WO2018171816A1 - 一种氘代的二肽硼酸或其酯类化合物及其合成方法与用途 - Google Patents

一种氘代的二肽硼酸或其酯类化合物及其合成方法与用途 Download PDF

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WO2018171816A1
WO2018171816A1 PCT/CN2018/087761 CN2018087761W WO2018171816A1 WO 2018171816 A1 WO2018171816 A1 WO 2018171816A1 CN 2018087761 W CN2018087761 W CN 2018087761W WO 2018171816 A1 WO2018171816 A1 WO 2018171816A1
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deuterated
compound
boronic acid
acid
pharmaceutically acceptable
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PCT/CN2018/087761
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English (en)
French (fr)
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朱永强
雷萌
白恩赫
冯华云
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南京陵瑞医药科技有限公司
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    • 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
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/001—Acyclic or carbocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/005—Sugars; Derivatives thereof; Nucleosides; Nucleotides; Nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • the invention belongs to the field of drug synthesis, and particularly relates to a deuterated dipeptide boronic acid or an ester compound thereof, a synthesis method and use thereof.
  • the Ubiquitin-Proteasome Pathway is the main pathway for the degradation of intracellular protein systems and is involved in many physiologically important cellular processes including signal transduction, immune response, unfolded protein response and cell cycle. progress. This pathway has important relationships with cardiovascular and cerebrovascular diseases, cancer, and the pathogenesis of neurodegenerative diseases.
  • bortezomib PS-341
  • PS-341 the first proteasome inhibitor in 2003, was approved by the FDA for the treatment of recurrent myeloma.
  • the drug was approved for marketing in the European Union for multiple myeloma.
  • September 2005 the drug was introduced by Xi'an Yangsen and was first listed in Guangzhou, China.
  • the object of the present invention is to provide a novel class of compounds having proteasome inhibitory activity and better pharmacodynamic properties, as well as a synthesis method and use thereof.
  • the first aspect of the present invention discloses a deuterated dipeptide boronic acid or boric acid ester compound, or a crystal form thereof, or a pharmaceutically acceptable hydrate or solvate thereof, which has the structure of the following formula (a) Show,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from hydrogen, deuterium or halogen, or one or more deuterated or fully deuterated C 1 -C 4 alkyl groups; 1 , at least one of R 2 , R 3 , R 4 , R 5 , and R 6 is deuterated or deuterated;
  • the P group is selected from the following fragments:
  • B is a boron atom
  • Z 1 and Z 2 are independently selected from a hydroxyl group, a C 1-10 alkoxy group or an aryloxy group
  • the P group is a heterocyclic group containing N, S and/or O formed by the following fragment and other compounds,
  • B is a boron atom and the atom attached to formula (a) is B.
  • both R 1 and R 4 are halogen, and two of R 2 , R 3 , R 5 and R 6 and two hydrazines.
  • both R 1 and R 4 are chlorine, R 2 and R 3 are all hydrogen, and R 5 and R 6 are both hydrazine.
  • the P group is a boronic acid ester or anhydride group formed from the following fragment with an alpha-hydroxycarboxylic acid or a beta-hydroxycarboxylic acid,
  • B is a boron atom and the atom attached to formula (a) is B.
  • the P group is a borate group formed from a compound having two or more independent hydroxyl structures which are not on the same carbon atom,
  • B is a boron atom and the atom attached to formula (a) is B.
  • the deuterium isotope content of the deuterated substitution site is at least greater than the natural rhenium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%. More preferably greater than 95%, more preferably greater than 99%.
  • the compound of formula (I) contains at least one deuterium atom, more preferably two deuterium atoms.
  • R 1 and R 4 are selected from halogen; more preferably chlorine;
  • R 2 and R 3 are hydrogen
  • R 5 and R 6 are each independently selected from the group consisting of hydrogen, deuterium, deuterated methyl, deuterated ethyl, and halogen.
  • the borate moiety is a five-membered ring. In other embodiments, the borate moiety is a six-membered ring. In other embodiments, the borate moiety is a mixture of a five-membered ring and a six-membered ring.
  • the term "independently selected from” is that a plurality of groups are each selected from certain substituents, and that the groups are not related to each other, for example, "R 1 , R 2 are independently selected from hydrogen, deuterium or halogen. ",” means that R 1 is selected from hydrogen, deuterium or halogen, R 2 is selected from hydrogen, deuterium or halogen, and the R 1 and R 2 groups are not related to each other.
  • alpha-hydroxycarboxylic acid refers to a compound containing a hydroxyl group attached directly to a carbon atom relative to the alpha position of the carboxylic acid group.
  • alpha-hydroxycarboxylic acid is not intended to be limited to compounds having only one hydroxyl group and one carboxylic acid group.
  • ⁇ -hydroxycarboxylic acid refers to a compound containing a hydroxyl group attached directly to a carbon atom relative to the beta position of the carboxylic acid group.
  • ⁇ -hydroxycarboxylic acid is not intended to be limited to compounds having only one hydroxyl group and one carboxylic acid group.
  • the beta-hydroxy carboxylic acid is selected from the group consisting of malic acid, citric acid, 3-hydroxybutyric acid, beta-hydroxyisovaleric acid, and salicylic acid. In some other embodiments, the beta-hydroxy carboxylic acid is selected from the group consisting of malic acid, citric acid, 3-hydroxybutyric acid, beta-hydroxyisovaleric acid, tartaric acid, and salicylic acid. In certain embodiments, the beta-hydroxy carboxylic acid is citric acid. Some other non-limiting examples of beta-hydroxycarboxylic acids include glucoheptonic acid, maltoic acid, lactobionic acid, and galactosuccinic acid. Some other non-limiting examples of beta-hydroxycarboxylic acids include diquat, 1-hydroxy-2-naphthoic acid, and 3-hydroxy-2-naphthoic acid.
  • the alpha-hydroxy acid or beta-hydroxy acid is selected from the group consisting of glycolic acid, malic acid, hexahydromandelic acid, 2-hydroxyisobutyric acid, capric acid, mandelic acid, lactic acid, 3-hydroxy-butyric acid , ⁇ -hydroxyisovalerate, 2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid,-hydroxyisohexanoic acid, tartaric acid, salicylic acid and benzoic acid.
  • the compound having two or more independent hydroxyl structures that are not on the same carbon atom is selected from the group consisting of diethanolamine, tartaric acid, alpha-D-glucose, D-ribose, and the like.
  • the structure of the formula (a) may be of the following two structures:
  • formula (a) may also be represented by formula (3) or (4):
  • the carbon atom to which the R 5 and R 6 groups in the compound of the present invention are attached may be a racemate or may be optically active.
  • the compound of interest is a preferred compound selected from the group consisting of;
  • a process for the preparation of the deuterated dipeptide boronic acid or borate ester compound, or a crystalline form thereof, or a pharmaceutically acceptable hydrate or solvate thereof comprising the following reaction process:
  • the P group is a heterocyclic group containing N, S and/or O formed by the following fragment and other compounds,
  • B is a boron atom and the atom attached to formula (a) is B.
  • a particularly preferred compound of the invention is ((R)-1-(2-(2,5-benzamide)-2,2-diacetylacetylamino)-3-methylbutyl)borate Ethanolamine ester (compound NNU-458).
  • Another particularly preferred compound of the invention is ((R)-1-(2-(2,5-benzamide)-2,2-dioxaacetylamino)-3-methylbutyl)boronic acid Phthalate
  • the present invention provides methods for their preparation.
  • the method includes:
  • NNU-455 can be reacted with decanoic acid in a similar manner to form NNU-459.
  • the compound (VIII) is produced by the following method:
  • the compound (VII) is reacted under the action of an inert solvent, heavy water, DMSO-d 6 and a base to form the compound (VIII).
  • the compound (VII) is produced by the following method:
  • the compound (VI) is produced by the following method:
  • the compound (IV) and the compound (V) are dehydrated and condensed by a condensing agent and a base to form the compound (VI).
  • the compound (IV) is obtained by the following method:
  • the compound (III) is obtained by the following method:
  • the base is selected from the group consisting of potassium carbonate, cesium carbonate, sodium hydride, potassium hydride, potassium hydroxide, sodium hydroxide or a combination.
