WO2019228299A1 - 一种肽硼酸酯类化合物的合成与用途 - Google Patents

一种肽硼酸酯类化合物的合成与用途 Download PDF

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WO2019228299A1
WO2019228299A1 PCT/CN2019/088506 CN2019088506W WO2019228299A1 WO 2019228299 A1 WO2019228299 A1 WO 2019228299A1 CN 2019088506 W CN2019088506 W CN 2019088506W WO 2019228299 A1 WO2019228299 A1 WO 2019228299A1
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compound
borate
alkyl
peptide
cancer
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PCT/CN2019/088506
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English (en)
French (fr)
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秦艳茹
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江苏正大丰海制药有限公司
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Priority to AU2019277933A priority Critical patent/AU2019277933B2/en
Priority to CA3101824A priority patent/CA3101824C/en
Priority to JP2021517089A priority patent/JP7314259B2/ja
Priority to CN201980036098.7A priority patent/CN112384519B/zh
Priority to CN202211551468.0A priority patent/CN115785137A/zh
Priority to KR1020207036576A priority patent/KR102558265B1/ko
Priority to US17/059,066 priority patent/US11542283B2/en
Priority to EP19811456.3A priority patent/EP3805237A4/en
Publication of WO2019228299A1 publication Critical patent/WO2019228299A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/04—Esters of boric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/027—Organoboranes and organoborohydrides
    • 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 belongs to the field of pharmaceutical synthesis, and particularly relates to a series of preparation methods of novel peptide borate compounds and their application in pharmacodynamics.
  • cancer is one of the most important diseases that endanger human health.
  • cancer treatment has made great progress in the fields of surgery, chemotherapy, radiation therapy, etc., it is not yet possible to fundamentally treat cancer.
  • the currently available anti-cancer drugs have certain effects, they have serious toxic and side effects. Therefore, in-depth discussion and research on how to study targeted new anticancer drugs from effective tumor targets has become a top priority for medical workers.
  • Ubiquitin-Proteasome Pathway is the main pathway for the degradation of intracellular protein systems and participates in many physiologically important cellular processes, including signal transduction, immune responses, unfolded protein responses, and the cell cycle progress. This pathway has important relations with cardiovascular and cerebrovascular diseases, cancer, and neurological degenerative diseases. The use of some effective inhibitors to inhibit the excessive degradation of important proteins in this pathway will provide new ideas for the treatment of these diseases.
  • PS-341 the first proteasome inhibitor bortezomib
  • PS-341 was approved by the FDA in 2003 for the treatment of recurrent myeloma. In 2004, the drug was approved for marketing in the European Union and was used for multiple myeloma.
  • the purpose of the present invention is to synthesize a series of novel peptide borate compounds with novel structure and proteasome inhibitory function that can be taken orally.
  • 20S proteasome inhibitors they can effectively block tumor cell proliferation and induce tumor cell apoptosis, and thus they are used for the prevention and treatment of human and animal diseases such as malignant tumors.
  • the object of the present invention is to include a pharmaceutically acceptable carrier and a peptide borate compound of the present invention, optionally in combination with one or more other therapeutic agents simultaneously, separately or sequentially.
  • Another object of the present invention is to provide a use of a peptide borate compound in preparing a proteasome inhibitor.
  • the tumors of the present invention include solid tumors and hematomas, wherein the solid tumors are selected from non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cells Cancer, gastrointestinal stromal tumor, or nasopharyngeal carcinoma; hematoma is selected from leukemia, multiple myeloma, mantle cell lymphoma, or histiocytic lymphoma.
  • Another object of the present invention is to provide a method for preparing the above-mentioned peptide borate compounds.
  • the object of the present invention can be achieved by the following measures:
  • a peptide borate compound or a pharmaceutically acceptable salt thereof has a structure shown in Formula I,
  • R 1 is selected from C 1-10 alkyl, C 1-10 alkoxy, C 1-10 alkoxy C 1-10 alkyl, C 3-6 cycloalkyl, phenyl, naphthyl, tetrahydronaphthalene , 2,5-dichlorophenyl, or heterocyclyl, or optionally C 1 to 4 alkyl, C 1 to 4 alkoxy, C 1 to 4 cycloalkyl, halogen or halogenated C 1 to 4 alkyl substitution; R 1 is preferably C 1-10 alkyl, C 1-10 alkoxy, C 1-10 alkoxymethyl, C 1-10 alkoxyethyl, C 3-6 cycloalkyl, phenyl, 2,5-dichlorophenyl, pyrazinyl, pyridyl, naphthyl, tetrahydronaphthyl, oxazolyl or isoxazolyl, or optionally substituted C 1 ⁇ 4 -alky
  • R 1 is preferably:
  • R 3 , R 4 , R 5 and R 6 are independently selected from hydrogen, methyl, methoxy, ethyl, ethoxy, chlorine, bromine, fluorine or trifluoromethyl.
  • R 2 is selected from H, phenyl, methoxy, methylthio, cyclohexyl, 2,3-dihydro-1,4-benzodioxane, or is optionally selected from one or more C 1 to 4 Alkyl, C 1-4 alkoxy, nitro, halogen or trifluoromethyl.
  • B, Z 1 and Z 2 together form a heterocyclic group containing N, S or O, or B together with Z 1 and Z 2 forms a heterocyclic group containing O, and an oxygen atom is connected to the boron atom.
  • B, Z 1 and Z 2 together form a boric acid- ⁇ -pinene glycol ester, or B together with Z 1 and Z 2 forms a borate ester, and an oxygen atom is connected to the boron atom.
  • B, Z 1 and Z 2 together form a boric acid- ⁇ -pinene glycol ester, or B, Z 1 and Z 2 together form a diethanolamine borate, a citrate borate, a tartrate borate, Malic acid borate, ⁇ -hydroxy-glutaric acid borate, and other prodrugs such as glucose borate with the ortho-hydroxy structure of glucose.
  • R 1 and R 2 groups of the present invention means that the groups of R 1 and R 2 may be substituted by these groups or may not be substituted by these groups, that is, they are not limited to Cases in which these listed groups are substituted also include cases in which they are not substituted.
  • R 1 is substituted or unsubstituted C 1-10 alkyl, C 3-6 cycloalkyl or heterocycloalkyl, phenyl, naphthyl or indolyl, where the substituent is "C 1-4 alkyl, C 1-4 alkoxy, cyano, hydroxy, mercapto, amino, or halogen" are expressed in the same way, but the definition of substitution or non-substitution is not limited to C 1-10 Alkyl, but extended to all said groups, that is, including substituted or unsubstituted C 3 to 6 cycloalkyl or heterocycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted naphthyl group, a substituted or unsubstituted indole group and the like, wherein the substituent is C 1-4 alkyl, C 1-4 alkoxy, cyano, hydroxy, mercapto, amino or halogen.
  • alkyl is used to indicate a saturated hydrocarbon group
  • a C 1-10 alkyl group refers to a saturated hydrocarbon group containing 1 to 10 carbon atoms
  • a C 1 to 4 alkyl group refers to a saturated hydrocarbon group containing 1 to 4 carbon atoms.
  • cycloalkyl refers to non-aromatic carbocyclic groups, including cyclized alkyl groups. Cycloalkyl can include bicyclic or polycyclic systems. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a C 3 to 6 cycloalkyl group means a cycloalkyl group having 3 to 6 carbon atoms.
  • benzyl refers to benzyl, substituted benzyl means that at least one hydrogen atom on the benzene ring of benzyl is substituted with a non-hydrogen moiety.
  • the substituent of benzyl may be halogen, -CN, -OH,- SH, -NH 2, 1-6 carbon straight-chain or branched alkyl, substituted linear or branched alkyl group having 1-6 carbon atoms.
  • heterocycloalkyl refers to non-aromatic heterocarbocyclic groups, including cyclized alkyl groups, in which one or more ring-forming carbon atoms are replaced with heteroatoms such as O, N or S atoms.
  • Heterocycloalkyl preferably has 3, 4, 5, 6, or 7 ring-forming atoms.
  • heterocyclyl refers to a cyclic group containing heteroatoms O, N or S, including aromatic heterocyclic groups or non-aromatic heterocyclic groups such as furan, thiophene, pyrrole, thiazole, imidazole, pyridine , Pyridazine, pyrimidine, pyrazine, quinoline, isoquinoline, indole, benzofuran, purine, acridine, oxazolyl, isoxazolyl, and the like.
  • Alkoxy refers to an -O-alkyl group, which generally has 1 to 10 carbon atoms. Examples of alkoxy include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy and the like.
  • Aryl refers to an aromatic carbocyclic group, including monocyclic or polycyclic aromatic hydrocarbons such as phenyl, naphthyl, anthracenyl, phenanthryl and the like.
  • Aryloxy refers to -O-aryl, and the concept of aryl is as described above. The most preferred example of aryloxy is phenoxy.
  • Halogen includes fluorine, chlorine, bromine and iodine.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof is selected from:
  • the compounds of the present invention can be used to prepare antitumor drugs.
  • the overall preparation route is:
  • each group R 1 , R 2 , Z 1 and Z 2 in this reaction formula are as described above.
  • the first route is phthalimide and ethyl chloroacetate to form formula (II-1), formula ( II-1) reacts with alanine to form formula (II-2), formula (II-2) reacts with 8-aminoquinoline to formula (II-3), and formula (II-3) reacts with aryl iodide to generate formula (II-4), formula (II-4) is reacted under the condition of boron trifluoride ether to form formula (II-5), and formula (II-5) is reacted under the condition of ethylenediamine to form formula (II); Formula (III-1) is reacted with methanol under the action of SOCl 2 to obtain formula (III).
  • R 1 , R 2 , Z 1 and Z 2 are as described above.
  • the method for preparing compound II includes the following steps:
  • a compound of the formula (II-2) first forms an acid chloride under the action of SOCl 2 and then forms a compound of the formula (II-3) with 8-aminoquinoline under basic conditions;
  • a compound of the formula (II-3) and an aryl iodide form a compound of the formula (II-4) under the conditions of palladium and silver tetrafluoroborate;
  • a compound of the formula (II-4) is formed with a compound of the formula (II-5) with methanol under the condition of boron trifluoride diethyl ether;
  • a compound of the formula (II-5) has a compound of the formula (II) with methanol under the condition of ethylenediamine.
  • the method for preparing compound (III) includes the following steps:
  • the method for preparing compound (I) includes the following steps:
  • a compound of the formula (I-1) undergoes a saponification reaction under basic conditions to form its sodium salt, and then generates an compound (I-2) under acidic conditions;
  • a compound of formula (IV) is generated under boiling ethyl acetate to form a compound of formula (I)
  • a common peptide condensing agent in the above reaction is N, N-dicyclohexyl-carbodiimide (abbreviated as DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (Abbreviated as EDC.HCl), 1-hydroxybenzotriazole (abbreviated as HOBt) or isobutyl chloroformate.
  • DCC N, N-dicyclohexyl-carbodiimide
  • EDC.HCl 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
  • HOBt 1-hydroxybenzotriazole
  • isobutyl chloroformate isobutyl chloroformate.
  • R 1 , R 2 , Z 1 and Z 2 are as described above.
