PCTCN2017111666-appb-100001
    其中，
    A选自(CH2)eN(R7)C(O)N(R8)(CH2)f和CH2C(O)NR7，其中e和f独立为0或1，其中各个基团由其左端连接R3和R4取代的环；
    L为-[Cm(O)(Z)n(NH)q]-，其中m,q分别为0或1，n为0～11，p为0～8；Z选自-CR10-，-CR10-O-CR10-，-S-S-，-CR10＝CR10-，-CR10≡CR10-，-Ar，-CO-NH-和-N＝CR10-中的任意一种基团或几种基团以常规方式相连接；
    R1和R2独立选自氢、烷氧基、烷氧基烷氧基、烷氧基烷基、烷基、芳氧基、芳氧基烷基、卤基、卤代烷氧基、卤代烷基、杂环基、杂环基烯基、杂环基烷氧基、杂环基烷基、杂环基氧基烷基、羟基、羟基烷氧基、羟基烷基、(NRaRb)烷氧基、(NRaRb)烯基、(NRaRb)烷基、(NRaRb)羰基烯基和(NRaRb)羰基烷基；
    R3和R4独立选自氢、烷氧基、烷基、卤基、卤代烷氧基、卤代烷基和羟基；
    R5和R6独立选自氢、烷氧基、烷氧基烷基、烷氧基羰基、烷基、芳氧基、芳基烷基、羧基、氰基、卤基、卤代烷氧基、卤代烷基、羟基、羟基烷基、硝基和-NRcRd；
    R7和R8独立选自氢和烷基；
    R9选自氢、羟基，氨基，烯基、炔基，烷氧基，烷胺基，烷氧基烷基、烷基、烷氧基羰基、芳基、杂环烷基；
    R10选自氢、烷基、烷氧基、芳氧基、链烯氧基、硝基、卤基、伯胺基、仲胺基和叔胺基；
    Ra和Rb独立选自氢、烷基、烷基羰基、烷基磺酰基、芳基磺酰基、卤代烷基磺酰基和杂环基磺酰基；
    Rc和Rd独立选自氢、烷基、烷基羰基、芳基、芳基烷基、环烷基、环烷基烷基、杂环基和杂环基烷基。
  2. 如权利要求1所述的化合物、其药学上可接受的盐、立体异构体、溶剂化物、多晶形物，其结构式为：
    Figure PCTCN2017111666-appb-100002
    Figure PCTCN2017111666-appb-100003
    Figure PCTCN2017111666-appb-100004
  3. 如权利要求1或2所述的化合物、其药学上可接受的盐、立体异构体、溶剂化物、多晶形物的制备方法，其特征在于，所述方法包括如下步骤；
    步骤a，将多肽与带苄基保护的L在催化剂和缩合剂存在的条件下进行反应，得到带保护基团的中间体化合物1；
    步骤b，所述中间体化合物1在极性溶剂中进行催化氢化脱除保护基团得到中间体化合物2；
    步骤c，中间体化合物2与Linifanib或其衍生物在催化剂和缩合剂存在的条件下进行反应，得到带保护基团的中间体化合物3；
    步骤d，所述中间体化合物3在酸性条件下脱除保护基团得到式I化合 物。
  4. 如权利要求3所述的制备方法，其特征在于，步骤a中所述反应温度为-20℃～125℃；所述催化剂为1-羟基苯并三唑；所述缩合剂为1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐、1,3-二环己基碳二亚胺或4-二甲氨基吡啶中的任意一种或几种。
  5. 如权利要求3所述的制备方法，其特征在于，步骤b中所述反应温度为-20℃～125℃；所述催化剂为钯碳，氢氧化钯干性或湿性。
  6. 如权利要求3所述的制备方法，其特征在于，步骤c中所述反应温度为-20℃～125℃；所述催化剂为1-羟基苯并三唑；所述缩合剂为1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐、1,3-二环己基碳二亚胺或4-二甲氨基吡啶中的任意一种或几种。
  7. 如权利要求3所述的制备方法，其特征在于，步骤d中所述反应温度为-20℃～125℃；所述酸性试剂为甲酸，乙酸，三氟乙酸。
  8. 如权利要求1或2所述的化合物、其药学上可接受的盐、立体异构体、溶剂化物、多晶形物在制备具有抗癌作用的药物中的应用；所述癌症包括食管癌、子宫内膜癌、恶性淋巴瘤、多发性骨髓瘤、胃肠道间质瘤、结肠癌、直肠癌、乳腺癌、肝癌、胃癌、卵巢癌、子宫癌、宫颈癌、阴道癌、肺癌、肾癌、前列腺癌、膀胱癌、胰腺癌、脑癌和黑色素瘤。
  9. 如权利要求8所述的应用，其特征在于，所述癌症为肝癌。
  10. 一种治疗癌症的方法，包括给予受试者治疗有效量的权利要求1或2所述的化合物、其药学上可接受的盐、立体异构体、溶剂化物或多晶形物。
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