  • a peptide condensing agent commonly used 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.
  • each reaction temperature is carried out at -20 to 100 °C.
  • a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to a compound of the first aspect of the invention, or a pharmaceutically acceptable crystalline form thereof The hydrate or solvate is mixed to form a pharmaceutical composition.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable hydrate or a solvent Compound.
  • the pharmaceutical composition is an injection, a sachet, a tablet, a pill, a powder or a granule.
  • the pharmaceutical composition further comprises an additional therapeutic agent, which is a medicament for cancer, cardiovascular disease, inflammation, immune disease, kidney disease, angiogenesis, prostate disease.
  • an additional therapeutic agent which is a medicament for cancer, cardiovascular disease, inflammation, immune disease, kidney disease, angiogenesis, prostate disease.
  • the therapeutic agents include, but are not limited to, 5-fluorouracil, AV412, avastin (bevacizumab), bexarotene, bortezomib, ossification Calcitriol, canertinib, capecitabine, carboplatin, celecoxib, cetuximab, CHR-2797, cis Platinum (cisplatin), dasatinib, digoxin, enzastaurin, erlotinib, etoposide, everolimus, fulvestrant Fulvestrant), gefitinib, 2,2-difluorodeoxycytidine, genistein, imatinib, irinotecan , lapatinib, lenalidomide, letrozole, leucovorin, matuzumab, oxaliplatin, paclitaxel Paclitaxel), panitumumab, pegfilgrastin, peglated al
  • a compound as described in the first aspect of the invention or a crystalline form, a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the preparation of a proteasome inhibiting substance Pharmaceutical composition.
  • the compounds are useful for the treatment and prevention of diseases associated with proteasome targets.
  • the pharmaceutical composition is for treating and preventing a disease: cancer, cardiovascular disease, inflammation, immune disease, kidney disease, angiogenesis, or prostate disease.
  • the cancer includes, but is not limited to, multiple myeloma, non-small cell lung cancer, uterine cancer, rectal, brain cancer, head cancer, neck cancer, skin cancer, prostate cancer, breast Cancer, solid tumor, kidney cancer, blood cancer, liver cancer, stomach cancer, or pancreatic cancer.
  • a method of treatment comprising the step of administering a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable hydrate or solvent, to a subject in need of treatment
  • the composition, or the pharmaceutical composition described in the third aspect of the invention is administered to inhibit the proteasome.
  • the disease comprises cancer, cardiovascular disease, inflammation, immune disease, kidney disease, angiogenesis or prostate disease.
  • the deuterated dipeptide boronic acid and boric acid ester of the present invention have more excellent pharmacokinetic and/or pharmacodynamic properties than the undeuterated compound, and thus are more suitable as a compound for inhibiting proteasome. It is further suitable for the preparation of a medicament for treating cancer and related diseases.
  • the present invention has been completed on this basis.
  • NNU-459 also showed better activity than the control compound MLN-9708 on the IC 50 (nM) of tumor cell line U266, RPMI-8266 and breast cancer cell line MAD-MB-231. .
  • deuterated refers to the replacement of one or more hydrogens in a compound or group by deuterium. Deuterated can be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms “one or more deuterated” are used interchangeably with “one or more deuterated”.
  • the deuterated substitution of germanium in the unknown isotope content is at least greater than the natural rhenium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%. More preferably greater than 95%, more preferably greater than 99%.
  • the compound of the formula contains at least one deuterium atom, more preferably two deuterium atoms.
  • compound of the invention refers to a compound of formula (a).
  • the term also encompasses various crystalline forms, pharmaceutically acceptable hydrates or solvates of the compounds of formula (a).
  • the preparation methods of the undeuterated dipeptide boronic acid and its esters used in the present invention are known.
  • the dipeptide boronic acid and its ester corresponding to the deuterated can be used as the starting material of the corresponding deuterated boric acid compound. Synthesize using the same route.
  • a preferred preparation procedure is as follows.
  • compositions and methods of administration are provided.
  • the compound of the present invention has excellent inhibitory activity against proteasome, the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable hydrate or solvate thereof, and pharmaceutical composition containing the compound of the present invention as a main active ingredient It can be used to treat, prevent, and alleviate diseases mediated by the proteasome.
  • the compounds of the invention are useful in the treatment of diseases such as cancer, cardiovascular disease, obesity, diabetes and the like.
  • compositions of the present invention comprise a safe or effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical compositions contain from 1 to 2000 mg of the compound of the invention per agent, more preferably from 10 to 200 mg of the compound of the invention per agent.
  • the "one dose" is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity.
  • “compatibility” it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • carrier used interchangeably herein and include any and all solvents, diluents and other liquid carriers, dispersion or suspension aids, surfactants, pH adjusters, Isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., are suitable for the particular dosage form desired.
  • any conventional carrier medium is incompatible with a compound of the invention, for example, by causing any undesirable biological effects or interaction with any other component of the pharmaceutical composition to render the compound ineffective or ineffective, or to produce a hazardous substance, its use is It is considered to be within the scope of the invention.
  • Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, carbonates, magnesium hydroxide and aluminum glycine, Sorbic acid or potassium sorbate, a mixture of partial glycerides of saturated plant fatty acids, water, pyrogen-free water, salts or electrolytes such as protamine sulfate, disodium phosphate, disodium hydrogen phosphate, sodium chloride and zinc salts, colloidal dioxide Silicon, magnesium trisilicate, polyvinylpyrrolidone, polyacrylate, polyethylene-polyoxypropylene block polymer, lanolin, sugars such as lactose, glucose, sucrose and mannitol, starches such as corn starch and potato starch, cellulose And derivatives thereof such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, powdered tragacanth; malt
  • compositions of the present invention can be prepared by methods well known in the art, such as conventional granulation, mixing, dissolving, encapsulating, lyophilizing or emulsifying methods and the like.
  • the compositions can be produced in a variety of forms including granules, pellets or granules, powders, including freeze-dried, spin-dried or spray-dried powders, amorphous powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, mashes, and mixtures. Suspending agent or solution.
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and according to a preferred embodiment, the composition of the present invention is formulated for administration to a mammal, preferably to a human.
  • These pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implantable kit.
  • the term "digestive administration" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic and intracranial injection or infusion techniques.
  • the composition is administered orally, intravenously or subcutaneously.
  • the formulations of the invention can be designed to be short acting, fast release or long lasting. Additionally, the compounds can be administered in a local rather than systemic manner, such as at the site of a tumor (eg, by injection).
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl benzoate, propylene glycol, 1,3-butanediol, cyclodextrin, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin , tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters, sorbitan and mixtures thereof.
  • the oral compositions may also contain adj
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example , starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants , for example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; An absorption accelerator, for example, a quaternary amine compound; (g) a wetting agent, for example, cetylene glycol
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • Injectable preparations for example, sterile injectable aqueous or oily suspensions, may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, a compound sodium chloride injection, and an isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium, and any of the ⁇ RTIgt; Further, a fatty acid such as oleic acid is used for the preparation of an injection.
  • the injectable preparation may be sterilized by, for example, filtration through a bacterial retention filter or by incorporating a sterilizing agent in the form of a sterile solid composition which may be dissolved or dispersed in sterile water or otherwise before use.
  • sterile injectable media Compositions formulated for parenteral administration may be administered by bolus injection or by timed bolus injection, or may be administered by continuous infusion.
  • Dosage forms for topical or transdermal administration of a compound of the invention include ointments, powders, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers which may be required.
  • Ophthalmic formulations, ear drops and eye drops are also considered to be within the scope of the invention.
  • the present invention contemplates the use of transdermal patches that have the added advantage of providing controlled delivery of a compound to the body.
  • Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • the invention provides pharmaceutical compositions of the compounds and other excipients described herein. In some other embodiments, the invention provides pharmaceutical compositions of a compound of NNU-455 and other excipients described herein. In other embodiments, the invention provides a pharmaceutical composition comprising a decanoate (NNU-459) and diethanolamine (NNU-458) of NNU 455 and other excipients described herein.
  • the pharmaceutical formulations of the present invention are prepared by using excipients having low or low moisture content and using dry or non-aqueous formulation methods to provide a stable solid oral dosage form of the active compound.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 20 to 500 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the compound of the present invention has excellent inhibitory property against a proteasome.