  • the compounds of the present invention have good proteasome inhibitory activity and antitumor activity, and some compounds exhibit good proteasome inhibitory activity and antitumor effect at nanomolar level, and have the ability to prepare proteasome inhibitors or Application value of antitumor drugs.
  • the peptide borate compounds have better pharmacokinetic properties than the peptide borate compounds.
  • the compound preparation method designed by the present invention has high yield, simple process, and can be suitable for industrial production.
  • Figure 1 shows the growth trend of ARH-77 xenograft tumors after administration of different compounds
  • Figure 2 shows the results of proteasome activity detection in blood cells.
  • the compound of the present invention can be prepared according to the following process:
  • Phthalimide was dissolved in DMF, triethylamine was added, and ethyl chloroacetate was added dropwise to the reaction system at 0 ° C. After slowly rising to room temperature, the reaction was performed for 2 hours. The reaction solution was poured into ice water and filtered The filter cake was washed with ice water and dried under vacuum to obtain a pure compound of formula (II-1).
  • Compound II-3 is dissolved in tert-butanol, palladium acetate, silver tetrafluoroborate and alkyl iodide are added, and the mixture is refluxed for 24 hours. After returning to room temperature, it is diluted with CH 2 Cl 2 and triethylamine is added to react for 3 hours. After passing through diatomaceous earth, the solvent was distilled off under reduced pressure, and the compound II-4 was isolated by column chromatography.
  • the compound (III-1 was dissolved in MeOH, and SOCl 2 was added dropwise under an ice-salt bath. After the dropwise addition, the temperature was raised to room temperature overnight, and the pressure was reduced.
  • R 1 -COOH was dissolved in CH 2 Cl 2 , and 1-hydroxybenzotriazole (HOBt) was added. After reacting at -5 ° C for 10 minutes, a peptide condensing agent (EDC.HCl) was added, and the reaction was conducted for 20 minutes. Add compound II or III. After 10 minutes, add N, N-diisopropylethylamine (DIPEA). After half an hour of reaction, stir at room temperature overnight. The reaction solution was washed with acid (10% hydrochloric acid), alkaline (5% sodium bicarbonate) and saturated brine, and dried with desiccant (anhydrous sodium sulfate and anhydrous magnesium sulfate). The drying agent was filtered off, the solvent was evaporated to dryness under reduced pressure, and the compound I-1 was isolated by column chromatography.
  • HOBt 1-hydroxybenzotriazole
  • Phthalimide (7.36 g, 50 mmol) was dissolved in DMF (25 mL), triethylamine (9 mL, 65 mmol) was added, and ethyl chloroacetate (5.7 mL, 60 mmol) was added dropwise to the reaction system at 0 ° C. ), After slowly warming to room temperature, the reaction was detected by TLC, the reaction was completed after 2 hours, the reaction solution was poured into ice water, filtered, the filter cake was washed with ice water, and vacuum dried to obtain pure N-ethyl phthalimide Amine 8.67 g, yield 79.1%, mp 81.4-83.6 ° C.
  • II-4b Synthesis of II-3 and 1-iodo-3,5-bis (trifluoromethyl) benzene using the method of Example (1);
  • II-4c Synthesis of II-3 and 6-iodo-1, 4-Benzodioxane was synthesized by the method of Example (1);
  • II-4d II-3 and 2,4-dimethoxyiodobenzene were synthesized by the method of Example (1).
  • II-5b Synthesize II-4b by the method of Example (2)
  • II-5c Synthesize II-4c by the method of Example (2)
  • II-5d Adopt II-4d by Example (2) Method of synthesis.
  • IIb Synthesis of II-5b by the method of Example (3)
  • IIc Synthesis of I-5c by the method of Example (3)
  • IId Synthesis of II-5d by the method of Example (3).
  • hydrochloride salts of other amino acid methyl esters used in the present invention can be prepared by using the above steps.
  • Compound IIIb synthesized using D-cyclohexylglycine according to the method for synthesizing compound IIIa
  • compound IIIc synthesized using L-cyclohexylglycine according to the method for synthesizing compound IIIa
  • compound IIId synthesized using phenylalanine according to the method for synthesizing compound IIIa.
  • Compound I-1b Synthesized by 2.5-dichlorobenzoic acid and IIb according to EDC.HCl
  • Compound I-1c Synthesized by 2.5-dichlorobenzoic acid and IIc according to EDC.HCl
  • EDC.HCl condensation was synthesized using 2.5-dichlorobenzoic acid and IId.
  • the other amino-protected amino acid methyl esters used in the present invention can be prepared by the EDC.HCl condensation method described in Example (1). All the methyl esters have not been purified and are directly used in the next reaction.
  • Methoxyacetic acid (280 mg, 3.13 mmol) was dissolved in CH 2 Cl 2 (6 mL), and SOCl 2 (0.25 mL, 3.45 mmol) was added dropwise at -10 ° C. After the dropwise addition was completed, the temperature was raised to room temperature and reacted for 2h. The methoxyacetyl chloride solution was directly sent to the next step.
  • IIId (0.67 g, 3.13 mmol) was dissolved in 3 mL of CH 2 Cl 2 , triethylamine (1.58 g, 15.65 mmol) was added, and a methoxyacetyl chloride solution was added dropwise to react overnight. The reaction was detected by TLC, washed with water, dried over Na 2 SO 4 and separated by column chromatography to obtain 0.69 g of the target compound as an oil with a yield of 87.3%.
  • Compound I-1p Synthesized using 3-methoxypropionic acid and IIId as acid chloride
  • Compound I-1q Synthesized using butyric acid and IIId as acid chloride
  • Compound I-1r Cyclopropyl as acid chloride Synthesis of formic acid and IIId
  • compound I-1s synthesis using cyclopentylcarboxylic acid and IIId according to the acid chloride condensation method.
  • the other amino-protected amino acid methyl esters and alkyl acids used in the present invention can be prepared by the condensation method described in Example (2). All the methyl esters have not been purified and are directly used in the next reaction.
  • amino-protected amino acids used in the present invention can be prepared by the method described in Example 2.
  • Compound I-2b I-1b was synthesized by the method of Example 2; Compound I-2c: I-1c was synthesized by the method of Example 2; Compound I-2d: I-1d was synthesized by the method of Example 2 ; Compound I-2e: I-1e was synthesized by the method of Example 2; Compound I-2f: I-1f was synthesized by the method of Example 2; Compound I-2g: I-1g was adopted by the method of Example 2 Synthesis; Compound I-2h: Synthesis of I-1h using the method of Example 2; Compound I-2i: Synthesis of I-1i using the method of Example 2; Compound I-2j: Using I-1j of Example 2 Method Synthesis; Compound I-2k: I-1k was synthesized by the method of Example 2; Compound I-2l: I-1l was synthesized by the method of Example 2; Compound I-2m: I-1m was adopted by Example 2; Compound I-2n: I-1n was synthesized by the method of Example 2; Compound I
  • amino-protected amino acids used in the present invention can be prepared by the method described in Example 3.
  • boric acid compounds can be reacted with citric acid to form boric acid ester compounds for use as prodrugs.
  • the preparation method is described in the following example, but is not limited to this example:
  • the other diethanolamine borate and citrate borate prodrug compounds of the present invention can be synthesized by the methods of Examples 5 and 6. The specific compounds are shown in the following table.
  • the present invention uses a fluorescent polypeptide substrate Suc-Leu-Leu-Val-Tyr-AMC (abbreviation Suc-LLVY-AMC, Suc stands for succinyl and AMC stands for 7-amide-4-methylcoumarin) to determine the proteasome Chymotrypsin-like enzyme activity.
  • Suc-LLVY-AMC fluorescent polypeptide substrate
  • Suc stands for succinyl
  • AMC stands for 7-amide-4-methylcoumarin
  • the proteasome used in the present invention is a human red blood cell 20S proteasome, and the enzyme, fluorescent substrate, and test buffer were purchased from Enzo Company.
  • the experimental system is 16 ⁇ L, including 8 ⁇ L of substrate, 4 ⁇ L (0.8 ng) of proteasome, final concentration of 50 ⁇ M, 4 ⁇ L of drug (inhibitor), final concentration of 2 ⁇ 10 -6 M ⁇ 4.88 ⁇ 10 -10 M, the last concentration It is 0M.
  • the actual concentration is 8 ⁇ 10 -6 M ⁇ 1.95 ⁇ 10 -9 M.
  • the last concentration is 0M.
  • the specific experimental process is as follows:
  • the drug was weighed and dissolved in DMSO to a concentration of 10 -2 M. Pipette 2 ⁇ L to 98 ⁇ L DMSO to obtain 2 ⁇ 10 -4 M, then extract 8 ⁇ L from 2 ⁇ 10 -4 M concentration drug and add 198 ⁇ L H 2 O to obtain 8 ⁇ 10 -6 M. Use the same method Get 2 ⁇ 10 -6 M, 5 ⁇ 10 -7 M, 1.25 ⁇ 10 -7 M, 3.12 ⁇ 10 -8 M, 7.8 ⁇ 10 -9 M, 1.95 ⁇ 10 -9 M, and the last concentration is 0M For no medication.
  • 25mg of fluorescent peptide substrate was dissolved in 654 ⁇ L of DMSO to obtain a 50mM stock solution, which was stored at -20 ° C and diluted 500-fold during use. 8 ⁇ L was added to each sample so that the final substrate concentration in the reaction system was 50 ⁇ M.
  • the marketed drug Wan Ke is a positive control drug, which reacts at 37 ° C for 15 minutes. After the reaction was completed, 8 ⁇ L of fluorescent substrate was added to each well, and the reaction was performed at 37 ° C in the dark for 1 hour, and the fluorescence value was detected using a 360nm / 460nm fluorescence microplate reader (BMG PLALAB POLARstar OPTIMA Microplate Reader).
  • the detection solution used in the present invention is a single solution cell proliferation detection box from Promega Company; the cells used are U266, RPMI8226, ARH77.
  • the experimental system is 110uL, which contains 90 ⁇ L of cell suspension, 10 ⁇ L of detection solution, and 10 ⁇ L of drug (inhibitor).
  • the final concentration is 4.54 ⁇ 10 -8 M ⁇ 1.77 ⁇ 10 -9 M, the last concentration is 0M, the actual concentration It is 5 ⁇ 10 -7 M ⁇ 1.95 ⁇ 10 -8 M, and the last concentration is 0M.
  • the specific experimental process is as follows:
  • U266 was diluted to 1 ⁇ 10 4 cells / well, and RPMI8226 and ARH77 were both 1 ⁇ 10 4 cells / well.
  • the two groups were given IV-9 and V-9A by tail vein injection according to the following table, and were administered before and after 10min, 20min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and 36h.
  • Approximately 0.200 mL of blood was collected from the jugular vein, placed in a test tube containing EDTA-K2, centrifuged at 7800 ⁇ g for 15 minutes, and the plasma was separated and stored at -15 ° C to -35 ° C. It is used to compare the pharmacokinetics of IV-9 and V-9A intravenously.
  • the other two groups were given IV-9 and V-9A by gavage at the dosages shown in the table below, and before and 5min, 10min, 20min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and At 36 hours, approximately 0.200 mL of blood was collected from the jugular vein, placed in a test tube containing EDTA-K2, centrifuged at 7800 ⁇ g for 15 minutes, and the plasma was separated and stored at -15 ° C to -35 ° C. To compare the pharmacokinetics of IV-9 and V-9A administered orally.
