TW201938539A - Anticancer compound and preparation method and application thereof which can be converted in vitro biologically into the active drug Linifanib - Google Patents

Abstract

This invention provides a compound of Formula I, its preparation method and its use in treating cancer. The compound of the present invention has inhibitory effect to multiple cancer cell types and can be converted in vitro (in plasma) biologically into the active drug Linifanib so as to use lower dosage to inhibit the proliferation of tumor cells particularly hepatoma cells.

Description

Compound with anti-cancer effect, preparation method and application thereof (2)

The invention relates to a compound, a preparation method and application thereof, and particularly to a compound that is selectively converted into a stronger anticancer activity in vivo, and a preparation method and application thereof.

Background technique

The use of anti-tumor drugs to selectively kill tumor cells with less toxicity to normal cells has always been a difficult problem in tumor treatment. Targeted therapies that have emerged in recent years have focused on mutations in specific targets in tumor cells, bringing the gospel to cancer patients. However, targeted therapy also has many limitations, such as a small group of beneficiaries and rapid drug resistance after administration. This has forced biomedical research and development to find another way to provide new treatment options for more patients.

Linifanib is a multi-target anti-cancer compound. Its targets are mostly angiogenesis-related kinases, which have a good inhibitory effect on VEGFRs, PDGFRs, CSF-1R and Flt-1 / 3. In a large randomized phase III clinical trial for liver cancer, it was found that Tini (time to progression) and ORR (overall response rate) for liver cancer patients were significantly better than the only approved targeted drugs for liver cancer. Sorafenib (TTP 5.4 months vs. 4.0 months, ORR 13.0% vs 6.9%), but its toxic and side effects are also greater than Sorafenib, so its overall efficacy is not stronger than Sorafenib, so it has not passed FDA approval ( J Clin Oncol, 2014, 33 172-179 ).

In order to solve the above problems, this application connects Linifanib or a derivative thereof to a polypeptide through a multi-carbon chain to form a compound Linifanib-Cx-AAy (that is, a compound of Formula I of the present application). Membrane antigen) is highly expressed in tumor endothelial cells and some tumor cells of solid tumors, and is specifically degraded at the tumor site Linifanib-Cx-AAy forms the active anticancer compound Linifanib or its derivative, thereby specifically enriching the anticancer compound at the tumor site while reducing its systemic toxicity.

As one aspect of the present application, the present application provides a compound having the structure of Formula I, a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof: Where X is selected from O, S and NR ₉ ; 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 , where e and f are independently 0 or 1, wherein each group is a ring substituted by R ₃ and R _{4 at} its left end; L is-[C _m (O) (Z) _n (NH) _q ]-, where m, q 0 or 1, respectively, n is 0 to 11, p is 0 to 8; Z is selected from -CR _10- , -CR ₁₀ -O-CR _10- , -SS-, -CR ₁₀ = CR _10- , -CR _10≡ CR _10- , -Ar, -CO-NH- and -N = CR ₁₀ -any one or several groups are connected in a conventional manner; R ₁ and R _{2 are} independently selected from hydrogen, alkoxy Alkyl, alkoxyalkoxy, alkoxyalkyl, alkyl, aryloxy, aryloxyalkyl, halo, haloalkoxy, haloalkyl, heterocyclyl, heterocyclylalkenyl, heterocyclic Alkoxy, heterocyclylalkyl, heterocyclyloxyalkyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, (NR _a R _b ) alkoxy, (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 hydrogen, alkoxy, alkyl, halo, haloalkoxy group, a haloalkyl group and a hydroxyl group; R ₅ and R _{6 are} independently selected from hydrogen, alkoxy, alkoxyalkyl Alkoxycarbonyl, alkyl, aryloxy, arylalkyl, carboxy, cyano, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, nitro, and -NR _c R _d; R ₇ and R _{8 is} independently selected from hydrogen and alkyl; R _{9 is} selected from hydrogen, alkenyl, alkoxyalkyl, alkyl, alkoxycarbonyl, aryl, heterocyclylalkyl, hydroxyalkyl, and (NR _a R _b ) alkyl; R _{10 is} selected from hydrogen, alkyl, alkoxy, aryloxy, alkenyloxy, nitro, halo, primary amine, secondary amine, and tertiary amine; R _{11 is} selected from hydrogen , a hydroxyl group, an amino group, an alkenyl group, an alkynyl group, an alkoxy group, an alkoxy group, an alkoxyalkyl group, an alkyl group, an alkoxycarbonyl group, an aryl group, a heterocyclic group; R _a and R _{b are} independently selected from hydrogen , Alkyl, alkylcarbonyl, alkylsulfonyl, arylsulfonyl, haloalkylsulfonyl and heterocyclylsulfonyl; _Rc and _{Rd are} independently selected from hydrogen, alkyl, alkylcarbonyl, Aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl.