  • the dosage can be varied and a long acting formulation can be formed, which can also improve the suitability in the form of a long acting formulation.
  • Substituting hydrazine for the hydrogen atom in the compound, due to its isotope effect, can increase the drug concentration of the compound in the animal to improve the efficacy of the drug.
  • Step III Take the compound obtained in Step III (129mg, 0.31mmol) was dissolved with 2.5mLMeOH, was added LiOH ⁇ H 2 O (39mg, 0.92mmol) and H 2 O (0.8mL), TLC detection, reaction was complete after 2h.
  • the organic phase was dried, the aqueous phase was extracted with diethyl ether (2 ⁇ 1 mL), and aqueous hydrochloric acid was added dropwise to pH 2-3 to yield a large white solid, ethyl acetate.
  • -N-(2,5-dichlorobenzoyl)glycine 106 mg, yield 86.0%, mp 169.3-170.8 °C.
  • the compound VII (500 mg, 1.38 mmol) was dissolved in 16 ml of anhydrous tetrahydrofuran, potassium carbonate (191.4 mg, 1.38 mmol), DMSO-d6 (3.75 ml), and water (2.5 ml) were added, and the reaction was stirred for 5 hours in an oil bath at 65 ° C. . After the completion of the reaction by LC-MS, the tetrahydrofuran was evaporated under reduced pressure, and a small amount of water was added and the mixture was extracted with ethyl acetate. The organic phase was washed twice with water and brine, and the solvent was evaporated. Pure product 278mg, yield 55.6%.
  • the citric acid (192.12 mg, 0.39 mmol) was dissolved in 2 mL of ethyl acetate, and the temperature was raised to 74 ° C. After the decanoic acid was completely dissolved, Compound VII (363.03 mg, 0.36 mmol) dissolved in 1 mL of ethyl acetate was added. Slowly cool to 60 ° C, react for 3 h, then slowly cool to 25 ° C overnight. The reaction was detected by TLC, filtered, and the cake was vacuum dried to give a pure product (yield: 90.0 mg).
  • Two groups were administered with 0.100 mg ⁇ mL -1 NNU-459 and MLN-9708 in the tail vein at a dose of 0.500 mg ⁇ kg -1 , respectively, before administration and 10 min, 20 min, 30 min, 1 h, 2 h, 4 h after administration.
  • blood was collected from the jugular vein by about 0.200mL, placed in a test tube containing EDTA-K 2 , centrifuged at high speed (7800 ⁇ g) for 5 min, and the plasma was separated and stored at -15 ° C to -35 ° C. . Used to compare the pharmacokinetic differences between NNU-459 and MLN-9708 administered intravenously.
  • the other two groups were administered 0.150 mg ⁇ mL -1 NNU-459 and MLN-9708 at a dose of 1.50 mg ⁇ kg -1 , respectively, before administration and 5 min, 10 min, 20 min, 30 min, 1 h, 2 h after administration.
  • blood was collected from the jugular vein by about 0.200mL, placed in a test tube containing EDTA-K 2 , and centrifuged at high speed (7800 ⁇ g) for 15 minutes, and the plasma was separated at -15 ° C ⁇ -35 °C save. Used to compare the pharmacokinetic differences between NNU-459 and MLN-9708 administered orally.
  • the compound of the present invention has better pharmacokinetics in animals and thus has better pharmacodynamics and therapeutic effects.
  • the metabolism of the compounds of the present invention in organisms is altered by deuteration. This leads to a reduction in the first-pass effect.
  • the dosage can be varied and a long acting formulation can be formed, which can also improve the suitability in the form of a long acting formulation.
  • the pharmacokinetic effect is also altered by deuteration because the deuterated compound completely forms another hydrate membrane such that the distribution in the organism is significantly different from the undeuterated compound.
  • Example 4 Liver microsome stability experiment of NNU-455 and MLN-2238
  • NNU-455 and MLN-2238 of NNU-459 and MLN-9708 was determined in liver microsomes of different species.
  • Human liver microsome working solution (2 ⁇ ) 0.3 mL of human liver microsome stock solution (20 mg/mL) was added to 5.7 mL of phosphate buffer to obtain a working solution of 1.0 mg/mL.
  • Rat liver microsome working solution (2 ⁇ ) 0.3 mL of rat liver microsome stock solution (20 mg/mL) was added to 5.7 mL of phosphate buffer to obtain a working solution of 1.0 mg/mL.
  • Mouse liver microsome working solution (2 ⁇ ) 0.3 mL of mouse liver microsome stock solution (20 mg/mL) was added to 5.7 mL of phosphate buffer to obtain a working solution of 1.0 mg/mL.
  • Canine liver microsome working solution (2 ⁇ ) 0.3 mL of canine liver microsome stock solution (20 mg/mL) was added to 5.7 mL of phosphate buffer to obtain a working solution of 1.0 mg/mL.
  • v. Monkey liver microsome working solution (2 ⁇ ) 0.3 mL of monkey liver microsome stock solution (20 mg/mL) was added to 5.7 mL of phosphate buffer to obtain a working solution of 1.0 mg/mL.
  • Working solution of the test article (10 ⁇ ) firstly prepare a 10 mM stock solution with DMSO, then dilute to 100 ⁇ M with 50% methanol-water, and then dilute to a working solution at a concentration of 10 ⁇ M with a phosphate buffer.
  • Stop Solution 100 ng/mL tolbutamide was formulated with methanol as a stop solution containing an internal standard.
  • the matrix required for the reaction was added in the order of Table 1 below, and the test article was made in 3 parallels at each time point, and the positive control was made in 3 parallels at each time point.
  • the concentration of the compound at different incubation time points is expressed as Aa/Ai (the ratio of the compound to the peak area of the internal standard mass spectrometry signal).
  • the residual rate is obtained by dividing the concentration of each compound at each time point by the concentration at time 0, and the calculation formula is as follows:
  • Vd is the apparent partition coefficient
  • the detection solution used in this patent is a single-solution cell proliferation assay kit from Promega; the cells used are U266, RPMI8226.
  • the experimental system is 110 ⁇ L, 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 to 1.77 ⁇ 10 -9 M, and the last concentration is 0 M. It is 5 ⁇ 10 -7 M to 1.95 ⁇ 10 -8 M, and the last concentration is 0M.
  • the specific experimental process is as follows:
  • the drug was accurately weighed and dissolved in DMSO to 10 -2 M.
  • the dilution configuration U266 was 1 ⁇ 10 4 /well
  • the RPMI 8226 was 1 ⁇ 10 4 /well.
  • NNU-459, NNU-458 are similar to the IC 50 MLN-9708, showed the same activity or better inhibition of tumor.
  • the medicament of the present invention may take, but is not limited to, the following pharmaceutical composition:
  • Dosage per unit dose (mg) Dosage (%w/w or w/w) The compound 4 1.3 Microcrystalline cellulose 231.8 77.3 Pregelatinized starch 60 20 Silica 1.2 0.4 Magnesium stearate 3 1 Content weight 300 100
  • Dosage per unit dose (mg) Dosage (%w/w or w/w)
  • the medicament 4 1.3 Microcrystalline cellulose 246.8 82.3 Mannitol 45 15 talcum powder 1.2 0.4 Magnesium stearate 3 1 Content weight 300 100
  • Dosage per unit dose (mg) Dosage (%w/w or w/w) The compound 3 1.5 Microcrystalline cellulose 150 75 corn starch 45 22.5 Magnesium stearate 2 1 Content weight 200 100
  • Dosage per unit dose (mg) Dosage (%w/w or w/w) The compound 3 1.5 Mannitol 120 60 lactose 45 37.5 Magnesium stearate 2 1 Content weight 200 100
  • Mannitol 100 50 Microcrystalline cellulose 95 47.5 Magnesium stearate 2 1 Content weight 200 100
  • Dosage per unit dose (mg) Dosage (%w/w or w/w) The compound 2.3 1.15 Microcrystalline cellulose 195 97.5 talcum powder 0.7 0.35 Magnesium stearate 2 1 Content weight 200 100
  • Dosage per unit dose (mg) Dosage (%w/w or w/w) The compound 2.3 1.15 Silicified microcrystalline cellulose 155 77.5 Mannitol 40 20 talcum powder 0.7 0.35 Magnesium stearate 2 1 Content weight 200 100
  • the above pharmaceutical composition was uniformly mixed in a usual manner, and then placed in an opaque white gelatin capsule.