  • a human multiple myeloma cell line ARH-77 was used to implant tumors subcutaneously in Balb / cnude mice to establish a transplanted tumor model.
  • the specific experimental process is as follows:
  • Balb / cnude mice were purchased from Shanghai Bikai Experimental Animal Co., Ltd., and transferred to a barrier system to adapt to the environment for one week.
  • volume (mm 3 ) 0.5 ⁇ (length (mm) ⁇ width (mm) 2 )
  • the compound to be used is prepared with a 5% ⁇ -sulfobutylcyclodextrin sodium aqueous solution to an appropriate concentration, and then sonicated until thawed, and stored in a refrigerator at 4 ° C. for later use.
  • the mode of administration is oral administration, the frequency of administration in the positive drug group is twice a week, the dosage is 5 mg / kg, the experiment group is daily, the dosage is 1 mg / kg, and the administration is continued for three weeks. The experiment was terminated after sacrifice. Tumor volume was measured and recorded twice a week during this period. GraphPad Prism software was used to calculate the inhibitory effect of the drug on tumor growth. The specific experimental results are shown in Figure 1 of the accompanying drawings.
  • the compounds designed in the present invention can be used to treat malignant tumors of the blood system, so the subject evaluates the compound's efficacy by detecting the activity of proteasomes in the blood after a single administration, and can be performed by blood collection testing at different time points. In vivo pharmacodynamic studies.
  • the 8-week-old female ICR mice used in the experiment were purchased from Shanghai Bikai Experimental Animal Co., Ltd., and transferred to a barrier system to accommodate the environment for one week.
  • each animal took 100 ⁇ L of blood from the orbital venous plexus as its own blank control.
  • the proteasome activity measured by the blood cells in this sample was set to 100%, and the timing was started after administration, one hour after administration.
  • the proteasome activity in blood cells was detected by taking blood from the orbital venous plexus at 24 hours. Compared with the zero-time data, we can know the inhibitory effect and recovery of proteasome activity in blood by the drug.
  • the administration methods are all oral administration, the dosage of MLN9708 is 5mg / kg; the dosage of IV-9 and V-9A groups are 2mg / kg.
  • the kit for detecting proteasome activity in blood cells was purchased from Promega. The specific experimental steps are as follows: draw 100 ⁇ L of whole blood, add 500 ⁇ L of PBS to wash and collect red blood cells, wash again and add 100 ⁇ L of PBS to resuspend, and draw 50 ⁇ L from the suspension. Cell lysate was added for protein quantification to correct the detection value. Another 20 ⁇ L was diluted with PBS 5 times to 100 ⁇ L, and the diluted solution was further reacted with a fluorescent peptide substrate to detect the proteasome activity by a microplate reader.
  • the therapeutic dose of the compound designed in the present invention can be determined according to the mode of administration, the purpose of treatment, the health condition of the patient, and the doctor's prescription.
  • concentration and proportion of the compound designed according to the present invention in the combination medicine will vary depending on a variety of factors, including the route of administration, the dosage and the chemical characteristics.
  • Some conventional doses range from about 1 ⁇ g / kg to 1 g / kg per day. In specific embodiments, the dosage ranges from about 10 ⁇ g / kg body weight to 100 mg / kg body weight per day.
  • the dosage will vary depending on the route of administration, the patient's state of health, the type and extent of the disease or disorder, the relative biological potency of the compound, and the formulation of the excipient. Effective doses can be calculated from dose-response curves of in vitro or animal model test systems.

Abstract

一种肽硼酸酯类化合物或其药用盐，及其制备方法及在药学上的应用。所述肽硼酸酯类化合物或其药用盐结构如式(I)所示，所述化合物可以用于制备蛋白酶体抑制剂，可以应用于治疗实体瘤和血液瘤。

Description

一种肽硼酸酯类化合物的合成与用途 技术领域
本发明属于药物合成领域，具体涉及一系列新型肽硼酸酯类化合物的制备方法及其在药效学上的应用。
背景技术
目前，癌症是危害人类健康最主要的疾病之一。目前癌症的治疗虽然在手术治疗、化学治疗、放射治疗等领域已经取得了很大的进步，但还不能从根本上治疗癌症。虽然目前上市的抗癌药物具有一定的疗效，但它们具有严重的毒副作用。因此，深入探讨和研究如何从有效的肿瘤靶点出发来研究靶向性的新型抗癌药物成为医药工作者的当务之急。
泛素-蛋白酶体途径(Ubiquitin-Proteasome Pathway，简称UPP)是细胞内蛋白质系统降解的主要途径，并参与许多生理上重要的细胞进程，包括信号转导，免疫应答，未折叠蛋白反应和细胞周期进展。这一途径与心脑血管疾病、癌症及神经系统退行性疾病的发病等都有着重要的关系。使用一些有效的抑制剂来抑制这一途径中过度降解重要的蛋白质将会为上述疾病的治疗提供新的思路。针对这一新型靶点，2003年第一个蛋白酶体抑制剂硼替佐米(PS-341)获FDA批准上市，用于治疗复发性骨髓瘤。2004年该药又被批准在欧盟上市，又用于多发性骨髓瘤。2005年9月，该药由西安杨森引进，首次在我国广州上市。2005年，该药同时在法国、荷兰和比利时获得具有医药界的诺贝尔奖称号的“PrixGalien”奖。在2007年7月11日又被美国FDA批准用于治疗复发或难治性套细胞淋巴瘤(Mantle Cell Lymphoma，简称MCL)，成为目前唯一获FDA批准用于治疗MCL的药物。FDA批准万珂可以进行皮下给药，这不仅使万珂的吸收为容易，也大大的提高了患者对万珂的耐受性，减少了副作用。
2014年万珂的销售额达到30.69亿美元成为全球最畅销的肿瘤药物top20之一。万珂在中国市场售价为每3.5毫克约1万三千元，治疗一个周期费用约4万元，如此昂贵的费用对于许多患者是非常沉重的经济负担。而且，目前的临床数据显示，这类药物也存在比较多的副作用，如疲劳乏力、恶心、腹泻以及神经病变等。因此，如何开发出一种价格低廉、毒副作用低的高疗效蛋白酶体抑制剂药物是我们目前需要重点解决的问题。
针对这一疗效确证的靶点，我们设计了一系列结构全新的肽硼酸酯类化合物蛋白酶体抑制剂。
发明内容
本发明的目的是合成一系列可口服的结构新颖且具有蛋白酶体抑制功能的新型肽硼酸酯 类化合物。作为20S蛋白酶体抑制剂，它们能够有效阻断肿瘤细胞增殖，诱发肿瘤细胞凋亡，从而用于人和动物多种疾病如恶性肿瘤的预防和治疗。
本发明的目的是包含药用载体及本发明的肽硼酸酯类化合物，任选同时、分别或依序与一种或多种其他治疗剂组合。