Specifically, the structure of each compound is as follows:

Response route:

First, the polypeptide (reactant 1) is reacted with L (reactant 2) with benzyl protection in the presence of a catalyst and a condensing agent to obtain an intermediate compound 1 with a protective group. The intermediate compound 1 is further Intermediate compound 2 is removed by catalytic hydrogenation in a polar solvent to remove the protective group; intermediate compound 2 is reacted with Linifanib or its derivative in the presence of a catalyst and a condensing agent to obtain intermediate compound 3 with a protective group The intermediate compound 3 further removes the protecting group under acidic conditions to obtain a compound of formula I.

Reaction Roadmap:

Further, in the above method for preparing intermediate compound 1, the reaction temperature is performed at -20 ° C to 125 ° C; the organic solvent is selected from the group consisting of an ether, an alcohol, an alkane, an aromatic hydrocarbon, and a ketone containing 1-20 carbon atoms. , Haloalkane, amidine, nitrile, ester, or a mixture thereof; the catalyst is 1-hydroxybenzotriazole (HOBT); and the condensing agent is 1-ethyl-3- (3-dimethylaminepropyl) carbon Any one or more of diimine hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) or 4-dimethylaminopyridine (DMAP). In this step, the molar ratios of the reactants 1 and 2 in the reaction are 1: 1 to 1:10, and the molar ratios of the reactant 1 to the condensing agent are 1: 0.1 to 1:10; The ear ratio is 1: 0.1 ~ 1: 10.

Further, in the above method for preparing intermediate compound 2, the reaction temperature is performed at -20 ° C to 250 ° C; the organic solvent is selected from the group consisting of an ether, alcohol, haloalkane, amidine, nitrile containing 1-20 carbon atoms Or a mixture thereof, or a mixture with water in various proportions; the catalyst is palladium carbon, palladium hydroxide, dry or wet. In the above preparation method, the molar ratio of the reaction between the intermediate compound 2 and the catalyst is 1: 0.1 to 1:10.

Further, in the above method for preparing intermediate compound 3, the above reaction temperature is performed at -20 ° C to 125 ° C; the organic solvent is selected from the group consisting of an ether, an alcohol, an alkane, an aromatic hydrocarbon, a ketone, Haloalkane, amidine, nitrile, ester, or mixture thereof; the catalyst is 1-hydroxybenzotriazole (HOBT); and the condensing agent is 1-ethyl-3- (3-dimethylaminepropyl) carbodicarbonate Any one or more of imine hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) or 4-dimethylaminopyridine (DMAP). In this step, the molar ratio of Linifanib or its derivative to intermediate compound 2 is 1: 1 to 1:10, and the molar ratio of Linifanib or its derivative to the condensing agent is 1: 0.1 to 1:10; The molar ratio is 1: 0.1 ~ 1: 10.

Further, in the above method for preparing a compound of formula 1, the reaction temperature is performed at -20 ° C to 125 ° C; the organic solvent is an ether, alcohol, alkane, aromatic hydrocarbon, ketone, haloalkane containing 1-20 carbon atoms , Amidine, nitrile, ester, or a mixture thereof in various ratios; the acidic reagents are formic acid, acetic acid, and trifluoroacetic acid. In the above preparation method, the molar ratio of the intermediate compound 3 to the acidic reagent is 1: 1 to 1:10.

As another aspect of the present application, the present application provides a pharmaceutical composition comprising the compound of formula I described above or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, or prodrug thereof, And pharmaceutically acceptable carriers. The pharmaceutical composition includes, but is not limited to, oral dosage forms, parenteral dosage forms, external dosage forms, and rectal dosage forms. In some embodiments, the pharmaceutical composition may be oral tablets, capsules, pills, powders, sustained release preparations, solutions and suspensions, sterile solutions, suspensions or emulsions for parenteral injection, with Ointments or creams for external use, or suppositories for rectal administration. In some embodiments, the pharmaceutical composition and the at least one therapeutic agent are combined in separate dosage forms into a combined product, such as a kit.

As another aspect of the present application, the present application provides the compound of formula I, pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs thereof in the preparation of a medicament with anticancer effect. Applications. The cancer includes 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, vagina Cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer, melanoma, etc. Preferably, the effect is the best for liver cancer.

As another aspect of the present application, the present application provides a method for treating cancer, which method comprises treating a therapeutically effective amount of the compound of formula I, a pharmaceutically acceptable salt thereof, stereoisomers, solvates, polymorphs Administered to the individual in need thereof. In some embodiments, the cancer includes esophageal cancer, endometrial cancer, malignant lymphoma, multiple myeloma, gastrointestinal stromal tumor, colon cancer, rectal cancer, breast cancer, liver cancer, gastric cancer, ovarian cancer, uterus Cancer, cervical cancer, vaginal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, brain cancer, melanoma, etc. Preferably, the effect is the best for liver cancer.

"Pharmaceutically acceptable salt" as used herein refers to a salt that retains the biological potency of the free acid and free base of the specified compound and has no adverse effects biologically or otherwise. The salt in the present application refers to an acidic salt formed using an organic acid / inorganic acid, and a basic salt formed using an organic base / inorganic base.