  • the compound includes the compound NNU-455 and its phthalate ester (NNU-459) and diethanolamine ester (NNU-458).

Abstract

本发明公开了一种氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其结构如下式(a)所示，其中：R1、R2、R3、R4、R 5、R6独立地选自氢、氘或卤素，或一个或多个氘代的或全氘代的C1-C4烷基；并且R1、R2、R3、R4、R5、R6中至少一个是氘代的或是氘。本发明化合物可以有效抑制蛋白酶体，有效治疗或预防癌症、心血管疾病、炎症、免疫性疾病、肾病、血管发生或前列腺疾病。

Description

一种氘代的二肽硼酸或其酯类化合物及其合成方法与用途 技术领域
本发明属于药物合成领域，具体涉及一种氘代的二肽硼酸或其酯类化合物及其合成方法与用途。
背景技术
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。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，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。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)。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前20之一。万珂在中国市场售价为每3.5毫克约13000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。2015年12月美国FDA又批准了第一个口服蛋白酶体抑制剂Ixazomib上市，用于治疗多发性骨髓瘤。但是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。而且，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口服药物是我们目前需要重点解决的问题。
发明内容
发明目的：针对上述技术问题，本发明目的提供一类新型的具有蛋白酶体抑制活性和更好药效学性能的化合物及其合成方法和用途。
技术方案：本发明第一方面公开了一种氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其结构如下式(a)所示，
Figure PCTCN2018087761-appb-000001
其中：
R 1、R 2、R 3、R 4、R 5、R 6独立地选自氢、氘或卤素，或一个或多个氘代的或全氘代的C 1-C 4烷基；并且R 1、R 2、R 3、R 4、R 5、R 6中至少一个是氘代的或是氘；
P基团选自以下片段：
Figure PCTCN2018087761-appb-000002
其中，B为硼原子，Z 1、Z 2独立地选自羟基、C 1-10的烷氧基或芳氧基；
或者P基团是由以下片段与其他化合物所形成的含有N、S和/或O的杂环基团，
Figure PCTCN2018087761-appb-000003
其中B为硼原子，并且与式(a)连接的原子为B。
优选，所述R 1和R 4均为卤素，R 2、R 3、R 5、R 6中有两个氢和两个氘。
优选，所述R 1和R 4均为氯，R 2和R 3均为氢、R 5和R 6均为氘。
优选，P基团是由以下片段与α-羟基羧酸或β-羟基羧酸形成硼酸酯或酸酐基团，
Figure PCTCN2018087761-appb-000004
其中B为硼原子，并且与式(a)连接的原子为B。
优选，P基团是由以下片段与具有不在同一碳原子上的两个或两个以上独立羟基结构的化合物形成硼酸酯基团，
Figure PCTCN2018087761-appb-000005
其中B为硼原子，并且与式(a)连接的原子为B。
在另一优选例中，氘代在氘取代位置的氘同位素含量至少是大于天然氘同位素含量(0.015％)，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。
在另一优选例中，式(I)化合物至少含有1个氘原子，更佳地2个氘原子。
在另一优选例中，R 1、R 4选自卤素；更佳的是氯；
在另一优选例中，R 2、R 3为氢；
在另一优选例中，R 5、R 6分别独立地选自氢、氘、氘代甲基、氘代乙基、卤素。
在一些实施例中硼酸酯部分是五元环。在另一些实施例中，硼酸酯部分是六元环。在其他一些实施例中，硼酸酯部分是五元环和六元环的混合物。
如本文所用，术语“独立地选自”是多个基团分别选自某些取代基，并且各基团之间没有相互联系，例如“R 1、R 2独立地选自氢、氘或卤素”，是指R 1选自氢、氘或卤素，R 2选自氢、氘或卤素，并且R 1和R 2基团之间没有相互联系。
如本文所用，术语“α-羟基羧酸”是指含有直接连接到相对于羧酸基团的α位置的碳原子上的羟基的化合物。如本文所用，术语“α-羟基羧酸”不旨在限于仅具有一个羟基和一个羧酸基团的化合物。
如本文所用，术语“β-羟基羧酸”是指含有直接连接到相对于羧酸基团的β位的碳原子上的羟基的化合物。如本文所用，术语“β-羟基羧酸”不旨在限于仅具有一个羟基和一个羧酸基团的化合物。
在一些实施方案中，β-羟基羧酸选自苹果酸，枸橼酸，3-羟基丁酸，β-羟基异戊酸和水杨酸。在一些其它实施方案中，β-羟基羧酸选自苹果酸，枸橼酸，3-羟基丁酸，β-羟基异戊酸，酒石酸和水杨酸。在某些实施方案中，β-羟基羧酸是枸橼酸。β-羟基羧酸的一些其它非限制性实例包括葡庚糖酸，麦芽糖酸，乳糖酸和半乳糖二酸。β-羟基羧酸的一些其它非限制性实例包括敌草酸，1-羟基-2-萘甲酸和3-羟基-2-萘甲酸。
在一些实施方案中，α-羟基酸或β-羟基酸选自乙醇酸，苹果酸，六氢扁桃酸，2-羟基异丁酸，枸橼酸，扁桃酸，乳酸，3-羟基-丁酸，β-羟基异戊酸，2-羟基-3,3-二甲基丁酸，2-羟基-3-甲基丁酸，-羟基异己酸，酒石酸，水杨酸和苯甲酸。
在一些实施方案中，具有不在同一碳原子上的两个或两个以上独立羟基结构的化合物选自二乙醇胺，酒石酸，α-D-葡萄糖，D-核糖等。
当上述α-羟基羧酸或β-羟基羧酸是枸橼酸时，式(a)的结构可以是以下两种结构：
Figure PCTCN2018087761-appb-000006
在另一些实施方案中，α-羟基羧酸或β-羟基羧酸是枸橼酸时，羧酸与硼原子之间可以形成另外的键。不受任何化学键合理论的限制，在这样的实施方案中，式(a)也可以由式(3)或(4)表示：
Figure PCTCN2018087761-appb-000007
还存在其他不受任何化学键和理论的限制的，可以用于描述如(3)或(4)羟基羧酸与硼酸进一步键和的其他形式，本文不再一一描述。
本发明化合物中的R 5、R 6基团所连接的碳原子可以为消旋体，也可以具有光学活性。
另一优选例中，所属化合物是选自下组的优选化合物；
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸；
Figure PCTCN2018087761-appb-000008
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸-(+)-α-蒎烷二醇酯
Figure PCTCN2018087761-appb-000009
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸枸橼酸酯
Figure PCTCN2018087761-appb-000010
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-氘代二甲基-乙酰氨基)-3-甲基丁基)硼酸枸橼酸酯
Figure PCTCN2018087761-appb-000011
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸酒石酸酯
Figure PCTCN2018087761-appb-000012
酒石酸((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸酯
Figure PCTCN2018087761-appb-000013
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸α-羟基戊二酸酯
Figure PCTCN2018087761-appb-000014
((R)-1-((S)2-(2,5-苯甲酰胺基)-2-氘-乙酰氨基)-3-甲基丁基)硼酸苹果酸酯
Figure PCTCN2018087761-appb-000015
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸二乙醇胺酯
Figure PCTCN2018087761-appb-000016
((R)-1-((R)2-(2,5-苯甲酰胺基)-2,2-二氘-乙酰氨基)-3-甲基丁基)硼酸葡萄糖酯
Figure PCTCN2018087761-appb-000017
((R)-1-((R)2-(2,5-苯甲酰胺基)-2,2-二氘-乙酰氨基)-3-甲基丁基)硼酸二葡萄糖酯
Figure PCTCN2018087761-appb-000018