本发明还有一个目的是提供一种肽硼酸酯类化合物在制备蛋白酶体抑制剂中的用途。
本发明还有一个目的是提供一种肽硼酸酯类化合物在制备抗肿瘤药物方面的应用。本发明所述肿瘤包括实体瘤和血液瘤，其中实体瘤选自非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、胰腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、或鼻咽癌；血液瘤选自白血病、多发性骨髓瘤、套细胞淋巴瘤或组织细胞性淋巴癌。
本发明的另一目的是提供一种上述肽硼酸酯类化合物的制备方法。
本发明的目的具体可以通过以下措施来达到：
一种肽硼酸酯类化合物或其药用盐，其结构如式I所示，
Figure PCTCN2019088506-appb-000001
其中：
R 1选自C 1～10烷基、C 1～10烷氧基、C 1～10烷氧基C 1～10烷基、C 3～6环烷基、苯基、萘基、四氢萘基、2,5-二氯苯基或杂环基，或者任选地被C 1～4烷基、C 1～4烷氧基、C 1～4环烷基、卤素或卤代C 1～4烷基取代；R 1优选为C 1～10烷基、C 1～10烷氧基、C 1～10烷氧甲基、C 1～10烷氧乙基、C 3～6环烷基、苯基、2,5-二氯苯基、吡嗪基、吡啶基、萘基、四氢萘基、噁唑基或异噁唑基，或者任选地被C 1～4烷基、C 1～4烷氧基、卤素或卤代C 1～4烷基取代。
更进一步，R 1优选为：
Figure PCTCN2019088506-appb-000002
Figure PCTCN2019088506-appb-000003
其中R 3、R 4、R 5和R 6独立地选自氢、甲基、甲氧基、乙基、乙氧基、氯、溴、氟或三氟甲基。
R 2选自H、苯基、甲氧基、甲硫基、环己基、2,3-二氢-1,4-苯并二噁烷，或者任选地被一个或多个C 1～4烷基、C 1～4烷氧基、硝基、卤素或三氟甲基取代。
B、Z 1和Z 2一起形成含有N、S或O的杂环基团，或者B与Z 1和Z 2一起形成含有O杂环的基团，并且与硼原子相连的是氧原子。优选地，B、Z 1和Z 2一起形成硼酸-α-蒎烷二醇酯，或者B与Z 1和Z 2一起形成硼酸酯，并且与硼原子相连的是氧原子。进一步优选地，B、Z 1和Z 2一起形成硼酸-α-蒎烷二醇酯，或者B、Z 1和Z 2一起形成二乙醇胺硼酸酯、柠檬酸硼酸酯、酒石酸硼酸酯、苹果酸硼酸酯、α-羟基-戊二酸硼酸酯，以及与葡萄糖邻羟基结构形成葡萄糖硼酸酯等其他前药。
本发明R 1、R 2基团中的“任选地被…取代”是指R 1、R 2的基团可以被这些基团取代，也可以不被这些基团取代，即并不仅限于被所列举的这些基团取代的情况，也包括不被所列举的这些基团取代的情况。这种表达方式与“R 1为取代或非取代的C 1～10的烷基、C 3～6的环烷基或杂环烷基、苯基、萘基或吲哚基，其中取代基为C 1～4的烷基、C 1～4的烷氧基、氰基、羟基、巯基、氨基或卤素”的表达方式相同，但是取代或非取代的限定并非仅狭义地指C 1～10的烷基，而是扩大到所有所述的基团，即包括取代或非取代的C 3～6的环烷基或杂环烷基、取代或非取代的苄基、取代或非取代的萘甲基、取代或非取代的吲哚甲基等，其中取代基为C 1～ 4的烷基、C 1～4的烷氧基、氰基、羟基、巯基、氨基或卤素。
术语“烷基”用于表示饱和烃基，C 1～10的烷基是指含有1～10个碳原子的饱和烃基，C 1～ 4的烷基是指含有1～4个碳原子的饱和烃基。
术语“环烷基”指非芳族碳环基，包括环化的烷基。环烷基可以包括二环或多环系统。环烷基的例子包括环丙基，环丁基，环戊基，环己基，环庚基，C 3～6的环烷基是指含有3～6个碳原子的环烷基。
术语“苄基”是指苯甲基，取代的苄基是指在苄基的苯环上至少一个氢原子被非氢部分取代，苄基的取代基可以是卤素，-CN，-OH，-SH，-NH 2，1-6个碳的直链或支链烷基，1-6个碳的取代的直链或支链烷基。
术语“杂环烷基”是指非芳族杂碳环基，包括环化的烷基，其中一个或多个成环碳原子被杂原子例如O，N或S原子取代。杂环烷基优选具有3，4，5，6或7个成环原子。
术语“杂环基”是指含有杂原子O、N或S的环状基团，包括芳族杂环基团或者非芳族杂环基团，如呋喃、噻吩、吡咯、噻唑、咪唑、吡啶、哒嗪、嘧啶、吡嗪、喹啉、异喹啉、吲哚、苯并呋喃、嘌呤、吖啶、噁唑基、异噁唑基等。
“1-萘甲基”是指
Figure PCTCN2019088506-appb-000004
“2-萘甲基”是指
Figure PCTCN2019088506-appb-000005
“吲哚甲基”是指
Figure PCTCN2019088506-appb-000006
“2,3-二氢-1,4-苯并二噁烷”是指
Figure PCTCN2019088506-appb-000007
“四氢萘基”是指
Figure PCTCN2019088506-appb-000008
“噁唑基”是指
Figure PCTCN2019088506-appb-000009
“异噁唑基”是指
Figure PCTCN2019088506-appb-000010
“二乙醇胺硼酸酯”是指
Figure PCTCN2019088506-appb-000011
“柠檬酸硼酸酯”是指
Figure PCTCN2019088506-appb-000012
“酒石酸硼酸酯”是指
Figure PCTCN2019088506-appb-000013
“苹果酸硼酸酯”是指
Figure PCTCN2019088506-appb-000014
“α-羟基-戊二酸硼酸酯”是指
Figure PCTCN2019088506-appb-000015
“葡萄糖硼酸酯”是指
Figure PCTCN2019088506-appb-000016
“烷氧基”是指-O-烷基基团，其碳原子数一般为1～10个。烷氧基的例子包括甲氧基，乙氧基，丙氧基(如，n-丙氧基和异丙氧基)，t-丁氧基等。
“芳基”是指芳族碳环基，包括单环或多环芳烃例如苯基，萘基，蒽基，菲基等。
“芳氧基”是指-O-芳基，而芳基的概念如上所述，芳氧基最优选的例子是苯氧基。
“卤素”包括氟，氯，溴和碘。
本发明的化合物或其药用盐，选自：
Figure PCTCN2019088506-appb-000017
Figure PCTCN2019088506-appb-000018
Figure PCTCN2019088506-appb-000019
本发明化合物可以用以制备抗肿瘤药物，其总的制备路线为：
Figure PCTCN2019088506-appb-000020
该反应式中各基团R 1，R 2，Z 1和Z 2的定义如前所述，路线一为邻苯二甲酰亚胺与氯乙酸乙酯生成式(II-1)，式(II-1)与丙氨酸反应生成式(II-2)，式(II-2)与8-氨基喹啉生成式(II-3)， 式(II-3)与芳基碘反应生成式(II-4)，式(II-4)在三氟化硼乙醚条件下反应生成式(II-5)，式(II-5)在乙二胺条件下反应生成式(II)；路线二为式(III-1)在SOCl 2作用下与甲醇反应得到式(III)。路线一的式(II)或路线二的式(III))化合物在肽缩合剂下分别与R 1-COOH生成式(I-1)化合物，式(I-1)化合物皂化再酸化生成式(I-2)化合物，式(I-2)化合物在肽缩合剂下与硼酸酯的氨基盐酸盐或者三氟乙酸盐缩合生成式(I-3)化合物，式(I-3)化合物在酸性条件下生成式(IV)化合物，最后式(IV)化合物在热乙酸乙酯条件下生成式(I)化合物。
R 1，R 2，Z 1和Z 2的定义如上所述。
以下提供一种更为具体的详述本发明化合物的制备方法：
1、化合物II的制备方法包括如下的步骤：
Figure PCTCN2019088506-appb-000021
1)邻苯二甲酰亚胺与氯乙酸乙酯在三乙胺作用下反应得到式(II-1)结构的化合物；
2)式(II-1)结构的化合物与丙氨酸在Na 2CO 3和H 2O的条件下生成式(II-2)结构的化合物；
3)式(II-2)结构的化合物先在SOCl 2的作用下生成酰氯，然后和8-氨基喹啉在碱性条件下生成式(II-3)的化合物；
4)式(II-3)结构的化合物与芳基碘在钯和四氟硼酸银的条件下生成式(II-4)的化合物；
5)式(II-4)结构的化合物在三氟化硼乙醚的条件下与甲醇生成式(II-5)的化合物；
6)式(II-5)结构的化合物在乙二胺的条件下与甲醇生成式(II)的化合物。
2、化合物(III)的制备方法包括如下的步骤：
Figure PCTCN2019088506-appb-000022
式(III-1)结构的氨基酸在SOCl 2作用下与甲醇反应得到式(III)结构的化合物。
3、化合物(I)的制备方法包括如下的步骤：
Figure PCTCN2019088506-appb-000023
1)式(II)或式(III)所示化合物在肽缩合剂下与R 1-COOH生成式(I-1)化合物；
2)式(I-1)结构的化合物在碱性条件下发生皂化反应生成其钠盐，然后在酸性条件下生成化合物(I-2)；
3)式(I-2)化合物在肽缩合剂下与硼酸酯的氨基盐酸盐或三氟乙酸盐缩合生成式(I-3)化合物；
4)式(I-3)化合物在异丁基硼酸条件下生成式(IV)化合物。
5)式(IV)化合物在沸腾乙酸乙酯条件下生成式(I)化合物
上述反应中常见的肽缩合剂为N,N-二环己基-碳二亚胺(缩写为DCC)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(缩写为EDC.HCl)，1-羟基苯并三氮唑(缩写为HOBt)或氯甲酸异丁酯。
R 1，R 2，Z 1和Z 2的定义如上所述。
通过实验证实，本发明中的化合物具有良好的抑制蛋白酶体的活性和抗肿瘤活性，部分化合物在纳摩尔水平下表现出较好的蛋白酶体抑制活性和抗肿瘤作用，具有制备蛋白酶体抑 制剂或抗肿瘤药物方面的应用价值。本发明中肽硼酸酯类化合物相比于肽硼酸类化合物具有更好的药代动力学性质。
同时，本发明所设计的化合物制备方法产率较高，工艺简单，可适合于工业化生产。
附图说明
图1为给予不同化合物后ARH-77异种移植瘤的生长趋势；
图2为血细胞中蛋白酶体活性检测结果。
具体实施方式
第一部分化合物的合成
本发明的化合物的制备可按照如下过程实施：
一、化合物(II)的制备
Figure PCTCN2019088506-appb-000024
1、N-乙酸乙酯邻苯二甲酰亚胺II-1的制备：
将邻苯二甲酰亚胺溶解于DMF中，加入三乙胺，0℃下向反应体系中滴加氯乙酸乙酯，缓慢升至室温后反应2小时，将反应液倾倒入冰水中，过滤，滤饼用冰水洗涤，真空烘干得到纯的(式II-1)化合物。
2、N-邻苯二甲酰亚保护的丙氨酸II-2的制备：
将化合物II-1和L-丙氨酸溶于H 2O中，加入Na 2CO 3反应2小时，加1N HCl调节PH值至2，过滤，真空烘干即得纯(式II-2)化合物。
3、化合物II-3的制备：
将化合物II-2溶于CH 2Cl 2中，加入SOCl 2冷凝回流6小时，减压蒸除溶剂。将8-氨基喹 啉与DIPEA溶于CH 2Cl 2中，在-20℃下滴加CH 2Cl 2溶解的酰氯，缓慢升至室温反应过夜。减压蒸除溶剂，经柱层析分离得化合物II-3。
3、化合物II-4的制备：
将化合物II-3溶于叔丁醇中，加入醋酸钯、四氟硼酸银和烷基碘，冷凝回流24小时，恢复到室温后，用CH 2Cl 2稀释，加入三乙胺反应3小时，过硅藻土，减压蒸除溶剂，经柱层析分离得化合物II-4。
4、化合物II-5的制备：
将化合物II-4在厚壁耐压瓶中溶于MeOH中，滴加三氟化硼乙醚溶液，100℃反应过夜，加入三乙胺搅拌，减压蒸除溶剂，溶于CH 2Cl 2后，分别用酸洗(10％盐酸)，碱洗(5％碳酸氢钠)及饱和食盐水洗，干燥剂干燥(无水硫酸钠和无水硫酸镁)。滤除干燥剂，减压蒸干溶剂，经柱层析分离得化合物II-5。
4、化合物II的制备：
将化合物II-5溶于MeOH中，加入乙二胺，70℃冷凝回流反应5小时，过滤，滤液减压蒸干溶剂，经柱层析分离得化合物II。
二、化合物(III)的制备
Figure PCTCN2019088506-appb-000025
将化合物(III-1溶于MeOH中，冰盐浴下滴加SOCl 2，滴加完毕后升至室温过夜，减压