The “solvate” described herein refers to a combination of a compound of the present application and a solvent molecule formed by solvation. Such as hydrate, ethanol solvate, methanol solvate and so on.

The "polymorphs" or "polymorphs" mentioned herein refer to the compounds of the present application in different lattice forms.

The “stereoisomers” mentioned in the present application refer to isomers produced by different arrangement of atoms in a molecule in space.

The "pharmaceutical composition" mentioned in the present application refers to a biologically active compound optionally mixed with at least one pharmaceutically acceptable chemical component, and the pharmaceutically acceptable chemical component includes, but is not limited to, a carrier, a stabilizer, and a diluent. , Dispersants, suspending agents, thickeners and / or excipients. The "carrier" refers to a relatively non-toxic chemical agent that facilitates the introduction of a compound into a cell or tissue.

The "alkyl" mentioned in this application refers to a straight or branched saturated hydrocarbon chain containing 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-dimethylpentyl, n-heptyl, n-octyl, n-nonyl and n-decyl.

The "aryl" refers to an aromatic carbocyclic group containing 6 to 14 carbon ring atoms. Aryl may be monocyclic or polycyclic. In the case of polycyclic aromatic rings, only one ring in the polycyclic ring system needs to be unsaturated, and the remaining one or more rings may be saturated, partially saturated, or unsaturated. Examples of aryl include phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl.

The "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. Specifically, the "heteroaryl group" refers to an aromatic heterocyclic group containing 5 to 14 ring atoms. Heteroaryl groups can be monocyclic or 2 or 3 fused rings. Examples of 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 substituents 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, benzoisoxazolyl, benzooxazole Base, imidazolyl, indolyl, benzimidazolyl, pyrrolo [2,3-b] pyridyl, purinyl; and 6/6 membered fused rings such as benzopyranyl, quinolinyl, isopropyl Quinolinyl, perylene, quinazolinyl and benzoxazinyl.

The "cycloalkenyl" refers to a monocyclic or bridged hydrocarbon ring system. A monocyclic cycloalkenyl has 4, 5, 6, 7, or 8 carbon atoms and 0 heteroatoms. A four-membered ring system has one double bond, a five- or six-membered ring system has one or two double bonds, and a 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.

The "heterocycloalkyl" refers to a saturated ring structure containing a total of 3 to 14 ring atoms. At least one of the ring atoms is a heteroatom (ie, oxygen, nitrogen, or sulfur), and the remaining ring atoms are independently selected from carbon, oxygen, nitrogen, and sulfur. Such as tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolyl , Triazolyl, tetrazolyl.

By "effective amount" is meant a non-toxic, but sufficient amount of a drug or agent that provides the desired effect. In the pharmaceutical composition or kit of the present invention, an "effective amount" of an ingredient or a preparation unit means an amount of the ingredient that is effective to provide a desired effect when used in combination with other ingredients. The "effective amount" will vary Subjects vary, depending on age and general conditions of the individual, specific active drugs, etc. Therefore, it is not always possible to refer to an exact "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.

The "subject" may refer to a patient or other animals, particularly mammals, such as humans, dogs, monkeys, animals, etc. who receive a compound or pharmaceutical composition of the present invention to treat, prevent, alleviate and / or alleviate the diseases of the present invention. Cattle, horses, etc.

For in vitro experiments, the present application also synthesizes a compound of formula I, a metabolite of formula II, the reaction route of which is: First, Linifanib or its derivative reacts with L (reactant 3) with Boc protection under the conditions of a condensing agent and a catalyst Intermediate compound Ma is formed, and the intermediate compound Ma is deprotected from Boc under the action of trifluoroacetic acid to form intermediate compound Mb. The intermediate compound Mb is further condensed with aspartic acid with a protective group to obtain intermediate compound Mc. The intermediate compound Mc is removed from the Boc protection under the condition of trifluoroacetic acid to generate the intermediate compound Md, and Md is catalyzed by hydrogenation to remove the benzyl group under the condition of a noble metal catalyst to obtain a metabolite compound of formula 2.

Reaction Roadmap:

Where X is selected from O, S and NR ₉ ; 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 , where e and f are independently 0 or 1, wherein each group is a ring substituted by R ₃ and R _{4 at} its left end; L is-[C _m (O) (Z) _n (NH) _q ]-, where m, q 0 or 1, respectively, n is 0 to 11, p is 0 to 8; Z is selected from -CR _10- , -CR ₁₀ -O-CR _10- , -SS-, -CR ₁₀ = CR _10- , -CR _10≡ CR _10- , -Ar, -CO-NH- and -N = CR ₁₀ -any one or several groups are connected in a conventional manner; R ₁ and R _{2 are} independently selected from hydrogen, alkoxy , Alkoxyalkoxy, alkoxyalkyl, alkyl, aryloxy, aryloxyalkyl, halo, haloalkoxy, haloalkyl, heterocyclyl, heterocyclylalkenyl, heterocyclyl Alkoxy, heterocyclylalkyl, heterocyclyloxyalkyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, (NR _a R _b ) alkoxy, (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 hydrogen, alkoxy, alkyl, halo, haloalkoxy , Haloalkyl and hydroxy; R ₅ and R _{6 are} independently selected from hydrogen, alkoxy, alkoxyalkyl, Alkoxycarbonyl, alkyl, aryloxy, arylalkyl, carboxy, cyano, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, nitro, and -NR _c R _d ; R ₇ and R _{8 is} independently selected from hydrogen and alkyl; R _{9 is} selected from hydrogen, alkenyl, alkoxyalkyl, alkyl, alkoxycarbonyl, aryl, heterocyclylalkyl, hydroxyalkyl, and (NR _a R _b) alkyl; R ₁₀ is selected from hydrogen, alkyl, alkoxy, aryloxy, alkenyloxy, nitro, halo, primary amino, secondary amino and tertiary amino group; R _a and R _b are Independently selected from hydrogen, alkyl, alkylcarbonyl, alkylsulfonyl, arylsulfonyl, haloalkylsulfonyl, and heterocyclylsulfonyl; _Rc and _{Rd are} independently selected from hydrogen, alkyl, Alkylcarbonyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl.

Specifically, the structure of each metabolite is as follows:

The research of the present invention shows that the compound of formula I has inhibitory effect on a variety of cancer cells, and can be biologically transformed into the active drug Linifanib in plasma in vitro, and can inhibit the proliferation of tumor cells, especially liver cancer cells, at a lower dose.

detailed description Examples 1 to 4: Preparation of target compound 8 (Linifanib-C ₁₂ -AA ₅ ) Example 1 Preparation of Intermediate Compound 1

Weigh 404 mg (1.18 mmol) of benzyl- (12-amino) dodecanoate hydrochloride, 238 mg (1.76 mmol) of HOBT, 192 mg (1.76 mmol) of EDCI and dissolve in 250 ml of dichloromethane, and stir to dissolve at room temperature. Control the reaction temperature from 20 to 40 ° C and slowly add Asp (Boc) -Glu (OtBu) -Glu (OtBu) -Glu (OtBu) -Glu (OtBu)-(OtBu) 1267mg (1.23mmol). After the addition, maintain the reaction temperature. The reaction was stirred for 4 h. TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. 100 ml of dichloromethane was added to the reaction solution for dilution, and the reaction solution was washed twice with 250 ml of deionized water to separate an organic phase. The organic phase was washed with 150 ml of saturated saline and separated, and the organic phase was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a brown oil. The oil The material was subjected to silica gel column chromatography (petroleum ether / acetone = 10: 1 to 2: 1) to obtain 553 mg of a yellow solid powder with a yield of 35.6%.

Example 2 Preparation of Intermediate Compound 2

14000 mg (3.0 mmol) of the intermediate compound prepared in Example 1 was weighed, dissolved in 100 ml of anhydrous methanol, 10% Pd / C 50 mg was added under the protection of nitrogen, and hydrogen was substituted for 3 times, and the pressure was controlled to 2 MPa in a hydrogen atmosphere. The reaction was carried out at 20 ~ 65 ℃ for 6 ~ 12h. TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. The reaction solution was filtered under a nitrogen protective condition to recover palladium on carbon. The filtrate was concentrated at low temperature to give a yellow-brown oil. This oil was subjected to preparative chromatography to obtain 1595 mg of a pale yellow solid powder with a yield of 42.8%. ¹ H NMR (CDCl ₃ ) δ 1.27 (brs, 14H), 1.46 to 1.47 (m, 54H), 1.65 to 1.85 (m, 8H), 2.34 to 2.35 (brs, 16H), 3.06 to 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). 13 C NMR (CDCl 3) δ 192.97,190.34,173.02,172.22,172.00, 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, 28.71, 28.48, 28.33, 28.10, 28.01, 27.98, 27.76, 27.65, 26.68, 24.61, 12.10.

The structural formula is:

Example 3 Preparation of Intermediate Compound 3

Weigh 1-N Boc Linifanib 760mg (1.6mmol), HOBT 324mg (2.4mmol), EDCI 460mg (2.4mmol) dissolved in 250ml dichloromethane, stir the reaction for 0.5h, control the reaction temperature 20 ~ 40 ℃ slowly add the example 2340 mg (1.9 mmol) of the intermediate compound 2 prepared in 2 was added at the end, and 516 mg (4.0 mmol) of DIPEA was added. The reaction temperature was stirred for 12 h while maintaining the reaction temperature. TLC (DCM / MeOH = 40: 1) was used to detect the reaction was complete. 100 ml of dichloromethane was added to the reaction solution for dilution, and the reaction solution was washed twice with 250 ml of deionized water to separate an organic phase. Organic phase reuse 150 It was washed with ml of saturated brine, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a brown oil. This oil was subjected to silica gel column chromatography (DCM: MeOH = 0: 1 to 100: 1) to obtain 882 mg of an off-white solid powder with a yield of 32.7%.