((R)-1-((R)2-(2,5-苯甲酰胺基)-2-二氘-乙酰氨基)-3-甲基丁基)硼酸核糖酯
Figure PCTCN2018087761-appb-000019
本发明第二方面提供了所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物的制备方法，包括以下所示反应过程：
Figure PCTCN2018087761-appb-000020
其中，P基团是由以下片段与其他化合物所形成的含有N、S和/或O的杂环基团，
Figure PCTCN2018087761-appb-000021
其中B为硼原子，并且与式(a)连接的原子为B。
本发明的一种特别优选的化合物是((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸二乙醇胺酯(化合物NNU-458)。
Figure PCTCN2018087761-appb-000022
本发明的另一种特别优选的化合物是((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸枸橼酸酯
Figure PCTCN2018087761-appb-000023
本发明提供了它们的制备方法。
所述方法包括：
在沸腾的乙酸乙酯中，化合物(Ⅷ)即NNU-455，与二乙醇胺(DEA)反应，通过缓慢降温析晶得到终产物NNU-458；
Figure PCTCN2018087761-appb-000024
在另一优选例中，可以通过类似方法让NNU-455与枸橼酸反应，生成NNU-459。
在另一优选例中，化合物(Ⅷ)通过以下方法制备：
在惰性溶剂、重水、DMSO-d 6以及碱的作用下，化合物(Ⅶ)反应形成所述化合物(Ⅷ)。
Figure PCTCN2018087761-appb-000025
在另一优选例中，化合物(Ⅶ)通过以下方法制备：
化合物(Ⅵ)在酸的作用下与异丁基硼酸反应生成化合物(Ⅶ)；
Figure PCTCN2018087761-appb-000026
在另一优选例中，化合物(Ⅵ)通过以下方法制备：
在惰性溶剂中，化合物(Ⅳ)与化合物(Ⅴ)在缩合剂以及碱的作用下，脱水缩合形成所述化合物(Ⅵ)。
Figure PCTCN2018087761-appb-000027
在另一优选例中，化合物(Ⅳ)通过以下方法得到：
在甲醇和水的溶液中，在碱的作用下化合物(Ⅲ)脱甲基得到化合物(Ⅳ)
Figure PCTCN2018087761-appb-000028
在另一优选例中，化合物(Ⅲ)通过以下方法得到：
在惰性溶剂中，2,5-二氯苯甲酸(Ⅰ)和化合物(Ⅱ)在缩合剂和碱的作用下，得到化合物(Ⅲ)
Figure PCTCN2018087761-appb-000029
在另一优选例中，所述的碱选自碳酸钾、碳酸铯、氢化钠、氢化钾、氢氧化钾、氢氧化钠或组合。
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。
在另一优选例中，各反应温度在-20～100℃下进行。
在本发明的第三方面，提供了一种制备药物组合物的方法，包括步骤：将药学上可接受的载体与本发明第一方面中所述的化合物，或其药学上可接受的晶型、水合物或溶剂合物进行混合，从而形成药物组合物。
在本发明的第四方面，提供了一种药物组合物，它含有药学上可接受的载体和本发明第一方面中所述的化合物，或其晶型、药学上可接受的水合物或溶剂合物。
在另一优选例中，所述的药物组合物为注射剂、囊剂、片剂、丸剂、散剂或颗粒剂。
在另一优选例中，所述的药物组合物还含有另外的治疗药物，所述的另外的治疗药物为癌症、心血管疾病、炎症、免疫性疾病、肾病、血管发生、前列腺疾病的药物。
更佳地，所述的治疗药物包括(但并不限于):5-氟尿嘧啶、AV412、阿瓦斯丁TM(avastin,bevacizumab)、贝沙罗汀(bexarotene)、硼替佐米(bortezomib)、骨化三醇(calcitriol)、卡奈替尼(canertinib)、卡培他滨(capecitabine),碳铂(carboplatin),塞来考昔(celecoxib)、西妥昔单抗(cetuximab),CHR-2797、顺铂(cisplatin)、达沙替尼(dasatinib)、 地高辛(digoxin),enzastaurin、埃罗替尼(Erlotinib)、依托泊试(etoposide)、依维莫司(everolimus),氟维司群(fulvestrant)、吉非替尼(gefitinib),2,2-二氟脱氧胞嚓咤核昔(gemcitabine)、金雀异黄素(genistein)、伊马替尼(imatinib)、依立替康(irinotecan)、拉帕替尼(lapatinib)、来那度胺(lenalidomide),来曲哩(letrozole)、亚叶酸(leucovorin)、马妥珠单抗((matuzumab)、奥沙利铂(oxaliplatin)、紫杉醇(paclitaxel)、帕尼单抗(panitumumab),PEG化的粒细胞集落刺激因子(pegfilgrastin),PEG化的α-干扰素(peglatedalfa-interferon)、培美曲(pemetrexed),Polyphenon E、沙铂(satraplatin)、西罗莫司(sirolimus)、舒尼替尼(sutent,sunitinib)、舒林酸(sulindac)、泰索帝(taxotere)、替莫哩胺(temodar,temozomolomide)、Torisel(temsirolimus)、TGOltipifarnib、曲妥单抗(trastuzumab)、丙戊酸(valproic acid)、长春氟宁(vinflunine)、Volociximab,vorinostat和XL647。
在本发明的第五方面，提供了本发明第一方面中所述的化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物的用途，它们被用于制备抑制蛋白酶体的药物组合物。
在另一优选例中，所述的化合物可以用于治疗和预防与蛋白酶体靶点相关的疾病。
在另一优选例中，所述的药物组合物用于治疗和预防以下疾病:癌症、心血管疾病、炎症、免疫性疾病、肾病、血管发生、或前列腺疾病。
在另一优选例中，所述的癌症包括(但并不限于):多发性骨髓瘤、非小细胞肺癌、子宫癌、直肠、脑癌、头癌、颈癌、皮肤癌、前列腺癌、乳腺癌、实体肿瘤、肾癌、血癌、肝癌、胃癌、或胰腺癌。
在本发明的第六方面，提供了一种治疗方法，它包括步骤：给需要治疗的对象施用本发明第一方面中所述的化合物，或其晶型、药学上可接受的水合物或溶剂合物，或施用本发明第三方面中所述的药物组合物，从而抑制蛋白酶体。较佳地，所述的疾病包括：癌症、心血管疾病、炎症、免疫性疾病、肾病、血管发生或前列腺疾病。
应理解，在本发明范围中，上述的技术特征和在下文中具体描述的各技术特征可以相互组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
经研究发现，本发明的氘代二肽硼酸及硼酸酯与未氘代的化合物相比，具有更优异的药物动力学和/或药效学性能，因此更适合作为抑制蛋白酶体的化合物，进而更适用于制备治疗癌症及相关疾病的药物。在此基础上完成了本发明。
以((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸枸橼酸酯(化合物NNU-459)为例。
Figure PCTCN2018087761-appb-000030
大鼠体内药代动力学实验结果显示，NNU-459比MLN9708的半衰期T 2/1延长，曲线下面积AUC显著增加，NNU-459表观清除率也比MLN9708显著减少。同时在人的肝微粒体稳定性实验中，化合物NNU-459的体内活性成分(NNU-455)的半衰期(T 1/2＝108.3min)也要比MLN9708的活性成分(MLN2238)的半衰期(T 1/2＝57.3min)显著增长。
此外在体外实验中，对肿瘤细胞系U266、RPMI-8266以及乳腺癌细胞系 MAD-MB-231抑制的IC 50(nM)上，NNU-459也比对照化合物MLN-9708表现出更好的活性。
Figure PCTCN2018087761-appb-000031
定义
如本文所用，“氘代”指化合物或基团中的一个或多个氢被氘取代。氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。
在另一优选例中，氘代在氘取代未知的氘同位素含量至少是大于天然氘同位素含量(0.015％)，较佳地大于30％，更佳的大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。
在另一优选例中，式化合物至少含有1个氘原子，更佳的为2个氘原子。
活性成分
如本文所用术语“本发明化合物”指式(a)所示化合物。该术语还包括及式(a)化合物的各种晶型形式、药学上可接受的水合物或溶剂合物。
制备方法
下面更具体地描述本发明式(a)结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便的制得，这样的组合可由本发明所属领域的技术人员容易的进行。
本发明使用的未氘代的二肽硼酸及其酯类的制备方法是已知的。对应氘代的二肽硼酸及其酯类可以用相应的氘代硼酸化合物为原料。用同样的路线合成。
以NNU-459为例，一种优选的制备流程如下。
合成路线：
Figure PCTCN2018087761-appb-000032