蒸干溶剂，即得化合物III)。
三、化合物(I)的制备
Figure PCTCN2019088506-appb-000026
Figure PCTCN2019088506-appb-000027
1、化合物I-1的制备：
将R 1-COOH溶于CH 2Cl 2中，加入1-羟基苯并三氮唑(HOBt)，在-5℃下反应10分钟后，加入肽缩合剂(EDC.HCl)，反应20分钟，加入化合物II或者III，10分钟后加入N,N-二异丙基乙胺(DIPEA)，反应半小时后室温下搅拌过夜。反应液分别用酸洗(10％盐酸)，碱洗(5％碳酸氢钠)及饱和食盐水洗，干燥剂干燥(无水硫酸钠和无水硫酸镁)。滤除干燥剂，减压蒸干溶剂，经柱层析分离得化合物I-1。
2、化合物I-2的制备：
将化合物I-1溶于MeOH中，加入LiOH·H 2O和H 2O，反应3小时后，减压蒸除MeOH，加1N HCl调节pH值至2，用乙酸乙酯萃取分液，减压蒸干溶剂即得化合物I-2。
3、化合物I-3的制备：
将化合物I-2溶于CH 2Cl 2中，加入1-羟基苯并三氮唑(HOBt)，在-5℃下反应10分钟后，加入肽缩合剂(EDC.HCl)，反应20分钟，加入硼酸酯的盐酸盐或三氟乙酸盐，10分钟后加入N,N-二异丙基乙胺(DIPEA)，反应半小时后室温下搅拌过夜。反应液分别用酸洗(10％盐酸)，碱洗(5％碳酸氢钠)及饱和食盐水洗，干燥剂干燥(无水硫酸钠和无水硫酸镁)。滤除干燥剂，减压蒸干溶剂，经柱层析分离得化合物I-3。
4、化合物IV的制备
将化合物I-3溶于MeOH中，加入异丁基硼酸、正己烷和1N HCl，反应过夜。分液，用MeOH中萃取正己烷相2次，再用正己烷洗涤甲醇相1次，减压蒸除甲醇，用CH 2Cl 2萃取水相2次，用饱和食盐水洗涤有机相至水相呈中性。减压蒸干溶剂，经柱层析分离得化合物IV。
5、化合物I的制备
在74℃下将含有二醇的胺或者酸溶于乙酸乙酯，加入化合物IV，降温至60℃，反应3小时，再降温至25℃反应过夜。过滤，真空烘干得到纯的(式I)化合物。
以下以具体化合物的合成来描述本发明的化合物制备过程：
一、式(II)中化合物的制备：
Figure PCTCN2019088506-appb-000028
R 2：
Figure PCTCN2019088506-appb-000029
1、N-乙酸乙酯邻苯二甲酰亚胺(化合物II-1)的制备
将邻苯二甲酰亚胺(7.36g，50mmol)溶解于DMF(25mL)中，加入三乙胺(9mL，65mmol)，0℃下向反应体系中滴加氯乙酸乙酯(5.7mL，60mmol)，缓慢升至室温后，TLC检测反应，2h反应结束，将反应液倾倒入冰水中，过滤，滤饼用冰水洗涤，真空烘干得到纯的N-乙酸乙酯邻苯二甲酰亚胺8.67g，收率79.1％，mp 81.4-83.6℃。 1H NMR(400MHz,CDCl 3)δ1.44(-CH 3,t,J＝7.1Hz,3H),4.48(-CH 2,q,J＝7.1Hz,2H),7.80–7.85(-Ph,m,2H),7.93–7.99(-Ph,m,2H)。MS(ESI)：m/z 220.1[M+H] +。
2、N-邻苯二甲酰亚保护的丙氨酸(化合物II-2)的制备：
将化合物II-1(21.9g，100mmol)和L-丙氨酸(8.9g，100mmol)溶于H 2O(100mL)中，加入Na 2CO 3(10.6g，100mmol)，TLC检测反应，2h反应结束，加1N HCl调节PH值至2，过滤，真空烘干即得纯(式II-2)化合物17.4g，收率79.3％，mp 145.8-146.6℃。 1H NMR(400MHz,CDCl 3)δ1.71(-CH 3,d,J＝7.4Hz,3H),5.02(-CH,q,J＝7.4Hz,1H),7.69–7.75(-Ph,m,2H),7.82–7.88(-Ph,m,2H)。MS(ESI)：m/z218.2[M-H] -。
3、(S)-2-(邻苯二甲酰亚胺基)-N-(8喹啉基)丙酰胺(化合物II-3)的制备：
将化合物II-2(17.37g，79.25mmol)溶于CH 2Cl 2(80mL)中，加入SOCl 2(29mL，396.25mmol)冷凝回流6小时，减压蒸除溶剂。将8-氨基喹啉(11.4g，79.25mmol)与DIPEA(20.5g，158.5mmol) 溶于CH 2Cl 2(103mL)中，在-20℃下滴加CH 2Cl 2(31mL)溶解的酰氯，滴加完毕后缓慢升至室温反应过夜。TLC检测反应，减压蒸除溶剂，经柱层析分离得II-3化合物21.2g，收率77.42％，mp 180.0-181.9℃。 1H NMR(400MHz,CDCl 3)δ1.98(-CH 3,d,J＝7.3Hz,3H),5.27(-CH,q,J＝7.5Hz,1H),7.42(-Ph,dd,J 1＝4.2Hz,J 2＝8.3Hz,1H),7.51(-Ph,s,1H),7.53(-Py,d,J＝9.0,1H),7.65–7.85(-Ph,m,2H),7.90(-Ph,dt,J 1＝3.6,J 2＝7.1Hz,2H),8.15(-Py,d,J＝8.3Hz,1H),8.69(-Ph,d,J＝4.2Hz,1H),8.73(-Py,dd,J 1＝4.7,J 2＝8.9Hz,1H),10.33(-CONH,s,1H)。MS(ESI)：m/z346.0[M+H] +。
4、(S)-2-氨基-3-(4-(三氟甲基)苯基)丙酸甲酯(II-4a)的制备
(1)(S)-2-(邻苯二甲酰亚胺基)-N-(8-喹啉基)-3-(4-(三氟甲基)苯基)丙酰胺(II-4a)的制备
Figure PCTCN2019088506-appb-000030
将化合物II-3(5.2g，15mol)溶于叔丁醇(105mL)中，加入醋酸钯(331mg，1.5mmol)、四氟硼酸银(3.65g，18.75mmol)和4-碘三氟甲苯(6.12g，22.5mmol)，85℃冷凝回流24h，TLC检测反应，升温到室温后，用CH 2Cl 2(100mL)稀释，加入三乙胺(10mL)搅拌3h，反应液过硅藻土，减压蒸除溶剂，经柱层析分离得固体产物5.3g，收率72.1％，mp 124.0-125.5℃。 1H NMR(400MHz,CDCl 3)δ3.77–3.95(-CH 2,m,2H),5.47(-CH,dd,J 1＝6.9Hz,J 2＝9.7Hz,1H),7.39(-Ph,dd,J 1＝4.3Hz,J 2＝8.3Hz,1H),7.42(-Ph,d,J＝8.1Hz,2H),7.49(-Ph,d,J＝8.2Hz,2H),7.51(-Ph,s,1H),7.53(-Py,t,J＝5.5Hz,1H),7.68–7.78(-Ph,m,2H),7.78–7.91(-Ph,m,2H),8.12(-Py,dt,J 1＝7.1Hz,J 2＝14.1Hz,1H),8.58(-Ph,dd,J 1＝1.5Hz,,J 2＝4.2Hz,1H),8.67–8.79(-Py,m,1H),10.28(-CONH,s,1H)。MS(ESI)：m/z487.1[M-H] -。
其他类似的化合物均可以采用上述步骤制备。
II-4b：将II-3和1-碘-3,5-双(三氟甲基)苯采用实施例(1)的方法合成；II-4c：将II-3和6-碘-1,4-苯并二氧烷采用实施例(1)的方法合成；II-4d：将II-3和2,4-二甲氧基碘苯采用实施例(1)的方法合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000031
Figure PCTCN2019088506-appb-000032
(2)(S)-2-(邻苯二甲酰亚胺基)-3-(4-(三氟甲基)苯基)丙酸甲酯(II-5a)的制备
Figure PCTCN2019088506-appb-000033
将化合物II-5a(2g，4.1mmol)于厚壁耐压瓶中溶于MeOH(94mL)中，缓慢滴加三氟化硼乙醚溶液(5.2mL，40.9mmol)，在100℃下反应过夜，TLC检测反应，加入三乙胺(8.6mL，61.3mmol)搅拌一段时间，减压蒸除溶剂，用CH 2Cl 2(30mL)溶解，分别用酸洗(10％盐酸)，碱洗(5％碳酸氢钠)及饱和食盐水洗，干燥剂干燥(无水硫酸钠和无水硫酸镁)。滤除干燥剂，减压蒸干溶剂，经柱层析分离得油状产物1.3g，收率83.2％。 1H NMR(400MHz,CDCl 3)δ3.55–3.71(-CH 2,m,2H),3.78(-CH 3,s,3H),5.18(-CH,dd,J 1＝5.8Hz,J 2＝10.7Hz,1H),7.30 (-Ph,d,J＝8.0Hz,2H),7.46(-Ph,d,J＝8.0Hz,2H),7.67–7.75(-Ph,m,2H),7.79(-Ph,dt,J 1＝3.6Hz,J 2＝7.1Hz,2H)。MS(ESI):m/z 378.3[M+H] +。
其他类似的化合物均可以采用上述步骤制备。
II-5b：将II-4b采用实施例(2)的方法合成；II-5c：将II-4c采用实施例(2)的方法合成；II-5d：将II-4d采用实施例(2)的方法合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000034
(3)(S)-2-氨基-3-(4-(三氟甲基)苯基)丙酸甲酯(IIa)的制备
Figure PCTCN2019088506-appb-000035
将化合物II-5a(745mg，1.9mmol)溶于MeOH(19mL)中，加入乙二胺(297mg，4.9mmol)，70℃下冷凝回流，TLC检测反应，5h反应结束，过滤，滤液减压蒸干溶剂，经柱层析分离得 油状目标化合物311mg，收率62.4％。 1H NMR(400MHz,DMSO)δ2.84(-CH 2,dd,J 1＝7.7Hz,J 2＝13.3Hz,1H),2.95(-CH 2,dt,J 1＝9.5Hz,J 2＝19.0Hz,1H),3.59(-CH 3,s,3H),3.61(-CH,d,J＝6.9Hz,1H),7.40(-Ph,t,J＝11.9Hz,2H),7.59(-Ph,t,J＝21.6Hz,2H)。MS(ESI):m/z 248.1[M+H] +。
其他类似的化合物均可以采用上述步骤制备。
IIb：将II-5b采用实施例(3)的方法合成；IIc：将I-5c采用实施例(3)的方法合成；IId：将II-5d采用实施例(3)的方法合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000036
二、式(III)中化合物的制备：
Figure PCTCN2019088506-appb-000037
R 2：a＝H；
Figure PCTCN2019088506-appb-000038
1、甘氨酸甲酯盐酸盐(IIIa)的制备
Figure PCTCN2019088506-appb-000039
将化合物IIIa(3g，40mmol)溶于MeOH(30mL)中，冰盐浴冷却到-10℃，搅拌下缓慢滴加SOCl 2(29mL，400mmol)，滴加结束后反应10min，撤去冰盐浴，室温下反应过夜，TLC检测反应，减压浓缩，再加入20mLCH 2Cl 2，反复减压浓缩两次，旋干溶剂，干燥，得产物5g，收率99.5％，产品未经纯化，直接用于下步反应。
本发明中所用到的其它氨基酸甲酯的盐酸盐均可以采用上述步骤制备。化合物IIIb：按合成化合物IIIa的方法采用D-环己基甘氨酸合成；化合物IIIc：按合成化合物IIIa的方法采用L-环己基甘氨酸合成；化合物IIId：按合成化合物IIIa的方法采用苯丙氨酸合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000040
2、L-O-甲基丝氨酸甲酯盐酸盐(IIIe)的制备
Figure PCTCN2019088506-appb-000041
(1)BOC-L-O-甲基丝氨酸甲酯(IIIe-1)的制备
