WO2017198194A1 - Boronic acid and borate ester compound and applications thereof - Google Patents

Boronic acid and borate ester compound and applications thereof Download PDF

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WO2017198194A1
WO2017198194A1 PCT/CN2017/084924 CN2017084924W WO2017198194A1 WO 2017198194 A1 WO2017198194 A1 WO 2017198194A1 CN 2017084924 W CN2017084924 W CN 2017084924W WO 2017198194 A1 WO2017198194 A1 WO 2017198194A1
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compound
crystal form
compound according
group
drug
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PCT/CN2017/084924
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French (fr)
Chinese (zh)
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广兵
阳泰
董韧涵
刘进
覃传军
谢建
彭向阳
钟月玲
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成都奥璟生物科技有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—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

Definitions

  • the invention belongs to the field of chemical medicines, and in particular relates to an antitumor compound and a pharmaceutical composition thereof.
  • Ubiquitin-proteasome pathway-mediated protein degradation is an important mechanism by which the body regulates intracellular protein levels and functions. Once the proteasome exceeds normal levels, it causes a decrease in growth inhibition, a decrease in apoptosis, and promotes angiogenesis, thereby causing various tumor diseases, and thus the proteasome is an important target for drugs such as cancer. Proteasome inhibitors inhibit tumor cell growth and promote apoptosis by blocking cellular proteasome degradation.
  • MM Multiple myeloma
  • MM is a plasma cell carcinoma found in the bone marrow.
  • multiple myeloma a group of plasma cells or myeloma cells are transformed into cancer cells and proliferate, resulting in a higher than normal number of plasma cells.
  • plasma cells migrate extensively in the body, it is likely to involve most of the bones in the body, which may lead to compression fractures, osteolytic lesions and related pain.
  • Multiple myeloma can cause several serious health problems, involving red blood cell counts in the bones, immune system, kidneys, and individuals. Some of the more common symptoms include bone pain and fatigue.
  • Mantle cell lymphoma follicular lymphoma is a relatively common non-Hodgkin's lymphoma.
  • Mantle cell lymphoma accounts for 6% of non-Hodgkin's lymphoma, and the first-line treatment regimen does not achieve a completely satisfactory result.
  • Follicular lymphoma accounts for 22% of non-Hodgkin's lymphoma, and despite various treatments, there is still unmet clinical need.
  • Lung cancer, colon cancer, breast cancer, kidney cancer, cervical cancer, nasopharyngeal cancer, etc. are also high-risk tumor types. Although there are already a large number of therapeutic drugs and means, high-value therapeutic drugs in this field are still unmet clinical needs.
  • Ixazomib (boric acid moiety).
  • WO 2012/177835 discloses derivatives of the following structures of Ixazomib: (code name Ixazomib DEA)
  • Bilgicer et al (Journal of Medicinal Chemistry, 2014, 57:5282) reported different prodrugs of bortezomib, the prodrug of which was designed to form hydroxyl groups in boric acid structural units into various cyclic esters.
  • the prior art discloses the above-mentioned boric acid cyclic ester as a prodrug, and the release of the boric acid moiety is effective.
  • the present invention provides a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
  • ring A represents one of the following structures:
  • R 1 is selected from H or C 1-6 alkyl
  • R 2 is selected from C 1-6 alkyl
  • R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with the inserted oxygen and boron atoms form an optionally substituted 5-20 membered ring, the ring further comprising 0-2 selected from nitrogen and oxygen. Or a ring hetero atom of sulfur.
  • R 1 is selected from H.
  • R 2 is selected from isopropyl.
  • X 1 and X 2 are selected from hydroxyl, or X 1 and X 2 together form a portion of the acid functional groups formed by removing a hydrogen atom esterifying agent.
  • X 1 and X 2 are selected from a hydroxyl group.
  • the borating agent is selected from the group consisting of compounds containing at least one hydroxyl group or at least one carboxyl group.
  • the borating agent is a borate esterifying agent containing N atoms.
  • the ring formed is a 5-12 membered ring.
  • the borating agent is selected from the group consisting of a monosaccharide or a polyhydric alcohol.
  • the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the number of hydroxyl groups and carboxyl groups is 2 to 4.
  • the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine, 3-((2-hydroxyethyl) Amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N Any one of bis(2-hydroxyethyl)glycine; or the boronic acid esterifying agent is optionally C1-6 alkyl, C3-C6 cycloalkyl, C2-C6 carboxyalkyl , C1-C6 hydroxyalkyl substitution.
  • ring A is selected from the following structures:
  • the borating agent is selected from the group consisting of dipropanolamine.
  • the borating agent is selected from the group consisting of diethanolamine.
  • the compound is one of the following compounds or one of its mannitol esters:
  • the compound is one of the following compounds:
  • the compound is one of the following compounds:
  • the present inventors have surprisingly found that the different substituents on the benzene ring and the cyclic esters of boric acid have higher anti-multiple myeloma cell activity, and thus have a good application prospect.
  • the present invention also discloses a novel synthetic route for the above compounds, as follows:
  • CN200780100142 discloses a method for synthesizing a compound similar to the patent; which uses TBTU or the like as a condensing agent to form an amide reaction of a substituted intermediate S1 of a substituted benzoic acid with glycine with an amino boronate S2, but the present inventors are surprised It has been found that the preparation of the compounds disclosed herein using the above-disclosed patents will result in the predominantly by-product S4-1, which is predominantly deboronated.
  • the TBTU is replaced with other such as DCC, EDCI.HCl, etc., or the reaction solvent system is changed, but the target intermediate is surprisingly less or completely unavailable. It indicates that the difference of the substituents on the benzene ring directly affects the feasibility of the preparation method and is non-obvious.
  • a method of activating a carboxyl group such as preparing an activated ester (herein, a mixed acid anhydride), can obtain a target in a high yield.
  • the carboxyl activating agent is selected from any one or more selected from the group consisting of ethyl chloroformate, propyl chloroformate and isobutyl chloroformate.
  • the key is to keep the system alkaline
  • the base may be selected from any one or more of N-methylmorpholine, triethylamine and N-ethyldiisopropylamine.
  • the salt of the compound represented by S2 is a trifluoroacetate or a hydrochloride.
  • the present invention also provides the use of the compound, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof, or a boric anhydride, for the preparation of an antitumor drug or a proteasome inhibitor.
  • proteasome inhibitor drug is a proteasome chymotrypsin-like protease inhibitor drug or a proteasome caspase-like protease inhibitor drug.
  • the antitumor drug is a drug for preventing and/or treating plasmacytoma.
  • the plasmacytoma is multiple myeloma.
  • the compounds of the present invention also exhibit inhibitory effects against a variety of other cancers.
  • the antitumor drug is a drug for preventing and/or treating lymphoma.
  • the medicament is a medicament for preventing and/or treating non-Hodgkin's lymphoma.
  • the non-Hodgkin's lymphoma is a mantle cell lymphoma.
  • the non-Hodgkin's lymphoma is a follicular lymphoma.
  • the tumor is a mantle cell lymphoma or a follicular lymphoma.
  • the tumor is breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer, nasopharyngeal cancer.
  • the C 1 -C 6 alkyl group means a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 alkyl group, that is, a straight chain or a branch having 1 to 6 carbon atoms.
  • Alkyl of the chain such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, and the like.
  • the ring hetero atom refers to a boron atomating agent which is ring-formed with a boron atom or an oxygen atom, and a hetero atom other than a carbon atom in the ring-forming atom, excluding a substituent group on the ring. atom.
  • the boric acid compound in the present invention, it refers to a compound containing a boronic acid -B (OH) 2 moiety.
  • the boric acid compound can form an oligomeric anhydride by partially dehydrating the boric acid.
  • the boric anhydride refers to a compound formed by combining two or more boric acid compound molecules while losing one or more water molecules. When mixed with water, the boric anhydride compound is hydrated to release the free boric acid compound.
  • the boric anhydride may contain two, three, four or more boric acid units, and may have a cyclic or linear configuration. For example, the following ring structure:
  • n can be an integer from 0-10.
  • the borate esterifying agent refers to any compound having at least two functional groups, each of which can form a covalent bond with boron, such as a hydroxyl group and a carboxyl group.
  • boron such as a hydroxyl group and a carboxyl group.
  • mannitol, citric acid, malic acid, tartaric acid, glucose, aminodiethanol, aminodipropanol, triethanolamine, and tripropanolamine may be included.
  • the borate esterifying agent includes, in addition to the above, 3-((2-hydroxyethyl)amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2- ((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N-bis(2-hydroxyethyl)glycine, etc.; or, the borate esterifying agent is optionally C1 a -6 alkyl group, a C3-C6 cycloalkyl group, a C2-C6 carboxyalkyl group, a C1-C6 hydroxyalkyl group, or the like.
  • the present invention provides a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
  • ring A represents one of the following structures:
  • R 5 , R 6 , and R 7 are selected from the group consisting of H, F, Cl, Br, I, C 1-6 alkyl, C 1-6 alkoxy, trifluoromethyl;
  • R 1 is selected from the group consisting of H, C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl; the C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl The base can be further substituted;
  • R 2 is selected from C 1-6 alkyl
  • R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with the inserted oxygen and boron atoms form an optionally substituted 5-20 membered ring, the ring further comprising 0-2 selected from nitrogen and oxygen. Or a ring hetero atom of sulfur.
  • R 1 is selected from H or benzyl.
  • R 2 is selected from isopropyl.
  • X 1 and X 2 are selected from a hydroxyl group, or X 1 and X 2 together form a portion after dehydrogenation of the two functional groups of the borate esterifying agent.
  • X 1 and X 2 are selected from a hydroxyl group.
  • the borating agent is selected from the group consisting of compounds containing at least one hydroxyl group or at least one carboxyl group.
  • the borating agent is a borate esterifying agent containing N atoms.
  • the ring formed is a 5-10 membered ring.
  • the borating agent is selected from the group consisting of a monosaccharide or a polyhydric alcohol.
  • the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the amounts of the hydroxyl group and the carboxyl group is 2-8.
  • the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine, 3-((2-hydroxyethyl) Amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N Any one of bis(2-hydroxyethyl)glycine; or the boronic acid esterifying agent is optionally C1-6 alkyl, C3-C6 cycloalkyl, C2-C6 carboxyalkyl And a C1-C6 hydroxyalkyl group or the like.
  • the compound of the present invention may be one of the following structures:
  • the present inventors have surprisingly found that the different substituents of the benzene ring moiety, whether the boronic acid structural unit is esterified, and the different cyclic esters and different combinations in the above structure produce unexpected activity effects.
  • N-containing heterocyclic cyclic ester having a different boric acid structural unit although it may be slowly decomposed into a boric acid moiety, is not only a prodrug, but has an unexpected biological activity by itself.
  • the present invention also provides a pharmaceutical composition which is prepared by using the compound or a pharmaceutically acceptable salt thereof or boric anhydride as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant is selected from any one or more of a diluent, a filler, a colorant, a glidant, a lubricant, a binder, a stabilizer, a suspending agent or a buffer.
  • the preparation is a tablet, a capsule, an oral solution, an injection, a transdermal agent, an aerosol solid preparation, a liposome preparation or a controlled release preparation.
  • the prodrugs are derivatives of the aforementioned compounds which may themselves have weak or no activity, but are converted to corresponding conditions under physiological conditions (for example by metabolism, solvolysis or otherwise) after administration. Biologically active form.
  • the key intermediates and compounds of the present invention are isolated and purified in a manner that is commonly used in organic chemistry for separation and purification.
  • One or more compounds of the invention may be used in combination with one another, or the compounds of the invention may be used in combination with any other active agent for the preparation of an anti-tumor or proteasome inhibitor. If a group of compounds is used, the compounds can be administered to the subject simultaneously, separately or sequentially.
  • the compound of the present invention can be combined with other drugs based on the principle of anti-tumor synergistic mechanism, including sequential administration or simultaneous administration, to improve anti-tumor efficacy, delay drug resistance, and reduce drug toxicity.
  • the combination of the present invention which comprises the compound of any of the preceding, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof, for simultaneous or separate administration of the same or different unit preparations Or a boric anhydride and an antitumor drug or a drug for adjuvant treatment of a tumor, and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for preventing and/or treating a tumor, which comprises administering to a tumor patient a compound according to any one of the above, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof Or boric anhydride.
  • the tumor patient is a patient of plasmacytoma, lymphoma, breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer and/or nasopharyngeal cancer.
  • the patient is a multiple myeloma patient; the patient is a non-Hodgkin's lymphoma patient, and further a mantle cell lymphoma and/or follicular lymphoma patient.
  • the X-ray powder diffraction of the crystal form has characteristic peaks at 8.38, 11.4, 12.1, 13.29, 18.27, 19.62, 20.47, 23.32, and 24.74 degrees.
  • the relative intensity values of the characteristic peaks of the 2 ⁇ diffraction angle are:
  • the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
  • the present invention also provides a crystal form of a borate compound, wherein the X-ray powder diffraction of the crystal form has a 2 ⁇ diffraction angle of 6.19, 7.98, 10.05, 14.73, 14.92, 17.16, 18.8, 20.03, 21.05 degrees. Characteristic peaks.
  • the relative intensity values of the characteristic peaks of the 2 ⁇ diffraction angle are:
  • the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
  • the present invention also provides a crystal form of a borate ester compound having X-ray powder diffraction having characteristic peaks at 2.23, 12.53, 14.18, 17.06, 20.82, 21.46, 22.62 degrees in X-ray powder diffraction.
  • the relative intensity values of the characteristic peaks of the 2 ⁇ diffraction angle are:
  • the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
  • the present invention also provides a crystal form of a borate compound in which X-ray powder diffraction has a 2 ⁇ diffraction angle of 10.59, 11.76, 13.19, 15.56, 17.76, 19.5, 20.26, 21.37, 22.2 degrees. Characteristic peaks.
  • the relative intensity values of the characteristic peaks of the 2 ⁇ diffraction angle are:
  • the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
  • the present invention also provides a crystal form of a borate ester compound, X-ray powder diffraction of the crystal form, 2 ⁇ derivative
  • the shooting angles have characteristic peaks at 7.06, 10.61, 12.24, 14.9, 17.23, 20.21, 23.49, and 26.48 degrees.
  • the relative intensity values of the characteristic peaks of the 2 ⁇ diffraction angle are:
  • the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
  • cytotoxic drugs such as carboplatin, cisplatin, irinotecan, paclitaxel, fluorouracil, cytarabine, lenalidomide, retinoic acid
  • Hormonal drugs such as dexamethas
  • the pharmaceutically acceptable excipient of the present invention means a substance which is contained in a dosage form in addition to the active ingredient.
  • the compound of the invention has good proteasome inhibitory activity and antitumor activity, and has low toxicity and excellent clinical application prospect.
  • alkyl includes a straight or branched alkyl group.
  • the term "compound of the present invention” means a compound represented by the formula (I).
  • the term also encompasses various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I).
  • the term "pharmaceutically acceptable salt” means that the compound of the present invention and an acid or a base are suitable for use as a medicine. Salt of matter.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with an alkali metal.
  • Alkali metals suitable for forming salts include, but are not limited to, lithium, sodium, potassium, calcium, magnesium, and the like.
  • the pharmaceutically acceptable excipient which has a certain physiological activity, but the addition of the ingredient does not change the dominance of the above-mentioned pharmaceutical composition in the course of the disease treatment, but only plays an auxiliary effect, and the auxiliary effect is only the ingredient
  • the use of known activities is an adjuvant treatment that is commonly used in the medical field. It is still within the scope of the present invention to use the above-mentioned excipients in combination with the pharmaceutical compositions of the present invention.
  • the compound of the present invention has an excellent therapeutic antitumor effect
  • the compound of the present invention and various crystal forms thereof, a pharmaceutically acceptable inorganic or organic salt, hydrate or solvate, and a compound containing the compound of the present invention are mainly active ingredients.
  • the pharmaceutical composition can be used for anti-tumor.
  • 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 1 to 20 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. By “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.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid).
  • magnesium stearate magnesium stearate
  • calcium sulfate vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as Tween ), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
  • vegetable oil such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyol such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifier such as Tween
  • a wetting agent such as sodium lauryl sulfate
  • a coloring agent such as a flavoring agent, a stabilizer, an antioxidant, a preservative
  • the mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
  • 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, alginate, gelatin, polyvinylpyrrolidone, sucrose and (A) humectant, 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; (f) an absorption accelerator such as a quaternary amine compound; (g) a wetting agent such as cetyl alcohol and glyceryl
  • 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.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethyl
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • Figure 1 is a mass spectrum of Compound 1-1-2.
  • Figure 2 is a mass spectrum of Compound 1-1-3.
  • Figure 3 is a mass spectrum of the compound I-2-2.
  • Figure 4 is an X-ray powder diffraction pattern of Compound 1-1-2.
  • Figure 5 is an X-ray powder diffraction pattern of Compound 1-1-3.
  • Figure 6 is an X-ray powder diffraction pattern of Compound 1-1-4.
  • Figure 7 is an X-ray powder diffraction pattern of Compound 1-1-5.
  • Figure 8 is an X-ray powder diffraction pattern of Compound I-2-2.
  • Figure 9 is an X-ray powder diffraction pattern of Compound I-2-3.
  • Figure 10 is a graph showing the results of hydrolysis experiments of Compound I-2-3 in PBS.
  • Figure 11 shows that the compounds I-1-3 and I-2-3 of the present invention can significantly inhibit the proteasome activity in tumor tissues of mouse multiple myeloma in vivo.
  • Figure 12 shows that the compounds I-1-3 and I-2-3 of the present invention are capable of inducing the formation of tumor apoptosis markers - Cleaved PARP and Cleaved Caspase-3 activated fragments.
  • Figure 13 is a mouse tumor tissue section: 1 negative control group; 2 oral Ixazomib-DEA group; 3 oral administration of compound I-1-3 of the present invention; 4 oral administration of compound I-2-3 of the present invention.
  • the starting material S1 is prepared by dehydrating the substituted benzoic acid and the glycine methyl ester to form an amide, and then hydrolyzing the methyl ester; the trifluoroacetate or hydrochloride intermediate of the S2 aminoborate is commercially available, and other synthetic
  • the reagents are all commercially available.
  • the target compounds obtained in the examples were all controlled to have a purity of 98% or more.
  • CN200780100142 discloses a synthesis method of a compound similar to the present invention, which uses TBTU or the like as a condensing agent to form an amide reaction of a condensed intermediate S1 of a substituted benzoic acid with glycine with an amino boronate S2, but the inventors have found
  • the compound disclosed in the present invention is prepared by the above-mentioned patent disclosure route, and the obtained product is mainly a by-product S4-1 of deboronate, and the specific operation is as follows:
  • the target product was not obtained by replacing the reaction solvent system or using other condensing agents such as EDCI.HCl, DCC, and the like.
  • WO2012/177835 also discloses a synthetic route, as shown below:
  • the inventors selected X as Cl, reacted the above starting material (S1-1) with thionyl chloride to prepare an acid chloride, and formed an amidation reaction with S2 under basic conditions. Attempts, the resulting product is extremely complex, with many by-products and no preparative value (HPLC monitoring target conversion is less than 10%).
  • the specific preparation method of the present invention is as follows: 3 g (10 mmol) of starting material (S-1-1) in 20 mL of dichloromethane solution, NMM (N-methylmorpholine) 2.6 g (26 mmol) is added, and the temperature is lowered to 5 degrees. After adding 1.34 g (12 mmol) of ethyl chloroformate, and reacting to obtain the activated ester intermediate M1-1, the mixture was filtered, and 4.69 g of S2 (12 mmol) was added to the filtrate, stirred at room temperature for 3 hours, and then quenched with ice water. The organic layer was dried and concentrated to give a pale yellow solid object S3-1.
  • chloroethyl chloroformate or butyl chloroformate (isobutyl ester) or the like is used instead of ethyl chloroformate, and NMM is replaced with other organic bases such as triethylamine or N-ethyldiisopropylamine. Similar results, the yields were all above 80%.
  • the intermediate obtained in the previous step 5.1 g of S3-1, 40 mL of methanol, 4.5 mL of 1N hydrochloric acid solution, 2.6 g of isobutylboric acid (2.5 eq), and 20 mL of n-hexane were stirred at room temperature overnight to give n-hexane.
  • the compound I-1-2 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 4, and the characteristic data is as follows:
  • the boric acid raw material (I-1-1) 2.25 g (5.55 mmol) was dissolved in 45 ml of ethyl acetate, stirred at room temperature for 5 min, and then diethanolamine (S-3-3) 0.61 g (5.82 mmol) was added dropwise, dropwise. A white solid precipitated in the middle reaction solution. After completion of the dropwise addition, stirring was continued for 2 hours, and suction filtration was carried out to obtain 2.4 g of an I-1-3 compound in a yield of 91%.
  • the compound I-1-3 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 5, and the characteristic data is as follows:
  • the compound I-1-4 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 6, and the characteristic data is as follows:
  • the compound I-1-5 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 7, and the characteristic data is as follows:
  • the synthesis route is similar to that in Example 1, and the main product is obtained by the amide method disclosed in the patent.
  • the amide group is linked by the mixed acid anhydride method as shown above to finally obtain the target compound I-2-1;
  • the preparation method of -1 mannitol ester and compound I-2-1 sodium salt is similar to that of Example 1.
  • the compound I-2-2 is obtained and detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 8.
  • the characteristic data is as follows:
  • the compound I-2-3 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C.
  • the crystal X-ray powder diffraction pattern is as shown in Fig. 9, and the characteristic data is as follows:
  • the synthesis route is similar to that in Example 1, and the main product is a boric acid removal by-product obtained by the condensing agent method disclosed in the patent; the target product I-3-1; I-3-1 mannitol ester is obtained by the mixed acid anhydride method as shown in the above figure. And the preparation method of the sodium salt of the I-3-1 compound is the same as in the first embodiment.
  • Example 1 As in the synthesis route of Example 1, similarly, the boring agent method disclosed in the patent is used to obtain a by-product of boric acid removal; the mixed acid anhydride method as shown above is used to obtain the target substance I-4-1; the preparation method of the mannitol ester is carried out in the same manner. example 1.
  • the synthesis route is similar to that in Example 1, and the main product is obtained by the amide method disclosed in the patent.
  • the amide group is linked by the mixed acid anhydride method as shown above to finally obtain the target compound I-5-1; the compound I-5.
  • the preparation method of -1 mannitol ester and compound I-5-1 sodium salt is similar to that of Example 1.
  • I-6-1, I-7-1, and I-10-1 are compounds disclosed in CN200780100142. Surprisingly, it was found that the iodine-containing compounds I-8-1 and I-9-1 on the benzene ring are extremely easy to decompose at room temperature, indicating that the difference in halogen substituents on the benzene ring directly affects the stability and preparation of the derivative. Sex.
  • the pharmaceutical capsule composition of Compound I-1-2 contains 3 g or 4 g of Compound I-1-2, 193 g or 192 g of microcrystalline cellulose, 4 g of micronized silica gel, and a total of 200 g and No. 2 hollow capsules.
  • the preparation method is:
  • the mixed powder was passed through a 120 mesh sieve, filled into No. 2 capsules and sealed, and a total of 1000 capsules were prepared.
  • Example 25 Pharmaceutical tablet composition of a compound of the invention
  • the pharmaceutical tablet composition of the compound I-2-2, the compound I-2-2 is 1 part by weight, the lactose is 0.1-0.5 part by weight, the hydroxypropylcellulose is 0.05-0.08 part by weight, and the sodium carboxymethyl starch is 0.008- 0.014 parts by weight, an appropriate amount of povidone K30, 0.01-0.05 parts by weight of magnesium stearate; tablets are prepared according to the above ratio, and each tablet contains 1-6 mg of each of 1-6.
  • Example 26 Pharmaceutical Injectable Composition of a Compound of the Invention
  • a pharmaceutical injection composition of the compound I-1-1 which contains 1 g of I-1-1 mannitol ester, 34 g of disodium hydrogen phosphate, and a sufficient amount of water for injection.
  • the preparation method is:
  • step b the solution obtained in step a is filtered, dispensed in 1000 bottles of 2mL vials, half-plugged;
  • compositions of the compounds I-2-1, I-3-1, and I-5-1 are the same as above.
  • the pharmaceutically acceptable liposome composition of Compound I-2-2 contains 5 g of Compound 1-1-2 and 6.7 g of lecithin, 3.3 g of cholesterol, 5 g of vitamin C, and a sufficient amount of PBS buffer solution (pH 7.4).