Figure PCTCN2018087761-appb-000033
2,5-二氯苯甲酸(化合物Ⅰ)和甘氨酸甲酯盐酸盐(化合物Ⅱ)，在肽缩合剂和碱的作用下反应得到(2,5-二氯苯甲酰基)甘氨酸甲酯(化合物Ⅲ)；然后化合物Ⅲ在碱的作用下发生皂化反应得到(2,5-二氯苯甲酰基)甘氨酸(化合物Ⅳ)；化合物Ⅳ与(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(化合物Ⅴ)在肽缩合剂和碱的作用下，反应生成((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸-(+)-α-蒎烷二醇酯(化合物Ⅵ)；在甲醇中，在1N盐酸的作用下化合物5与异丁基硼酸反应生成((R)-1-(2-(2,5-苯甲酰胺基)-乙酰氨基)-3-甲基丁基)硼酸(化合物Ⅶ)；然后在四氢呋喃中，在碳酸钾与氘代DMSO和重水的作用下，生成((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸(化合物Ⅷ)；化合物Ⅶ在沸腾的乙酸乙酯中，与枸橼酸反应得到化合物NNU-459。
具体的合成方法在实施例1中有详细的说明。
药物组合物和施用方法。
由于本发明化合物具有优异的对蛋白酶体的抑制活性，因此本发明化合物及其各种晶型，药学上可接受的水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解由对蛋白酶体介导的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：癌症，心血管疾病，肥胖病，糖尿病等等。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-200mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可以接受的载体”指的是:一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。
术语“载体”，“赋形剂”或“载体”在本文中可互换使用，并且包括任何和所有溶剂，稀释剂和其它液体载体，分散或悬浮助剂，表面活性剂，pH调节剂，等渗剂，增稠或乳化试剂，防腐剂，固体粘合剂，润滑剂等，适合于所需的特定剂型。除非任何常规载体介质与本发明化合物不相容，例如通过产生任何不期望的生物效应或与药物组合物的任何其它组分相互作用使化合物减效或失效，或产生有害物质，否则其用途被认为在本发明的范围。
药学上可以接受的载体包括但不限于离子交换剂，氧化铝，硬脂酸铝，卵磷脂，血清蛋白如人血清白蛋白，缓冲物质如磷酸盐，碳酸盐，氢氧化镁和铝甘氨酸，山梨酸或山梨酸钾，饱和植物脂肪酸的偏甘油酯混合物，水，无热原水，盐或电解质如硫酸鱼精蛋白，磷酸二钠，磷酸氢二钠，氯化钠和锌盐，胶体二氧化硅，三硅酸镁，聚乙烯吡咯烷酮，聚丙烯酸酯，聚乙烯-聚氧丙烯嵌段聚合物，羊毛脂，糖如乳糖，葡萄糖，蔗糖和甘露醇，淀粉如玉米淀粉和马铃薯淀粉，纤维素及其衍生物如羧甲基纤维素钠，乙基纤维素和乙酸纤维素，粉末黄蓍胶；麦芽，明胶，滑石粉，赋形剂如可可脂和栓剂蜡，油如花生油，棉籽油，葵花籽油，芝麻油，橄榄油，玉米油和大豆油，二醇如丙二醇和聚乙二醇，酯如油酸乙酯和月桂酸乙酯，琼脂，海藻酸，等渗盐水，复方氯化钠注射液，醇如乙醇，异丙醇醇，十六醇和甘油，环糊精如羟丙基β-环糊精和磺丁基醚β-环糊精，润滑剂如十二烷基硫酸钠和硬脂酸镁，石油烃如矿物油和凡士林。着色剂，脱模剂，包衣剂，甜味剂，调味剂和芳香剂，防腐剂和抗氧化剂也可以根据配方师的判断存在于组合物中。
本发明的药物组合物可以通过本领域公知的方法制备，例如常规的制粒，混合，溶解，包封，冻干或乳化方法等。组合物可以以各种形式生产，包括颗粒，沉淀或颗粒、粉末，包括冷冻干燥，旋转干燥或喷雾干燥粉末，无定形粉末，片剂，胶囊，糖浆，栓剂，注射剂，乳剂，酏剂，混悬剂或溶液。
本发明化合物或药物组合物的施用方式没有特别限制，根据优选的实施方案，将本发明的组合物配制用于对哺乳动物，优选地对人类的药物施用。本发明的这些药物组合物可以通过口服，消化道外给药，通过吸入喷雾，局部，直肠，鼻，颊，阴道或经由植入型药盒施用。本文所用的术语“消化道外给药”包括皮下，静脉内，肌内，关节内，滑膜内，胸骨内，鞘内，肝内和颅内注射或输注技术。优选地，组合物经口，静脉内或皮下施用。本发明的制剂可以设计为短效，快速释放或长效。另外，化合物可以局部而不是全身方式施用，例如在肿瘤部位施用(例如通过注射)。
用于口服给药的液体剂型包括但不限于药学上可接受的乳剂，微乳剂，溶液剂，混悬剂，糖浆剂和酏剂。除了活性化合物之外，液体剂型可以含有本领域通常使用的惰性稀释剂，例如水或其它溶剂，增溶剂和乳化剂，例如乙醇，异丙醇，碳酸乙酯，乙酸乙酯，苄基醇，苯甲酸苄酯，丙二醇，1,3-丁二醇，环糊精，二甲基甲酰胺，油(特别是棉籽油，花生油，玉米油，胚芽油，橄榄油，蓖麻油和芝麻油)，甘油，四氢糠醇，聚乙二醇和脂肪酸酯，脱水山梨醇及其混合物。除惰性稀释剂外，口服组合物还可包括佐剂，例如润湿剂，乳化剂和悬浮剂，甜味剂，旋转剂和芳香剂。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如枸橼酸钠或磷酸二钙，或与下述成分混合:(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化 合物；(g)润湿剂，例如，鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
可注射制剂，例如无菌可注射水性或油性悬浮液，可根据已知技术使用合适的分散剂或润湿剂和悬浮剂来配制。无菌可注射制剂还可以是在胃肠外可接受的稀释剂或溶剂中的无菌可注射溶液、悬浮液或乳液，例如作为在1,3-丁二醇中的溶液。可以使用的可接受的载体和溶剂是水，复方氯化钠注射液，和等渗氯化钠溶液。此外，无菌的不挥发油通常用作溶剂或悬浮介质，可以使用任何温和的不挥发油，包括合成的甘油单酯或甘油二酯。此外，脂肪酸如油酸用于制备注射剂。可注射制剂可以通过例如通过细菌截留过滤器过滤或通过掺入无菌固体组合物形式的灭菌剂来灭菌，所述无菌固体组合物可在使用前溶解或分散于无菌水或其它无菌可注射介质中。配制用于胃肠外给药的组合物可通过推注或通过定时推注注射，或可通过连续输注给药。
用于局部或经皮施用本发明化合物的剂型包括软膏，散剂，霜剂，洗剂，凝胶，粉剂，溶液，喷雾剂，吸入剂或贴剂。将活性组分在无菌条件下与药学上可接受的载体和任何需要的防腐剂或可能需要的缓冲液混合。眼用制剂，滴耳剂和滴眼剂也被认为在本发明的范围内。此外，本发明考虑使用透皮贴剂，其具有向身体提供化合物的受控递送的附加优点。这样的剂型可以通过将化合物溶解或分散在适当的介质中来制备。吸收增强剂也可用于增加化合物穿过皮肤的通量。可以通过提供速率控制膜或通过将化合物分散在聚合物基质或凝胶中来控制速率。
在一些实施方案中，本发明提供了化合物和本文所述的其它赋形剂的药物组合物。在一些其它实施方案中，本发明提供了NNU-455的化合物和本文所述的其它赋形剂的药物组合物。在另一些实施方案中，本发明提供了包含NNU455的枸橼酸酯(NNU-459)和二乙醇胺酯(NNU-458)与本文所述的其它赋形剂的药物组合物。
在一些实施方案中，本发明的药物制剂通过使用具有低水或低水分含量的赋形剂，并使用干燥或非水制剂方法制备，提供活性化合物的稳定的固体口服剂型。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的化合物与现有技术中已知的不携带氘的化合物相比，具有一系列优点：
(1)本发明化合物对蛋白酶体具有优异的抑制性。
(2)通过氘化这一技术改变生物体中的代谢，使药物的代谢半衰期延长，这导致首过效应(First-pass effect)的降低。在这种情况下，可以改变剂量并形成长效制剂，其也可以长效制剂的形式改善适用性。
(3)通过氘化还改变了药物动力学作用，因为氘代化合物完全形成另一水合物膜，以 致在生物体中的分布明显不同于未氘代的化合物。
(4)用氘取代化合物中的氢原子，由于其同位素效应，能够提高化合物在动物体内的药物浓度，以提高药物疗效。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比。
实施例1：(S)-N-(2,5-二氯苯甲酰基)-2,2-二氘乙酰胺-D-亮氨酸硼酸枸橼酸酯(化合物NNU-459)。合成路线：
Figure PCTCN2018087761-appb-000034
(S)-N-(2,5-二氯苯甲酰基)甘氨酸(Ⅳ)的制备：
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Ⅱ(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％。
取上步所得化合物Ⅲ(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，得白色的(S)-N-(2,5-二氯苯甲酰基)甘氨酸106mg，收率86.0％，m.p.169.3-170.8℃。 1H NMR(400MHz,DMSO)δ3.91(-CH 2,d,J＝6.0Hz,2H),7.48(-CONH,d,J＝8.7Hz,1H),7.55(-Ph,d,J＝1.3Hz,2H),8.89(-Ph,t,J＝5.9Hz,1H),12.71(-COOH,s,1H).MS(ESI):m/z 246.1[M-H] -.