Example 4 Target Compound 8 (Linifanib-C ₁₂ -AA ₅ ) Preparation

1062 mg (0.63 mmol) of the intermediate compound 3 prepared in Example 3 was weighed and dissolved in 60 ml of dichloromethane, and the reaction temperature was controlled at -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added, and the reaction was stirred while maintaining the reaction temperature 20 ~ 24h, TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. 40 ml of dichloromethane was added to the reaction solution for dilution, and then washed twice with 120 ml of deionized water, twice with 60 ml of a 5% sodium bicarbonate solution, and then twice with 120 ml of deionized water. The organic phase was separated, and the organic phase was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a red-brown oil. This oil was subjected to preparative chromatography to obtain 240 mg of an off-white solid powder with a yield of 31.7%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.94 (s, 1H), 9.40 (s, 1H), 9.22 (s, 1H), 8.67 (s, 1H), 8.55 (s, 1H), 8.16 ( s, 1H), 8.04-8.07 (m, 4H), 7.44-7.48 (m, 3H), 7.33-7.40 (m, 3H), 7.10 (m, 1H), 6.98-6.99 (m, 1H), 6.995 ( m, 1H), 4.17 (m, 6H), 2.99 (m, 5H), 2.55 (m, 4H), 1.76-2.27 (m, 25H), 1.13-1.32 (m, 22H). HPLC purity: 99.831% ( 214nm), 98.242% (254nm) .MS (ESI): m / z 1204.5 [M + 1] ⁺

The structural formula is:

Examples 5-9: Preparation of the target compound 12 (Linifanib-C ₁₂ -Asp) Example 5 Preparation of Metabolite Intermediate Ma

Weigh 186 mg (0.59 mmol) of 12- (Boc-amino) dodecanoic acid, 107 mg (0.8 mmol) of HOBT, 152 mg (0.8 mmol) of EDCI in 10 ml of dichloromethane, stir the reaction for 0.5 h, and control the reaction temperature 20 ~ Slowly add 1-N Boc Linifanib 252mg (0.53mmol) at 40 ° C. Then 171 mg (1.3 mmol) of DIPEA was added. After the addition was complete, the reaction was stirred at the reaction temperature for 4 h. TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. 50 ml of dichloromethane was added to the reaction solution for dilution, and the mixture was washed twice with 100 ml of deionized water. The organic phase was separated and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a brown oil. This oil was subjected to silica gel column chromatography (DCM: MeOH = 1: 0 to 30: 1) to obtain 173 mg of a yellow oil with a yield of 42.2%.

Example 6 Preparation of metabolite intermediate Mb

255 mg (0.33 mmol) of the intermediate Ma prepared in Example 5 was weighed and dissolved in 20 ml of dichloromethane, and the reaction temperature was controlled at -5 to 5 ° C, and 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added, and the reaction temperature was maintained while stirring for 1.5 to 2h, TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. 50 ml of dichloromethane was added to the reaction solution for dilution, and then washed twice with 120 ml of deionized water, twice with 60 ml of a 5% sodium bicarbonate solution, and twice with 120 ml of deionized water. The organic phase was separated, and the organic phase was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a red-brown oil. This oil was subjected to preparative chromatography to obtain 155 mg of a yellow oil with a yield of 82%.

Example 7 Preparation of Metabolite Intermediate Mc

Weigh 137 mg (0.42 mmol) of Boc-L-aspartic acid 1-benzyl ester, 77.8 mg (0.58 mmol) of HOBT, 110 mg (0.58 mmol) of EDCI in 10 ml of dichloromethane, stir the reaction for 0.5 h, and control the reaction temperature 220 mg (0.38 mmol) of the intermediate Mb prepared in Example 6 was slowly added at 20-40 ° C. Finally, 124 mg (0.96 mmol) of DIPEA was added. After the addition was completed, the reaction was stirred and maintained for 4 h while maintaining the reaction temperature. TLC (DCM / MeOH = 40: 1) ) The detection reaction is complete. 50 ml of dichloromethane was added to the reaction solution for dilution, and the mixture was washed twice with 100 ml of deionized water. The organic phase was separated and dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a brown oil. This oil was subjected to silica gel column chromatography (DCM: MeOH = 1: 0 to 30: 1) to obtain 183 mg of a yellow oil with a yield of 54.9%.