  • the preparation method is:
  • the dry film container obtained in c, b was added to a PBS buffer solution (pH 7.4) to 500 mL, and vitamin C was added thereto, followed by ultrasonication.
  • I-1-3, I-2-3 compound S1:S2 1:3 without hydrolysis reaction, after the hydrolysis reaction, S2 in the measured nuclear magnetic spectrum contains the benzene ring hydrogen of the hydrolyzed product, but S1 remains It is a characteristic hydrogen of I-1-3 and I-2-3 compounds.
  • the calculation formula of the three hydrogen integral areas of the benzene ring after hydrolysis is: S2-3S1. Therefore, the molar ratio of the borate ester to its hydrolyzate boric acid is calculated as: 3S1/S2-3S1, and the time to be decomposed to half is t1/2.
  • the boric acid ester compound As a prodrug, combined with the results of other activity experiments of the inventors, Not only the boric acid moiety of the compound of the present invention has a strong activity, but also the borate ester compound can exert an active effect (not only the former Medicine), which plays an important role in the therapeutic effect of non-oral routes of administration.
  • the test sample was dissolved in DMSO and stored at low temperature. Before the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in the present embodiment.
  • Experimental method The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC (Try-AMC sequence) was used to detect the inhibitory activity of the sample on the proteasome ⁇ 5 subunit, and the inhibition of the enzyme activity by different compounds was observed. The hydrolysis of the proteasome ⁇ 5 subunit was observed. The Try-AMC sequence in the substrate releases AMC. Under the detection conditions of excitation wavelength 380 nm and emission wavelength 460 nm, the released AMC fluorescence absorption value can be detected, and the inhibitory activity of the compound on the enzyme can be observed and calculated.
  • the IC50 of inhibition of proteasome ⁇ 5 subunit activity by the test sample was calculated by Graphpad Prism 5.0, and the results were as follows:
  • the compound of the present invention has a good proteasome ⁇ 5 subunit-chymotrypsin-like protease inhibitory activity.
  • test sample was dissolved in DMSO and stored at low temperature. Test the stability of the sample by HPLC before the test.
  • the samples used in this embodiment were stable.
  • Multiple myeloma cells RPMI8226, U266, MM.1S, MM.1R (purchased from ATCC, USA) were added to a flat-bottom 96-well cell culture plate at 40,000 cells/well.
  • the highest concentration of the compound was 1 ⁇ M, Ixazomib Citrate, Ixazomib-DEA and Ixazomib were used as positive control groups. Dilute the drug concentration according to a 5-fold gradient.
  • the GI50 of the test sample against the proliferation inhibition activity of multiple myeloma cells was calculated by Graphpad Prism 5.0, and the results were as follows:
  • boric acid cyclic benzene ring substituents affects the compound's inhibition of the proliferation activity of multiple myeloma cells.
  • the order of activity is basically, diurylamine borate cyclic ester > triethanolamine cyclic ester of boric acid > diethanolamine cyclic ester of boric acid > citric acid cyclic ester of boric acid > boric acid compound ( Unesterified); substituents on the benzene ring, the order of activity is: 2-Cl-5-Br substitution >5-Cl-2-Br substitution >2,5-2-F-4-Cl substitution >4, 5-2-F-2-Cl substitution.
  • the compounds in Table 2-1 are all boric acid structures. It can be seen that Compounds I-1-1 and I-2-1 can effectively inhibit the proliferation of multiple myeloma cells, among which, in the inhibitory activity against MM1S, Compounds I-1-1 and I-2-1 were superior to the positive drug Ixazomib.
  • the compounds in Table 2-2 are all N-containing heterocyclic cyclic ester structures, and it can be seen that the compounds of the present invention are 1-1-2, I-1-3, I-2-2, and I-2-3. , I-2-5, I-3-2, I-3-3, I-4-2, and I-4-3 can effectively inhibit the proliferation of multiple myeloma cells, wherein compound I-1-2,
  • the inhibitory activities of I-1-3, I-2-2, I-2-3, I-2-5, and I-3-2 against all multiple myeloma cells were superior to the positive drug Ixazomib-DEA, Compound I. -3-3, I-4-2, and I-4-3 have better inhibitory activity against U266, MM1S, and MM1R than the positive drug Ixazomib-DEA.
  • the experimental results indicate the structure of the N-containing heterocyclic cyclic ester with 2-Cl-5-Br substitution, 5-Cl-2-Br substitution, 2,5-2-F-4-Cl substitution, 4,5-2-F
  • the compound formed by the substitution of -2-Cl has high inhibitory activity against multiple myeloma cells, among which, the structure containing N heterocyclic cyclic ester and 2-Cl-5-Br substituted, 5-Cl-2- are preferred.
  • Br replaces the compound formed by the complexation.
  • the compounds in Table 2-3 are all oxygen-containing heterocyclic cyclic ester structures, and it can be seen that the compounds of the present invention are 1-1-4, I-1-4-1, I-2-4, and I-3. -4, I-3-4-1, I-4-4, and I-4-4-1 can effectively inhibit the proliferation of multiple myeloma cells, among which, compounds I-1-4, I-1-4 -1, I-2-4 inhibited all of the multiple myeloma cells better than the positive drug IxazomibCitrate, compounds I-3-4, I-3-4-1, I-4-4, I-4 -4-1 has better inhibitory activity against MM1S than yang Sex drug IxazomibCitrate.
  • the experimental results indicate the structure of the oxygen-containing heterocyclic cyclic ester with 2-Cl-5-Br substitution, 5-Cl-2-Br substitution, 2,5-2-F-4-Cl substitution, 4,5-2-F
  • the compound formed by the substitution of -2-Cl has high inhibitory activity against multiple myeloma cells, and among them, an oxygen-containing heterocyclic cyclic ester structure and 2-Cl-5-Br substitution, 5-Cl-2- are preferred.
  • Br replaces the compound formed by the complexation.
  • MM.1S multiple myeloma cells were established using MM.1S multiple myeloma cells and severe combined immunodeficiency (SCID) mice: 3 ⁇ 10 7 MM.1S cells were resuspended in 100 ⁇ l of 1640 medium and mixed with 100 ⁇ l of Matrigel. The above 200 ⁇ l homogenate system was injected into the right abdomen of SCID mice (5 weeks old, female). After 6-7 days, a visible size (100 mm 3 ) was formed at the site of tumor injection, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control compound Ixazomib-DEA were orally administered orally. (The compound was dissolved with 5% HP ⁇ CD at a dose of 10 mg/kg).
  • tissue homogenate was performed by adding 200 ⁇ l of pre-cooled 1640 medium per 0.1 g of tumor tissue (on ice operation, keeping the tissue homogenization operation at a low temperature). 25 ⁇ l of the homogenate sample was added to Promega Proteasome-Glo TM Chymotrypsin-Like (for ⁇ 5 subunit assay), Caspase-Like (for ⁇ 1 subunit assay) and Trypsin-Like (for ⁇ 2 subunit assay) assay kit.
  • 25 ⁇ l of detection reagent was provided for detecting proteasome 20S- ⁇ 5, ⁇ 1 and ⁇ 2 subunit inhibitory activities, respectively.
  • tissue homogenate was added according to 1 ml of protein lysate per 0.1 g of tumor tissue, and then supernatant was centrifuged at 12,000 rpm for western-blot operation, using rabbit anti-PARP, rabbit anti-Caspase- 3 and rabbit anti-Cleaved Caspase-3 antibody were detected.
  • the activity of proteasome 20S- ⁇ 5 and ⁇ 1 subunit (caspase-like active subunit) in tumor tissues was significantly inhibited.
  • This example demonstrates that the compounds of the present invention can directly reach tumor tissues, inhibit proteasome activity, and thereby induce tumor tissue apoptosis.
  • administration of the compounds of the present invention, I-1-3 and I-2-3 induced the formation of a tumor-apoptable marker-Cleaved PARP and a Cleaved Caspase-3 activating fragment.
  • the 20S- ⁇ 1 subunit inhibitory activity of the proteasome was superior to the positive control drug Ixazomib-DEA, and the difference was extremely significant (P ⁇ 0.0001).
  • the compound of the present invention can reach the tumor tissue orally, and exerts a more remarkable activity than the Ixazomib-DEA proteasome inhibitory activity, meaning that the compound of the present invention has Stronger apoptosis-inducing activity. Furthermore, the administration of the compounds of the present invention, I-1-3 and I-2-3, was more effective than the Ixazomib-DEA in inducing the expression of the tumor apoptosis markers Cleaved PARP and Cleaved Caspase-3 (see Figure 12).
  • the experimental results indicate that the compounds of the present invention, I-1-3, I-2-3, can be prepared as proteasome inhibitor drugs, especially proteasome chymotrypsin-like (proteasome 20S- ⁇ 5) protease inhibitor drugs and Proteasome caspase-like (proteasome 20S- ⁇ 1) protease inhibitor drug.
  • the in vivo model of multiple myeloma mice was established according to the method described in Example 31, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control drug Ixazomib-DEA were orally administered orally. (The drug was dissolved with 5% HP ⁇ CD at a dose of 10 mg/kg). After 8 hours, the skin of the mice was peeled off, and the tumor tissues were taken out and fixed with 4% paraformaldehyde. The fixed tissue was dehydrated, embedded, and sliced, and the following operations were performed: the dewaxed sections were placed on a dyeing rack, and stained in a hematoxylin dyeing tank for 10-20 minutes.
  • Negative control group The number of tumor cells was large, and the growth was dense. Occasionally, the tumor cells were lysed, fragmented and disappeared, and the tumor cells showed a small amount of necrosis.
  • Ixazomib-DEA drugs the number of tumor cells is large, the growth is dense, the tumor cells are punctate or focal necrosis (the degree of tissue necrosis is ++), and the aggregated multiple tumor cells are concentrated and divided, and the necrotic area is occasionally seen. There are monocytes, neutrophil infiltration.
  • Oral administration of the compound of the present invention I-1-3 the number of tumor cells is small, the cells are arranged loosely, and the tumor cells are flaky necrosis (the degree of tissue necrosis is determined as +++), and most of the tumor cells are concentrated, divided, dissolved, and visible in the tissue. Protein-like substances and cell debris, a large number of neutrophils, plasma cells and monocytes can be seen in the necrotic area.
  • Oral administration of the compound of the present invention I-2-3 the number of tumor cells is small, the cells are arranged loosely, and the tumor cells are flaky necrosis (the degree of tissue necrosis is determined as +++), and most of the tumor cells are concentrated, divided, dissolved, and visible in the tissue. Protein Samples and cell debris, a large number of neutrophils, plasma cells and monocytes can be seen in the necrotic area.
  • this example demonstrates that the compounds of the present invention have superior apoptosis-inducing activity in tumor tissues in vivo than the control drug Ixazomib-DEA.
  • Example 33 TUNEL-POD method for detecting tumor tissue apoptosis in animals of the present invention
  • the in vivo model of multiple myeloma mice was established according to the above method, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control drug Ixazomib-DEA were administered orally by gavage (drug 5) %HP ⁇ CD was dissolved at a dose of 10 mg/kg). After 8 hours, the skin of the mice was peeled off, and the tumor tissue was taken out.
  • the BA200Digital digital trinocular camera micro-camera system micro-camera system was used to collect the images of the slices. Each slice was observed at 100 times before the whole tissue, and then one region was selected 400 times according to the size and expression of the tissue. Each slice was read, counted under the microscope, and the positive rate was counted. The results are shown in Table 4 below:
  • the test sample was dissolved in DMSO and stored at low temperature. Prior to the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in this embodiment.
  • the mantle cell lymphoma line JVM-2 and the follicular lymphoma line SU-DHL-6 were added to a flat-bottom 96-well cell culture plate at 10,000 cells/well. The highest concentration of the compound was 50 ⁇ M and Ixazomib-DEA was used as a control group. Dilute the drug concentration according to a 5-fold gradient. After the compound was applied for 48 hours, 10 ⁇ l of CCK-8 was added, and after incubation for 6 hours, the 450 nM wavelength absorption value was measured by a microplate reader.
  • the GI50 of the test sample for the proliferation inhibition activity of the mantle cell lymphoma cells was calculated by Graphpad Prism 5.0, and the results are shown in Table 5 below:
  • the experimental results demonstrate that the compounds of the present invention, I-1-3, I-2-3, and I-5-3, have the effect of inhibiting lymphoma, especially for mantle cell lymphoma and follicularity in non-Hodgkin's lymphoma.
  • the inhibition of lymphoma is significant.
  • Example 35 Inhibitory activity of the compound of the present invention against HT-29 human colon cancer cell line, breast cancer cell MDA-MB-231, lung cancer cell NCI-H460 and renal cancer cell line
  • Collect HT-29 human colon cancer cells in logarithmic growth phase count, resuspend the cells with complete medium, adjust the cell concentration to the appropriate concentration (determined according to the cell density optimization test results), inoculate 96-well plates, add 100 ⁇ l of cell suspension per well. liquid.
  • the cells were incubated for 24 hours at 37 ° C, 100% relative humidity, 5% CO 2 incubator.
  • the test compound was diluted with the medium to the corresponding concentration of action set, and added to the 96-well plate at 25 ⁇ l/well.
  • the final concentration of the compound was diluted from 100 ⁇ M to 0 ⁇ M in 4 fold gradients.
  • the cells were incubated for 72 hours at 37 ° C in a 100% relative humidity, 5% CO 2 incubator.
  • Test compound IC50(nM) Test compound IC50(nM) I-1-1 4.90 I-1-3 5.34 I-2-1 3.14 I-2-3 3.77 I-5-1 9.12 I-6-1 50.40 I-7-1 82.33 I-10-1 61.21 Ixazomib 55.35 Ixazomib-DEA 53.26
  • Test compound IC50(nM) Test compound IC50(nM) I-1-3 12.60 I-1-1 15.10 I-2-3 10.20 I-2-1 13.14 I-5-1 13.23 I-6-1 68.71 I-7-1 133.24 I-10-1 145.75 Ixazomib 58.82 Ixazomib-DEA 56.77
  • Test compound IC50(nM) Test compound IC50(nM) I-1-1 50.10 I-1-3 22.38 I-2-1 49.33 I-2-3 26.12 1-5-1 32.29 1-6-1 220.40 1-7-1 255.41 1-10-1 247.82 Ixazomib 160.91 Ixazomib-DEA 202.47
  • the results show that the compounds of the present invention are compared to the control compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, I-10-1 to NCI-H460 lung cancer cell line, MDA-MB-231 breast cancer cell line.
  • the proliferation of renal cancer cell lines has a more significant inhibitory effect.
  • Example 36 Inhibitory activity of the compound of the present invention on human cervical cancer and nasopharyngeal carcinoma cells
  • the test sample was dissolved in DMSO and stored at low temperature. Before the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in the present embodiment.
  • the cervical cancer cell line HeLa cells were added to a flat-bottom 96-well cell culture plate at 2000 cells/well. After the cells were cultured for 24 hours, the test compound was added. The highest concentration of the compound is 10 ⁇ M.
  • Ixazomib-DEA, Ixazomib, I-6-1, I-7-1, and I-10-1 were used as a control group. Dilute the drug concentration according to a 5-fold gradient.
  • the GI50 of the test sample against the proliferation inhibition activity of the cervical cancer cell line HeLa cells was calculated by Graphpad Prism 5.0, and the results are shown in Table 10 below:
  • Test compound GI50(nM) Test compound GI50(nM) I-1-1 9.11 I-1-3 29.48 I-2-1 8.05 I-2-3 30.02 I-5-1 18.31 I-6-1 170.53 I-7-1 116.26 I-10-1 182.93 Ixazomib 32.84 Ixazomib-DEA 51.16
  • the compounds of the present invention had more significant inhibitory effects on the cervical cancer cell line HeLa cells than the compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, and I-10-1.
  • Test compound GI50(nM) Test compound GI50(nM) I-1-1 603.97 I-1-3 616.03 I-2-1 614.80 I-2-3 670.52 I-5-1 683.52 I-6-1 1691.49 I-7-1 1922.08 I-10-1 1743.61 Ixazomib 803.52 Ixazomib-DEA 862.56
  • the benzene ring halogen substituent has a large influence on the activity, although those skilled in the art can consider Cl, Br, and I as electronic isosters to be mutually substituted, but surprisingly
  • the present inventors have found that a halogen change of a substituent on a benzene ring, and a different combination thereof, can give an unexpected difference in antitumor activity.
  • I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1 exhibited remarkably excellent antitumor activity.
  • Example 37 Determination of the effect of a compound of the invention on human hERG ion channels stably expressed in HEK293 cells using conventional patch clamps
  • the hERG ion channel steady-state expression HEK293 cells were transferred to a perfusion tank and perfused with extracellular fluid at room temperature, each cell being self-control.
  • Test compounds were all dissolved in DMSO and configured to a concentration gradient of 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, 100 ⁇ M.
  • the compounds were all perfused using a perfusion system using their own gravity. Test at least two cells per concentration. After the current is stable (or 5 minutes), the change in current magnitude before and after the compound is compared to calculate the blocking effect of the compound.
  • the positive control Cisapride was subjected to an IC50 test at a concentration gradient of 1 nM, 3 nM, 10 nM, 30 nM, 100 nM.
  • the method is the same as the test compound.
  • the test electrodes were drawn with PC-10 (Narishige, Japan).
  • the cells were clamped at –80 mV, then depolarized to 40 mV with a square wave lasting 4 seconds, and then hyperpolarized to -40 mV with a square wave lasting 2 seconds to obtain the hERG tail current. This procedure is repeated every 20 seconds.
  • the hERG tail current is a pure hERG current.
  • test compound for human hERG ion channel blockade expression in HEK293 cells is perfused, and when the reaction is stable, the blocking strength is calculated.
  • Table 12 The IC50 values of the test compounds for human hERG ion channel blockade expression in HEK293 cells are shown in Table 12 below:
  • Test compound IC50 ( ⁇ M) Test compound IC50 ( ⁇ M) I-1-1 >100 I-1-3 >100 I-2-1 >100 I-2-3 >100 Ixazomib 59.94 Ixazomib-DEA 56.43 Cisapride (positive control) 0.011
  • the compounds created by the present invention have a weaker blocking effect on the ion channel.
  • the compounds of the present invention have good proteasome inhibitory activity and antitumor activity, especially I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1.
  • Excellent anti-tumor activity superior to the existing compounds Ixazomib and Ixazomib-DEA, and the cardiotoxicity of I-1-1, I-1-3, I-2-1, I-2-3 is also extremely low, significantly lower than The existing compounds Ixazomib and Ixazomib-DEA have excellent clinical application prospects.

Abstract

Disclosed is a compound as represented by formula (I). Also provided in the present invention are a pharmaceutically acceptable salt of the compound, a preparation method for same, uses of same, and a pharmaceutical composition thereof. The compound provided in the present invention provides significant inhibitory effects against tumor cells, is applicable in preventing and/or treating tumor-related diseases, specifically breast cancer, colon cancer, lung cancer, and multiple myeloma, and has broad application prospects.

Description

一种硼酸和硼酸酯类化合物及其应用Boric acid and boric acid ester compound and application thereof 技术领域Technical field
本发明属于化学药物领域，具体涉及一种抗肿瘤化合物及其药物组合物。The invention belongs to the field of chemical medicines, and in particular relates to an antitumor compound and a pharmaceutical composition thereof.
背景技术Background technique
泛素蛋白酶体途径介导的蛋白降解是机体调节细胞内蛋白水平与功能的一个重要机制。一旦蛋白酶体超出正常水平，其会导致生长抑制的减弱、细胞凋亡的减少、以及促进血管生成，从而引发多种肿瘤疾病，因此蛋白酶体是抗癌等药物的重要靶点。蛋白酶体抑制剂通过阻断细胞蛋白酶体降解，从而抑制肿瘤细胞生长以及促进细胞凋亡。Ubiquitin-proteasome pathway-mediated protein degradation is an important mechanism by which the body regulates intracellular protein levels and functions. Once the proteasome exceeds normal levels, it causes a decrease in growth inhibition, a decrease in apoptosis, and promotes angiogenesis, thereby causing various tumor diseases, and thus the proteasome is an important target for drugs such as cancer. Proteasome inhibitors inhibit tumor cell growth and promote apoptosis by blocking cellular proteasome degradation.
多发性骨髓瘤(multiple myeloma，MM)是一种浆细胞癌，见于骨髓。多发性骨髓瘤中，一组浆细胞或骨髓瘤细胞转化为癌细胞并增生，使浆细胞的数目高于正常水平。由于浆细胞在体内广泛游走，有可能累及体内多数骨骼，可能导致压缩性骨折、骨溶解性病灶和相关疼痛。多发性骨髓瘤可导致若干严重健康问题，累及骨骼、免疫***、肾脏和个体的红细胞计数，部分较常见症状包括骨骼疼痛和疲乏。Multiple myeloma (MM) is a plasma cell carcinoma found in the bone marrow. In multiple myeloma, a group of plasma cells or myeloma cells are transformed into cancer cells and proliferate, resulting in a higher than normal number of plasma cells. Because plasma cells migrate extensively in the body, it is likely to involve most of the bones in the body, which may lead to compression fractures, osteolytic lesions and related pain. Multiple myeloma can cause several serious health problems, involving red blood cell counts in the bones, immune system, kidneys, and individuals. Some of the more common symptoms include bone pain and fatigue.
套细胞淋巴瘤、滤泡性淋巴瘤是较常见的非霍奇金淋巴瘤。套细胞淋巴瘤占非霍奇金淋巴瘤6％，且一线治疗方案不能达到完全满意的效果。滤泡性淋巴瘤占非霍奇金淋巴瘤22％，尽管已有多种治疗手段，但仍有未满足的临床需求。Mantle cell lymphoma, follicular lymphoma is a relatively common non-Hodgkin's lymphoma. Mantle cell lymphoma accounts for 6% of non-Hodgkin's lymphoma, and the first-line treatment regimen does not achieve a completely satisfactory result. Follicular lymphoma accounts for 22% of non-Hodgkin's lymphoma, and despite various treatments, there is still unmet clinical need.
肺癌、结肠癌、乳腺癌、肾癌、***、鼻咽癌等也是高发的肿瘤类型，尽管已经有大量的治疗药物和手段，但该领域高价值的治疗药物仍然是未满足的临床需求。Lung cancer, colon cancer, breast cancer, kidney cancer, cervical cancer, nasopharyngeal cancer, etc. are also high-risk tumor types. Although there are already a large number of therapeutic drugs and means, high-value therapeutic drugs in this field are still unmet clinical needs.
Bortezomib以及Ninlaro(Ixazomib Citrate)都是一种蛋白酶体抑制剂，其结构分别如下所示：Both Bortezomib and Ninlaro (Ixazomib Citrate) are proteasome inhibitors whose structures are as follows:
Figure PCTCN2017084924-appb-000001
Figure PCTCN2017084924-appb-000001
其中，Bortezomib主要以注射给药，Ixazomib Citrate以口服给药，并经过体内代谢成Ixazomib(硼酸部分)起效。Among them, Bortezomib is mainly administered by injection, Ixazomib Citrate is administered orally, and is metabolized in vivo to form Ixazomib (boric acid moiety).
WO2012/177835公开了Ixazomib如下结构的衍生物：(代号Ixazomib DEA) WO 2012/177835 discloses derivatives of the following structures of Ixazomib: (code name Ixazomib DEA)
Figure PCTCN2017084924-appb-000002
Figure PCTCN2017084924-appb-000002
上述专利研究了该类化合物作为Ixazomib的前药，通过口服吸收后代谢成Ixazomib，并与Ixazomib Citrate的口服生物利用度进行了对比，但并没有该化合物本身的活性报道。The above patents investigated the use of this compound as a prodrug of Ixazomib, which was metabolized by oral absorption into Ixazomib and compared with the oral bioavailability of Ixazomib Citrate, but without the activity of the compound itself.
Bilgicer等(Journal of Medicinal Chemistry,2014,57:5282)报道了硼替佐米不同的前药，其前药的设计方案采用了将硼酸结构单元中的羟基形成各种环状酯。换言之，现有公开的技术将上述硼酸环状酯视为前药，释放出硼酸结构部分起效。Bilgicer et al (Journal of Medicinal Chemistry, 2014, 57:5282) reported different prodrugs of bortezomib, the prodrug of which was designed to form hydroxyl groups in boric acid structural units into various cyclic esters. In other words, the prior art discloses the above-mentioned boric acid cyclic ester as a prodrug, and the release of the boric acid moiety is effective.