(S)-N-(2,5-二氯苯甲酰基)乙酰胺-D-亮氨酸硼酸-(+)-α-蒎烷二醇酯(Ⅵ)的制备
将化合物Ⅳ(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)δ0.83(-CH 3,s,3H),0.91(-CH 3,s,6H),1.19(-CH 2,d,J＝10.8Hz,1H),1.24(-CH 2,d,J＝7.1Hz,1H),1.27(-CH 3,s,3H),1.38(-CH 3,s,3H),1.59-1.69(-CH,m,1H),1.70(-CH,s,1H),1.77-1.85(-CH 2,m,1H),1.86-1.92(-CH 2,m,1H),1.96-2.01(-CH,m,1H),2.11-2.21(-CH 2,m,1H),2.25-2.36(-CH 2,m,1H),3.31(-CH,dd,J 1＝6.2Hz,J 2＝14.5Hz,1H),4.15(-CH 2,d,J＝5.3Hz,2H),4.28(-CH,dt,J 1＝6.3Hz,J 2＝12.5Hz,1H),6.39(-CONH,d,J＝5.1Hz,1H),7.24(-CONH,d,J＝4.6Hz,1H),7.34(-Ph,d,J＝1.4Hz,2H),7.58-7.65(-Ph,m,1H).MS(ESI):m/z 495.3[M+H] +.
((R)-1-(2-(2,5-苯甲酰胺基)-乙酰氨基)-3-甲基丁基)硼酸(Ⅶ)的制备：
将化合物Ⅵ(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,DMSO)δ0.82(-CH 3,s,3H),0.84(-CH 3,s,3H),1.19-1.28(-CH 2,m,2H),1.61(-CH,td,J 1＝6.6Hz,J 2＝13.2Hz,1H),2.66(-CH,s,1H),4.04(-CH 2,d,J＝5.6Hz,2H),7.55(-Ph,d,J＝1.3Hz,2H),7.66(-Ph,s,1H),8.82(-CONH,d,J＝46.3Hz,1H),8.99(-CONH,t,J＝5.7Hz,1H). 13C NMR(CDCl 3,100MHz)δ22.91,25.94,39.90,44.28,60.37,129.20,129.45,131.25,131.34,132.97,135.53,166.38,171.16.MS(ESI):m/z 359.2[M-H]-.HRMS(ESI):calcd for C 14H 19BCl 2N 2NaO 4[M+Na]+383.0710,found 383.0727.
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸(Ⅷ)的制备：
将化合物Ⅶ(500mg，1.38mmol)溶于16ml无水四氢呋喃中，加入碳酸钾(191.4mg，1.38mmol)、DMSO-d6(3.75ml)、重水(2.5ml)，65℃油浴搅拌反应5小时。LC-MS检测反应结束后，减压蒸除四氢呋喃，加入少量水，用乙酸乙酯萃取，然后有机相用水洗两次，饱和食盐水洗一次，减压蒸除溶剂，经柱层析分离得到产物纯品278mg，收率55.6％。1HNMR(400MHz,MeOD)δ0.93(-CH 3,s,3H).0.95(-CH 3,s,3H),1.29(-CH,s,1H),1.41-1.36(-CH 2，m,2H),2.78(-CH,dd,J 1＝8.9,J 2＝6.3Hz,1H),7.49(-Ph,d,J＝1.4Hz,2H),7.61(-Ph,t,J＝1.3Hz,1H),MS(ESI)：observed：m/z 375[M-H] -。
((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸枸橼酸酯(NNU-459)的制备：
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Ⅶ(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),7.78-7.44(-Ph,m,3H),9.12(-NH,s,1H),10.73(-NH,s,1H),12.15(-COOH,s,2H).MS(ESI):observed:m/z 519[M] +。
实施例2：((R)-1-(2-(2,5-苯甲酰胺基)-2,2-二氘乙酰氨基)-3-甲基丁基)硼酸二乙醇胺酯(化合物NNU-458)
Figure PCTCN2018087761-appb-000035
按照实施例1中所述方法，不同点在于，用二乙醇胺替换枸橼酸，从而制得目标化合物。 1H NMR(400MHz,DMSO)0.85-0.74(-CH 3,m,6H),1.33-1.16(-CH 2,m,2H),1.64-1.51(-CH,m,1H),2.98-2.63(-CH 2,m,4H),3.13(-CH,td,J 1＝14.0Hz,J 2＝7.1Hz,1H),3.67(-CH 2,dd,J 1＝11.4Hz,J 2＝5.8Hz,4H),5.24(-NH,t,J＝22.6Hz,1H),6.58(-NH,d,J＝28.1Hz,1H),7.01(-NH,d,J＝8.4Hz,1H),7.65-7.43(-Ph,m,3H),8.79(d,J＝36.3Hz,1H).MS(ESI):observed:m/z 432.3[M+H] +
实施例3：大鼠中药代动力学评价
SD雄性大鼠12只，体重220±20g，随机分为四组。
其中两组按0.500mg·kg -1剂量分别尾静脉注射给予0.100mg·mL -1NNU-459以及MLN-9708，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-K 2的试管中，高速离心(7800×g)5min后分离血浆，于-15℃～-35℃保存。用于比较NNU-459与MLN-9708静脉注射方式给药的药代动力学差异。
另外两组按1.50mg·kg -1的剂量分别灌胃给予0.150mg·mL -1NNU-459及MLN-9708，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-K 2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NNU-459与MLN-9708口服方式给药的药代动力学差异。
结果显示，不论是经静脉注射还是口服给药NNU-459的半衰期都要优于MLN-9708，尤其是经口服给药途径，NNU-459的半衰期为(13.2±7.97小时)明显高于MLN-9708的半衰期(5.80±1.33小时)。
从上面结果看出，本发明化合物在动物体内具有更好的药物动力学，因而具有更好的药效学和治疗效果。
另外，通过氘化，本发明化合物在生物体中的代谢过程有所改变。这导致首过效应(First-pass effect)的降低。在这种情况下，可以改变剂量并形成长效制剂，其也可以长效制剂的形式改善适用性。
另外，通过氘化还改变了药物动力学作用，因为氘代化合物完全形成另一水合物膜，以致在生物体中的分布明显不同于未氘代的化合物。
实施例4：NNU-455与MLN-2238的肝微粒体稳定性实验
测定在不同物种的肝微粒体中，NNU-459和MLN-9708的体内活性物质NNU-455与MLN-2238的稳定性。
肝微粒体和NADPH辅酶的准备：
i.人肝微粒体工作溶液(2×)：取0.3mL的人肝微粒体储存液(20mg/mL)加入到5.7mL磷酸盐缓冲液，得1.0mg/mL的工作溶液。
ii.大鼠肝微粒体工作溶液(2×)：取0.3mL的大鼠肝微粒体储存液(20mg/mL)加入到5.7mL磷酸盐缓冲液，得1.0mg/mL的工作溶液。
iii.小鼠肝微粒体工作溶液(2×)：取0.3mL的小鼠肝微粒体储存液(20mg/mL)加入到5.7mL磷酸盐缓冲液，得1.0mg/mL的工作溶液。
iv.犬肝微粒体工作溶液(2×)：取0.3mL的犬肝微粒体储存液(20mg/mL)加入到5.7mL磷酸盐缓冲液，得1.0mg/mL的工作溶液。
v.猴肝微粒体工作溶液(2×)：取0.3mL的猴肝微粒体储存液(20mg/mL)加入到5.7mL磷酸盐缓冲液，得1.0mg/mL的工作溶液。
vi.NADPH辅酶的工作溶液(2.5×)：取600μL的NADP(100mM)、600μL的G6P(200mM)、240μL的G6PDH(250U/mL)和480μL的氯化镁溶液(300mM)加入到18.08mL的磷酸缓冲液，得NADPH辅酶的工作溶液。
工作溶液的制备
i.阳性对照的工作溶液(10×)：维拉帕米作为阳性对照，用DMSO配成20mM储备液，再用磷酸缓冲液稀释为10μM工作溶液。
ii.测试品的工作溶液(10×)：先用DMSO配成10mM储备液，然后用50％的甲醇-水稀释到100μM，再用磷酸缓冲液稀释成10μM浓度的工作溶液。