Example 8 Preparation of metabolite intermediate Md

250 mg (0.29 mmol) of the intermediate compound Mc prepared in Example 7 was weighed and dissolved in 20 ml of dichloromethane, and the reaction temperature was controlled at -5 to 5 ° C. 3 ml (0.04 mmol) of trifluoroacetic acid was slowly added, and the reaction temperature was maintained at 1.5 with stirring. ~ 2h, TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. 50 ml of dichloromethane was added to the reaction solution for dilution, and then washed twice with 120 ml of deionized water, twice with 60 ml of a 5% sodium bicarbonate solution, and twice with 120 ml of deionized water. Separate The organic phase was dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated at low temperature to obtain a yellow oil. This oil was subjected to preparative chromatography to obtain 153 mg of a yellow oil with a yield of 67.6%.

Example 9 Target Compound 12 (Linifanib-C ₁₂ -Asp) Preparation

210 mg (0.27 mmol) of the intermediate Md prepared in Example 8 was weighed, dissolved in 30 ml of anhydrous methanol, and 10% Pd / C 25 mg was added under the protection of nitrogen. Hydrogen was substituted for 3 times, and 2 MPa was controlled under a hydrogen atmosphere. The reaction was carried out at 20 ~ 65 ℃ for 6 ~ 12h. TLC (DCM / MeOH = 40: 1) showed that the reaction was complete. The reaction solution was filtered under a nitrogen protective condition to recover palladium on carbon. The filtrate was concentrated at low temperature to give a pale yellow oil. This oil was subjected to preparative chromatography to obtain 91 mg of a white solid powder in a yield of 49.2%. ¹ H-NMR (DMSO) δ: 1.109-1.127 (m, 16H), 1.234-1.352 (m, 2H), 1.902-1.920 (m, 2H), 2.278 (s, 3H), 2.642-2.741 (m, 2H ), 2.993-3.040 (m, 2H), 3.740-3, 806 (m, 1H), 6.796-6.808 (m, 1H), 6.971-6.988 (m, 1H), 7.089-7.118 (m, 1H), 7.320-7.407 (m, 3H), 7.453-7.505 (m, 3H), 8.022-8.042 (d, J = 8Hz, 1H), 8.121-8.148 (t, J = 5.6Hz, 1H), 8.524 (s, 1H), 9.219 (s, 1H), 9.424 (s, 1H), 12.956 (s, 1H) .HPLC purity: 96.6% (214nm), 99.9% (254nm) .MS (ESI): m / z 688.4 [M + 1] +

The structural formula is:

The "precursor" described in the following Examples 10-11 is Linifanib-C ₁₂ -AA ₅ (Compound 8) prepared in Examples 1 to 4, and the "intermediate" is the Linifanib- prepared in Examples 5-9. C ₁₂ -Asp (compound 12), the "active drug" is Linifanib, and the metabolic pathway is Linifanib-C ₁₂ -AA ₅ → Linifanib-C ₁₂ -Asp → Linifanib.

Example 10 Effect of Linifanib Related Compounds on the Proliferation of Tumor Cell Lines

In the present application, three compounds (precursor Linifanib-C ₁₂ -AA ₅ , intermediate Linifanib-C ₁₂ -Asp, and active drug Linifanib) were assayed in 54 commercial tumor cell lines (including Inhibition concentration (IC ₅₀ value) on 26 hepatocellular carcinoma cell lines), and the difference between the activity of the precursor and the active drug Linifanib was examined.

Instruments and materials

Thermo 311 CO ₂ incubator; Haier biosafety cabinet; Molecular Devices microplate reader; Xiangyi L530 desktop low-speed centrifuge; Olympus IX51 inverted fluorescence microscope, DMEM, RPMI 1640, MEM, DMEM / F12 1: 1 Medium, fetal calf serum, 0.25% trypsin solution, phosphate buffered saline (Thermo Fisher Scientific Shanghai Co., Ltd.); sigma dimethylsulfine (DMSO), resazurin; 54 commercial tumor cell lines (containing 26 Strain of hepatocellular carcinoma).

Experimental drugs: the precursor Linifanib-C ₁₂ -AA ₅ (compound 8); the intermediate Linifanib-C ₁₂ -Asp (compound 12); the active drug Linifanib; the chemotherapeutic drug Doxorubicin (HY-15142; Shanghai Haoyuan) Biomedical Technology Co., Ltd.).

2. Experimental method

2.1 Culture of different cell lines

Fifty-four cell lines were cultured in a culture solution containing fetal bovine serum, and incubated in a 37 ° C, 5% CO ₂ incubator. Cells were grown in adherent state, and the growth condition was observed under an inverted microscope. Subculture was performed when the cell confluence reached 80% -90%. The proportion and number of passages are subject to the experimental requirements. The subculture ratio of this cell line is generally 1: 2 ~ 1: 3.