发明内容Summary of the invention
本发明提供了式(Ⅰ)所示的化合物或其晶型、或其药学上可接受的盐、或其溶剂合物或硼酸酐：The present invention provides a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
Figure PCTCN2017084924-appb-000003
Figure PCTCN2017084924-appb-000003
其中，环A表示如下结构之一：Among them, ring A represents one of the following structures:
Figure PCTCN2017084924-appb-000004
Figure PCTCN2017084924-appb-000004
R1选自H或C1-6烷基； R 1 is selected from H or C 1-6 alkyl;
R2选自C1-6烷基；R 2 is selected from C 1-6 alkyl;
R3和R4选自氢，或R3和R4与***的氧和硼原子共同形成一个任选取代的5-20元环，该环上还另外包括0-2个选自氮、氧或硫的环杂原子。R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with the inserted oxygen and boron atoms form an optionally substituted 5-20 membered ring, the ring further comprising 0-2 selected from nitrogen and oxygen. Or a ring hetero atom of sulfur.
进一步地，所述化合物如式(Ⅰa)所示：Further, the compound is as shown in the formula (Ia):
Figure PCTCN2017084924-appb-000005
Figure PCTCN2017084924-appb-000005
进一步地，R1选自H。Further, R 1 is selected from H.
进一步地，R2选自异丙基。Further, R 2 is selected from isopropyl.
进一步地，X1和X2选自羟基，或X1和X2共同形成与硼酸酯化剂的官能团脱去氢原子后的部分。Further, X 1 and X 2 are selected from hydroxyl, or X 1 and X 2 together form a portion of the acid functional groups formed by removing a hydrogen atom esterifying agent.
进一步地，X1和X2选自羟基。Further, X 1 and X 2 are selected from a hydroxyl group.
进一步地，所述硼酸酯化剂选自含有至少一个羟基或至少一个羧基的化合物。Further, the borating agent is selected from the group consisting of compounds containing at least one hydroxyl group or at least one carboxyl group.
进一步地，所述硼酸酯化剂为包含有N原子的硼酸酯化剂。Further, the borating agent is a borate esterifying agent containing N atoms.
进一步地，当X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分时，所形成的环为5-12元环。Further, when X 1 and X 2 together form a portion after dehydrogenation of the two functional groups of the borate esterifying agent, the ring formed is a 5-12 membered ring.
进一步地，所述硼酸酯化剂选自单糖或多元醇。Further, the borating agent is selected from the group consisting of a monosaccharide or a polyhydric alcohol.
进一步地，所述硼酸酯化剂选自C4-10的饱和硼酸酯化剂，其中，羟基和羧基的数量之和为2～4。Further, the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the number of hydroxyl groups and carboxyl groups is 2 to 4.
进一步地，所述硼酸酯化剂选自甘露醇、柠檬酸、苹果酸、酒石酸、葡萄糖、二乙醇胺、二丙醇胺、三乙醇胺、三丙醇胺、3-((2-羟基乙基)氨基)-1-丙醇、N-甲基二乙醇胺、N-丁基二乙醇胺、2-((2-羟基丙基)氨基)-1-丙醇、二异丙醇胺和N，N-双(2-羟乙基)甘氨酸中的任一种；或，所述硼酸酯化剂任选被C1-6的烷基、C3-C6的环烷基、C2-C6的羧烷基、C1-C6的羟烷基取代。Further, the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine, 3-((2-hydroxyethyl) Amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N Any one of bis(2-hydroxyethyl)glycine; or the boronic acid esterifying agent is optionally C1-6 alkyl, C3-C6 cycloalkyl, C2-C6 carboxyalkyl , C1-C6 hydroxyalkyl substitution.
进一步地，所述环A选自下述结构： Further, the ring A is selected from the following structures:
Figure PCTCN2017084924-appb-000006
Figure PCTCN2017084924-appb-000006
进一步地，所述硼酸酯化剂选自二丙醇胺。Further, the borating agent is selected from the group consisting of dipropanolamine.
进一步地，所述硼酸酯化剂选自二乙醇胺。Further, the borating agent is selected from the group consisting of diethanolamine.
进一步地，所述化合物为如下化合物之一或其甘露醇酯之一：Further, the compound is one of the following compounds or one of its mannitol esters:
Figure PCTCN2017084924-appb-000007
Figure PCTCN2017084924-appb-000007
进一步地，所述化合物为如下化合物之一：Further, the compound is one of the following compounds:
Figure PCTCN2017084924-appb-000008
Figure PCTCN2017084924-appb-000008
Figure PCTCN2017084924-appb-000009
Figure PCTCN2017084924-appb-000009
Figure PCTCN2017084924-appb-000010
Figure PCTCN2017084924-appb-000010
进一步地，所述化合物为如下化合物之一：Further, the compound is one of the following compounds:
Figure PCTCN2017084924-appb-000011
Figure PCTCN2017084924-appb-000011
本发明人惊奇地发现，苯环上不同的取代基，以及与硼酸成环状酯的不同，得到的上述衍生物具有较高的抗多发性骨髓瘤细胞活性，因此具有较好的应用前景。The present inventors have surprisingly found that the different substituents on the benzene ring and the cyclic esters of boric acid have higher anti-multiple myeloma cell activity, and thus have a good application prospect.
本发明还公开了上述化合物的新的合成路线，如下所示： The present invention also discloses a novel synthetic route for the above compounds, as follows:
Figure PCTCN2017084924-appb-000012
Figure PCTCN2017084924-appb-000012
CN200780100142公开了一类与本专利类似化合物的合成方法；其采用了TBTU等作为缩合剂，将取代苯甲酸与甘氨酸的缩合中间体S1与氨基硼酸酯S2进行成酰胺反应，但本发明人惊奇地发现，采用上述专利公开路线制备本发明公开的化合物，将得到主要以脱硼酸酯的副产物S4-1为主。将TBTU换成其他如DCC、EDCI·HCl等，或改变反应溶剂体系，但均出人意料地较少或完全不能得到目标中间体。说明苯环上取代基的不同，直接影响到制备方法的可行性，具有非显而易见性。CN200780100142 discloses a method for synthesizing a compound similar to the patent; which uses TBTU or the like as a condensing agent to form an amide reaction of a substituted intermediate S1 of a substituted benzoic acid with glycine with an amino boronate S2, but the present inventors are surprised It has been found that the preparation of the compounds disclosed herein using the above-disclosed patents will result in the predominantly by-product S4-1, which is predominantly deboronated. The TBTU is replaced with other such as DCC, EDCI.HCl, etc., or the reaction solvent system is changed, but the target intermediate is surprisingly less or completely unavailable. It indicates that the difference of the substituents on the benzene ring directly affects the feasibility of the preparation method and is non-obvious.
经过反复探索，本发明人发现，采用活化羧基的方法，如先制备活化酯(在此特指混合酸酐)，将可以高收率地得到目标物。After repeated investigations, the inventors have found that a method of activating a carboxyl group, such as preparing an activated ester (herein, a mixed acid anhydride), can obtain a target in a high yield.
一种制备所述化合物的方法，所述化合物如式(Ⅰaa)所示，它包括以下步骤：A method of preparing the compound, which is represented by the formula (Iaa), which comprises the steps of:
Figure PCTCN2017084924-appb-000013
Figure PCTCN2017084924-appb-000013
(1)在碱的存在下，将S1所示化合物与Z-X所示的羧基活化剂反应，制备得到M1所示的活化酯；其中，X表示羧基活化剂在反应中离去的部分，Z表示羧基活化剂在反应中进行取代的部分； (1) reacting a compound represented by S1 with a carboxyl activating agent represented by ZX in the presence of a base to prepare an activated ester represented by M1; wherein X represents a portion of the carboxyl activator which leaves in the reaction, and Z represents a moiety in which a carboxyl activator is substituted in the reaction;
Figure PCTCN2017084924-appb-000014
Figure PCTCN2017084924-appb-000014
(2)以M1所示的活化酯和S2所示的化合物或其盐为原料，制备得到式S3所示化合物；(2) using the activated ester represented by M1 and the compound represented by S2 or a salt thereof as a raw material to prepare a compound represented by the formula S3;
Figure PCTCN2017084924-appb-000015
Figure PCTCN2017084924-appb-000015
(3)将S3所示化合物水解，得到式(Ⅰaa)所示化合物。(3) Hydrolysis of the compound represented by S3 to give a compound of the formula (Iaa).
进一步地，步骤(1)中，所述羧基活化剂选自氯甲酸乙酯、氯甲酸丙酯和氯甲酸异丁酯中的任一种或多种。Further, in the step (1), the carboxyl activating agent is selected from any one or more selected from the group consisting of ethyl chloroformate, propyl chloroformate and isobutyl chloroformate.
进一步地，步骤(1)中，关键在于保持体系为碱性，所述碱可以选自N-甲基吗啉、三乙胺和N-乙基二异丙胺中的任一种或多种。Further, in the step (1), the key is to keep the system alkaline, and the base may be selected from any one or more of N-methylmorpholine, triethylamine and N-ethyldiisopropylamine.
进一步地，步骤(2)中，S2所示化合物的盐为三氟醋酸盐或盐酸盐。Further, in the step (2), the salt of the compound represented by S2 is a trifluoroacetate or a hydrochloride.
本发明还提供了所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐在制备抗肿瘤药物或蛋白酶体抑制剂类药物中的用途。The present invention also provides the use of the compound, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof, or a boric anhydride, for the preparation of an antitumor drug or a proteasome inhibitor.
进一步地，所述蛋白酶体抑制剂类药物是蛋白酶体胰凝乳蛋白酶样蛋白酶抑制剂类药物或者蛋白酶体胱天蛋白酶样蛋白酶抑制剂类药物。Further, the proteasome inhibitor drug is a proteasome chymotrypsin-like protease inhibitor drug or a proteasome caspase-like protease inhibitor drug.
进一步地，所述抗肿瘤药物是预防和/或治疗浆细胞瘤的药物。Further, the antitumor drug is a drug for preventing and/or treating plasmacytoma.
进一步地，所述浆细胞瘤是多发性骨髓瘤。Further, the plasmacytoma is multiple myeloma.
本发明的化合物除应用在多发性骨髓瘤之外，还表现出对其它多种癌症的抑制作用。In addition to application to multiple myeloma, the compounds of the present invention also exhibit inhibitory effects against a variety of other cancers.
进一步地，所述抗肿瘤药物是预防和/或治疗淋巴瘤的药物。再进一步地，所述药物是预防和/或治疗非霍奇金淋巴瘤的药物。再进一步地，所述非霍奇金淋巴瘤为套细胞淋巴瘤。再进一步地，所述非霍奇金淋巴瘤为滤泡性淋巴瘤。Further, the antitumor drug is a drug for preventing and/or treating lymphoma. Still further, the medicament is a medicament for preventing and/or treating non-Hodgkin's lymphoma. Still further, the non-Hodgkin's lymphoma is a mantle cell lymphoma. Still further, the non-Hodgkin's lymphoma is a follicular lymphoma.
进一步的，所述肿瘤是套细胞淋巴瘤、滤泡性淋巴瘤。 Further, the tumor is a mantle cell lymphoma or a follicular lymphoma.
进一步地，所述肿瘤为乳腺癌、结肠癌、肺癌、肾癌、***、鼻咽癌。Further, the tumor is breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer, nasopharyngeal cancer.
本发明中，所述C1～C6的烷基是指C1、C2、C3、C4、C5、C6的烷基，即具有1～6个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、仲丁基、戊基、己基等等。In the present invention, the C 1 -C 6 alkyl group means a C 1 , C 2 , C 3 , C 4 , C 5 , C 6 alkyl group, that is, a straight chain or a branch having 1 to 6 carbon atoms. Alkyl of the chain, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, and the like.
本发明中，所述的环杂原子是指硼酸酯化剂与硼原子、氧原子成环，成环的原子中除碳原子以外的其它杂原子，不包括环上的取代基团中的原子。In the present invention, the ring hetero atom refers to a boron atomating agent which is ring-formed with a boron atom or an oxygen atom, and a hetero atom other than a carbon atom in the ring-forming atom, excluding a substituent group on the ring. atom.
本发明中，所述硼酸是指含有-B(OH)2部分的化合物。硼酸化合物可通过使硼酸部分脱水而形成寡聚酸酐。In the present invention, it refers to a compound containing a boronic acid -B (OH) 2 moiety. The boric acid compound can form an oligomeric anhydride by partially dehydrating the boric acid.
本发明中，所述硼酸酐是指由两个或两个以上硼酸化合物分子结合同时失去一个或多个水分子所形成的化合物。当与水混合时，硼酸酐化合物被水化而释放游离硼酸化合物。在各种实施例中，硼酸酐可含有两个、三个、四个或四个以上硼酸单元，并且可具有环状或线性构型。例如下述的环状结构：In the present invention, the boric anhydride refers to a compound formed by combining two or more boric acid compound molecules while losing one or more water molecules. When mixed with water, the boric anhydride compound is hydrated to release the free boric acid compound. In various embodiments, the boric anhydride may contain two, three, four or more boric acid units, and may have a cyclic or linear configuration. For example, the following ring structure:
Figure PCTCN2017084924-appb-000016
Figure PCTCN2017084924-appb-000016
下述的线性结构：The linear structure described below:
Figure PCTCN2017084924-appb-000017
Figure PCTCN2017084924-appb-000017
n可以是0-10的整数。n can be an integer from 0-10.
本发明中，硼酸酯化剂指的是任何具有至少两个官能团的化合物，所述至少两个官能团各自可与硼形成共价键，例如羟基和羧基。作为一些更为具体的实施方式，可以包括甘露醇、柠檬酸、苹果酸、酒石酸、葡萄糖、氨基二乙醇、氨基二丙醇、三乙醇胺和三丙醇胺等。In the present invention, the borate esterifying agent refers to any compound having at least two functional groups, each of which can form a covalent bond with boron, such as a hydroxyl group and a carboxyl group. As some more specific embodiments, mannitol, citric acid, malic acid, tartaric acid, glucose, aminodiethanol, aminodipropanol, triethanolamine, and tripropanolamine may be included.
其中含有至少一个N杂原子的该类硼酸酯化剂中，N原子的孤对电子与硼原子的空轨道配位，从而形成稳定的环状酯化物。如下所示：In such a borate esterifying agent containing at least one N hetero atom, the lone pair of electrons of the N atom are coordinated with the empty orbital of the boron atom to form a stable cyclic ester compound. As follows:
Figure PCTCN2017084924-appb-000018
Figure PCTCN2017084924-appb-000018
其中，n1，n2＝0或1，R1为氢或其它取代基团。因此，硼酸酯化剂除上述几种之外，其它包括3-((2-羟基乙基)氨基)-1-丙醇，N-甲基二乙醇胺，N-丁基二乙醇胺，2-((2-羟基丙基)氨基)-1-丙醇、二异丙醇胺和N，N-双(2-羟乙基)甘氨酸等；或，所述硼酸酯化剂任选被C1-6的烷基、C3-C6的环烷基、C2-C6的羧烷基、C1-C6的羟烷基等取代。Wherein n1, n2=0 or 1, and R1 is hydrogen or another substituent group. Therefore, the borate esterifying agent includes, in addition to the above, 3-((2-hydroxyethyl)amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2- ((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N-bis(2-hydroxyethyl)glycine, etc.; or, the borate esterifying agent is optionally C1 a -6 alkyl group, a C3-C6 cycloalkyl group, a C2-C6 carboxyalkyl group, a C1-C6 hydroxyalkyl group, or the like.
本发明提供了式(Ⅰ)所示的化合物或其晶型、或其药学上可接受的盐、或其溶剂合物或硼酸酐：The present invention provides a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
Figure PCTCN2017084924-appb-000019
Figure PCTCN2017084924-appb-000019
其中，环A表示如下结构之一：Among them, ring A represents one of the following structures:
Figure PCTCN2017084924-appb-000020
Figure PCTCN2017084924-appb-000020
其中，R5、R6、R7选自H、F、Cl、Br、I、C1-6烷基、C1-6烷氧基，三氟甲基； Wherein R 5 , R 6 , and R 7 are selected from the group consisting of H, F, Cl, Br, I, C 1-6 alkyl, C 1-6 alkoxy, trifluoromethyl;
R1选自H、C1-6烷基、C1-6环烷基、烷氧基、苄基；所述C1-6烷基、C1-6环烷基、烷氧基、苄基可以被进一步取代；R 1 is selected from the group consisting of H, C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl; the C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl The base can be further substituted;
R2选自C1-6烷基；R 2 is selected from C 1-6 alkyl;
R3和R4选自氢，或R3和R4与***的氧和硼原子共同形成一个任选取代的5-20元环，该环上还另外包括0-2个选自氮、氧或硫的环杂原子。R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with the inserted oxygen and boron atoms form an optionally substituted 5-20 membered ring, the ring further comprising 0-2 selected from nitrogen and oxygen. Or a ring hetero atom of sulfur.
进一步地，所述化合物如式(Ⅰa)所示：Further, the compound is as shown in the formula (Ia):
Figure PCTCN2017084924-appb-000021
Figure PCTCN2017084924-appb-000021
进一步地，R1选自H或苄基。Further, R 1 is selected from H or benzyl.
进一步地，R2选自异丙基。Further, R 2 is selected from isopropyl.
进一步地，X1和X2选自羟基，或X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分。Further, X 1 and X 2 are selected from a hydroxyl group, or X 1 and X 2 together form a portion after dehydrogenation of the two functional groups of the borate esterifying agent.
进一步地，X1和X2选自羟基。Further, X 1 and X 2 are selected from a hydroxyl group.
进一步地，所述硼酸酯化剂选自含有至少一个羟基或至少一个羧基的化合物。Further, the borating agent is selected from the group consisting of compounds containing at least one hydroxyl group or at least one carboxyl group.
进一步地，所述硼酸酯化剂为包含有N原子的硼酸酯化剂。Further, the borating agent is a borate esterifying agent containing N atoms.
进一步地，当X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分时，所形成的环为5-10元环。Further, when X 1 and X 2 together form a portion after dehydrogenation of the two functional groups of the borate esterifying agent, the ring formed is a 5-10 membered ring.
进一步地，所述硼酸酯化剂选自单糖或多元醇。Further, the borating agent is selected from the group consisting of a monosaccharide or a polyhydric alcohol.
进一步地，所述硼酸酯化剂选自C4-10的饱和硼酸酯化剂，其中，羟基和羧基的数量之和为2～8。Further, the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the amounts of the hydroxyl group and the carboxyl group is 2-8.
进一步地，所述硼酸酯化剂选自甘露醇、柠檬酸、苹果酸、酒石酸、葡萄糖、二乙醇胺、二丙醇胺、三乙醇胺、三丙醇胺、3-((2-羟基乙基)氨基)-1-丙醇、N-甲基二乙醇胺、N-丁基二乙醇胺、2-((2-羟基丙基)氨基)-1-丙醇、二异丙醇胺和N，N-双(2-羟乙基)甘氨酸中的任一种；或，所述硼酸酯化剂任选被C1-6的烷基、C3-C6的环烷基、C2-C6的羧烷基、C1-C6的羟烷基等取代。Further, the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine, 3-((2-hydroxyethyl) Amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol, diisopropanolamine and N,N Any one of bis(2-hydroxyethyl)glycine; or the boronic acid esterifying agent is optionally C1-6 alkyl, C3-C6 cycloalkyl, C2-C6 carboxyalkyl And a C1-C6 hydroxyalkyl group or the like.
本发明化合物可以为如下结构的化合物之一： The compound of the present invention may be one of the following structures:
Figure PCTCN2017084924-appb-000022
Figure PCTCN2017084924-appb-000022
Figure PCTCN2017084924-appb-000023
Figure PCTCN2017084924-appb-000023
Figure PCTCN2017084924-appb-000024
Figure PCTCN2017084924-appb-000024
Figure PCTCN2017084924-appb-000025
Figure PCTCN2017084924-appb-000025
Figure PCTCN2017084924-appb-000026
Figure PCTCN2017084924-appb-000026
Figure PCTCN2017084924-appb-000027
Figure PCTCN2017084924-appb-000027
Figure PCTCN2017084924-appb-000028
Figure PCTCN2017084924-appb-000028
Figure PCTCN2017084924-appb-000029
Figure PCTCN2017084924-appb-000029
Figure PCTCN2017084924-appb-000030
Figure PCTCN2017084924-appb-000030
Figure PCTCN2017084924-appb-000031
Figure PCTCN2017084924-appb-000031
Figure PCTCN2017084924-appb-000032
Figure PCTCN2017084924-appb-000032
Figure PCTCN2017084924-appb-000033
Figure PCTCN2017084924-appb-000033
Figure PCTCN2017084924-appb-000034
Figure PCTCN2017084924-appb-000034
Figure PCTCN2017084924-appb-000035
Figure PCTCN2017084924-appb-000035
Figure PCTCN2017084924-appb-000036
Figure PCTCN2017084924-appb-000036
Figure PCTCN2017084924-appb-000037
Figure PCTCN2017084924-appb-000037
Figure PCTCN2017084924-appb-000038
Figure PCTCN2017084924-appb-000038
Figure PCTCN2017084924-appb-000039
Figure PCTCN2017084924-appb-000039
Figure PCTCN2017084924-appb-000040
Figure PCTCN2017084924-appb-000040
Figure PCTCN2017084924-appb-000041
Figure PCTCN2017084924-appb-000041
本发明人惊奇地发现，上述结构中苯环部分的不同取代基、硼酸结构单元是否成酯，以及不同的环状酯以及不同的组合，都会产生意想不到的活性效果。The present inventors have surprisingly found that the different substituents of the benzene ring moiety, whether the boronic acid structural unit is esterified, and the different cyclic esters and different combinations in the above structure produce unexpected activity effects.
本发明人还发现硼酸结构单元不同的含N杂环环状酯，尽管可能缓慢分解成硼酸结构部分，但其并不仅仅是前药，其本身也具有意想不到的生物活性。The present inventors have also found that a N-containing heterocyclic cyclic ester having a different boric acid structural unit, although it may be slowly decomposed into a boric acid moiety, is not only a prodrug, but has an unexpected biological activity by itself.
本发明还提供了一种药物组合物，它是以所述的化合物或其药学上可接受的盐或硼酸酐为活性成分，加上药学上可接受的辅料制备而成的制剂。The present invention also provides a pharmaceutical composition which is prepared by using the compound or a pharmaceutically acceptable salt thereof or boric anhydride as an active ingredient, together with a pharmaceutically acceptable adjuvant.
其中，所述药学上可接受的辅料选自稀释剂、填充剂、着色剂、助流剂、润滑剂、粘合剂、稳定剂、助悬剂或缓冲剂的任一种或多种。Wherein the pharmaceutically acceptable adjuvant is selected from any one or more of a diluent, a filler, a colorant, a glidant, a lubricant, a binder, a stabilizer, a suspending agent or a buffer.
进一步地，所述制剂是片剂、胶囊剂、口服液、注射剂、透皮剂、气雾剂固体制剂、脂质体制剂或缓控释制剂。Further, the preparation is a tablet, a capsule, an oral solution, an injection, a transdermal agent, an aerosol solid preparation, a liposome preparation or a controlled release preparation.
所述前药是前述化合物的衍生物，它们自身可能具有较弱的活性或甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。The prodrugs are derivatives of the aforementioned compounds which may themselves have weak or no activity, but are converted to corresponding conditions under physiological conditions (for example by metabolism, solvolysis or otherwise) after administration. Biologically active form.
本发明中的关键中间体和化合物进行分离和纯化，所使用的方式是有机化学中常用的分离和纯化方法。The key intermediates and compounds of the present invention are isolated and purified in a manner that is commonly used in organic chemistry for separation and purification.
本发明的一种或多种化合物可以彼此联合使用，也可选择将本发明的化合物与任何其它的活性试剂结合使用，用于制备抗肿瘤药物或蛋白酶体抑制剂类药物。如果使用的是一组化合物，则可将这些化合物同时、分别或有序地对受试对象进行给药。本发明的化合物基于抗肿瘤的协同机制等原则，可与其它药物联合用药，包括序贯给药或同时给药，以提高其抗肿瘤疗效、延缓耐药性的产生、减少药物毒性。One or more compounds of the invention may be used in combination with one another, or the compounds of the invention may be used in combination with any other active agent for the preparation of an anti-tumor or proteasome inhibitor. If a group of compounds is used, the compounds can be administered to the subject simultaneously, separately or sequentially. The compound of the present invention can be combined with other drugs based on the principle of anti-tumor synergistic mechanism, including sequential administration or simultaneous administration, to improve anti-tumor efficacy, delay drug resistance, and reduce drug toxicity.