将BOC-L-丝氨酸甲酯(5g，22.8mmol)溶于丙酮(110mL)中，加入碘甲烷(32mL，524mmol)和氧化银(8.2g，35.4mmol)，在59℃下避光冷凝回流过夜。TLC检测反应，过滤，减压蒸除溶剂，经柱层析分离得油状目标化合物1.8g，收率34.5％。 1H NMR(400MHz,CDCl 3)δ1.43(-CH 3,s,9H),3.32(-CH 3,s,3H),3.57(-CH 2,dd,J 1＝3.4Hz,J 2＝9.4Hz,1H),3.74(-CH 3,d,J＝6.3Hz,3H),3.78(-CH 2,dd,J 1＝3.1Hz,J 2＝9.4Hz,1H),4.35–4.44(-CH,m,1H),5.28–5.44(-CONH,m,1H)。MS(ESI):m/z 234.2[M+H] +。
(2)L-O-甲基丝氨酸甲酯盐酸盐(IIIe)的制备
将化合物IIIe-1(496mg，2.1mmol)溶于乙酸乙酯(2.5mL)中，在冰浴下滴加HCl的乙酸乙酯溶液(5.2mL,21.2mmol)，室温下反应，TLC检测反应，2h反应结束，过滤，滤饼真空烘干即得纯产物351mg，收率97.5％。 1H NMR(400MHz,CDCl 3)δ3.41(-CH 3,s,3H),3.83(-CH 3,s,3H),3.95(-CH 2,dd,J 1＝3.6Hz,J 2＝10.4Hz,1H),4.03(-CH 2,dd,J 1＝2.6Hz,J 2＝10.3Hz,1H),4.45(-CH,s,1H),8.70(-NH 3 +,s,3H)。
3、S-甲基-L-半胱氨酸甲酯盐酸盐(IIIf)的制备
Figure PCTCN2019088506-appb-000042
(1)S-甲基-L-半胱氨酸(IIIf-1)的制备
将L-半胱氨酸盐酸盐一水合物(3.1g，17.5mmol)溶于MeOH(45mL)中，冰浴下滴加30％的甲醇钠的甲醇溶液(11.2g，62mmol)，反应1h后，滴加碘甲烷(0.9mL，13.mmol)，升温至室温反应，TLC检测反应，2h反应结束，用10N HCl调pH值至5，加入40mL乙醚搅拌10min，过滤，滤饼用60mL乙醚洗涤，真空干燥，得到粗产品4.715g。
(2)S-甲基-L-半胱氨酸甲酯盐酸盐(IIIf)的制备
将S-甲基-L-半胱氨酸(4.715g，34.9mmol)溶于MeOH(25mL)中，冰盐浴冷却到-10℃，搅拌下缓慢滴加SOCl 2(25mL，348.8mmol)，滴加结束后反应10min，撤去冰盐浴，室温下反应过夜，TLC检测反应，过滤，滤饼用CH 2Cl 2洗涤，真空烘干即得纯S-甲基-L-半胱氨酸甲酯盐酸盐3.1g，收率95.4％。 1H NMR(400MHz,D 2O)δ4.44(-CH,dd,J＝7.7,4.6Hz,1H),3.90(-CH 3,s,3H),3.23(-CH 2,dd,J＝15.1,4.6Hz,1H),3.14–3.07(-CH 2,m,1H),2.18(-CH 3,s,3H)。
三、式(I)中化合物的制备：
Figure PCTCN2019088506-appb-000043
Figure PCTCN2019088506-appb-000044
R 1：
Figure PCTCN2019088506-appb-000045
Figure PCTCN2019088506-appb-000046
Z 1和Z 2：
Figure PCTCN2019088506-appb-000047
1、(S)-N-(2,5-二氯苯甲酰基)-3-(4-三氟甲基苯基)丙氨酸甲酯(I-1a)的制备
Figure PCTCN2019088506-appb-000048
将2,5-二氯苯甲酸(90mg，0.47mmol)和HOBt(92mg，0.7mmol)溶于CH 2Cl 2(8mL)中，在-10℃下反应10min，加入EDC.HCl(135mg，0.7mmol)反应30min，加入化合物IIa(116mg，0.47mmol)，反应10min后加入DIPEA(151mg，1.17mmol)，反应20min后升至室温反应过夜。TLC检测反应，分别用10％的盐酸溶液(10mL)，5％的NaHCO 3溶液(10mL)和饱和食盐水(2×10mL)洗涤，CH 2Cl 2层用无水Na 2SO 4干燥，过滤，减压蒸除溶剂，得油状化合物166mg，，收率84.3％。产品未经纯化，直接用于下步反应。
鉴于EDC.HCl缩合法得到的产品产率高，所以本发明中所用到的其它氨基未保护的氨基酸甲酯可以采用实施例1中所述的EDC.HCl缩合法制备，所有甲酯均未纯化，直接用于下步反应。
化合物I-1b：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIb合成；化合物I-1c：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIc合成；化合物I-1d：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IId合成。
(1)、(S)-N-(2,5-二氯苯甲酰基)甘氨酸甲酯(I-1e)的制备
Figure PCTCN2019088506-appb-000049
将2,5-二氯苯甲酸(7.6g，40mmol)和HOBt(8.1g，40mmol)溶于CH 2Cl 2(200mL)中，在-10℃下反应10min，加入EDC.HCl(11.5g，60mmol)反应30min，加入化合物IIIa(5g，40mmol)，反应10min后加入DIPEA(18.1g，140mmol)，反应20min后升至室温反应过夜。TLC检测反应，分别用10％的盐酸溶液(200mL)，5％的NaHCO 3溶液(200mL)和饱和食盐水(2×200mL)洗涤，CH 2Cl 2层用无水Na 2SO 4干燥，过滤，减压蒸除溶剂，得油状化合物9.32g，，收率88.9％。产品未经纯化，直接用于下步反应。
化合物I-1f：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIIb合成；化合物I-1g：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIIc合成；化合物I-1h：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIIe合成；化合物I-1i：按EDC.HCl缩合法利用2.5-二氯苯甲酸和IIIf合成；化合物I-1j：按EDC.HCl缩合法利用2-吡嗪甲酸和IIa合成；化合物I-1k：按EDC.HCl缩合法利用2-吡嗪甲酸和IIc合成；化合物I-1l：按EDC.HCl缩合法利用5,6,7,8-四氢-1-萘甲酸和IIc合成；化合物I-1m：按EDC.HCl缩合法利用5-甲基异恶唑-3-甲酸和IIIa合成。化合物I-1n：按EDC.HCl缩合法利用5-甲基-2-吡嗪甲酸和IIId合成。
本发明中所用到的其它氨基保护的氨基酸甲酯可以采用实施例(1)中所述的EDC.HCl缩合法制备，所有甲酯均未纯化，直接用于下步反应。
(2)、(S)-N-(甲氧基乙酰基)苯丙氨酸甲酯(I-1o)的制备
Figure PCTCN2019088506-appb-000050
将甲氧基乙酸(280mg,3.13mmol)溶解在CH 2Cl 2(6mL)，-10℃下滴加SOCl 2(0.25mL，3.45mmol)。滴加完毕后升至室温反应2h。甲氧基乙酰氯溶液直接投下一步。将IIId(0.67g,3.13mmol)溶于3mL CH 2Cl 2中，加入三乙胺(1.58g，15.65mmol)，滴加甲氧基乙酰氯溶液，反应过夜。TLC检测反应，用水洗涤，Na 2SO 4干燥，经柱层析分离得油状目标化合物0.69g，收率87.3％。
化合物I-1p：按酰氯缩合法利用3-甲氧基丙酸和IIId合成；化合物I-1q：按酰氯缩合法利用丁酸和IIId合成；化合物I-1r：按酰氯缩合法利用环丙基甲酸和IIId合成；化合物I-1s：按酰氯缩合法利用环戊基甲酸和IIId合成。
本发明中所用到的其它氨基保护的氨基酸甲酯与烷基酸可以采用实施例(2)中所述的缩合法制备，所有甲酯均未纯化，直接用于下步反应。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000051
Figure PCTCN2019088506-appb-000052
Figure PCTCN2019088506-appb-000053
2、(S)-N-(2,5-二氯苯甲酰基)-3-(4-三氟甲基苯基)丙氨酸(I-2a)的制备
Figure PCTCN2019088506-appb-000054
将化合物I-1a(129mg，0.31mmol)用2.5mLMeOH溶解，加入LiOH·H 2O(39mg，0.92mmol)和H 2O(0.8mL)，TLC检测，2h后反应完毕。旋干有机相，用乙醚(2×1mL)萃取水相，水相滴加盐酸至pH值为2～3，产生大量白色固体，乙酸乙酯萃取，，减压蒸除溶剂，得白色产品106mg，收率86.0％，mp 185.1-186.9℃。 1H NMR(400MHz,DMSO)δ3.02(-CH 2,dd,J 1＝10.6Hz,J 2＝13.8Hz,1H),3.30(-CH 2,dd,J 1＝4.6Hz,J 2＝13.9Hz,1H),4.68(-CH,ddd,J 1＝4.7Hz,J 2＝8.4Hz,J 3＝10.4Hz,1H),7.15(-Ph,d,J＝1.8Hz,1H),7.49(-Ph,t,J＝4.9Hz,2H),7.52(-Ph,d,J＝6.0Hz,2H),7.66(-Ph,d,J＝8.1Hz,2H),8.90(-CONH,d,J＝8.2Hz,1H),13.12(-COOH,s,1H)。MS(ESI):m/z 403.9[M-H] -。
本发明中所用到的其它氨基保护的氨基酸可以采用实施例2中所述的方法制备。
化合物I-2b：将I-1b采用实施例2的方法合成；化合物I-2c：将I-1c采用实施例2的方法合成；化合物I-2d：将I-1d采用实施例2的方法合成；化合物I-2e：将I-1e采用实施例2的方法合成；化合物I-2f：将I-1f采用实施例2的方法合成；化合物I-2g：将I-1g采用实施例2的方法合成；化合物I-2h：将I-1h采用实施例2的方法合成；化合物I-2i：将I-1i采用实施例2的方法合成；化合物I-2j：将I-1j采用实施例2的方法合成；化合物I-2k：将I-1k采用实施例2的方法合成；化合物I-2l：将I-1l采用实施例2的方法合成；化合物I-2m：将 I-1m采用实施例2的方法合成；化合物I-2n：将I-1n采用实施例2的方法合成；化合物I-2o：将I-1o采用实施例2的方法合成；化合物I-2p：将I-1p采用实施例2的方法合成；化合物I-2q：将I-1q采用实施例2的方法合成；化合物I-2r：将I-1r采用实施例2的方法合成；化合物I-2s：将I-1s采用实施例2的方法合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000055
Figure PCTCN2019088506-appb-000056
Figure PCTCN2019088506-appb-000057
3、(S)-N-(2,5-二氯苯甲酰基)-3-(4-三氟甲基苯基)丙酰胺-D-亮氨酸硼酸-(+)-α-蒎烷二醇酯(I-3a)的制备
Figure PCTCN2019088506-appb-000058