终止液的制备：用甲醇配置100ng/mL甲苯磺丁脲作为含内标的终止液。
按照下表1顺序加入反应所需基质，测试品每个时间点做3个平行，阳性对照物每个时间点做3个平行。
Figure PCTCN2018087761-appb-000036
反应完成后，将样品在4℃，4000rpm离心20min。取100μL的上清加上200μL甲醇用于质谱定量分析。化合物在不同孵育时间点的浓度用Aa/Ai(化合物与内标质谱信号峰面积的比值)表示。将同一化合物每个时间点的浓度除以0时间点浓度得到剩余率，计算公式如下：
半衰期：
将不同时间点的剩余百分比以指数回归模型作图，用下列一级动力学公式计算半衰期:
Figure PCTCN2018087761-appb-000037
清除率(CL)：
Figure PCTCN2018087761-appb-000038
其中，Vd为表观分配系数
Figure PCTCN2018087761-appb-000039
实验结果见表1，在人肝微粒体中，NNU-455的半衰期(108.3min)要显著长于MLN-2238的半衰期(57.3min)，同时Y455的清除率(12.8μL/min/mg)也明显少于MLN-2238(24.2μL/min/mg)。
表1 在不同物种肝微粒体中NNU-455的稳定性实验结果
Figure PCTCN2018087761-appb-000040
以上实验结果表明，本发明中的化合物NNU-459在体内更不容易被代谢清除，从
Figure PCTCN2018087761-appb-000041
而拥有更好的代谢动力学性质以及药效。
实施例5：化合物抑制体外肿瘤细胞的活性检测
本专利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。实验体系为110μL，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，使用化合物MLN-9708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。
5、结果
如表2所示，在体外培养的两种不同多发性骨髓瘤细胞系(U266、RPMI8226)，以及人乳腺癌细胞系MAD-MB-231细胞中。NNU-459、NNU-458的IC 50均与MLN-9708相似，显示出相同或更好的抑制肿瘤的活性。
表2 化合物NNU-458、NNU-459以及MLN-9708对不同肿瘤细胞系的活性
Figure PCTCN2018087761-appb-000042
实施例6：药物组合物
本发明中的药物可以采取但不限于如下的药物组合物：
药物组合物1
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 4 1.3
硅化微晶纤维素 292.4 97.5
滑石粉 0.6 0.2
硬脂酸镁 3 1
内容物重量 300 100
药物组合物2
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 4 1.3
微晶纤维素 231.8 77.3
预胶化淀粉 60 20
二氧化硅 1.2 0.4
硬脂酸镁 3 1
内容物重量 300 100
药物组合物3
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述药剂 4 1.3
微晶纤维素 246.8 82.3
甘露醇 45 15
滑石粉 1.2 0.4
硬脂酸镁 3 1
内容物重量 300 100
药物组合物4
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 3 1.5
微晶纤维素 150 75
玉米淀粉 45 22.5
硬脂酸镁 2 1
内容物重量 200 100
药物组合物5
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 3 1.5
甘露醇 120 60
乳糖 45 37.5
硬脂酸镁 2 1
内容物重量 200 100
药物组合物6
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 3 1.5
甘露醇 100 50
微晶纤维素 95 47.5
硬脂酸镁 2 1
内容物重量 200 100
药物组合物7
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 2.3 1.15
微晶纤维素 195 97.5
滑石粉 0.7 0.35
硬脂酸镁 2 1
内容物重量 200 100
药物组合物8
成分 每单位剂量用量(mg) 用量(％w/w或w/w)
所述化合物 2.3 1.15
硅化微晶纤维素 155 77.5
甘露醇 40 20
滑石粉 0.7 0.35
硬脂酸镁 2 1
内容物重量 200 100
按常规方法，将上述药物组合物按比例混合均匀以后，装入不透明的白色明胶胶囊中。
在本实施例中，“所述化合物”包括化合物NNU-455及其枸橼酸酯(NNU-459)和二乙醇胺酯(NNU-458)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (9)

  1. 一种氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其结构如下式(a)所示，
    Figure PCTCN2018087761-appb-100001
    其中：
    R 1、R 2、R 3、R 4、R 5、R 6独立地选自氢、氘或卤素，或一个或多个氘代的或全氘代的C 1-C 4烷基；并且R 1、R 2、R 3、R 4、R 5、R 6中至少一个是氘代的或是氘；
    P基团选自以下片段：
    Figure PCTCN2018087761-appb-100002
    其中，B为硼原子，Z 1、Z 2独立地选自羟基、C 1-10的烷氧基、芳氧基；
    或者P基团是由以下片段与其他化合物所形成的含有N、S和/或O的杂环基团，
    Figure PCTCN2018087761-appb-100003
    其中B为硼原子，并且与式(a)连接的原子为B。
  2. 根据权利要求1所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其特征在于，所述R 1和R 4均为卤素，R 2、R 3、R 5、R 6中有两个氢和两个氘。
  3. 根据权利要求1所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其特征在于，所述R 1和R 4均为氯，R 2和R 3均为氢、R 5和R 6均为氘。
  4. 根据权利要求1所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其特征在于，P基团是由以下片段与α-羟基羧酸或β-羟基羧酸形成硼酸酯或酸酐基团，
    Figure PCTCN2018087761-appb-100004
    其中B为硼原子，并且与式(a)连接的原子为B。
  5. 根据权利要求1所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物，其特征在于，P基团是由以下片段与具有不在同一碳原子上的两个或两个以上独立羟基结构的化合物形成硼酸酯基团，
    Figure PCTCN2018087761-appb-100005
    其中B为硼原子，并且与式(a)连接的原子为B。
  6. 权利要求1-5任一项所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物的制备方法，其特征在于，包括以下所示反应过程：
    Figure PCTCN2018087761-appb-100006
    其中，P基团是由以下片段与其他化合物所形成的含有N、S和/或O的杂环基团，
    Figure PCTCN2018087761-appb-100007
    其中B为硼原子，并且与式(a)连接的原子为B。
  7. 权利要求1-5任一项所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物在制备抑制蛋白酶体药物中的应用。
  8. 权利要求1-5任一项所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物在制备治疗和预防蛋白酶体相关疾病药物中的应用。
  9. 权利要求1-5任一项所述的氘代的二肽硼酸或硼酸酯类化合物、或其晶型、或药学上可接受的水合物或溶剂合物在制备治疗或预防癌症、心血管疾病、炎症、免疫性疾病、肾病、血管发生或前列腺疾病药物中的应用。
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