本发明联合用药物，它含有相同或不同规格单位制剂的用于同时或者分别给药的前述任一项所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐与抗肿瘤药物或辅助***的药物，以及药学上可接受的载体。The combination of the present invention, which comprises the compound of any of the preceding, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof, for simultaneous or separate administration of the same or different unit preparations Or a boric anhydride and an antitumor drug or a drug for adjuvant treatment of a tumor, and a pharmaceutically acceptable carrier.
本发明还提供了一种肿瘤的预防和/或治疗方法，它是对肿瘤患者给予前述任一项所述的化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐。The present invention also provides a method for preventing and/or treating a tumor, which comprises administering to a tumor patient a compound according to any one of the above, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof Or boric anhydride.
其中，所述肿瘤患者是浆细胞瘤、淋巴瘤、乳腺癌、结肠癌、肺癌、肾癌、***和/或鼻咽癌的患者。Wherein the tumor patient is a patient of plasmacytoma, lymphoma, breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer and/or nasopharyngeal cancer.
进一步地，所述患者是多发性骨髓瘤患者；所述患者是非霍奇金淋巴瘤患者，再进一步地是套细胞淋巴瘤和/或滤泡性淋巴瘤患者。 Further, the patient is a multiple myeloma patient; the patient is a non-Hodgkin's lymphoma patient, and further a mantle cell lymphoma and/or follicular lymphoma patient.
本发明硼酸酯类化合物的晶型，所述晶型的X射线粉末衍射中，2θ衍射角度在8.38、11.4、12.1、13.29、18.27、19.62、20.47、23.32、24.74度处有特征峰。In the crystal form of the borate ester compound of the present invention, the X-ray powder diffraction of the crystal form has characteristic peaks at 8.38, 11.4, 12.1, 13.29, 18.27, 19.62, 20.47, 23.32, and 24.74 degrees.
优选地，该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：Preferably, in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
Figure PCTCN2017084924-appb-000042
Figure PCTCN2017084924-appb-000042
进一步优选地，该晶型具有基本如图5所示的X射线粉末衍射图谱。Further preferably, the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
本发明还提供了一种硼酸酯类化合物的晶型，所述晶型的X射线粉末衍射中，2θ衍射角度在6.19、7.98、10.05、14.73、14.92、17.16、18.8、20.03、21.05度处有特征峰。The present invention also provides a crystal form of a borate compound, wherein the X-ray powder diffraction of the crystal form has a 2θ diffraction angle of 6.19, 7.98, 10.05, 14.73, 14.92, 17.16, 18.8, 20.03, 21.05 degrees. Characteristic peaks.
优选地，该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：Preferably, in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
Figure PCTCN2017084924-appb-000043
Figure PCTCN2017084924-appb-000043
进一步优选地，该晶型具有基本如图6所示的X射线粉末衍射图谱。Further preferably, the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
本发明还提供了一种硼酸酯类化合物的晶型，所述晶型的X射线粉末衍射中，2θ衍射角度在9.23、12.53、14.18、17.06、20.82、21.46、22.62度处有特征峰。The present invention also provides a crystal form of a borate ester compound having X-ray powder diffraction having characteristic peaks at 2.23, 12.53, 14.18, 17.06, 20.82, 21.46, 22.62 degrees in X-ray powder diffraction.
优选地，该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：Preferably, in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
Figure PCTCN2017084924-appb-000044
Figure PCTCN2017084924-appb-000044
进一步优选地，该晶型具有基本如图7所示的X射线粉末衍射图谱。Further preferably, the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
本发明还提供了一种硼酸酯类化合物的晶型，所述晶型的X射线粉末衍射中，2θ衍射角度在10.59、11.76、13.19、15.56、17.76、19.5、20.26、21.37、22.2度处有特征峰。The present invention also provides a crystal form of a borate compound in which X-ray powder diffraction has a 2θ diffraction angle of 10.59, 11.76, 13.19, 15.56, 17.76, 19.5, 20.26, 21.37, 22.2 degrees. Characteristic peaks.
优选地，该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：Preferably, in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
Figure PCTCN2017084924-appb-000045
Figure PCTCN2017084924-appb-000045
进一步优选地，该晶型具有基本如图8所示的X射线粉末衍射图谱。Further preferably, the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
本发明还提供了一种硼酸酯类化合物的晶型，所述晶型的X射线粉末衍射中，2θ衍 射角度在7.06、10.61、12.24、14.9、17.23、20.21、23.49、26.48度处有特征峰。The present invention also provides a crystal form of a borate ester compound, X-ray powder diffraction of the crystal form, 2θ derivative The shooting angles have characteristic peaks at 7.06, 10.61, 12.24, 14.9, 17.23, 20.21, 23.49, and 26.48 degrees.
优选地，该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：Preferably, in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
Figure PCTCN2017084924-appb-000046
Figure PCTCN2017084924-appb-000046
进一步优选地，该晶型具有基本如图9所示的X射线粉末衍射图谱。Further preferably, the crystal form has an X-ray powder diffraction pattern substantially as shown in FIG.
本领域公知，由于测量的操作人员、测量的环境、样品的纯度等诸多因素会导致峰位置的些许偏移，各个2θ值具有±0.1度的误差。It is well known in the art that due to factors such as the operator of the measurement, the environment of the measurement, the purity of the sample, and the like, a slight shift in the peak position is caused, and each 2θ value has an error of ±0.1 degrees.
这些可以与本专利化合物联合用药的药物包括但不仅限于，细胞毒类药物，如卡铂、顺铂、伊立替康、紫杉醇、氟脲嘧啶、阿糖胞苷、来拉度胺、维甲酸，激素类药物，如***、氟维司群、他莫昔芬等，分子靶向药物，如厄洛替尼、拉帕替尼、曲妥珠单抗，辅助治疗类药物，如重组人粒细胞集落刺激因子、***、帕米膦酸二钠、唑来膦酸等。These drugs which may be combined with the compounds of the present invention include, but are not limited to, cytotoxic drugs such as carboplatin, cisplatin, irinotecan, paclitaxel, fluorouracil, cytarabine, lenalidomide, retinoic acid, Hormonal drugs such as dexamethasone, fulvestrant, tamoxifen, etc., molecularly targeted drugs such as erlotinib, lapatinib, trastuzumab, adjuvant therapy, such as recombinants Granulocyte colony-stimulating factor, erythropoietin, disodium pamidronate, zoledronic acid, and the like.
本发明所述药学上可接受的辅料，是指除活性成分以外包含在剂型中的物质。The pharmaceutically acceptable excipient of the present invention means a substance which is contained in a dosage form in addition to the active ingredient.
本发明化合物具有良好的蛋白酶体抑制活性和抗肿瘤活性，而且毒性低，临床应用前景优良。The compound of the invention has good proteasome inhibitory activity and antitumor activity, and has low toxicity and excellent clinical application prospect.
本发明中，英文缩写对应的中文全称如下表所示：In the present invention, the full Chinese name corresponding to the English abbreviation is as follows:
TBTUTBTU O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸O-benzotriazole-N,N,N',N'-tetramethylureatetrafluoroboric acid
DIEADIEA N,N-二异丙基乙胺N,N-diisopropylethylamine
NMMNMM N-甲基吗啉N-methylmorpholine
DMFDMF N,N-二甲基甲酰胺N,N-dimethylformamide
DCCDCC 二环己基碳二亚胺Dicyclohexylcarbodiimide
EDCI·HClEDCI·HCl 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride
本发明中，“烷基”包括直链或支链的烷基。In the present invention, "alkyl" includes a straight or branched alkyl group.
活性成分Active ingredient
本发明中，术语“本发明化合物”指式(I)所示的化合物。该术语还包括及式(I)化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。In the present invention, the term "compound of the present invention" means a compound represented by the formula (I). The term also encompasses various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I).
本发明中，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药 物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与碱金属形成的盐。适合形成盐的碱金属包括但并不限于：锂、钠，钾、钙、镁等。In the present invention, the term "pharmaceutically acceptable salt" means that the compound of the present invention and an acid or a base are suitable for use as a medicine. Salt of matter. Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with an alkali metal. Alkali metals suitable for forming salts include, but are not limited to, lithium, sodium, potassium, calcium, magnesium, and the like.
辅料Excipient
所述药学上可接受的辅料，它具有一定生理活性，但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位，而仅仅发挥辅助功效，这些辅助功效仅仅是对该成分已知活性的利用，是医药领域惯用的辅助治疗方式。若将上述辅料与本发明药物组合物配合使用，仍然应属于本发明保护的范围。The pharmaceutically acceptable excipient, which has a certain physiological activity, but the addition of the ingredient does not change the dominance of the above-mentioned pharmaceutical composition in the course of the disease treatment, but only plays an auxiliary effect, and the auxiliary effect is only the ingredient The use of known activities is an adjuvant treatment that is commonly used in the medical field. It is still within the scope of the present invention to use the above-mentioned excipients in combination with the pharmaceutical compositions of the present invention.
药物组合物和施用方法Pharmaceutical composition and method of administration
由于本发明化合物具有优异的治疗抗肿瘤作用，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，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抗肿瘤。Since the compound of the present invention has an excellent therapeutic antitumor effect, the compound of the present invention and various crystal forms thereof, a pharmaceutically acceptable inorganic or organic salt, hydrate or solvate, and a compound containing the compound of the present invention are mainly active ingredients. The pharmaceutical composition can be used for anti-tumor.
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1-20mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。The pharmaceutical 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. By "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. In general, the pharmaceutical compositions contain from 1 to 2000 mg of the compound of the invention per agent, more preferably from 1 to 20 mg of the compound of the invention per agent. Preferably, the "one dose" is a capsule or tablet.
“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。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2017084924-appb-000047
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"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. By "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. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as Tween
Figure PCTCN2017084924-appb-000047
), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肠胃外(静脉内、肌肉内或皮下)、和局部给药。The mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
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，如柠檬酸钠或磷酸二钙，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 ***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；和(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。Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, 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, alginate, gelatin, polyvinylpyrrolidone, sucrose and (A) humectant, 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; (f) an absorption accelerator such as a quaternary amine compound; (g) a wetting agent such as cetyl alcohol and glyceryl monostearate; (h) an adsorbent such as kaolin; Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.
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。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.
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
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。In addition to these inert diluents, the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants. The active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
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。The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention.
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。 但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.
附图说明DRAWINGS
图1为化合物I-1-2的质谱图。Figure 1 is a mass spectrum of Compound 1-1-2.
图2为化合物I-1-3的质谱图。Figure 2 is a mass spectrum of Compound 1-1-3.
图3为化合物I-2-2的质谱图。Figure 3 is a mass spectrum of the compound I-2-2.
图4为化合物I-1-2的X射线粉末衍射图。Figure 4 is an X-ray powder diffraction pattern of Compound 1-1-2.
图5为化合物I-1-3的X射线粉末衍射图。Figure 5 is an X-ray powder diffraction pattern of Compound 1-1-3.
图6为化合物I-1-4的X射线粉末衍射图。Figure 6 is an X-ray powder diffraction pattern of Compound 1-1-4.
图7为化合物I-1-5的X射线粉末衍射图。Figure 7 is an X-ray powder diffraction pattern of Compound 1-1-5.
图8为化合物I-2-2的X射线粉末衍射图。Figure 8 is an X-ray powder diffraction pattern of Compound I-2-2.
图9为化合物I-2-3的X射线粉末衍射图。Figure 9 is an X-ray powder diffraction pattern of Compound I-2-3.
图10为化合物I-2-3在PBS中水解实验的结果图。Figure 10 is a graph showing the results of hydrolysis experiments of Compound I-2-3 in PBS.
图11为本发明化合物I-1-3以及I-2-3能显著抑制小鼠多发性骨髓瘤体内模型肿瘤组织中蛋白酶体活性。Figure 11 shows that the compounds I-1-3 and I-2-3 of the present invention can significantly inhibit the proteasome activity in tumor tissues of mouse multiple myeloma in vivo.
图12为本发明化合物I-1-3以及I-2-3能诱导肿瘤凋亡标志-Cleaved PARP以及Cleaved Caspase-3活化片段的生成。Figure 12 shows that the compounds I-1-3 and I-2-3 of the present invention are capable of inducing the formation of tumor apoptosis markers - Cleaved PARP and Cleaved Caspase-3 activated fragments.
图13为小鼠肿瘤组织切片：①阴性对照组；②口服Ixazomib-DEA组；③口服本发明化合物I-1-3组；④口服本发明化合物I-2-3组。Figure 13 is a mouse tumor tissue section: 1 negative control group; 2 oral Ixazomib-DEA group; 3 oral administration of compound I-1-3 of the present invention; 4 oral administration of compound I-2-3 of the present invention.
具体实施方式detailed description
起始原料S1的制备方法是，从取代苯甲酸与甘氨酸甲酯脱水成酰胺后，水解甲酯自制；S2氨基硼酸酯的三氟醋酸盐或盐酸盐中间体为市售，其他合成试剂均为市售。实施例中得到的目标化合物均控制在HPLC纯度98％以上。The starting material S1 is prepared by dehydrating the substituted benzoic acid and the glycine methyl ester to form an amide, and then hydrolyzing the methyl ester; the trifluoroacetate or hydrochloride intermediate of the S2 aminoborate is commercially available, and other synthetic The reagents are all commercially available. The target compounds obtained in the examples were all controlled to have a purity of 98% or more.
实施例1本发明中间体S3-1及目标化合物I-1-1的制备Example 1 Preparation of Intermediate S3-1 of the Invention and Target Compound I-1-1
合成路线如下图所示： The synthetic route is shown below:
Figure PCTCN2017084924-appb-000048
Figure PCTCN2017084924-appb-000048
CN200780100142公开了一种与本发明类似化合物的合成方法；其采用了TBTU等作为缩合剂，将取代苯甲酸与甘氨酸的缩合中间体S1与氨基硼酸酯S2进行成酰胺反应，但本发明人发现，采用上述专利公开路线制备本发明公开的化合物，得到的产物主要是脱硼酸酯的副产物S4-1，具体操作如下：CN200780100142 discloses a synthesis method of a compound similar to the present invention, which uses TBTU or the like as a condensing agent to form an amide reaction of a condensed intermediate S1 of a substituted benzoic acid with glycine with an amino boronate S2, but the inventors have found The compound disclosed in the present invention is prepared by the above-mentioned patent disclosure route, and the obtained product is mainly a by-product S4-1 of deboronate, and the specific operation is as follows:
将起始原料(S-1-1)0.205克(0.70mmol)的10mLDMF溶液中，加入缩合剂TBTU0.248g(0.74mmol)以及0.267gS2(0.70mmol，1eq),降温至0度左右，滴入DIEA0.367mL(2.1mmol)，反应结束后，有机层加入100mL水，二氯甲烷分次萃取，硫酸钠干燥，减压浓缩得到淡黄色固体，但核磁共振谱图等证明完全生成副产物S4-1，而无法得到我们的目标化合物的中间产物S3-1。S4-1氢谱的图谱数据如下：To a solution of 0.205 g (0.70 mmol) of starting material (S-1-1) in 10 mL of DMF, condensing agent TBTU 0.248 g (0.74 mmol) and 0.267 g of S2 (0.70 mmol, 1 eq) were added, and the temperature was lowered to about 0 degree. DIEA 0.367 mL (2.1 mmol). After the reaction was completed, the organic layer was added with 100 mL of water, and the mixture was evaporated. 1, can not get the intermediate product S3-1 of our target compound. The map data of the S4-1 hydrogen spectrum is as follows:
1H NMR(300MHz,CDCl3)δ(ppm)7.78(brs,1H),7.48-7.51(m,1H),7.27-7.30(m,2H),6.41(brs,1H),4.14(d,2H),3.30-3.32(m,2H),1.60-1.64(m,1H),1.40-1.42(m,2H),0.90(d,6H)。 1 H NMR (300MHz, CDCl3) δ (ppm) 7.78 (brs, 1H), 7.48-7.51 (m, 1H), 7.27-7.30 (m, 2H), 6.41 (brs, 1H), 4.14 (d, 2H) , 3.30-3.32 (m, 2H), 1.60-1.64 (m, 1H), 1.40-1.42 (m, 2H), 0.90 (d, 6H).
更换反应溶剂体系或者采用其他如EDCI.HCl、DCC等缩合剂，均未能得到目标产物。The target product was not obtained by replacing the reaction solvent system or using other condensing agents such as EDCI.HCl, DCC, and the like.
WO2012/177835也公开了一种合成路线，如下图：WO2012/177835 also discloses a synthetic route, as shown below:
Figure PCTCN2017084924-appb-000049
Figure PCTCN2017084924-appb-000049
如图所示，其中X为离去基团。 As shown, where X is a leaving group.
本发明人根据报道的合成路线，选择X为Cl，将上述起始原料(S1-1)与二氯亚砜反应制备得到酰氯，与S2在碱性条件下进行成酰胺化反应，经多次尝试，得到的产物极其复杂，副产物很多，无制备价值(HPLC监测目标物转化率低于10％)。According to the reported synthetic route, the inventors selected X as Cl, reacted the above starting material (S1-1) with thionyl chloride to prepare an acid chloride, and formed an amidation reaction with S2 under basic conditions. Attempts, the resulting product is extremely complex, with many by-products and no preparative value (HPLC monitoring target conversion is less than 10%).
实验证明，采用WO2012/177835公开的合成路线，选择X为Cl，也难以有效制备得到目标中间体S3-1。Experiments have shown that it is difficult to efficiently prepare the target intermediate S3-1 by using the synthetic route disclosed in WO2012/177835 and selecting X to be Cl.
本发明特定的制备方法如下：将起始原料(S-1-1)3g(10mmol)20mL二氯甲烷溶液中,加入NMM(N-甲基吗啉)2.6g(26mmol),降温至5度，滴入氯甲酸乙酯1.34g(12mmol),待反应完全得到活化酯中间体M1-1之后，过滤，在滤液中加入4.69gS2(12mmol),室温搅拌3小时后，加入冰水淬灭，有机层干燥后，浓缩得到淡黄色固体目标物S3-1。将上述体系中，用氯甲酸丙酯或氯甲酸丁酯(异丁酯)等代替氯甲酸乙酯，NMM换成三乙胺或N-乙基二异丙胺等其他有机碱，均能重复得到相似的结果，收率均在80％以上。The specific preparation method of the present invention is as follows: 3 g (10 mmol) of starting material (S-1-1) in 20 mL of dichloromethane solution, NMM (N-methylmorpholine) 2.6 g (26 mmol) is added, and the temperature is lowered to 5 degrees. After adding 1.34 g (12 mmol) of ethyl chloroformate, and reacting to obtain the activated ester intermediate M1-1, the mixture was filtered, and 4.69 g of S2 (12 mmol) was added to the filtrate, stirred at room temperature for 3 hours, and then quenched with ice water. The organic layer was dried and concentrated to give a pale yellow solid object S3-1. In the above system, chloroethyl chloroformate or butyl chloroformate (isobutyl ester) or the like is used instead of ethyl chloroformate, and NMM is replaced with other organic bases such as triethylamine or N-ethyldiisopropylamine. Similar results, the yields were all above 80%.
中间体S3-1获得之后，进行硼酸酯的水解反应，如下路线所示：After the intermediate S3-1 is obtained, the hydrolysis reaction of the borate ester is carried out as shown in the following route:
Figure PCTCN2017084924-appb-000050
Figure PCTCN2017084924-appb-000050
如上路线所示，将上步得到的中间体5.1gS3-1,甲醇40mL,1N盐酸溶液4.5mL,异丁基硼酸2.6g(2.5eq),正己烷20mL混合后，室温搅拌过夜，将正己烷分去，减压浓缩甲醇，加入20mL水,1N氢氧化钠中和并调节pH为8-9，二氯甲烷20mL分次萃取，剩下碱水液体用1N盐酸溶液调酸后，二氯甲烷萃取多次，干燥后，浓缩干得到淡黄色固体；固体中含有I-1-1化合物以及形成的三聚体；进一步地纯化采用甲醇/水混合溶解后，反相高效液相色谱纯化(流动相采用甲醇/水梯度洗脱，UV检测)，冻干得到目标物白色粉末化合物I-1-1。As shown in the above route, the intermediate obtained in the previous step, 5.1 g of S3-1, 40 mL of methanol, 4.5 mL of 1N hydrochloric acid solution, 2.6 g of isobutylboric acid (2.5 eq), and 20 mL of n-hexane were stirred at room temperature overnight to give n-hexane. Distilled, concentrated methanol under reduced pressure, added 20 mL of water, neutralized with 1N sodium hydroxide and adjusted to pH 8-9, 20 mL of dichloromethane was extracted in portions, and then the aqueous alkali liquid was acidified with 1N hydrochloric acid solution, dichloromethane The mixture is extracted several times, dried, and concentrated to obtain a pale yellow solid; the solid contains the I-1-1 compound and the formed trimer; further purification is dissolved by methanol/water mixture, and purified by reversed-phase high performance liquid chromatography (flow) The phase was eluted with a methanol/water gradient, UV detection), and lyophilized to give the object white powder compound I-1-1.
1H NMR(300MHz,DMSO-d6)δ(ppm)：8.97(brs,1H),7.78(m,1H),7.63-7.66(m,2H),7.47-7.50(m,1H),4.03(m，2H),2.66(m,1H),1.62(m,1H),1.28-1.34(m,2H),0.83(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm): 8.97 (brs, 1H), 7.78 (m, 1H), 7.63-7.66 (m, 2H), 7.47-7.50 (m, 1H), 4.03 (m , 2H), 2.66 (m, 1H), 1.62 (m, 1H), 1.28-1.34 (m, 2H), 0.83 (d, 6H).
将上述白色粉末0.1克(0.25mmol)分散于9mL叔丁醇、15mL水中，加入0.9gD-甘露醇(4.9mmol)，温热溶解，冷冻干燥得到0.88g白色疏松状粉末I-1-1.20D-甘露醇。化合物I-1-1分散溶解于定量稀氢氧化钠水溶液中，冷冻干燥得到化合物I-1-1钠盐。 0.1 g (0.25 mmol) of the above white powder was dispersed in 9 mL of t-butanol, 15 mL of water, 0.9 g of D-mannitol (4.9 mmol) was added, and the mixture was warmly dissolved, and lyophilized to obtain 0.88 g of white loose powder I-1-1.20D. - Mannitol. The compound I-1-1 was dispersed and dissolved in a quantitative dilute aqueous sodium hydroxide solution, and lyophilized to obtain a sodium salt of the compound I-1-1.
实施例2本发明化合物I-1-2的制备Example 2 Preparation of Compound I-1-2 of the Invention
Figure PCTCN2017084924-appb-000051
Figure PCTCN2017084924-appb-000051
将硼酸起始原料(I-1-1)5克(12.3mmol)、二丙醇胺(S-3-2,分子量133.19)1.95克(14.6mmol)、20mL乙酸乙酯，室温搅拌过夜，析出白色固体，过滤得到4.8克化合物I-1-2，收率78％，即2-氯-5-溴-N-[(R)-1-[1,3,7,2]-二氧氮杂-2-硼基-3-甲基-丁羰酰胺基]-甲基]-苯甲酰胺。5 g (12.3 mmol) of boric acid starting material (I-1-1), 1.95 g (14.6 mmol) of dipropanolamine (S-3-2, molecular weight 133.19), and 20 mL of ethyl acetate were stirred at room temperature overnight to precipitate. White solid, filtered to give 4.8 g of compound I-1-2, yield 78%, 2-chloro-5-bromo-N-[(R)-1-[1,3,7,2]-diox Hetero-2-boryl-3-methyl-butancarbonylamido]-methyl]-benzamide.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.95(brs,1H),7.63-7.68(m,2H),7.48-7.50(m,1H),6.63(d,1H,J＝8.61Hz),4.81(m,1H),3.87-3.92(m,1H),3.75(m,1H),3.65(m,4H),3.20-3.34(m,3H),2.66(m,2H),1.89-1.99(m,1H),1.63-1.66(m,1H),1.49(m,2H),1.29-1.34(m,1H),1.15-1.23(m,1H),0.94-0.98(m,1H),0.83(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.95 (brs, 1H), 7.63-7.68 (m, 2H), 7.48-7.50 (m, 1H), 6.63 (d, 1H, J = 8.61Hz) , 4.81 (m, 1H), 3.87-3.92 (m, 1H), 3.75 (m, 1H), 3.65 (m, 4H), 3.20-3.34 (m, 3H), 2.66 (m, 2H), 1.89-1.99 (m, 1H), 1.63-1.66 (m, 1H), 1.49 (m, 2H), 1.29-1.34 (m, 1H), 1.15 - 1.23 (m, 1H), 0.94-0.98 (m, 1H), 0.83 (d, 6H).