2.2 Inhibition of proliferation of different tumor cell lines

Cell test: Take 54 cell lines in the logarithmic growth phase, and inoculate the cells in a 96-well culture plate with 500 ~ 1 × 10 ⁴ / well (the optimal seeding density of each cell line is determined in the pre-experiment). After incubation at 37 ° C for 4 hours in a humidified CO ₂ incubator, 10 μL of the precursor Linifanib-C ₁₂ -AA ₅ , the intermediate Linifanib-C ₁₂ -Asp and the active drug Linifanib were added to each well. Each compound was tested for 9 drug concentrations Gradient (diluted 3.16 times from the highest test concentration). The initial concentration is 100 or 30 μM, depending on the solubility of each compound. The QC reference compound Doxorubicin was added simultaneously during the test of each cell line, and the final drug concentrations were 10, 3.16, 1, 0.31, 0.1, 0.03, 0.01, 0.003, and 0.001 μM, respectively. In addition, a positive control group (100% inhibition) and a negative control group (0% inhibition) are set at the same time. Each concentration of the drug group is repeated for 2 wells, and the positive control group and the negative control group are repeated for 6 wells. AlamarBlue test operation;

AlamarBlue test operation: Add 10 μL AlamarBlue reagent to each well and incubate for 1-4 hours, shake for 1-2 minutes, measure the fluorescence value at the MD microplate reader EX: 560nm, EM: 590nm, record the results, and calculate the cell inhibition rate of the compound of the present invention (%) = (A _{0% inhibition-} A _{administration} ) / (A _{0% inhibition-} A _{100% inhibition} ) × 100%, and then use GraphPad Prism 5.0 or MATILAB software to adopt non-linear regression method (four-parameter simulation is often used) plotting curve equation co) dose response curves obtained, to thereby obtain IC ₅₀ values of the compounds to act on the cell line of the present invention.

3. Results and analysis

3.1 The IC ₅₀ summary results of 3 test samples (compounds 8, 12 and Linifanib) on 54 commercial tumor cell lines are shown in Table 1.

Note: Cell lines 29-54 in the table indicate the response of liver cancer cell lines to each compound

From the results in Table 1, it can be seen that compounds 8 and 12 successfully blocked Linifanib's activity to inhibit tumor cell proliferation. Most of the tumor cells of these two compounds and an IC ₅₀ of Linifanib difference more than 5 times. There are 3 strains sensitive to Linifanib, namely KASUMI-1 (leukemia cells), NCI-H1703 (lung cancer cells) and AN3-CA (endometrial tumor cells), with IC50 of 0.01, 0.02 and 0.19 μM, respectively. The IC ₅₀ for the precursor Linifanib-C ₁₂ -AA ₅ (Compound 8) was 0.39, 0.19, and 5.68 μM, respectively, with differences of 39, 8.5, and 29.9 times, respectively.

Of the 54 commercial tumor cell lines, 26 are hepatocellular carcinoma cell lines. Nearly half of the hepatocellular carcinoma cell lines (12/26, 46%) are moderately sensitive to Linifanib, with an IC50 of <5 μM. At the same time, most hepatoma cell lines (23 / 26, 88%) The IC50 of the precursor Linifanib-C ₁₂ -AA ₅ (Compound 8) and Linifanib is more than 8 times, and almost all liver cancer cells have no obvious response to the intermediate (Compound 12). See Table 1 .

Example 11 Plasma Stability Study

The purpose of this example is to investigate the incubation stability of compound 8 in rat plasma (compound 8 is metabolized to form intermediate compound 12 and Linifanib, and intermediate compound 12 is further metabolized to form Linifanib), and quantitative analysis of the metabolites is performed with positive The stability of the incubation system is verified by drugs, which provides a reference for the evaluation of compound drugability.

1 instruments and materials

Instrument: API3000 LC / MS, ABI

Materials: Male SD rats (200-250g), Beijing Weitonglihua

Test article: positive drug and its metabolites M1 and M2, compound 8 and its metabolite compounds 12 and Linifanib.

2 Compound 8 plasma stability study

Test animal

Species: SD rats; Number: 2

Gender: Male; Weight: 200-250g;

Experimental steps

1 Blood was collected from an animal, and the blood sample was placed in an EDTA anticoagulation tube, and separated at 3000 g at 4 ° C. Heart for 15min, plasma was separated, 2 blood samples were mixed in equal volume;

2 Weigh out a certain amount of compound Compound 8 dissolved in DMSO: MeOH (2: 8), prepare a 200 μM mother liquor by purity conversion, add the compound to the plasma to make the final concentration 1 μg / mL, and the organic ratio in the system should not exceed 0.5 %.

3 Incubate at 37 ° C in a water bath. Set the sampling points to 0, 0.5, 1, 2, 4, 6, 8h. At each sampling point, 100 μL of sample was added to 300 μL of acetonitrile (including internal standard) for precipitation treatment, centrifuged at 12000 rpm for 5 minutes, and 200 μL of the supernatant was taken for analysis by LC-MS / MS.

4 Configure a standard curve for quantitative detection of Compound 8, Compound 12 and Linifanib

3 Results of plasma stability studies

The positive drug is expected to be metabolized to its metabolites in plasma over time, which indicates that the plasma system is stable and the subsequent test results are reliable.

The results of the plasma stability of the test compound 8 are shown in Table 2.