将化合物I-2a(340mg，0.84mmol)和HOBt(218g，1.67mmol)溶于CH 2Cl 2(18mL)中，在-10℃下反应10min，加入EDC.HCl(321mg，1.67mmol)反应30min，加入(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐(317mg，0.84mmol)，反应10min后加入DIPEA(433mg，3.35mmol)，反应20min后升至室温反应过夜。TLC检测反应，分别用10％的盐酸溶液(20mL)，5％的NaHCO 3溶液(20mL)和饱和食盐水(2×20mL)洗涤，CH 2Cl 2层用无水Na 2SO 4干燥，过滤，减压蒸除溶剂，经柱层析分离得油状目标化合物480mg，收率87.6％。 1H NMR(400MHz,CDCl 3)δ0.82(-CH 3,s,3H),0.85(-CH 3,s,6H),1.16(-CH 2,dd,J 1＝7.8Hz J 2＝10.8Hz,,1H),1.28(-CH 3,s,3H),1.32(-CH 2,d,J＝14.3Hz,1H),1.39(-CH 3,s,3H),1.41–1.52(-CH,m,1H),1.63(-CH,s,1H),1.81(-CH 2,dd,J 1＝2.8Hz,J 2＝14.5Hz,1H),1.90(-CH 2,d,J＝2.4Hz,1H),1.98–2.05(-CH,m,1H),2.12–2.23(-CH 2,m,1H),2.25–2.38(-CH 2,m,1H),3.19(-CH,dd,J 1＝8.5Hz,J 2＝13.7Hz,1H),3.23–3.31(-CH 2,m,2H),4.21–4.34(-CH,m,1H),4.75–4.94(-CH,m,1H),5.90(-CONH,dd,J 1＝5.6Hz,J 2＝22.2Hz,1H),6.94(-CONH,d,J＝7.7Hz,1H),7.29–7.36(-Ph,m,2H),7.41(-Ph,dd,J 1＝4.2Hz,J 2＝7.8Hz,2H),7.51(-Ph,s,1H),7.55(-Ph,dd,J 1＝3.7Hz,J 2＝8.0Hz,2H)。MS(ESI):m/z 653.2[M+H] +。
本发明中所用到的其它氨基保护的氨基酸可以采用实施例3中所述的方法制备。
化合物I-3b：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2b合成；化合物I-3c：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2c合成；化合物I-3d：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2d合成；化合物I-3e：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2e合成；化合物I-3f：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2f合成；化合物I-3g：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2g合成；化合物I-3h：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2h合成；化合物I-3i：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2i合成；化合物I-3j：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2j合成；化合物I-3k：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2k合成；化合物I-3l：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2l合成；化合物I-3m：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2m合成；化合物I-3n：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2n合成；化合物I-3o：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2o合成；化合物I-3p：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2p合成；化合物I-3q：按EDC.HCl缩合法利用(aR,3aS,4S,6S,7aR)-六氢-3a,8,8-三甲基-alpha-(2-甲基丙基)-4,6-甲桥-1,3,2-苯并二氧硼烷-2-甲胺2,2,2-三氟乙酸盐和I-2q合成。
合成的具体化合物及其性质如下表。
Figure PCTCN2019088506-appb-000059
Figure PCTCN2019088506-appb-000060
Figure PCTCN2019088506-appb-000061
Figure PCTCN2019088506-appb-000062
Figure PCTCN2019088506-appb-000063
4、(S)-N-(2,5-二氯苯甲酰基)-3-(4-三氟甲基苯基)丙酰胺-D-亮氨酸硼酸(IV-1)的制备
将化合物I-3a(317mg，0.49mmol)溶于3mL的MeOH中，依次加入异丁基硼酸(247mg，2.43mmol)、正己烷(3mL)和1N HCl(1.2mL，1.2mmol)，反应搅拌过夜。TLC检测反应，正己烷相用MeOH(2×3mL)萃取2次，正己烷(3mL)洗涤甲醇相1次，减压蒸除甲醇，CH 2Cl 2(2×2mL)萃取水相2次，用饱和食盐水(3×5mL)洗涤有机相至水相呈中性。减压蒸除溶剂，经柱层析分离得到纯产物193mg，收率76.5％。 1H NMR(400MHz,CDCl 3)δ 1.18(-CH 3,s,3H),1.25(-CH 3,s,3H),2.13–2.41(-CH 2,m,2H),2.45–2.61(-CH,m,1H),3.20–3.58(-CH 2,m,2H),3.58–3.71(-CH,m,1H),5.21–5.62(-CH,m,1H),7.62–7.75(-Ph,m,4H),7.84(-Ph,t,J＝13.5Hz,3H)。 13C NMR(CDCl 3,100MHz)δ22.67,27.21,31.90,35.60,51.28,54.80,125.37,125.55,128.92,129.05,129.47,129.76,129.85,131.34,131.60,133.12,133.17,139.92,165.18,170.98。MS(ESI):m/z 517.1[M-H] -,calcd:518.1。HRMS(ESI):calcd for C 22H 24BCl 2F 3N 2NaO 4[M+Na] +541.1054,found 541.1118.
本发明的其它的硼酸类化合物的合成可以采用上述的方法。
具体的化合物如下表所示。
Figure PCTCN2019088506-appb-000064
Figure PCTCN2019088506-appb-000065
Figure PCTCN2019088506-appb-000066
Figure PCTCN2019088506-appb-000067
以上所述的硼酸类化合物可以和柠檬酸等反应生成硼酸酯类化合物作为前药使用，制备方法如下例所述，但不限于本例：
5、(S)-N-(2,5-二氯苯甲酰基)-3-甲氧基丙酰胺-D-亮氨酸硼酸二乙醇胺酯(V-8A)的制备
Figure PCTCN2019088506-appb-000068
将二乙醇胺(160mg，1.52mmol)溶解于8mL乙酸乙酯中，升温至74℃，加入溶于1.5mL 乙酸乙酯的IV-1(500mg，1.38mmol)，缓慢降温至60℃，反应3h，再缓慢降温至25℃过夜。TLC检测反应，过滤，滤饼真空烘干得纯产物557mg，收率85.4％。1H NMR(400MHz,DMSO)δ0.80(-CH3,dd,J1＝6.7Hz,J2＝9.7Hz,6H),1.12-1.39(-CH2,m,2H),1.59(-CH,d,J＝5.5Hz,1H),2.75(-CH2,dd,J1＝6.4Hz,J2＝26.3Hz,2H),2.85-3.04(-CH2,m,2H),3.08-3.20(-CH,m,1H),3.26(-CH3,s,3H),3.59(-CH2,dt,J1＝8.1Hz,J2＝22.2Hz,4H),3.69(-CH2,d,J＝5.3Hz,2H),4.59(-CH,dd,J1＝6.7Hz,J2＝12.9Hz,1H),6.56(-NH,s,1H),6.99(-CONH,d,J＝8.2Hz,1H),7.45(-Ph,d,J＝13.7Hz,1H),7.54(-Ph,s,2H),8.69-8.82(-CONH,m,J＝7.9Hz,1H).HRMS(ESI):calcd for C 20H 30BCl 2N 3O 5[M+Na]+496.1548,found:497.1546.
6、(S)-N-(2,5-二氯苯甲酰基)-3-甲氧基丙酰胺-D-亮氨酸硼酸枸橼酸酯(V-8B)的制备
Figure PCTCN2019088506-appb-000069
将枸橼酸(192.12mg，0.39mmol)溶解于2mL乙酸乙酯中，升温至74℃，待枸橼酸完全溶解后，加入溶于1mL乙酸乙酯的化合物IV-1(363.03mg，0.36mmol)，缓慢降温至60℃，反应3h，再缓慢降温至25℃过夜。TLC检测反应，过滤，滤饼真空烘干得纯产物90.0mg，收率48.6％。 1H NMR(400MHz,DMSO)δ0.86(-CH 3,d,J＝6.3Hz,6H),1.39-1.21(-CH 2，m,2H),1.70(-CH,d,J＝26.1Hz,1H),2.81-2.52(-CH 2,m,4H),2.88(-CH,s,1H),3.15(-CH 3,s,3H)3.30-3.55(-CH 2,m,2H),4.46(-CH,m,1H),7.78-7.44(-Ph,m,3H),9.12(-NH,s,1H),10.73(-NH,s,1H),12.15(-COOH,s,2H).HRMS(ESI):calcd for C 20H 30BCl 2N 3O 5[M+Na] +583.1028,found:583.1030.
本发明的其它的二乙醇胺硼酸酯、柠檬酸硼酸酯前药化合物均可以采用如实施例5与实施例6方法合成。具体化合物如下表所示。
Figure PCTCN2019088506-appb-000070
Figure PCTCN2019088506-appb-000071
Figure PCTCN2019088506-appb-000072
Figure PCTCN2019088506-appb-000073
第二部分抑制蛋白酶体活性测定
蛋白酶体抑制活性
本发明利用荧光多肽底物Suc-Leu-Leu-Val-Tyr-AMC(简写Suc-LLVY-AMC，Suc表示琥珀酰基，AMC表示7-酰胺-4-甲基香豆素)来测定蛋白酶体的糜蛋白酶样的酶活性。
本发明所用的蛋白酶体为人红细胞20S蛋白酶体，酶、荧光底物及测试缓冲液均购自Enzo公司。实验体系为16μL，其中底物8μL，蛋白酶体4μL(0.8ng)，最终浓度为50μM，药物(抑制剂)4μL，最终浓度为2×10 -6M～4.88×10 -10M,最后一个浓度是0M，实际配置浓度为8×10 -6M～1.95×10 -9M，最后一个浓度是0M。具体实验过程如下：
1、药物配置：
称取药物，加入DMSO溶解至浓度为10 -2M。用移液枪吸取2μL加至98μL DMSO得到2×10 -4M，然后再从2×10 -4M浓度药物中吸取8μL加入198μL H 2O中得到8×10 -6M，利用同样的方法得到2×10 -6M、5×10 -7M、1.25×10 -7M、3.12×10 -8M、7.8×10 -9M、1.95×10 -9M浓度的药物，最后一个浓度0M为不加药。
2、底物制备：
将25mg荧光多肽底物溶解于654μL DMSO中，得到50mM储备液，于-20℃保存，使用时稀释500倍，每份样品中加入8μL，使得反应体系中的最终底物浓度为50μM。
3、反应体系制备：
用缓冲溶液将20S蛋白酶体(2ng/μL)稀释成浓度为8ng/μL的溶液，加入到384孔荧光酶标板中，每孔加入4μL，再在每孔中加入4μL待测样品，使用已上市药物万珂为阳性对照药，37℃下反应15min。反应结束后，每孔加入8μL荧光底物，37℃避光反应1小时，利用360nm/460nm荧光酶标仪(BMG LABTECH POLARstar OPTIMA Microplate Reader)检测荧光值。
4、数据处理
计算扣除本底物后不同浓度的药物作用下所得产物的荧光值,运用GraphPad Prism软件，计算药物对蛋白酶体抑制的IC 50浓度。