ESI m/z：500.0[M-H]-。ESI m/z: 500.0 [MH] - .
按上述制备工艺得到化合物I-1-2，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图4，其特征数据如下所示：The compound I-1-2 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 4, and the characteristic data is as follows:
Figure PCTCN2017084924-appb-000052
Figure PCTCN2017084924-appb-000052
实施例3本发明化合物I-1-3的制备Example 3 Preparation of Compound I-1-3 of the Invention
Figure PCTCN2017084924-appb-000053
Figure PCTCN2017084924-appb-000053
将硼酸原料(I-1-1)2.25g(5.55mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入二乙醇胺(S-3-3)0.61g(5.82mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-1-3化合物2.4g，收率91％。The boric acid raw material (I-1-1) 2.25 g (5.55 mmol) was dissolved in 45 ml of ethyl acetate, stirred at room temperature for 5 min, and then diethanolamine (S-3-3) 0.61 g (5.82 mmol) was added dropwise, dropwise. A white solid precipitated in the middle reaction solution. After completion of the dropwise addition, stirring was continued for 2 hours, and suction filtration was carried out to obtain 2.4 g of an I-1-3 compound in a yield of 91%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.85(brs,1H),7.64-7.69(m,2H),7.48-7.52(m,1H),7.00(d,1H,J＝7.62Hz),6.59(m,1H),3.80-3.85(m,2H),3.68(m,3H),3.58(m,1H),3.14(m,1H), 2.99(m,2H),2.74-2.79(m,2H),1.59(m,1H),1.29-1.32(m,1H),1.19-1.13(m,2H),0.82(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.85 (brs, 1H), 7.64-7.69 (m, 2H), 7.48-7.52 (m, 1H), 7.00 (d, 1H, J = 7.62Hz) , 6.59 (m, 1H), 3.80-3.85 (m, 2H), 3.68 (m, 3H), 3.58 (m, 1H), 3.14 (m, 1H), 2.99 (m, 2H), 2.74-2.79 (m , 2H), 1.59 (m, 1H), 1.29-1.32 (m, 1H), 1.19-1.13 (m, 2H), 0.82 (d, 6H).
ESI m/z：475.9[M+H]+。ESI m/z: 475.9 [M+H] + .
按上述制备工艺得到化合物I-1-3，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图5，其特征数据如下所示：The compound I-1-3 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 5, and the characteristic data is as follows:
2θ(°)2θ(°) 8.388.38 11.411.4 12.112.1 13.2913.29 18.2718.27 19.6219.62 20.4720.47 23.3223.32 24.7424.74
强度(％)strength(%) 63.463.4 35.635.6 24.424.4 22.322.3 100100 35.935.9 39.839.8 29.829.8 35.135.1
实施例4本发明化合物I-1-4的制备Example 4 Preparation of Compound I-1-4 of the Invention
Figure PCTCN2017084924-appb-000054
Figure PCTCN2017084924-appb-000054
将硼酸原料(I-1-1)2.25g(5.55mmol)溶于45ml乙酸乙酯，室温下搅拌5min后加入无水柠檬酸1.12g(5.82mmol)。混合后继续搅拌2h，析出白色固体，抽滤得I-1-4化合物2.39g，收率77％。2.25 g (5.55 mmol) of the boric acid starting material (I-1-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 1.12 g (5.82 mmol) of anhydrous citric acid was added. After stirring, stirring was continued for 2 hours, and a white solid was precipitated, which was filtered to afford 2.39 g of the compound I-1-4, yield 77%.
1H NMR(300MHz,DMSO-d6)δ(ppm)12.16(brs,2H),10.71(brs,1H),9.12(brs,1H),7.78(s,1H),7.68(m,1H),7.48(m,1H),4.26(brs，2H),2.72-2.87(m,5H),1.67(m,1H),1.08-1.23(m,2H),0.87(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 12.16 (brs, 2H), 10.71 (brs, 1H), 9.12 (brs, 1H), 7.78 (s, 1H), 7.68 (m, 1H), 7.48 (m, 1H), 4.26 (brs, 2H), 2.72-2.87 (m, 5H), 1.67 (m, 1H), 1.08-1.23 (m, 2H), 0.87 (d, 6H).
ESI m/z：583.1[M+Na]+。ESI m/z: 583.1 [M+Na] + .
按上述制备工艺得到化合物I-1-4，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图6，其特征数据如下所示：The compound I-1-4 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 6, and the characteristic data is as follows:
Figure PCTCN2017084924-appb-000055
Figure PCTCN2017084924-appb-000055
将硼酸原料(I-1-1)2.25g(5.55mmol)溶于45ml乙酸乙酯，加热回流，加入无水柠檬酸1.12g(5.82mmol)。混合后继续搅拌2h，逐渐缓慢降温(控制每3分钟降温1度)，待室温后，抽滤得到析出的固体I-1-4-1，纯化后得到化合物2.1g，收率68％。经过X衍射证明，为下述结构： 2.25 g (5.55 mmol) of the boric acid starting material (I-1-1) was dissolved in 45 ml of ethyl acetate, and the mixture was heated to reflux, and then, 1.12 g (5.82 mmol) of anhydrous citric acid was added. After mixing, stirring was continued for 2 hours, and the temperature was gradually lowered (control was lowered by 1 degree every 3 minutes). After room temperature, the precipitated solid I-1-4-1 was obtained by suction filtration, and 2.1 g of the compound was obtained after purification, yield 68%. It is proved by X-ray diffraction that it is the following structure:
Figure PCTCN2017084924-appb-000056
Figure PCTCN2017084924-appb-000056
1H NMR(300MHz,DMSO-d6)δ(ppm)12.16(brs,2H),10.71(brs,1H),9.12(brs,1H),7.78(s,1H),7.68(m,1H),7.48(m,1H),4.26(brs，2H),2.72-2.87(m,5H),1.67(m,1H),1.08-1.23(m,2H),0.87(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 12.16 (brs, 2H), 10.71 (brs, 1H), 9.12 (brs, 1H), 7.78 (s, 1H), 7.68 (m, 1H), 7.48 (m, 1H), 4.26 (brs, 2H), 2.72-2.87 (m, 5H), 1.67 (m, 1H), 1.08-1.23 (m, 2H), 0.87 (d, 6H).
ESI m/z：583.1[M+Na]+。ESI m/z: 583.1 [M+Na] + .
实施例5本发明化合物I-1-5的制备Example 5 Preparation of Compound I-1-5 of the Invention
Figure PCTCN2017084924-appb-000057
Figure PCTCN2017084924-appb-000057
将硼酸原料(I-1-1)2.25g(5.55mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入三乙醇胺0.87g(5.82mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-1-5化合物2.53g，收率88％。2.25 g (5.55 mmol) of boric acid starting material (I-1-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 0.87 g (5.82 mmol) of triethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After completion of the dropwise addition, stirring was continued for 2 hours, and suction filtration was carried out to obtain 2.53 g of the compound I-1-5, yield 88%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.98(brs,1H),7.68(m,1H),7.61(m,1H),7.49(m,1H),6.51(d,1H,J＝9.36Hz),4.88(brs，1H),3.73-3.76(m,8H),3.17(m,3H),3.02(m,2H),2.87(m,2H),1.51(m,1H),1.21(m,2H),0.79-0.86(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.98 (brs, 1H), 7.68 (m, 1H), 7.61 (m, 1H), 7.49 (m, 1H), 6.51 (d, 1H, J = 9.36 Hz), 4.88 (brs, 1H), 3.73-3.76 (m, 8H), 3.17 (m, 3H), 3.02 (m, 2H), 2.87 (m, 2H), 1.51 (m, 1H), 1.21. m, 2H), 0.79-0.86 (m, 6H).
ESI m/z：519.2[M+H]+。ESI m/z: 519.2 [M+H] + .
按上述制备工艺得到化合物I-1-5，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图7，其特征数据如下所示：The compound I-1-5 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 7, and the characteristic data is as follows:
Figure PCTCN2017084924-appb-000058
Figure PCTCN2017084924-appb-000058
实施例6本发明化合物I-2-1及其甘露醇酯的制备 Example 6 Preparation of Compound I-2-1 of the Invention and Its Mannitol Ester
Figure PCTCN2017084924-appb-000059
Figure PCTCN2017084924-appb-000059
如实施例1合成路线类似，如采用专利公开的缩合剂成酰胺方法得到主要为副产物；采用如上图示混合酸酐法链接酰胺基，最终得到目标物化合物I-2-1；化合物I-2-1甘露醇酯以及化合物I-2-1钠盐的制备方法类似实施例1。The synthesis route is similar to that in Example 1, and the main product is obtained by the amide method disclosed in the patent. The amide group is linked by the mixed acid anhydride method as shown above to finally obtain the target compound I-2-1; The preparation method of -1 mannitol ester and compound I-2-1 sodium salt is similar to that of Example 1.
实施例7本发明化合物I-2-2的制备Example 7 Preparation of Compound I-2-2 of the Invention
Figure PCTCN2017084924-appb-000060
Figure PCTCN2017084924-appb-000060
将硼酸起始原料(I-2-1)5克(12.3mmol)、二丙醇胺(S-3-2,分子量133.19)1.95克(14.6mmol)、20mL乙酸乙酯，室温搅拌过夜，析出白色固体，过滤得到5.13克化合物I-2-2，收率83％，5 g (12.3 mmol) of boric acid starting material (I-2-1), 1.95 g (14.6 mmol) of dipropanolamine (S-3-2, molecular weight 133.19), and 20 mL of ethyl acetate were stirred at room temperature overnight to precipitate. White solid, filtered to give 5.13 g of compound I-2-2, yield 83%.
1H NMR(300MHz,DMSO-d6)δ(ppm)9.03(brs,1H),7.69-7.72(m,1H),7.48(brs,2H),6.63(d,1H,J＝8.97Hz),4.87(brs,1H),3.88-3.91(m,1H),3.73(m,1H),3.65(m,4H),3.19-3.43(m,3H),2.67(m,2H),1.87-1.99(m,1H),1.64-1.67(m,1H),1.49(m,2H),1.29-1.33(m,1H),1.15-1.23(m,1H),0.95-0.98(m,1H),0.83(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 9.03 (brs, 1H), 7.69-7.72 (m, 1H), 7.48 (brs, 2H), 6.63 (d, 1H, J = 8.97Hz), 4.87 (brs, 1H), 3.88-3.91 (m, 1H), 3.73 (m, 1H), 3.65 (m, 4H), 3.19-3.43 (m, 3H), 2.67 (m, 2H), 1.87-1.99 (m , 1H), 1.64-1.67 (m, 1H), 1.49 (m, 2H), 1.29-1.33 (m, 1H), 1.15-1.23 (m, 1H), 0.95-0.98 (m, 1H), 0.83 (m) , 6H).
ESI m/z：500.0[M-H]-。ESI m/z: 500.0 [MH] - .
按上述制备工艺得到化合物I-2-2，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图8，其特征数据如下所示：According to the above preparation process, the compound I-2-2 is obtained and detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 8. The characteristic data is as follows:
Figure PCTCN2017084924-appb-000061
Figure PCTCN2017084924-appb-000061
实施例8本发明化合物I-2-3的制备 Example 8 Preparation of Compound I-2-3 of the Invention
Figure PCTCN2017084924-appb-000062
Figure PCTCN2017084924-appb-000062
将硼酸原料(I-2-1)2.25g(5.55mmol)溶于45mL乙酸乙酯，室温下搅拌5min后逐滴加入二乙醇胺0.61g(5.82mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-2-3化合物2.21g，收率83％。2.25 g (5.55 mmol) of boric acid raw material (I-2-1) was dissolved in 45 mL of ethyl acetate, and stirred at room temperature for 5 min, then 0.61 g (5.82 mmol) of diethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After the completion of the dropwise addition, stirring was continued for 2 hours, and 2.21 g of an I-2-3 compound was obtained by suction filtration, yield 83%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.87(brs,1H),7.65-7.68(m,1H),7.44-7.48(m,2H),6.96(d,1H,J＝8.07Hz),6.47(m,1H),3.80-3.85(m,2H),3.68(m,3H),3.58(m,1H),3.14(m,1H),2.99(m,2H),2.74-2.79(m,2H),1.51(m,1H),1.19-1.13(m,2H),0.79(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.87 (brs, 1H), 7.65-7.68 (m, 1H), 7.44-7.48 (m, 2H), 6.96 (d, 1H, J = 8.07Hz) , 6.47 (m, 1H), 3.80-3.85 (m, 2H), 3.68 (m, 3H), 3.58 (m, 1H), 3.14 (m, 1H), 2.99 (m, 2H), 2.74-2.79 (m , 2H), 1.51 (m, 1H), 1.19-1.13 (m, 2H), 0.79 (m, 6H).
ESI m/z：475.9[M+H]+。ESI m/z: 475.9 [M+H] + .
按上述制备工艺得到化合物I-2-3，在温度为20～25℃下检测，该晶型X射线粉末衍射图如附图9，其特征数据如下所示：The compound I-2-3 was obtained according to the above preparation process and was detected at a temperature of 20 to 25 ° C. The crystal X-ray powder diffraction pattern is as shown in Fig. 9, and the characteristic data is as follows:
Figure PCTCN2017084924-appb-000063
Figure PCTCN2017084924-appb-000063
实施例9本发明化合物I-2-4的制备Example 9 Preparation of Compound I-2-4 of the Invention
Figure PCTCN2017084924-appb-000064
Figure PCTCN2017084924-appb-000064
将硼酸原料(I-2-1)2.25g(5.55mmol)溶于45mL乙酸乙酯，室温下搅拌5min后加入无水柠檬酸1.12g(5.82mmol)。混合后继续搅拌2h，析出白色固体，抽滤得I-2-4化合物2.49g，收率80％。2.25 g (5.55 mmol) of the boric acid starting material (I-2-1) was dissolved in 45 mL of ethyl acetate, and stirred at room temperature for 5 min, then 1.12 g (5.82 mmol) of anhydrous citric acid was added. After stirring, stirring was continued for 2 hours, and a white solid was precipitated, which was filtered, and then evaporated to yield 2.
1H NMR(300MHz,DMSO-d6)δ(ppm)12.07(brs,1H),10.71(brs,1H),9.13(brs,1H),7.71(m,1H),7.63(s,1H),7.49(m,1H),4.26(brs,2H),2.88(m,1H),2.72(m,4H),1.67(m,1H),1.05-1.21(m,2H),0.87(d,J＝4.2Hz,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 12.07 (brs, 1H), 10.71 (brs, 1H), 9.13 (brs, 1H), 7.71 (m, 1H), 7.63 (s, 1H), 7.49 (m,1H), 4.26 (brs, 2H), 2.88 (m, 1H), 2.72 (m, 4H), 1.67 (m, 1H), 1.05-1.21 (m, 2H), 0.87 (d, J = 4.2) Hz, 6H).
ESI m/z：583.1[M+Na]+。ESI m/z: 583.1 [M+Na] + .
同实施例4类似的反应条件，得到化合物I-2-4-1： Similar to the reaction conditions of Example 4, the compound I-2-4-1 was obtained:
Figure PCTCN2017084924-appb-000065
Figure PCTCN2017084924-appb-000065
实施例10本发明化合物I-2-5的制备Example 10 Preparation of Compound I-2-5 of the Invention
Figure PCTCN2017084924-appb-000066
Figure PCTCN2017084924-appb-000066
将硼酸原料(I-2-1)2.25g(5.55mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入三乙醇胺0.87g(5.82mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-2-5化合物2.57g，收率89％。2.25 g (5.55 mmol) of boric acid raw material (I-2-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 0.87 g (5.82 mmol) of triethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After the completion of the dropwise addition, stirring was continued for 2 hours, and suction filtration was performed to obtain 2.57 g of the compound I-2-5, yield 89%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.96(brs,1H),7.71(m,1H),7.46-7.49(m,2H),6.54(d,1H,J＝9.27Hz),4.88(brs，1H),3.73-3.76(m,8H),3.17(m,3H),3.03(m,2H),2.87(m,2H),1.52(m,1H),1.21(m,2H),0.79-0.86(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.96 (brs, 1H), 7.71 (m, 1H), 7.46-7.49 (m, 2H), 6.54 (d, 1H, J = 9.27Hz), 4.88 (brs, 1H), 3.73-3.76 (m, 8H), 3.17 (m, 3H), 3.03 (m, 2H), 2.87 (m, 2H), 1.52 (m, 1H), 1.21 (m, 2H), 0.79-0.86 (m, 6H).
ESI m/z：519.2[M+H]+。ESI m/z: 519.2 [M+H] + .
实施例11本发明化合物I-3-1及其甘露醇酯的制备Example 11 Preparation of Compound I-3-1 of the Invention and Its Mannitol Ester
Figure PCTCN2017084924-appb-000067
Figure PCTCN2017084924-appb-000067
如实施例1合成路线类似，如采用专利公开的缩合剂方法得到主要为硼酸脱掉副产物；采用如上图示混合酸酐法，得到目标物I-3-1；I-3-1甘露醇酯以及I-3-1化合物钠盐制备方法同实施例1。The synthesis route is similar to that in Example 1, and the main product is a boric acid removal by-product obtained by the condensing agent method disclosed in the patent; the target product I-3-1; I-3-1 mannitol ester is obtained by the mixed acid anhydride method as shown in the above figure. And the preparation method of the sodium salt of the I-3-1 compound is the same as in the first embodiment.
实施例12本发明化合物I-3-2的制备 Example 12 Preparation of Compound I-3-2 of the Invention
Figure PCTCN2017084924-appb-000068
Figure PCTCN2017084924-appb-000068
将硼酸原料(I-3-1)2.25g(6.20mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入二丙醇胺0.87g(6.51mmol)。滴加完成后继续搅拌2h，抽滤得白色固体2.46g，即化合物I-3-2，收率80％。2.25 g (6.20 mmol) of a boric acid starting material (I-3-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 0.87 g (6.51 mmol) of dipropanolamine was added dropwise. After the completion of the dropwise addition, stirring was continued for 2 hours, and filtered to give a white solid, 2.46 g, Compound I-3-2, yield 80%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.67(brs,1H),7.83-7.85(m,1H),7.69-7.71(m,1H),6.79(d,1H,J＝9.18Hz),4.83(m,1H),3.97-4.01(m,1H),3.77(m,1H),3.64(m,4H),3.34(m,2H),3.20-3.24(m,1H),2.66(m,2H),1.90-1.99(m,1H),1.62-1.66(m,1H),1.50(m,2H),1.28-1.32(m,1H),1.16-1.24(m,1H),0.94-0.98(m,1H),0.83(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.67 (brs, 1H), 7.83-7.85 (m, 1H), 7.69-7.71 (m, 1H), 6.79 (d, 1H, J = 9.18Hz) , 4.83 (m, 1H), 3.97-4.01 (m, 1H), 3.77 (m, 1H), 3.64 (m, 4H), 3.34 (m, 2H), 3.20-3.24 (m, 1H), 2.66 (m) , 2H), 1.90-1.99 (m, 1H), 1.62-1.66 (m, 1H), 1.50 (m, 2H), 1.28-1.32 (m, 1H), 1.16-1.24 (m, 1H), 0.94-0.98 (m, 1H), 0.83 (d, 6H).
ESI m/z：460.0[M+H]+。ESI m/z: 460.0 [M+H] + .
实施例13本发明化合物I-3-3的制备Example 13 Preparation of Compound I-3-3 of the Invention
Figure PCTCN2017084924-appb-000069
Figure PCTCN2017084924-appb-000069
将硼酸原料(I-3-1)2.25g(6.20mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入三乙醇胺0.97g(6.51mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-3-3化合物2.53g，收率86％。2.25 g (6.20 mmol) of boric acid raw material (I-3-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 0.97 g (6.51 mmol) of triethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After the completion of the dropwise addition, stirring was continued for 2 hours, and 2.53 g of an I-3-3 compound was obtained by suction filtration, yield 86%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.76(brs,1H),7.82-7.84(m,1H),7.64-7.66(m,1H),6.56(d,1H,J＝9Hz),4.88(brs，1H),3.69-3.81(m,8H),3.16(m,3H),3.01(m,2H),2.85(m,2H),1.50(m,1H),1.20(m,2H),0.79-0.86(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.76 (brs, 1H), 7.82-7.84 (m, 1H), 7.64-7.66 (m, 1H), 6.56 (d, 1H, J = 9Hz), 4.88 (brs, 1H), 3.69-3.81 (m, 8H), 3.16 (m, 3H), 3.01 (m, 2H), 2.85 (m, 2H), 1.50 (m, 1H), 1.20 (m, 2H) , 0.79-0.86 (m, 6H).
ESI m/z：476.1[M+H]+。ESI m/z: 476.1 [M+H] + .
实施例14本发明化合物I-3-4的制备 Example 14 Preparation of Compound I-3-4 of the Invention
Figure PCTCN2017084924-appb-000070
Figure PCTCN2017084924-appb-000070
将硼酸原料(I-3-1)2.25g(6.20mmol)溶于45ml乙酸乙酯，室温下搅拌5min后加入无水柠檬酸1.25g(6.51mmol)。混合后继续搅拌2h，析出白色固体，抽滤得I-3-4化合物2.48g，收率77％。2.25 g (6.20 mmol) of the boric acid starting material (I-3-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 1.25 g (6.51 mmol) of anhydrous citric acid was added. After the mixture was stirred for 2 hours, a white solid was precipitated, and then filtered, to give the compound I-3-4, 2.48 g, yield 77%.
1H NMR(300MHz,DMSO-d6)δ(ppm)12.12(brs,1H),10.65(brs,1H),8.90(brs,1H),7.82-85(m,1H),7.77-7.79(m,1H),7.49(m,1H),4.28(brs,2H),2.72-2.87(m,1H),2.71(m,4H),1.66(m,1H),1.17-1.20(m,2H),0.86(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 12.12 (brs, 1H), 10.65 (brs, 1H), 8.90 (brs, 1H), 7.82-85 (m, 1H), 7.77-7.79 (m, 1H), 7.49 (m, 1H), 4.28 (brs, 2H), 2.72-2.87 (m, 1H), 2.71 (m, 4H), 1.66 (m, 1H), 1.17-1.20 (m, 2H), 0.86 (d, 6H).
ESI m/z：541.1[M+Na]+ ESI m/z: 541.1 [M+Na] +
同实施例4类似的反应条件，得到化合物I-3-4-1：Similar to the reaction conditions of Example 4, the compound I-3-4-1 was obtained:
Figure PCTCN2017084924-appb-000071
Figure PCTCN2017084924-appb-000071
实施例15本发明化合物I-4-1及其甘露醇酯的制备Example 15 Preparation of Compound I-4-1 of the Invention and Its Mannitol Ester
Figure PCTCN2017084924-appb-000072
Figure PCTCN2017084924-appb-000072
如实施例1合成路线类似，如采用专利公开的缩合剂方法得到主要为硼酸脱掉副产物；采用如上图示混合酸酐法，得到目标物I-4-1；其甘露醇酯制备方法同实施例1。As in the synthesis route of Example 1, similarly, the boring agent method disclosed in the patent is used to obtain a by-product of boric acid removal; the mixed acid anhydride method as shown above is used to obtain the target substance I-4-1; the preparation method of the mannitol ester is carried out in the same manner. example 1.