It can be known from Table 2 that Compound 8 is relatively stable in incubation in plasma, and the amount of intermediate compound 12 and Linifanib produced is very small, and its peak molar concentration is only equivalent to 1/100 and 1/1000 of Compound 8.

From the results of the above plasma stability studies, it can be seen that the stability of Compound 8 in plasma is very good, and the amount of Compound 12 and Linifanib produced by metabolism is very small.

Claims (10)

A compound having the structure of formula I, a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof, Where X is selected from O, S and NR ₉ ; 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 , where e and f are independently 0 or 1, wherein each group is a ring substituted by R ₃ and R _{4 at} its left end; L is-[C _m (O) (Z) _n (NH) _q ]-, where m, q 0 or 1, respectively, n is 0 to 11, p is 0 to 8; Z is selected from -CR _10- , -CR ₁₀ -O-CR _10- , -SS-, -CR ₁₀ = CR _10- , -CR _10≡ CR _10- , -Ar, -CO-NH- and -N = CR ₁₀ -any one or several groups are connected in a conventional manner; R ₁ and R _{2 are} independently selected from hydrogen, alkoxy Alkyl, alkoxyalkoxy, alkoxyalkyl, alkyl, aryloxy, aryloxyalkyl, halo, haloalkoxy, haloalkyl, heterocyclyl, heterocyclylalkenyl, heterocyclic Alkoxy, heterocyclylalkyl, heterocyclyloxyalkyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, (NR _a R _b ) alkoxy, (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 hydrogen, alkoxy, alkyl, halo, haloalkoxy , haloalkyl and hydroxy; R ₅ and R _{6 are} independently selected from hydrogen, alkoxy, alkoxyalkyl Alkoxycarbonyl, alkyl, aryloxy, arylalkyl, carboxy, cyano, halo, haloalkoxy, haloalkyl, hydroxy, hydroxyalkyl, nitro, and -NR _c R _d; R ₇ and R _{8 is} independently selected from hydrogen and alkyl; R _{9 is} selected from hydrogen, alkenyl, alkoxyalkyl, alkyl, alkoxycarbonyl, aryl, heterocyclylalkyl, hydroxyalkyl, and (NR _a R _b ) alkyl; R _{10 is} selected from hydrogen, alkyl, alkoxy, aryloxy, alkenyloxy, nitro, halo, primary amine, secondary amine, and tertiary amine; R _{11 is} selected from hydrogen , a hydroxyl group, an amino group, an alkenyl group, an alkynyl group, an alkoxy group, an alkoxy group, an alkoxyalkyl group, an alkyl group, an alkoxycarbonyl group, an aryl group, a heterocyclic group; R _a and R _{b are} independently selected from hydrogen , Alkyl, alkylcarbonyl, alkylsulfonyl, arylsulfonyl, haloalkylsulfonyl and heterocyclylsulfonyl; _Rc and _{Rd are} independently selected from hydrogen, alkyl, alkylcarbonyl, Aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl. The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof, having the structural formula: or, or, or, or, or, or, or, or, The method for preparing a compound according to claim 1 or 2, a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof, wherein the method comprises the following steps; step a, the polypeptide React with benzyl-protected L in the presence of a catalyst and a condensing agent to obtain an intermediate compound 1 with a protective group; step b, the intermediate compound 1 is subjected to catalytic hydrogenation in a polar solvent to remove the protective group Group to obtain intermediate compound 2; step c, intermediate compound 2 reacts with Linifanib or its derivative in the presence of a catalyst and a condensing agent to obtain intermediate compound 3 with a protecting group; step d, the intermediate Compound 3 is protected under acidic conditions to give a compound of formula I. The preparation method according to claim 3, wherein the reaction temperature in step a is -20 ° C to 125 ° C; the catalyst is 1-hydroxybenzotriazole; and the condensation agent is 1-ethyl Any one or more of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1,3-dicyclohexylcarbodiimide, or 4-dimethylaminopyridine. The preparation method according to claim 3, wherein the reaction temperature in step b is -20 ° C to 125 ° C; and the catalyst is palladium carbon and palladium hydroxide is dry or wet. The preparation method according to claim 3, wherein the reaction temperature in step c is -20 ° C to 125 ° C; the catalyst is 1-hydroxybenzotriazole; and the condensation agent is 1-ethyl Any one or more of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1,3-dicyclohexylcarbodiimide, or 4-dimethylaminopyridine. The preparation method according to claim 3, wherein the reaction temperature in step d The temperature is -20 ° C to 125 ° C; the acidic reagents are formic acid, acetic acid, and trifluoroacetic acid. Application of the compound according to claim 1 or 2, its pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs in the preparation of a medicament with an anticancer effect; the cancer includes esophageal cancer, uterus 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, and melanoma. The application according to claim 8, wherein the cancer is liver cancer. A method for treating cancer, comprising administering to a subject a therapeutically effective amount of a compound according to claim 1 or 2, a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.