部分化合物的结果如下表：
化合物编号 IC 50(nM)
IV-8 7.517
IV-9 4.862
V-8A 7.023
V-8B 8.197
V-9A 5.686
V-9B 6.597
Velcade 9.916
MLN-9708 7.468
其中Velcade以及MLN9708的化学结构式为：
Figure PCTCN2019088506-appb-000074
细胞株抑制活性
本发明利用的检测液为单溶液细胞增殖检测盒，来自Promega公司；所用的细胞为U266，RPMI8226，ARH77。实验体系为110uL，其中含有细胞悬液90μL，检测液10μL，药物(抑制剂)10μL，其终浓度为4.54×10 -8M～1.77×10 -9M,最后一个浓度是0M，实际配置浓度为5×10 -7M～1.95×10 -8M，最后一个浓度是0M。具体实验过程如下：
1、药物配置：
准确称量药物，加入DMSO溶解至10 -2M。用移液器吸取1μL加至199μL DMSO得到5×10 -5M，然后从5×10 -5M浓度药物中吸取3.3μL加326.7μL无血清的RPMI1640培养基得到5×10 -7M，1.5倍梯度稀释，得到3.3×10 -7M、2.2×10 -7M、1.48×10 -7M、9.87×10 -8M、6.58×10 -8M、4.38×10 -8M、2.92×10 -8M、1.95×10 -8M浓度的药物，最后一个浓度0M为不加药。
2、细胞悬液配置：
细胞分别计数后，稀释配置U266为1×10 4个/孔，RPMI8226和ARH77均为1×10 4个/孔。
3、反应体系制备：
96孔荧光酶标板中每孔加入细胞悬液90μL，孵育24h；然后每孔中加入10μL待测样品，使用已上市药物万珂为阳性对照药，孵育24h；反应完毕后，每孔加入10μL检测液，孵育2-3h，,490nm荧光酶标仪(BMG LABTECH POLARstar OPTIMA Microplate Reader)检测吸光度。
4、数据处理
计算扣除本底后不同浓度药物作用下所得产物的吸光度,运用GraphPad Prism软件，计算药物对细胞毒性的IC 50浓度。
部分化合物的结果如下表：
编号 RPMI8226 ARH-77 U266B1 编号 RPMI8226 ARH-77 U266B1
IV-8 8.99 9.10 6.75 V-9A 8.66 8.96 6.54
IV-9 8.97 8.85 6.45 V-9B 8.17 9.34 6.42
IV-10 150.4 58.03 79.89 V-8A 8.43 8.21 7.02
IV-11 43.44 29.26 30.26 V-8B 8.15 8.93 7.14
Velcade 11.2 9.57 11.63 MLN2238 55.32 65.50 52.15
MLN9708 49.74 43.25 67.1        
通过体外酶活和细胞毒性结果可以发现，本发明中化合物及其前药与目前上市蛋白酶体药物相比，在体外酶活和多种细胞中都显示了更好的活性。
体内药代动力学评价
SD雄性大鼠12只，体重220±20g，随机分为四组。
其中两组按下表剂量分别尾静脉注射给予IV-9以及V-9A，并于给药前及给药后10min、20min、30min、1h、2h、4h、8h、12h、24h和36h，由颈静脉采血约0.200mL，置于装有EDTA-K2的试管中，高速离心(7800×g)15min后分离血浆，于-15℃～-35℃保存。用于比较IV-9与V-9A静脉注射方式给药的药代动力学差异。
另外两组按如下表所示剂量分别灌胃给予IV-9以及V-9A，并于给药前及给药后5min、10min、20min、30min、1h、2h、4h、8h、12h、24h和36h，由颈静脉采血约0.200mL，置于装有EDTA-K2的试管中，高速离心(7800×g)15min后分离血浆，于-15℃～-35℃保存。用于比较IV-9与V-9A口服方式给药的药代动力学差异。
V-9A及其前药(IV-9)药代动力学参数比较
Figure PCTCN2019088506-appb-000075
由以上药代动力学数据可知，将化合物IV-9制成二乙醇胺硼酸酯前药(V-9A)后，化合物的药代动力学性质有了明显的提升，半衰期(T 1/2)有所延长，并且口服生物利用度从11％(IV-9)提升至24.9％(V-9A)。由此可知，本发明中所优选的一系列肽硼酸酯类化合物相比于肽硼酸类化合物具有更好的药代动力学性质。
动物异种移植模型评价
利用人源多发性骨髓瘤细胞系ARH-77，在Balb/cnude小鼠皮下种植肿瘤，建立移植瘤模型。具体实验过程如下：
1、4-6周Balb/cnude小鼠购自上海必凯实验动物有限公司，转入屏障系统适应环境饲养一周。
2、培养细胞至对数生长期，将细胞消化离心后，按1×10 7个/ml的密度用基质胶重悬，置于冰上备用。进入屏障系统内，按每只老鼠接种1×10 6个细胞将肿瘤细胞接种于动物右前肢腋下，每只动物接种体积为100微升。
3、继续饲养动物并使用游标卡尺测量肿瘤体积，至平均体积增至100～150mm 3时，将动物随机分为三组，分别为空白对照组、阳性药组(化合物MLN-9708)和实验组(化合物V-2A)，每组6只。
肿瘤体积计算公式为：体积 (mm 3 )＝0.5×(长 (mm)×宽 (mm) 2)
4、所用化合物使用5％β-磺丁基环糊精钠水溶液配制为适宜浓度，超声至融清后放入4℃冰箱保存备用。
5、给药方式为口服给药，阳性药组给药频率为每周两次，给药剂量为5mg/kg，实验组为每天给药，给药剂量为1mg/kg，连续给药三周后处死结束实验。期间每周测量记录两次肿瘤体积。并运用GraphPad Prism软件，计算药物对肿瘤生长的抑制效果。具体实验结果如附图1所示。
给药后对ARH-77异种移植瘤生长情况的影响
Figure PCTCN2019088506-appb-000076
通过以上结果得知，化合物V-9A在1mg/kg剂量下，抑瘤率达到63.48％，显著高于已上市的口服蛋白酶体抑制剂MLN-9708 5mg/kg的抑瘤率(25.28％)。上述研究结果证明，与已上市产品相比，V-9A在更低的剂量下体现出更好的体内药效。
体内血细胞药效动力学研究
本发明中所设计化合物可以用于治疗血液系统的恶性肿瘤，因此课题通过检测单次给药后血液中蛋白酶体的活性来评价化合物的药效，并且可以通过在不同时间点的采血检测来进行体内药效动力学研究。
具体实验过程如下：
1、试验所用8周龄雌性ICR小鼠购自上海必凯实验动物有限公司，转入屏障系统适应环境饲养一周。
2、将动物分为三组，分别为阳性对照组(MLN9708)和两个实验组(IV-9、V-9A)每 组3只。
3、给药前，各个动物分别通过眼眶静脉丛取血100μL作为自身空白对照，将此样品中血细胞所测得蛋白酶体活性设为100％，给药后开始计时，分别于给药后1小时、24小时通过眼眶静脉丛取血检测血细胞中蛋白酶体活性，与零时数据对比可知药物对血液中蛋白酶体活性的抑制效果以及恢复情况。
4、给药方式均为口服给药，MLN9708给药剂量为5mg/kg；IV-9组、V-9A组给药剂量均为2mg/kg。
5、检测血细胞中蛋白酶体活性的试剂盒购自Promega公司，具体实验步骤如下：吸取100μL全血，加入500μL PBS洗涤收集红细胞，再次洗涤后加入100μL PBS重悬，自混悬液中吸取50μL，加入细胞裂解液进行蛋白定量用以修正检测值，另外吸取20μL用PBS稀释5倍至100μL，使用该稀释液进一步与荧光肽底物反应通过酶标仪检测蛋白酶体活性。
实验所得具体数据如附图2所示：由研究结果可知，给药1小时后，化合物IV-9达到与MLN9708基本相当的抑制活性，而V-9A具有更好的抑制活性；而给药后24小时，MLN9708组的蛋白酶体活性已经恢复至对照组活性的80％，IV-9只恢复至对照组活性的约60％，而V-9A组只恢复至对照组活性的约40％。以上研究数据说明，由于本发明化合物具有更优良的药效动力学性质，在体内其具有更优的药效，并且药效作用更持久。
本发明中所设计的化合物的治疗剂量可以根据给药的方式、治疗的用途、病人的健康状况以及医生的处方而决定。组合药物中本发明所设计的化合物的浓度和所占比例将随多种因素而改变，包括给药途径、给药剂量以及化学特性，例如，本发明所设计的化合物可以提供于用于非肠道给药的含大约0.1到10％w/v化合物的水性生理缓冲液中。一些常规的剂量范围为每天大约1μg/kg到1g/kg。在具体实施方案中，剂量范围从每天大约10μg/kg体重到100mg/kg体重。剂量会根据给药途径、病人的健康状态、疾病或失调的类型和进展程度、化合物的相对生物学效价以及赋形剂的配方而改变。有效的剂量可以从体外或动物模型测试系统的剂量反应曲线推算出。

Claims (10)

  1. 一种肽硼酸酯类化合物或其药用盐，其结构如下式所示，
    Figure PCTCN2019088506-appb-100001
    其中：
    R 1选自C 1～10烷基、C 1～10烷氧基、C 1～10烷氧基C 1～10烷基、C 3～6环烷基、苯基、萘基、四氢萘基、2,5-二氯苯基或杂环基，或者任选地被C 1～4烷基、C 1～4烷氧基、C 1～4环烷基、卤素或卤代C 1～4烷基取代；
    R 2选自H、苯基、甲氧基、甲硫基、环己基、2,3-二氢-1,4-苯并二噁烷，或者任选地被一个或多个C 1～4烷基、C 1～4烷氧基、硝基、卤素或三氟甲基取代；
    B、Z 1和Z 2一起形成含有N、S或O的杂环基团，或者B与Z 1和Z 2一起形成含有O杂环的基团，并且与硼原子相连的是氧原子。
  2. 根据权利要求1所述的肽硼酸酯类化合物或其药用盐，其特征在于所述R 1选自C 1～10烷基、C 1～10烷氧基、C 1～10烷氧甲基、C 1～10烷氧乙基、C 3～6环烷基、苯基、2,5-二氯苯基、吡嗪基、吡啶基、萘基、四氢萘基、噁唑基或异噁唑基，或者任选地被C 1～4烷基、C 1～4烷氧基、卤素或卤代C 1～4烷基取代。
  3. 根据权利要求1所述的肽硼酸酯类化合物或其药用盐，其特征在于所述R 1选自：
    Figure PCTCN2019088506-appb-100002
    其中R 3、R 4、R 5和R 6独立地选自氢、甲基、甲氧基、乙基、乙氧基、氯、溴、氟或三氟甲基。
  4. 根据权利要求1所述的肽硼酸酯类化合物或其药用盐，其特征在于所述R 2选自苯基、甲 氧基、甲巯基、环己基、2,3-二氢-1,4-苯并二噁烷，或者任选地被一个或多个C 1～4烷基、C 1～ 4烷氧基、卤素或三氟甲基取代。
  5. 根据权利要求1所述的肽硼酸酯类化合物或其药用盐，其特征在于所述B、Z 1和Z 2一起形成硼酸-α-蒎烷二醇酯，或者B与Z 1和Z 2一起形成硼酸酯，并且与硼原子相连的是氧原子。
  6. 根据权利要求1所述的肽硼酸酯类化合物或其药用盐，其特征在于所述B、Z 1和Z 2一起形成硼酸-α-蒎烷二醇酯，或者B、Z 1和Z 2一起形成二乙醇胺硼酸酯、柠檬酸硼酸酯、酒石酸硼酸酯、苹果酸硼酸酯、α-羟基-戊二酸硼酸酯，或与葡萄糖邻羟基结构形成葡萄糖硼酸酯。
  7. 化合物或其药用盐，选自：
    Figure PCTCN2019088506-appb-100003
    Figure PCTCN2019088506-appb-100004
  8. 药物组合物，其包含药用载体及权利要求1-7中任一项的化合物，任选同时、分别或依序与一种或多种其他治疗剂组合。
  9. 权利要求1-7中任一项的化合物在制备蛋白酶体抑制剂中的用途。
  10. 权利要求1-7中任一项的化合物在制备治疗疾病药物中的用途，其特征在于所述疾病选自：实体瘤和血液瘤，其中实体瘤选自非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、胰腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、胃肠间质瘤、或鼻咽癌；血液瘤选自白血病、多发性骨髓瘤、套细胞淋巴瘤或组织细胞性淋巴癌。
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