实施例16本发明化合物I-4-2的制备 Example 16 Preparation of Compound I-4-2 of the Invention
Figure PCTCN2017084924-appb-000073
Figure PCTCN2017084924-appb-000073
将硼酸原料(I-4-1)2.25g(6.20mmol)溶于45mL乙酸乙酯，室温下搅拌5min后逐滴加入三乙醇胺0.97g(6.51mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-4-2化合物2.53g，收率86％。2.25 g (6.20 mmol) of boric acid raw material (I-4-1) was dissolved in 45 mL of ethyl acetate, and stirred at room temperature for 5 min, then 0.97 g (6.51 mmol) of triethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After the completion of the dropwise addition, stirring was continued for 2 hours, and 2.53 g of an I-4-2 compound was obtained by suction filtration, yield 86%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.80(brs,1H),7.83-7.86(m,1H),7.62-7.65(m,1H),7.01(d,1H,J＝8.7Hz),6.56(brs，1H),3.67-3.90(m,8H),3.34(m,3H),2.98(m,2H),2.69(m,2H),1.60(m,1H),1.24(m,2H),0.80-0.82(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.80 (brs, 1H), 7.83-7.86 (m, 1H), 7.62-7.65 (m, 1H), 7.01 (d, 1H, J = 8.7Hz) , 6.56 (brs, 1H), 3.67-3.90 (m, 8H), 3.34 (m, 3H), 2.98 (m, 2H), 2.69 (m, 2H), 1.60 (m, 1H), 1.24 (m, 2H) ), 0.80-0.82 (m, 6H).
ESI m/z：476.1[M+H]+。ESI m/z: 476.1 [M+H] + .
实施例17本发明化合物I-4-3的制备Example 17 Preparation of Compound I-4-3 of the Invention
Figure PCTCN2017084924-appb-000074
Figure PCTCN2017084924-appb-000074
将硼酸原料(I-4-1)2.25g(6.20mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入二乙醇胺0.68g(6.51mmol)。滴加完成后继续搅拌2h，抽滤得白色固体2.20g，即化合物I-4-3，收率82％。2.25 g (6.20 mmol) of a boric acid starting material (I-4-1) was dissolved in 45 ml of ethyl acetate, and the mixture was stirred at room temperature for 5 min, and then, then, diethyldiamine (0.68 g) (6.51 mmol) was added dropwise. After the completion of the dropwise addition, stirring was continued for 2 hours, and filtered to give a white solid, 2.20 g, Compound I-4-3, yield 82%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.81(brs,1H),7.83-7.86(m,1H),7.62-7.65(m,1H),7.01-7.03(m,1H),6.56(m,1H),6.47(m,1H),4.01(m，2H),3.77-3.90(m,4H),3.14(m,1H),2.99(m,3H),2.68(m,2H),1.60(m,1H),0.98-1.31(m,3H),0.81-0.83(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.81 (brs, 1H), 7.83-7.86 (m, 1H), 7.62-7.65 (m, 1H), 7.01-7.03 (m, 1H), 6.56 ( m,1H), 6.47 (m, 1H), 4.01 (m, 2H), 3.77-3.90 (m, 4H), 3.14 (m, 1H), 2.99 (m, 3H), 2.68 (m, 2H), 1.60 (m, 1H), 0.98-1.31 (m, 3H), 0.81 - 0.83 (m, 6H).
ESI m/z：454.4[M+Na]+。ESI m/z: 454.4 [M+Na] + .
实施例18本发明化合物I-4-4的制备 Example 18 Preparation of Compound I-4-4 of the Invention
Figure PCTCN2017084924-appb-000075
Figure PCTCN2017084924-appb-000075
将硼酸原料(I-4-1)2.25g(6.20mmol)溶于45ml乙酸乙酯，室温下搅拌5min后加入无水柠檬酸1.25g(6.51mmol)。混合后继续搅拌2h，析出白色固体，抽滤得I-4-4化合物2.35g，收率73％。2.25 g (6.20 mmol) of a boric acid starting material (I-4-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 1.25 g (6.51 mmol) of anhydrous citric acid was added. After the mixture was stirred for 2 hours, a white solid was precipitated, and then filtered, to give 2.35 g of compound I-4-4, yield 73%.
1H NMR(300MHz,DMSO-d6)δ(ppm)12.23(brs,1H),10.70(brs,1H),9.12(brs,1H),7.70-7.87(m,2H),4.27(brs,2H),2.76-2.88(m,2H),2.71(m,4H),1.67(m,1H),1.20(m,2H),0.86(m,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 12.23 (brs, 1H), 10.70 (brs, 1H), 9.12 (brs, 1H), 7.70-7.87 (m, 2H), 4.27 (brs, 2H) , 2.76-2.88 (m, 2H), 2.71 (m, 4H), 1.67 (m, 1H), 1.20 (m, 2H), 0.86 (m, 6H).
ESI m/z：541.1[M+Na]+ ESI m/z: 541.1 [M+Na] +
同实施例4类似的反应条件，得到化合物I-4-4-1：Similar to the reaction conditions of Example 4, the compound I-4-4-1 was obtained:
Figure PCTCN2017084924-appb-000076
Figure PCTCN2017084924-appb-000076
实施例19本发明化合物I-5-1及其甘露醇酯的制备Example 19 Preparation of Compound I-5-1 of the Invention and Its Mannitol Ester
Figure PCTCN2017084924-appb-000077
Figure PCTCN2017084924-appb-000077
如实施例1合成路线类似，如采用专利公开的缩合剂成酰胺方法得到主要为副产物；采用如上图示混合酸酐法链接酰胺基，最终得到目标物化合物I-5-1；化合物I-5-1甘露醇酯以及化合物I-5-1钠盐的制备方法类似实施例1。The synthesis route is similar to that in Example 1, and the main product is obtained by the amide method disclosed in the patent. The amide group is linked by the mixed acid anhydride method as shown above to finally obtain the target compound I-5-1; the compound I-5. The preparation method of -1 mannitol ester and compound I-5-1 sodium salt is similar to that of Example 1.
实施例20本发明化合物I-5-2的制备 Example 20 Preparation of Compound I-5-2 of the Invention
Figure PCTCN2017084924-appb-000078
Figure PCTCN2017084924-appb-000078
将硼酸起始原料(I-5-1)2.25克(5.00mmol)、二丙醇胺(S-3-2,分子量133.19)0.70克(5.25mmol)、45mL乙酸乙酯，室温搅拌过夜，析出白色固体，过滤得到2.29克化合物I-5-2，收率84％，2.25 g (5.00 mmol) of boric acid starting material (I-5-1), 0.70 g (5.25 mmol) of dipropanolamine (S-3-2, molecular weight 133.19), 45 mL of ethyl acetate, and stirred at room temperature overnight, and precipitated. White solid, filtered to give 2.29 g of compound I-5-2, yield 84%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.84(brs,1H)，7.62-7.64(m,1H)，7.57-7.59(m,2H)，6.60(d,1H,J＝10.00Hz)，4.71(m,1H)，3.84-3.90(m,1H)，3.71-3.76(m,1H)，3.65(m,4H)，3.41-3.43(m,2H)，3.20-3.22(m,1H)，2.67(m,2H)，1.86-1.89(m,1H)，1.63-1.66(m,1H)，1.49(m,2H)，1.30-1.33(m,1H)，1.16-1.23(m,1H)，0.93-0.99(m,1H)，0.83(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.84 (brs, 1H), 7.62-7.64 (m, 1H), 7.57-7.59 (m, 2H), 6.60 (d, 1H, J = 10.00Hz) , 4.71 (m, 1H), 3.84-3.90 (m, 1H), 3.71-3.76 (m, 1H), 3.65 (m, 4H), 3.41-3.43 (m, 2H), 3.20-3.22 (m, 1H) , 2.67 (m, 2H), 1.86-1.89 (m, 1H), 1.63-1.66 (m, 1H), 1.49 (m, 2H), 1.30-1.33 (m, 1H), 1.16-1.23 (m, 1H) , 0.93-0.99 (m, 1H), 0.83 (d, 6H).
ESI m/z：545.0[M-H]-。ESI m/z: 545.0 [MH] - .
实施例21本发明化合物I-5-3的制备Example 21 Preparation of Compound I-5-3 of the Invention
Figure PCTCN2017084924-appb-000079
Figure PCTCN2017084924-appb-000079
将硼酸原料(I-5-1)2.25g(5.00mmol)溶于45mL乙酸乙酯，室温下搅拌5min后逐滴加入二乙醇胺0.55g(5.25mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-5-3化合物2.24g，收率86％。2.25 g (5.00 mmol) of boric acid raw material (I-5-1) was dissolved in 45 mL of ethyl acetate, and stirred at room temperature for 5 min, then 0.55 g (5.25 mmol) of diethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After the completion of the dropwise addition, stirring was continued for 2 hours, and filtered to give 2.24 g of the compound I-5-3, yield 86%.
1H NMR(300MHz,DMSO-d6)δ(ppm)8.80(brs,1H),7.61-7.63(m,1H),7.56-7.59(m,2H),6.97(d,1H,J＝8.40Hz),6.55(m,1H),3.76-3.85(m,2H),3.70(m,3H),3.58(m,1H),3.12-3.15(m,1H),2.97-3.01(m,2H),2.69-2.78(m,2H),1.59(m,1H),1.29-1.36(m,1H),1.16-1.23(m,1H),0.81(d,6H)。 1 H NMR (300MHz, DMSO- d6) δ (ppm) 8.80 (brs, 1H), 7.61-7.63 (m, 1H), 7.56-7.59 (m, 2H), 6.97 (d, 1H, J = 8.40Hz) , 6.55 (m, 1H), 3.76-3.85 (m, 2H), 3.70 (m, 3H), 3.58 (m, 1H), 3.12-3.15 (m, 1H), 2.97-3.01 (m, 2H), 2.69 -2.78 (m, 2H), 1.59 (m, 1H), 1.29-1.36 (m, 1H), 1.16-1.23 (m, 1H), 0.81 (d, 6H).
ESI m/z：520.1[M+H]+。ESI m/z: 520.1 [M+H] + .
实施例22本发明化合物I-5-4的制备 Example 22 Preparation of Compound I-5-4 of the Invention
Figure PCTCN2017084924-appb-000080
Figure PCTCN2017084924-appb-000080
将硼酸原料(I-5-1)2.25g(5.00mmol)溶于45mL乙酸乙酯，室温下搅拌5min后加入无水柠檬酸1.01g(5.25mmol)。混合后继续搅拌2h，析出白色固体，抽滤得I-5-4化合物2.38g，收率78％。2.25 g (5.00 mmol) of the boric acid starting material (I-5-1) was dissolved in 45 mL of ethyl acetate, and stirred at room temperature for 5 min, and then, then, 1.01 g (5.25 mmol) of anhydrous citric acid was added. After the mixture was stirred for 2 hours, a white solid was precipitated, and then filtered, to give 2.
ESI m/z：628.9[M+Na]+。ESI m/z: 628.9 [M+Na] + .
同实施例4类似的反应条件，得到化合物I-5-4-1：Similar to the reaction conditions of Example 4, Compound I-5-4-1 was obtained:
Figure PCTCN2017084924-appb-000081
Figure PCTCN2017084924-appb-000081
实施例23本发明化合物I-5-5的制备Example 23 Preparation of Compound I-5-5 of the Invention
Figure PCTCN2017084924-appb-000082
Figure PCTCN2017084924-appb-000082
将硼酸原料(I-5-1)2.25g(5.00mmol)溶于45ml乙酸乙酯，室温下搅拌5min后逐滴加入三乙醇胺0.78g(5.25mmol)，滴加过程中反应液中即析出白色固体。滴加完成后继续搅拌2h，抽滤得I-5-5化合物2.41g，收率86％。2.25 g (5.00 mmol) of boric acid starting material (I-5-1) was dissolved in 45 ml of ethyl acetate, and stirred at room temperature for 5 min, then 0.78 g (5.25 mmol) of triethanolamine was added dropwise, and white precipitated in the reaction mixture during the dropwise addition. solid. After completion of the dropwise addition, stirring was continued for 2 hours, and suction filtration was carried out to obtain 2.4 g of the compound I-5-5, yield 86%.
ESI m/z：563.8[M+H]+。ESI m/z: 563.8 [M+H] + .
为研究苯环取代基以及不同酯化剂对抗肿瘤活性的影响，按照上述合成方法或文献报道方法制备得到对比化合物Ixazomib-DEA、I-6-1、I-7-1、I-8-1、I-9-1，I-10-1，其结构分别如下： In order to study the effects of phenyl ring substituents and different esterification agents on tumor activity, comparative compounds Ixazomib-DEA, I-6-1, I-7-1, I-8-1 were prepared according to the above synthetic methods or literature reports. , I-9-1, I-10-1, their structures are as follows:
Figure PCTCN2017084924-appb-000083
Figure PCTCN2017084924-appb-000083
其中，I-6-1、I-7-1、I-10-1是CN200780100142公开的化合物。令人惊奇的发现，苯环上含碘化合物I-8-1、I-9-1在室温下极其容易分解，说明苯环上卤素取代基的不同，直接影响到衍生物的稳定性和成药性。Among them, I-6-1, I-7-1, and I-10-1 are compounds disclosed in CN200780100142. Surprisingly, it was found that the iodine-containing compounds I-8-1 and I-9-1 on the benzene ring are extremely easy to decompose at room temperature, indicating that the difference in halogen substituents on the benzene ring directly affects the stability and preparation of the derivative. Sex.
实施例24本发明化合物的药用胶囊剂组合物Example 24 Medicinal Capsule Composition of the Compound of the Invention
化合物I-1-2，的药用胶囊剂组合物，含有3g或者4g化合物I-1-2、193g或者192g微晶纤维素、4g微粉硅胶，共计200g及2号空心胶囊。制备方法为：The pharmaceutical capsule composition of Compound I-1-2 contains 3 g or 4 g of Compound I-1-2, 193 g or 192 g of microcrystalline cellulose, 4 g of micronized silica gel, and a total of 200 g and No. 2 hollow capsules. The preparation method is:
a，使用常规方法混合化合物I-1-2，微晶纤维素和微粉硅胶；a, mixing the compound I-1-2, microcrystalline cellulose and microsilica gel using a conventional method;
b，将混合粉末过120目筛后装填入2号胶囊并封口，共制1000粒。b. The mixed powder was passed through a 120 mesh sieve, filled into No. 2 capsules and sealed, and a total of 1000 capsules were prepared.
化合物I-1-3、I-1-4、I-1-5、I-2-2、I-2-3、I-2-4、I-2-5、I-3-2、I-3-3、I-4-2、I-4-3、I-4-4、I-5-2、I-5-3的药用胶囊剂组合物同上述。Compounds I-1-3, I-1-4, I-1-5, I-2-2, I-2-3, I-2-4, I-2-5, I-3-2, I The pharmaceutical capsule compositions of -3-3, I-4-2, I-4-3, I-4-4, I-5-2, and I-5-3 are the same as above.
实施例25本发明化合物的药用片剂组合物Example 25 Pharmaceutical tablet composition of a compound of the invention
化合物I-2-2的药用片剂组合物，化合物I-2-2分别为1重量份，乳糖0.1-0.5重量份，羟丙纤维素0.05-0.08重量份，羧甲基淀粉钠0.008-0.014重量份，聚维酮K30适量，硬脂酸镁0.01-0.05重量份；按照上述比例制备成片剂，每片含化合物1-6分别为0.5-10毫克。The pharmaceutical tablet composition of the compound I-2-2, the compound I-2-2 is 1 part by weight, the lactose is 0.1-0.5 part by weight, the hydroxypropylcellulose is 0.05-0.08 part by weight, and the sodium carboxymethyl starch is 0.008- 0.014 parts by weight, an appropriate amount of povidone K30, 0.01-0.05 parts by weight of magnesium stearate; tablets are prepared according to the above ratio, and each tablet contains 1-6 mg of each of 1-6.
化合物I-1-2、I-1-3、I-1-4、I-1-5、I-2-3、I-2-4、I-2-5、I-3-2、I-3-3、I-4-2、I-4-3、I-4-4、I-5-2、I-5-3的药用片剂组合物同上述。Compounds I-1-2, I-1-3, I-1-4, I-1-5, I-2-3, I-2-4, I-2-5, I-3-2, I Pharmaceutical tablet compositions of -3-3, I-4-2, I-4-3, I-4-4, I-5-2, I-5-3 are as described above.
实施例26本发明化合物的的药用注射剂组合物Example 26 Pharmaceutical Injectable Composition of a Compound of the Invention
化合物I-1-1的药用注射剂组合物，含有1gI-1-1甘露醇酯、34g磷酸氢二钠及足量注射用水。制备方法为： A pharmaceutical injection composition of the compound I-1-1, which contains 1 g of I-1-1 mannitol ester, 34 g of disodium hydrogen phosphate, and a sufficient amount of water for injection. The preparation method is:
a，用注射用水溶解磷酸氢二钠、I-1-1甘露醇酯，用注射用水配置到2000mL；a, dissolving disodium hydrogen phosphate, I-1-1 mannitol ester with water for injection, and dissolving to 2000 mL with water for injection;
b，a步骤所得溶液滤过，分装于1000瓶2mL西林瓶中，半加塞；b, the solution obtained in step a is filtered, dispensed in 1000 bottles of 2mL vials, half-plugged;
c，b中所得西林瓶置入冻干机中制备为冻干粉末，全加塞；The obtained vials in c, b are placed in a lyophilizer to prepare a lyophilized powder, which is fully stoppered;
d，c中所得西林瓶加铝盖并作检查。The obtained vials in d, c were covered with aluminum lids and examined.
化合物I-2-1、I-3-1、I-5-1的药用注射剂组合物同上述。The pharmaceutical injection compositions of the compounds I-2-1, I-3-1, and I-5-1 are the same as above.
实施例27本发明化合物的药用脂质体组合物Example 27 Pharmaceutical Liposomal Composition of a Compound of the Invention
化合物I-2-2的药用脂质体组合物，含有5g化合物I-1-2以及6.7g卵磷脂、3.3g胆固醇、5g维生素C、足量PBS缓冲溶液(pH7.4)。The pharmaceutically acceptable liposome composition of Compound I-2-2 contains 5 g of Compound 1-1-2 and 6.7 g of lecithin, 3.3 g of cholesterol, 5 g of vitamin C, and a sufficient amount of PBS buffer solution (pH 7.4).
制备方法为：The preparation method is:
a,，混合化合物I-1-2、卵磷脂和胆固醇，溶于氯仿；a, mixed compound I-1-2, lecithin and cholesterol, dissolved in chloroform;
b，a中所得溶液于37℃充氮气减压浓缩制得干膜；The solution obtained in b, a is concentrated under vacuum at 37 ° C to obtain a dry film;
c，b中所得干膜容器加入PBS缓冲溶液(pH7.4)至500mL，加入维生素C，超声处理。The dry film container obtained in c, b was added to a PBS buffer solution (pH 7.4) to 500 mL, and vitamin C was added thereto, followed by ultrasonication.
化合物I-1-3、I-1-5、I-2-2、I-2-3、I-2-5、I-3-2、I-3-3、I-4-2、I-4-3、I-5-2、I-5-3的药用脂质体组合物同上述。Compounds I-1-3, I-1-5, I-2-2, I-2-3, I-2-5, I-3-2, I-3-3, I-4-2, I The pharmaceutically acceptable liposome compositions of -4-3, I-5-2, and I-5-3 are the same as above.
实施例28本发明化合物PBS溶液水解实验Example 28 Hydrolysis Experiment of Compound PBS Solution of the Present Invention
将化合物I-1-3、I-2-3(硼酸酯)分别用d6-DMSO溶解在核磁管中，加入PBS(磷酸缓冲溶液，pH7.4)后，每隔一定时间测定1HNMR图谱(Bruker300MHz核磁共振仪)，对I-1-3、I-2-3化合物7.0ppm附近的1个特征氢进行积分，其积分面积为S1；苯环上的3个氢化学位移在7.3-7.7ppm之间，其积分面积之和为S2。未发生水解反应的I-1-3、I-2-3化合物S1:S2＝1:3，水解反应发生后，测定的核磁谱图中S2包含了水解后产物的苯环氢，但S1仍然是I-1-3、I-2-3化合物的1个特征氢，水解后产物苯环3个氢积分面积计算公式为：S2-3S1。因此，硼酸酯与其水解产物硼酸的摩尔比例计算公式为：3S1/S2-3S1，分解到一半的时间计为t1/2。Compounds I-1-3 and I-2-3 (borate) were dissolved in a nuclear magnetic tube with d6-DMSO, and 1 H NMR spectra were measured at regular intervals after adding PBS (phosphate buffer solution, pH 7.4). (Bruker 300MHz NMR), integrating one characteristic hydrogen near 7.0ppm of I-1-3 and I-2-3 compounds, the integral area is S1; the three hydrogen chemical shifts on the benzene ring are 7.3-7.7 The sum of the integrated areas between ppm is S2. I-1-3, I-2-3 compound S1:S2=1:3 without hydrolysis reaction, after the hydrolysis reaction, S2 in the measured nuclear magnetic spectrum contains the benzene ring hydrogen of the hydrolyzed product, but S1 remains It is a characteristic hydrogen of I-1-3 and I-2-3 compounds. The calculation formula of the three hydrogen integral areas of the benzene ring after hydrolysis is: S2-3S1. Therefore, the molar ratio of the borate ester to its hydrolyzate boric acid is calculated as: 3S1/S2-3S1, and the time to be decomposed to half is t1/2.
如图10所示，I-2-3在PBS溶液中第3天，与水解后I-2-1比例为：3:2.89。实验表明，I-1-3、I-2-3在PBS中半数水解时间t1/2>3天，并且在7天之后仍然还有较多原型存在。不同于Bilgicer等对该类含N杂环硼酸酯在1.5分钟水解过半的报道，以及现有技术将该类硼酸酯化合物视为前药的偏见，结合本发明人其它活性实验结果，证明不仅本发明化合物的硼酸部分具有较强的活性，而且硼酸酯类化合物也能发挥活性效果(不仅仅是前 药)，这对于非口服给药途径发挥治疗作用具有重要意义。As shown in Fig. 10, the ratio of I-2-3 in the PBS solution on the third day to the I-2-1 after hydrolysis was: 3: 2.89. Experiments showed that I-1-3, I-2-3 had half hydrolysis time in PBS for t1/2>3 days, and there were still more prototypes after 7 days. Different from the report that Bilgicer et al. hydrolyzed half of the N-containing heterocyclic boronate in 1.5 minutes, and the prior art bias of the boric acid ester compound as a prodrug, combined with the results of other activity experiments of the inventors, Not only the boric acid moiety of the compound of the present invention has a strong activity, but also the borate ester compound can exert an active effect (not only the former Medicine), which plays an important role in the therapeutic effect of non-oral routes of administration.
实施例29蛋白酶体β5亚基-胰凝乳蛋白酶样蛋白酶(Proteasome Chymotrypsin-like Protease)体外抑制活性实验：Example 29 In vitro inhibitory activity of Proteasome Chymotrypsin-like Protease in Proteasome:
测试样品采用DMSO溶解，低温保存。试验前，利用HPLC检测样品稳定性，保证本实施方案所使用样品稳定。实验方法：采用荧光底物Suc-Leu-Leu-Val-Tyr-AMC(Try-AMC序列)检测样品对蛋白酶体β5亚基抑制活性，观察不同化合物对酶活性的抑制；蛋白酶体β5亚基水解底物中的Try-AMC序列，释放出AMC，在激发波长380nm以及发射光波长460nm检测条件下，可以检测到释放的AMC荧光吸收值，观察和计算化合物对酶的抑制活性。通过Graphpad Prism5.0计算测试样品对蛋白酶体β5亚基活性抑制的IC50，结果如下：The test sample was dissolved in DMSO and stored at low temperature. Before the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in the present embodiment. Experimental method: The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC (Try-AMC sequence) was used to detect the inhibitory activity of the sample on the proteasome β5 subunit, and the inhibition of the enzyme activity by different compounds was observed. The hydrolysis of the proteasome β5 subunit was observed. The Try-AMC sequence in the substrate releases AMC. Under the detection conditions of excitation wavelength 380 nm and emission wavelength 460 nm, the released AMC fluorescence absorption value can be detected, and the inhibitory activity of the compound on the enzyme can be observed and calculated. The IC50 of inhibition of proteasome β5 subunit activity by the test sample was calculated by Graphpad Prism 5.0, and the results were as follows:
表1本发明化合物及对照化合物对蛋白酶体β5亚基抑制活性Table 1 Inhibitory activity of the compound of the present invention and a control compound against the proteasome β5 subunit
化合物编号Compound number 类型Types of 单位unit 结果result 误差error
I-1-1I-1-1 IC50IC50 nMnM 1.991.99 0.440.44
I-1-2I-1-2 IC50IC50 nMnM 7.627.62 1.131.13
I-1-3I-1-3 IC50IC50 nMnM 5.395.39 0.280.28
I-1-5I-1-5 IC50IC50 nMnM 5.075.07 0.500.50
I-2-1I-2-1 IC50IC50 nMnM 2.322.32 0.240.24
I-2-2I-2-2 IC50IC50 nMnM 24.4624.46 4.264.26
I-2-3I-2-3 IC50IC50 nMnM 5.445.44 0.610.61
I-2-5I-2-5 IC50IC50 nMnM 6.426.42 2.102.10
I-3-1I-3-1 IC50IC50 nMnM 14.6014.60 2.342.34
I-3-2I-3-2 IC50IC50 nMnM 18.0018.00 1.981.98
I-3-3I-3-3 IC50IC50 nMnM 6.886.88 1.051.05
I-4-1I-4-1 IC50IC50 nMnM 7.977.97 0.530.53
I-4-2I-4-2 IC50IC50 nMnM 10.9110.91 1.761.76
I-4-3I-4-3 IC50IC50 nMnM 15.1115.11 2.712.71
I-5-1I-5-1 IC50IC50 nMnM 2.162.16 0.320.32
IxazomibIxazomib IC50IC50 nMnM 3.803.80 0.310.31
Ixazomib CitrateIxazomib Citrate IC50IC50 nMnM 10.1910.19 0.990.99
Ixazomib-DEAIxazomib-DEA IC50IC50 nMnM 10.7510.75 2.072.07
CarfilzomibCarfilzomib IC50IC50 nMnM 4.944.94 2.112.11
如上表所示，本发明化合物具有较好的蛋白酶体β5亚基-胰凝乳蛋白酶样蛋白酶抑制活性。As shown in the above table, the compound of the present invention has a good proteasome β5 subunit-chymotrypsin-like protease inhibitory activity.
实施例30本发明化合物抑制多发性骨髓瘤细胞增殖活性检测Example 30 Detection of Proliferative Activity of Multiple Myeloma Cells by Compounds of the Invention
测试样品采用DMSO溶解，低温保存。在试验前，利用HPLC检测样品稳定性，保 证本实施方案所使用样品稳定。多发性骨髓瘤细胞RPMI8226、U266、MM.1S、MM.1R(购于美国ATCC)按照40000个细胞/孔，加入到平底96孔细胞培养板中。化合物最高浓度为1μM，Ixazomib Citrate，Ixazomib-DEA以及Ixazomib作为阳性对照组。按照5倍梯度做药物浓度稀释。化合物作用48小时后加入10μl CCK-8，孵育6h后，利用酶标仪测定450nM波长吸收值。药物对肿瘤细胞生长抑制率(GI50)计算方法按照美国国家癌症研究所(National Cancer Institute,NCI)标准方法进行：当Ti(药物组，培养48h，CCK-8显色吸收OD值)≥Tz(不含药物组，培养起始时CCK-8显色吸收OD值)，肿瘤细胞存活率＝[(Ti-Tz)/(C-Tz)]×100，其中C为不含药物组48小时后CCK-8显色吸收OD值；当Ti<Tz时，肿瘤细胞存活率＝[(Ti-Tz)/Tz]×100。通过GraphpadPrism5.0计算测试样品对多发性骨髓瘤细胞增殖抑制活性的GI50，结果如下：The test sample was dissolved in DMSO and stored at low temperature. Test the stability of the sample by HPLC before the test. The samples used in this embodiment were stable. Multiple myeloma cells RPMI8226, U266, MM.1S, MM.1R (purchased from ATCC, USA) were added to a flat-bottom 96-well cell culture plate at 40,000 cells/well. The highest concentration of the compound was 1 μM, Ixazomib Citrate, Ixazomib-DEA and Ixazomib were used as positive control groups. Dilute the drug concentration according to a 5-fold gradient. After the compound was applied for 48 hours, 10 μl of CCK-8 was added, and after incubation for 6 hours, the 450 nM wavelength absorption value was measured by a microplate reader. The method for calculating the tumor cell growth inhibition rate (GI50) of the drug was carried out according to the National Cancer Institute (NCI) standard method: when Ti (drug group, cultured for 48 h, CCK-8 colorimetric absorption OD value) ≥ Tz ( The drug-free group, CCK-8 color absorbing OD value at the start of culture), tumor cell survival rate = [(Ti-Tz) / (C-Tz)] × 100, wherein C is 48 hours after the drug-free group CCK-8 color absorption OD value; when Ti < Tz, tumor cell survival rate = [(Ti-Tz) / Tz] × 100. The GI50 of the test sample against the proliferation inhibition activity of multiple myeloma cells was calculated by Graphpad Prism 5.0, and the results were as follows:
表2本发明化合物及对照化合物抑制多发性骨髓瘤细胞增殖试验结果Table 2 Results of the compound of the present invention and a control compound for inhibiting proliferation of multiple myeloma cells
Figure PCTCN2017084924-appb-000084
Figure PCTCN2017084924-appb-000084
Figure PCTCN2017084924-appb-000085
Figure PCTCN2017084924-appb-000085
从上表可以看出，硼酸成环状酯以及苯环取代基的不同，影响了化合物抑制多发性骨髓瘤细胞的增殖活性。就硼酸不同环状酯而言，活性强弱顺序基本为，硼酸二丙醇胺环状酯>硼酸三乙醇胺环状酯>硼酸二乙醇胺环状酯>硼酸柠檬酸环状酯>硼酸类化合物(未成酯)；苯环上的取代基，活性强弱顺序为：2-Cl-5-Br取代>5-Cl-2-Br取代>2,5-2-F-4-Cl取代>4,5-2-F-2-Cl取代。It can be seen from the above table that the difference between boric acid and cyclic benzene ring substituents affects the compound's inhibition of the proliferation activity of multiple myeloma cells. In the case of different cyclic esters of boric acid, the order of activity is basically, diurylamine borate cyclic ester > triethanolamine cyclic ester of boric acid > diethanolamine cyclic ester of boric acid > citric acid cyclic ester of boric acid > boric acid compound ( Unesterified); substituents on the benzene ring, the order of activity is: 2-Cl-5-Br substitution >5-Cl-2-Br substitution >2,5-2-F-4-Cl substitution >4, 5-2-F-2-Cl substitution.
在后期的动物实验中，化合物I-1-3，体内活性非常高，与前述体外实验结果不匹配，因此，申请人对其体外活性数据进行了多次重复验证实验，统计的平均结果如下表：In the later animal experiments, the compound I-1-3 was very active in vivo and did not match the above-mentioned in vitro results. Therefore, the applicant performed repeated verification experiments on its in vitro activity data. The statistical average results are shown in the following table. :
Figure PCTCN2017084924-appb-000086
Figure PCTCN2017084924-appb-000086
我们认为，表2中I-1-3的实验数据存在偏差，准确结果应以上表为准。We believe that the experimental data of I-1-3 in Table 2 is biased, and the exact results should be based on the above table.
前述表格中，包含三类不同结构的化合物的实验数据，这三类化合物的阳性对照药物各不相同，为了方便比较，将前述数据按照化合物的类型，拆分为如下3个表格：In the above table, the experimental data of three types of compounds with different structures are included. The positive control drugs of the three types of compounds are different. For the convenience of comparison, the above data are divided into the following three tables according to the type of the compound:
表2-1(硼酸结构化合物的比较)Table 2-1 (Comparison of boric acid structural compounds)
Figure PCTCN2017084924-appb-000087
Figure PCTCN2017084924-appb-000087
表2-1中的化合物均为硼酸结构，比较可以看出，化合物I-1-1和I-2-1可以有效抑制多发性骨髓瘤细胞的增殖，其中，在对MM1S的抑制活性上，化合物I-1-1和I-2-1优于阳性药物Ixazomib。The compounds in Table 2-1 are all boric acid structures. It can be seen that Compounds I-1-1 and I-2-1 can effectively inhibit the proliferation of multiple myeloma cells, among which, in the inhibitory activity against MM1S, Compounds I-1-1 and I-2-1 were superior to the positive drug Ixazomib.
实验结果说明2-Cl-5-Br取代和5-Cl-2-Br取代与硼酸搭配形成的化合物，对多发性骨髓瘤细胞的抑制活性较高。The experimental results indicate that the compound formed by the combination of 2-Cl-5-Br substitution and 5-Cl-2-Br substitution with boric acid has higher inhibitory activity against multiple myeloma cells.
表2-2(含N杂环环状酯类化合物的比较) Table 2-2 (Comparison of N-containing heterocyclic cyclic ester compounds)
Figure PCTCN2017084924-appb-000088
Figure PCTCN2017084924-appb-000088
表2-2中的化合物均为含N杂环环状酯类结构，比较可以看出，本发明化合物I-1-2、I-1-3、I-2-2、I-2-3、I-2-5、I-3-2、I-3-3、I-4-2、I-4-3可以有效抑制多发性骨髓瘤细胞的增殖，其中，化合物I-1-2、I-1-3、I-2-2、I-2-3、I-2-5、I-3-2对所有多发性骨髓瘤细胞的抑制活性均优于阳性药物Ixazomib-DEA，化合物I-3-3、I-4-2、I-4-3对U266、MM1S、MM1R的抑制活性优于阳性药物Ixazomib-DEA。The compounds in Table 2-2 are all N-containing heterocyclic cyclic ester structures, and it can be seen that the compounds of the present invention are 1-1-2, I-1-3, I-2-2, and I-2-3. , I-2-5, I-3-2, I-3-3, I-4-2, and I-4-3 can effectively inhibit the proliferation of multiple myeloma cells, wherein compound I-1-2, The inhibitory activities of I-1-3, I-2-2, I-2-3, I-2-5, and I-3-2 against all multiple myeloma cells were superior to the positive drug Ixazomib-DEA, Compound I. -3-3, I-4-2, and I-4-3 have better inhibitory activity against U266, MM1S, and MM1R than the positive drug Ixazomib-DEA.
实验结果说明含N杂环环状酯结构与2-Cl-5-Br取代、5-Cl-2-Br取代、2,5-2-F-4-Cl取代、4,5-2-F-2-Cl取代配合形成的化合物，对多发性骨髓瘤细胞的抑制活性均较高，其中，优选含N杂环环状酯结构与2-Cl-5-Br取代、5-Cl-2-Br取代配合形成的化合物。The experimental results indicate the structure of the N-containing heterocyclic cyclic ester with 2-Cl-5-Br substitution, 5-Cl-2-Br substitution, 2,5-2-F-4-Cl substitution, 4,5-2-F The compound formed by the substitution of -2-Cl has high inhibitory activity against multiple myeloma cells, among which, the structure containing N heterocyclic cyclic ester and 2-Cl-5-Br substituted, 5-Cl-2- are preferred. Br replaces the compound formed by the complexation.
表2-3(含氧杂环环状酯类化合物的比较)Table 2-3 (Comparison of oxygen-containing heterocyclic cyclic ester compounds)
Figure PCTCN2017084924-appb-000089
Figure PCTCN2017084924-appb-000089
表2-3中的化合物均为含氧杂环环状酯类结构，比较可以看出，本发明化合物I-1-4、I-1-4-1、I-2-4、I-3-4、I-3-4-1、I-4-4、I-4-4-1均可以有效抑制多发性骨髓瘤细胞的增殖，其中，化合物I-1-4、I-1-4-1、I-2-4对所有的多发性骨髓瘤细胞的抑制活性均优于阳性药物IxazomibCitrate，化合物I-3-4、I-3-4-1、I-4-4、I-4-4-1对MM1S的抑制活性均优于阳 性药物IxazomibCitrate。The compounds in Table 2-3 are all oxygen-containing heterocyclic cyclic ester structures, and it can be seen that the compounds of the present invention are 1-1-4, I-1-4-1, I-2-4, and I-3. -4, I-3-4-1, I-4-4, and I-4-4-1 can effectively inhibit the proliferation of multiple myeloma cells, among which, compounds I-1-4, I-1-4 -1, I-2-4 inhibited all of the multiple myeloma cells better than the positive drug IxazomibCitrate, compounds I-3-4, I-3-4-1, I-4-4, I-4 -4-1 has better inhibitory activity against MM1S than yang Sex drug IxazomibCitrate.
实验结果说明含氧杂环环状酯结构与2-Cl-5-Br取代、5-Cl-2-Br取代、2,5-2-F-4-Cl取代、4,5-2-F-2-Cl取代配合形成的化合物，对多发性骨髓瘤细胞的抑制活性均较高，其中，优选含氧杂环环状酯结构与2-Cl-5-Br取代、5-Cl-2-Br取代配合形成的化合物。The experimental results indicate the structure of the oxygen-containing heterocyclic cyclic ester with 2-Cl-5-Br substitution, 5-Cl-2-Br substitution, 2,5-2-F-4-Cl substitution, 4,5-2-F The compound formed by the substitution of -2-Cl has high inhibitory activity against multiple myeloma cells, and among them, an oxygen-containing heterocyclic cyclic ester structure and 2-Cl-5-Br substitution, 5-Cl-2- are preferred. Br replaces the compound formed by the complexation.
实施例31评估本发明化合物对动物体内肿瘤组织中蛋白酶体20S-β5，β1以及β2亚单位抑制活性以及凋亡诱导活性Example 31 Evaluation of Proteasome 20S-β5, β1 and β2 Subunit Inhibitory Activity and Apoptosis Inducing Activity of Tumors in Tumor Tissues of Animals in Vivo
利用MM.1S多发性骨髓瘤细胞和重症联合免疫缺陷(SCID)小鼠建立肿瘤模型：100μl1640培养基重悬3×107MM.1S细胞，并和100μl基质胶混匀。将上述200μl混匀体系注射至SCID小鼠(5周龄，雌性)右侧腹。6-7天后，在肿瘤注射部位形成可见大小(100mm3)，待肿瘤生长至直径大约2cm左右；口服灌胃给予本发明化合物I-1-3，I-2-3以及对照化合物Ixazomib-DEA(化合物用5％HPβCD进行溶解，给药剂量为10mg/Kg)。8小时后，剥离小鼠皮肤，取出肿瘤组织，按照每0.1g肿瘤组织加入200μl预冷1640培养基进行组织匀浆(冰上操作，保持组织匀浆操作低温进行)。取匀浆样品25μl加入Promega Proteasome-GloTM Chymotrypsin-Like(用于β5亚单位测定),Caspase-Like(用于β1亚单位测定)和Trypsin-Like(用于β2亚单位测定)检测试剂盒所提供检测试剂25μl，分别用于检测蛋白酶体20S-β5，β1以及β2亚单位抑制活性。对于肿瘤组织凋亡诱导活性检测：取肿瘤组织，按照每0.1g肿瘤组织加入1ml蛋白裂解液进行组织匀浆，然后12000rpm离心取上清进行western-blot操作，利用兔抗PARP、兔抗Caspase-3以及兔抗Cleaved Caspase-3抗体进行检测。如图11结果显示肿瘤组织中蛋白酶体20S-β5，β1亚单位(胱天蛋白酶(caspase)样活性亚基)活性均得到明显抑制。该实施例证明了本发明化合物能直接到达肿瘤组织内，抑制蛋白酶体活性，从而诱导肿瘤组织凋亡。如图12所示，给予本发明化合物I-1-3以及I-2-3能诱导肿瘤凋亡标志-Cleaved PARP以及Cleaved Caspase-3活化片段的生成。Tumor models were established using MM.1S multiple myeloma cells and severe combined immunodeficiency (SCID) mice: 3×10 7 MM.1S cells were resuspended in 100 μl of 1640 medium and mixed with 100 μl of Matrigel. The above 200 μl homogenate system was injected into the right abdomen of SCID mice (5 weeks old, female). After 6-7 days, a visible size (100 mm 3 ) was formed at the site of tumor injection, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control compound Ixazomib-DEA were orally administered orally. (The compound was dissolved with 5% HPβCD at a dose of 10 mg/kg). After 8 hours, the skin of the mice was peeled off, and the tumor tissues were taken out, and tissue homogenate was performed by adding 200 μl of pre-cooled 1640 medium per 0.1 g of tumor tissue (on ice operation, keeping the tissue homogenization operation at a low temperature). 25 μl of the homogenate sample was added to Promega Proteasome-Glo TM Chymotrypsin-Like (for β5 subunit assay), Caspase-Like (for β1 subunit assay) and Trypsin-Like (for β2 subunit assay) assay kit. 25 μl of detection reagent was provided for detecting proteasome 20S-β5, β1 and β2 subunit inhibitory activities, respectively. For the detection of apoptosis-inducing activity of tumor tissue: tumor tissue was taken, and tissue homogenate was added according to 1 ml of protein lysate per 0.1 g of tumor tissue, and then supernatant was centrifuged at 12,000 rpm for western-blot operation, using rabbit anti-PARP, rabbit anti-Caspase- 3 and rabbit anti-Cleaved Caspase-3 antibody were detected. As shown in Figure 11, the activity of proteasome 20S-β5 and β1 subunit (caspase-like active subunit) in tumor tissues was significantly inhibited. This example demonstrates that the compounds of the present invention can directly reach tumor tissues, inhibit proteasome activity, and thereby induce tumor tissue apoptosis. As shown in Figure 12, administration of the compounds of the present invention, I-1-3 and I-2-3, induced the formation of a tumor-apoptable marker-Cleaved PARP and a Cleaved Caspase-3 activating fragment.
从图11中可以看出，本发明化合物I-1-3在肿瘤组织中，对蛋白酶体20S-β5亚单位抑制活性均优于阳性对照药物Ixazomib-DEA，差异显著(P＝0.0245)；对蛋白酶体20S-β1亚单位抑制活性均优于阳性对照药物Ixazomib-DEA，差异极其显著(P＝0.0004)。本发明化合物I-2-3在肿瘤组织中，对蛋白酶体20S-β5亚单位抑制活性均优于阳性对照药物Ixazomib-DEA，差异极其显著(P＝0.0002)。对蛋白酶体20S-β1亚单位抑制活性均优于阳性对照药物Ixazomib-DEA，差异极其显著(P<0.0001)。 As can be seen from Fig. 11, the compound I-1-3 of the present invention has a higher inhibitory activity against the proteasome 20S-β5 subunit in the tumor tissue than the positive control drug Ixazomib-DEA, and the difference is significant (P=0.0245); The proteasome 20S-β1 subunit inhibitory activity was superior to the positive control drug Ixazomib-DEA, and the difference was extremely significant (P=0.0004). In the tumor tissue, the compound I-2-3 of the present invention had better inhibitory activity against the proteasome 20S-β5 subunit than the positive control drug Ixazomib-DEA, and the difference was extremely significant (P=0.0002). The 20S-β1 subunit inhibitory activity of the proteasome was superior to the positive control drug Ixazomib-DEA, and the difference was extremely significant (P<0.0001).
由于蛋白酶体20S-β5以及β1亚单位是抗多发性骨髓瘤的作用靶点，本发明化合物经过口服可以到达肿瘤组织中，发挥更显著优于Ixazomib-DEA蛋白酶体抑制活性，意味本发明化合物具有更强凋亡诱导活性。并且给予本发明化合物I-1-3以及I-2-3相比Ixazomib-DEA，能更加有效诱导肿瘤凋亡标志-Cleaved PARP以及Cleaved Caspase-3活化片段生成(详见图12)。Since the proteasome 20S-β5 and the β1 subunit are targets of action against multiple myeloma, the compound of the present invention can reach the tumor tissue orally, and exerts a more remarkable activity than the Ixazomib-DEA proteasome inhibitory activity, meaning that the compound of the present invention has Stronger apoptosis-inducing activity. Furthermore, the administration of the compounds of the present invention, I-1-3 and I-2-3, was more effective than the Ixazomib-DEA in inducing the expression of the tumor apoptosis markers Cleaved PARP and Cleaved Caspase-3 (see Figure 12).
实验结果说明，本发明化合物I-1-3，I-2-3可以制备成为蛋白酶体抑制剂类药物，尤其是蛋白酶体胰凝乳蛋白酶样(蛋白酶体20S-β5)蛋白酶抑制剂类药物和蛋白酶体胱天蛋白酶样(蛋白酶体20S-β1)蛋白酶抑制剂类药物。The experimental results indicate that the compounds of the present invention, I-1-3, I-2-3, can be prepared as proteasome inhibitor drugs, especially proteasome chymotrypsin-like (proteasome 20S-β5) protease inhibitor drugs and Proteasome caspase-like (proteasome 20S-β1) protease inhibitor drug.
实施例32组织病理学检测本发明化合物对动物体内肿瘤组织凋亡诱导活性Example 32 Histopathological Detection of Tumor Tissue Apoptosis Inducing Activity in Animals of the Invention
按照实施例31所述方法建立多发性骨髓瘤小鼠体内模型，待肿瘤生长至直径大约2cm左右；口服灌胃给予本发明化合物I-1-3、I-2-3以及对照药物Ixazomib-DEA(药物用5％HPβCD进行溶解，给药剂量为10mg/Kg)。8小时后，剥离小鼠皮肤，取出肿瘤组织，4％多聚甲醛固定。固定组织经全自动脱水机脱水，包埋，切片后进行如下操作：将脱蜡的切片放染色架上，放入苏木精染液缸染色10-20分钟。取出放搪瓷盅水洗，水洗取出放50℃的温水中或弱碱性水溶液反蓝，直到出现蓝色为止。镜检染色效果，细胞核呈蓝色，胞浆不着色。放伊红染液缸内染色1分钟。将染好色的片子依次放入不同浓度酒精中，去除吹干。二甲苯I：1～5分钟。二甲苯II：1～5分钟。用中性树胶封固，贴上相对应的标签。结果如下图13以及表3：The in vivo model of multiple myeloma mice was established according to the method described in Example 31, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control drug Ixazomib-DEA were orally administered orally. (The drug was dissolved with 5% HPβCD at a dose of 10 mg/kg). After 8 hours, the skin of the mice was peeled off, and the tumor tissues were taken out and fixed with 4% paraformaldehyde. The fixed tissue was dehydrated, embedded, and sliced, and the following operations were performed: the dewaxed sections were placed on a dyeing rack, and stained in a hematoxylin dyeing tank for 10-20 minutes. Take out the enamel, wash it, wash it out and put it in warm water at 50 °C or a weak alkaline solution against blue until blue appears. Microscopic examination of the staining effect, the nucleus is blue, and the cytoplasm is not colored. Dye in the red dye bath for 1 minute. The dyed tablets are placed in different concentrations of alcohol in turn, and dried. Xylene I: 1 to 5 minutes. Xylene II: 1 to 5 minutes. Seal with neutral gum and attach the corresponding label. The results are shown in Figure 13 and Table 3 below:
阴性对照组：肿瘤细胞数量较多，生长致密，偶见有肿瘤细胞核溶解、碎裂、消失，肿瘤细胞呈点状少量坏死。Negative control group: The number of tumor cells was large, and the growth was dense. Occasionally, the tumor cells were lysed, fragmented and disappeared, and the tumor cells showed a small amount of necrosis.
口服给予Ixazomib-DEA药物：肿瘤细胞数量较多，生长致密，肿瘤细胞呈点状或局灶性坏死(组织坏死程度判定为++)，聚集的多个肿瘤细胞核浓缩、***，坏死区域偶见有单核细胞、嗜中性粒细胞浸润。Oral administration of Ixazomib-DEA drugs: the number of tumor cells is large, the growth is dense, the tumor cells are punctate or focal necrosis (the degree of tissue necrosis is ++), and the aggregated multiple tumor cells are concentrated and divided, and the necrotic area is occasionally seen. There are monocytes, neutrophil infiltration.
口服给予本发明化合物I-1-3：肿瘤细胞数量较少，细胞排列疏松，肿瘤细胞呈片状坏死(组织坏死程度判定为+++)，多数肿瘤细胞核浓缩、***、溶解，组织中可见蛋白样物质及细胞碎片，坏死区域可见大量的嗜中性粒细胞、浆细胞和单核细胞。Oral administration of the compound of the present invention I-1-3: the number of tumor cells is small, the cells are arranged loosely, and the tumor cells are flaky necrosis (the degree of tissue necrosis is determined as +++), and most of the tumor cells are concentrated, divided, dissolved, and visible in the tissue. Protein-like substances and cell debris, a large number of neutrophils, plasma cells and monocytes can be seen in the necrotic area.
口服给予本发明化合物I-2-3：肿瘤细胞数量较少，细胞排列疏松，肿瘤细胞呈片状坏死(组织坏死程度判定为+++)，多数肿瘤细胞核浓缩、***、溶解，组织中可见蛋白 样物质及细胞碎片，坏死区域可见大量的嗜中性粒细胞、浆细胞和单核细胞。Oral administration of the compound of the present invention I-2-3: the number of tumor cells is small, the cells are arranged loosely, and the tumor cells are flaky necrosis (the degree of tissue necrosis is determined as +++), and most of the tumor cells are concentrated, divided, dissolved, and visible in the tissue. Protein Samples and cell debris, a large number of neutrophils, plasma cells and monocytes can be seen in the necrotic area.
表3.组织病理学检测本发明化合物对动物体内肿瘤组织凋亡诱导活性Table 3. Histopathological detection of tumor-induced apoptosis-inducing activity of the compounds of the present invention in animals
分组Grouping 组织名称name of association 结果result
阴性对照组Negative control group 肿瘤Tumor 肿瘤细胞点状坏死(+)Tumor cell point necrosis (+)
Ixazomib-DEAIxazomib-DEA 肿瘤Tumor 肿瘤细胞点状坏死(++)Tumor cell point necrosis (++)
I-1-3I-1-3 肿瘤Tumor 肿瘤细胞片状坏死(+++)Tumor cell plaque necrosis (+++)
I-2-3I-2-3 肿瘤Tumor 肿瘤细胞片状坏死(+++)Tumor cell plaque necrosis (+++)
综上，该实施例证实本发明化合物在体内肿瘤组织中凋亡诱导活性均优于对照药物Ixazomib-DEA。In conclusion, this example demonstrates that the compounds of the present invention have superior apoptosis-inducing activity in tumor tissues in vivo than the control drug Ixazomib-DEA.
实施例33TUNEL-POD法检测本发明化合物动物体内肿瘤组织凋亡效应Example 33 TUNEL-POD method for detecting tumor tissue apoptosis in animals of the present invention
按照上述方法建立多发性骨髓瘤小鼠体内模型，待肿瘤生长至直径大约2cm左右；口服灌胃给予本发明化合物I-1-3、I-2-3以及对照药物Ixazomib-DEA(药物用5％HPβCD进行溶解，给药剂量为10mg/Kg)。8小时后，剥离小鼠皮肤，取出肿瘤组织，4％多聚甲醛固定后进行如下操作：载玻片防脱片处理：使用APES侵泡，捞片后置烤箱60℃，60min以使切片紧密粘附；切片常规脱蜡至水；用胰蛋白酶K(Proteinase K)工作液在37℃处理组织25min；PBS漂洗3次；制备TUNEL反应混合液：50μl 1号液+450μl 2号液混匀；玻片干后，加50μl TUNEL反应混合液于标本上，加盖玻片在暗湿盒中反应37℃，1h；PBS漂洗3次；玻片干后加50μl 3号液(converter-POD)于标本上，加盖玻片或封口膜在暗湿盒中反应37℃，30min；PBS漂洗3次；滴加50-100μl DAB显色剂，观察反应25℃，10min；PBS漂洗3次；苏木素轻度复染，几秒后自来水冲洗；酒精脱水，二甲苯透明，中性树胶封片。采用BA200Digital数码三目摄像显微摄像***显微摄像***对切片进行图像采集，每张切片先于100倍下观察全部组织，再根据组织大小及表达情况分别选取1个区域400倍采集图像；对每张切片进行读片，镜下计数，统计阳性率，结果如下表4：The in vivo model of multiple myeloma mice was established according to the above method, and the tumor was grown to a diameter of about 2 cm; the compounds of the present invention I-1-3, I-2-3 and the control drug Ixazomib-DEA were administered orally by gavage (drug 5) %HPβCD was dissolved at a dose of 10 mg/kg). After 8 hours, the skin of the mice was peeled off, and the tumor tissue was taken out. After fixing 4% paraformaldehyde, the following operations were carried out: slide-proof treatment: using APES to infuse, and then placing the oven at 60 ° C for 60 min to make the slices tight Adhesion; section conventional dewaxing to water; treated with trypsin K (Proteinase K) working solution at 37 ° C for 25 min; PBS rinse 3 times; preparation of TUNEL reaction mixture: 50 μl No. 1 liquid + 450 μl No. 2 liquid mixed; After the slides were dried, add 50 μl of TUNEL reaction mixture to the specimen, cover the slides in a dark and humid box at 37 ° C for 1 h, rinse with PBS 3 times, and add 50 μl of 3 (converter-POD) to the slides. On the specimen, cover the slide or seal film in a dark and humid box for 37 ° C, 30 min; PBS rinse 3 times; add 50-100 μl DAB developer, observe the reaction at 25 ° C, 10 min; PBS rinse 3 times; hematoxylin light Degree of counterstaining, a few seconds after tap water rinse; alcohol dehydration, xylene transparent, neutral gum seal. The BA200Digital digital trinocular camera micro-camera system micro-camera system was used to collect the images of the slices. Each slice was observed at 100 times before the whole tissue, and then one region was selected 400 times according to the size and expression of the tissue. Each slice was read, counted under the microscope, and the positive rate was counted. The results are shown in Table 4 below:
表4.细胞凋亡检测(TUNEL-POD法)阳性细胞率Table 4. Apoptosis detection (TUNEL-POD method) positive cell rate
Figure PCTCN2017084924-appb-000090
Figure PCTCN2017084924-appb-000090
口服给予本发明化合物I-1-3，肿瘤组织中凋亡阳性细胞率明显多于Ixazomib-DEA， 差异极其显著(**P＝0.0021以及**P＝0.0018)。Oral administration of the compound of the present invention I-1-3, the rate of apoptosis-positive cells in tumor tissues is significantly higher than that of Ixazomib-DEA, The difference is extremely significant (**P=0.0021 and **P=0.0018).
口服给予本发明化合物I-2-3，肿瘤组织中凋亡阳性细胞率明显多于Ixazomib-DEA，差异极其显著(**P＝0.0006以及**P＝0.0005)The compound of the present invention I-2-3 was orally administered, and the rate of apoptosis-positive cells in tumor tissues was significantly higher than that of Ixazomib-DEA, and the difference was extremely significant (**P=0.0006 and **P=0.0005).
该实施例证实本发明化合物在体内肿瘤组织中凋亡诱导活性均优于对照化合物Ixazomib-DEA。This example demonstrates that the compounds of the invention are superior to the control compound Ixazomib-DEA in tumor-inducing activity in tumor tissues in vivo.
实施例34本发明化合物对套细胞淋巴瘤和滤泡性淋巴瘤抑制活性Example 34 Inhibitory Activity of the Compounds of the Invention on Mantle Cell Lymphoma and Follicular Lymphoma
测试样品采用DMSO溶解，低温保存。在试验前，利用HPLC检测样品稳定性，保证本实施方案所使用样品稳定。套细胞淋巴瘤系JVM-2以及滤泡性淋巴瘤系SU-DHL-6按照10000个细胞/孔加入平底96孔细胞培养板中。化合物最高浓度为50μM，Ixazomib-DEA作为对照组。按照5倍梯度做药物浓度稀释。化合物作用48小时后加入10μl CCK-8，孵育6h后，利用酶标仪测定450nM波长吸收值。药物对肿瘤细胞生长抑制率(GI50)计算方法按照美国国家癌症研究所(National Cancer Institute,NCI)标准方法进行：当Ti(药物组，培养48h，CCK-8显色吸收OD值)≥Tz(不含药物组，培养起始时CCK-8显色吸收OD值)，肿瘤细胞存活率＝[(Ti-Tz)/(C-Tz)]×100，其中C为不含药物组48小时后CCK-8显色吸收OD值；当Ti<Tz时，肿瘤细胞存活率＝[(Ti-Tz)/Tz]×100。通过Graphpad Prism5.0计算测试样品对套细胞淋巴瘤细胞增殖抑制活性的GI50，结果如下表5：The test sample was dissolved in DMSO and stored at low temperature. Prior to the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in this embodiment. The mantle cell lymphoma line JVM-2 and the follicular lymphoma line SU-DHL-6 were added to a flat-bottom 96-well cell culture plate at 10,000 cells/well. The highest concentration of the compound was 50 μM and Ixazomib-DEA was used as a control group. Dilute the drug concentration according to a 5-fold gradient. After the compound was applied for 48 hours, 10 μl of CCK-8 was added, and after incubation for 6 hours, the 450 nM wavelength absorption value was measured by a microplate reader. The method for calculating the tumor cell growth inhibition rate (GI50) of the drug was carried out according to the National Cancer Institute (NCI) standard method: when Ti (drug group, cultured for 48 h, CCK-8 colorimetric absorption OD value) ≥ Tz ( The drug-free group, CCK-8 color absorbing OD value at the start of culture), tumor cell survival rate = [(Ti-Tz) / (C-Tz)] × 100, wherein C is 48 hours after the drug-free group CCK-8 color absorption OD value; when Ti < Tz, tumor cell survival rate = [(Ti-Tz) / Tz] × 100. The GI50 of the test sample for the proliferation inhibition activity of the mantle cell lymphoma cells was calculated by Graphpad Prism 5.0, and the results are shown in Table 5 below:
表5.本发明化合物及对照化合物对套细胞淋巴瘤细胞抑制活性(GI50，nM)Table 5. Inhibitory activity of the compounds of the invention and control compounds against mantle cell lymphoma cells (GI50, nM)
待测化合物Test compound JVM-2套细胞淋巴瘤JVM-2 mantle cell lymphoma SU-DHL-6滤泡性淋巴瘤SU-DHL-6 follicular lymphoma
I-1-3I-1-3 37.5137.51 131.46131.46
I-2-3I-2-3 34.6834.68 129.57129.57
I-5-3I-5-3 21.0621.06 125.63125.63
Ixazomib-DEAIxazomib-DEA 84.7384.73 437.22437.22
结果显示，本发明化合物相比对照化合物Ixazomib-DEA对JVM-2套细胞淋巴瘤细胞及SU-DHL-6滤泡性淋巴瘤细胞增殖具有显著的抑制作用。The results showed that the compound of the present invention had a significant inhibitory effect on the proliferation of JVM-2 mantle cell lymphoma cells and SU-DHL-6 follicular lymphoma cells compared to the control compound Ixazomib-DEA.
实验结果证明，本发明化合物I-1-3、I-2-3、I-5-3具有抑制淋巴瘤的作用，尤其是对非霍奇金淋巴瘤中的套细胞淋巴瘤和滤泡性淋巴瘤的抑制作用显著。 The experimental results demonstrate that the compounds of the present invention, I-1-3, I-2-3, and I-5-3, have the effect of inhibiting lymphoma, especially for mantle cell lymphoma and follicularity in non-Hodgkin's lymphoma. The inhibition of lymphoma is significant.
实施例35本发明化合物对HT-29人结肠癌细胞株、乳腺癌细胞MDA-MB-231、肺癌细胞NCI-H460及肾癌细胞系抑制活性Example 35 Inhibitory activity of the compound of the present invention against HT-29 human colon cancer cell line, breast cancer cell MDA-MB-231, lung cancer cell NCI-H460 and renal cancer cell line
收集对数生长期HT-29人结肠癌细胞，计数，用完全培养基重新悬浮细胞，调整细胞浓度至合适浓度(依照细胞密度优化试验结果确定)，接种96孔板，每孔加100μl细胞悬液。细胞在37℃，100％相对湿度，5％CO2培养箱中孵育24小时后。用培养基将待测化合物稀释至所设置的相应作用浓度，按25μl/孔加入96孔板中。化合物作用终浓度从100μM至0μM，4倍梯度稀释。细胞置于37℃，100％相对湿度，5％CO2培养箱中孵育72小时。加入100μl含10％CCK-8的新鲜培养基，置于37℃培养箱中孵育2-4小时。轻轻震荡后在SpectraMax M5Microplate Reader上测定450nm波长处的吸光度，以650nm处吸光度作为参比，待测化合物对HT-29人结肠癌细胞株抑制活性IC50结果如下表6：Collect HT-29 human colon cancer cells in logarithmic growth phase, count, resuspend the cells with complete medium, adjust the cell concentration to the appropriate concentration (determined according to the cell density optimization test results), inoculate 96-well plates, add 100 μl of cell suspension per well. liquid. The cells were incubated for 24 hours at 37 ° C, 100% relative humidity, 5% CO 2 incubator. The test compound was diluted with the medium to the corresponding concentration of action set, and added to the 96-well plate at 25 μl/well. The final concentration of the compound was diluted from 100 μM to 0 μM in 4 fold gradients. The cells were incubated for 72 hours at 37 ° C in a 100% relative humidity, 5% CO 2 incubator. 100 μl of fresh medium containing 10% CCK-8 was added and incubated in a 37 ° C incubator for 2-4 hours. The absorbance at 450 nm was measured on a SpectraMax M5 Microplate Reader with a light shock. The absorbance at 650 nm was used as a reference. The IC50 results of the test compound against HT-29 human colon cancer cell line are shown in Table 6 below:
表6.本发明化合物及对照化合物对HT-29人结肠癌细胞株抑制活性(IC50，nM)Table 6. Inhibitory activity of the compounds of the invention and control compounds against HT-29 human colon cancer cell line (IC50, nM)
待测化合物Test compound IC50(nM)IC50(nM) 待测化合物Test compound IC50(nM)IC50(nM)
I-1-1I-1-1 4.904.90 I-1-3I-1-3 5.345.34
I-2-1I-2-1 3.143.14 I-2-3I-2-3 3.773.77
I-5-1I-5-1 9.129.12 I-6-1I-6-1 50.4050.40
I-7-1I-7-1 82.3382.33 I-10-1I-10-1 61.2161.21
IxazomibIxazomib 55.3555.35 Ixazomib-DEAIxazomib-DEA 53.2653.26
结果显示，本发明化合物相比对照化合物Ixazomib、Ixazomib-DEA、I-6-1、I-7-1、I-10-1对HT-29人结肠癌细胞株增殖具有更加显著的抑制作用。The results showed that the compounds of the present invention had a more pronounced inhibitory effect on the proliferation of HT-29 human colon cancer cell lines than the control compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, and I-10-1.
类似上述实验过程，测定了本发明化合物及对照化合物对肺癌细胞NCI-H460、乳腺癌细胞MDA-MB-231、肾癌细胞系抑制活性，如下表7、8、9：The inhibitory activities of the compound of the present invention and the control compound against lung cancer cells NCI-H460, breast cancer cells MDA-MB-231, and renal cancer cell lines were determined similarly to the above experimental procedures, as shown in Tables 7, 8, and 9 below:
表7.本发明化合物及对照化合物对NCI-H460肺癌细胞株抑制活性(IC50，nM)Table 7. Inhibitory activity of the compounds of the invention and control compounds against NCI-H460 lung cancer cell lines (IC50, nM)
待测化合物Test compound IC50(nM)IC50(nM) 待测化合物Test compound IC50(nM)IC50(nM)
I-1-3I-1-3 12.6012.60 I-1-1I-1-1 15.1015.10
I-2-3I-2-3 10.2010.20 I-2-1I-2-1 13.1413.14
I-5-1I-5-1 13.2313.23 I-6-1I-6-1 68.7168.71
I-7-1I-7-1 133.24133.24 I-10-1I-10-1 145.75145.75
IxazomibIxazomib 58.8258.82 Ixazomib-DEAIxazomib-DEA 56.7756.77
表8.本发明化合物及对照化合物对乳腺癌细胞MDA-MB-231细胞株抑制活性(IC50，nM) Table 8. Inhibitory activity of the compound of the present invention and a control compound against breast cancer cell line MDA-MB-231 (IC50, nM)
待测化合物Test compound IC50(nM)IC50(nM) 待测化合物Test compound IC50(nM)IC50(nM)
I-1-1I-1-1 50.1050.10 I-1-3I-1-3 22.3822.38
I-2-1I-2-1 49.3349.33 I-2-3I-2-3 26.1226.12
1-5-11-5-1 32.2932.29 1-6-11-6-1 220.40220.40
1-7-11-7-1 255.41255.41 1-10-11-10-1 247.82247.82
IxazomibIxazomib 160.91160.91 Ixazomib-DEAIxazomib-DEA 202.47202.47
表9本发明化合物及对照化合物对肾癌细胞系抑制活性(IC50，nM)Table 9 Inhibitory activity of the compound of the present invention and a control compound against renal cancer cell lines (IC50, nM)
Figure PCTCN2017084924-appb-000091
Figure PCTCN2017084924-appb-000091
结果显示，本发明化合物相比对照化合物Ixazomib、Ixazomib-DEA、I-6-1、I-7-1、I-10-1对NCI-H460肺癌细胞株、MDA-MB-231乳腺癌细胞株、肾癌细胞系增殖均具有更加显著的抑制作用。The results show that the compounds of the present invention are compared to the control compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, I-10-1 to NCI-H460 lung cancer cell line, MDA-MB-231 breast cancer cell line. The proliferation of renal cancer cell lines has a more significant inhibitory effect.
实验结果证明，本发明化合物I-1-1、I-1-3、I-2-1、I-2-3、I-5-1可以有效抑制结肠癌、肺癌、乳腺癌和肾癌。The experimental results demonstrate that the compounds of the present invention, I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1, can effectively inhibit colon cancer, lung cancer, breast cancer, and kidney cancer.
实施例36本发明化合物对人***、鼻咽癌细胞抑制活性Example 36 Inhibitory activity of the compound of the present invention on human cervical cancer and nasopharyngeal carcinoma cells
测试样品采用DMSO溶解，低温保存。试验前，利用HPLC检测样品稳定性，保证本实施方案所使用样品稳定。***细胞系HeLa细胞按照2000个细胞/孔，加入到平底96孔细胞培养板中。细胞培养24小时贴壁后，加入待测化合物。化合物最高浓度为10μM， Ixazomib-DEA、Ixazomib、I-6-1、I-7-1、I-10-1作为对照组。按照5倍梯度做药物浓度稀释。化合物作用48小时后加入10μl CCK-8，孵育6h后，利用酶标仪测定450nM波长吸收值。药物对肿瘤细胞生长抑制率(GI50)计算方法按照美国国家癌症研究所(National Cancer Institute,NCI)标准方法进行：当Ti(药物组，培养48h，CCK-8显色吸收OD值)≥Tz(不含药物组，培养起始时CCK-8显色吸收OD值)，肿瘤细胞存活率＝[(Ti-Tz)/(C-Tz)]×100，其中C为不含药物组48小时后CCK-8显色吸收OD值；当Ti<Tz时，肿瘤细胞存活率＝[(Ti-Tz)/Tz]×100。通过GraphpadPrism5.0计算测试样品对***细胞系HeLa细胞增殖抑制活性的GI50，结果如下表10：The test sample was dissolved in DMSO and stored at low temperature. Before the test, the stability of the sample was measured by HPLC to ensure the stability of the sample used in the present embodiment. The cervical cancer cell line HeLa cells were added to a flat-bottom 96-well cell culture plate at 2000 cells/well. After the cells were cultured for 24 hours, the test compound was added. The highest concentration of the compound is 10 μM. Ixazomib-DEA, Ixazomib, I-6-1, I-7-1, and I-10-1 were used as a control group. Dilute the drug concentration according to a 5-fold gradient. After the compound was applied for 48 hours, 10 μl of CCK-8 was added, and after incubation for 6 hours, the 450 nM wavelength absorption value was measured by a microplate reader. The method for calculating the tumor cell growth inhibition rate (GI50) of the drug was carried out according to the National Cancer Institute (NCI) standard method: when Ti (drug group, cultured for 48 h, CCK-8 colorimetric absorption OD value) ≥ Tz ( The drug-free group, CCK-8 color absorbing OD value at the start of culture), tumor cell survival rate = [(Ti-Tz) / (C-Tz)] × 100, wherein C is 48 hours after the drug-free group CCK-8 color absorption OD value; when Ti < Tz, tumor cell survival rate = [(Ti-Tz) / Tz] × 100. The GI50 of the test sample against the proliferation inhibition activity of the cervical cancer cell line HeLa cells was calculated by Graphpad Prism 5.0, and the results are shown in Table 10 below:
表10.本发明化合物及对照化合物对***细胞抑制活性(GI50，nM)Table 10. Inhibitory activity of compounds of the invention and control compounds against cervical cancer cells (GI50, nM)
待测化合物Test compound GI50(nM)GI50(nM) 待测化合物Test compound GI50(nM)GI50(nM)
I-1-1I-1-1 9.119.11 I-1-3I-1-3 29.4829.48
I-2-1I-2-1 8.058.05 I-2-3I-2-3 30.0230.02
I-5-1I-5-1 18.3118.31 I-6-1I-6-1 170.53170.53
I-7-1I-7-1 116.26116.26 I-10-1I-10-1 182.93182.93
IxazomibIxazomib 32.8432.84 Ixazomib-DEAIxazomib-DEA 51.1651.16
结果显示，本发明化合物相比照化合物Ixazomib、Ixazomib-DEA、I-6-1、I-7-1、I-10-1对***细胞系HeLa细胞有更加显著的抑制作用。The results showed that the compounds of the present invention had more significant inhibitory effects on the cervical cancer cell line HeLa cells than the compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, and I-10-1.
类似上述实验过程，测定了本发明化合物及对照化合物对鼻咽癌细胞系6-10B细胞抑制活性，结果如下表11：The inhibitory activity of the compound of the present invention and the control compound against the nasopharyngeal carcinoma cell line 6-10B was measured in the same manner as the above experimental procedure. The results are shown in Table 11 below:
表11.本发明化合物及对照化合物对鼻咽癌细胞抑制活性(GI50，nM)Table 11. Inhibitory activity of nasopharyngeal carcinoma cells by compounds of the invention and control compounds (GI50, nM)
待测化合物Test compound GI50(nM)GI50(nM) 待测化合物Test compound GI50(nM)GI50(nM)
I-1-1I-1-1 603.97603.97 I-1-3I-1-3 616.03616.03
I-2-1I-2-1 614.80614.80 I-2-3I-2-3 670.52670.52
I-5-1I-5-1 683.52683.52 I-6-1I-6-1 1691.491691.49
I-7-1I-7-1 1922.081922.08 I-10-1I-10-1 1743.611743.61
IxazomibIxazomib 803.52803.52 Ixazomib-DEAIxazomib-DEA 862.56862.56
结果显示，本发明化合物相比照化合物Ixazomib、Ixazomib-DEA、I-6-1、I-7-1、I-10-1对鼻咽癌细胞系6-19B细胞有更加显著的抑制作用。The results showed that the compounds of the present invention had a more pronounced inhibitory effect on the nasopharyngeal carcinoma cell line 6-19B cells than the compounds Ixazomib, Ixazomib-DEA, I-6-1, I-7-1, and I-10-1.
实验结果证明，本发明化合物I-1-1、I-1-3、I-2-1、I-2-3、I-5-1可以有效抑制***和鼻咽癌。 The experimental results demonstrate that the compounds I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1 of the present invention can effectively inhibit cervical cancer and nasopharyngeal carcinoma.
从实施例34-36的结果可以看出，苯环卤素取代基对活性影响较大，尽管本领域技术人员把Cl、Br、I视为电子等排体可以互相置换，但令人惊奇的是，本发明人发现，苯环上取代基的的卤素变化、其不同的组合，会得到令人意想不到的抗肿瘤活性差异。其中，I-1-1、I-1-3、I-2-1、I-2-3、I-5-1表现出显著优异的抗肿瘤活性。As can be seen from the results of Examples 34-36, the benzene ring halogen substituent has a large influence on the activity, although those skilled in the art can consider Cl, Br, and I as electronic isosters to be mutually substituted, but surprisingly The present inventors have found that a halogen change of a substituent on a benzene ring, and a different combination thereof, can give an unexpected difference in antitumor activity. Among them, I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1 exhibited remarkably excellent antitumor activity.
实施例37利用传统膜片钳测定本发明化合物对稳态表达于HEK293细胞的人类hERG离子通道的作用Example 37 Determination of the effect of a compound of the invention on human hERG ion channels stably expressed in HEK293 cells using conventional patch clamps
将hERG离子通道稳态表达HEK293细胞转移到灌流槽中，于室温下用细胞外液进行灌流，每个细胞以自身为对照。测试化合物均用DMSO溶解，配置为0.3μM、1μM、3μM、10μM、30μM、100μM的浓度梯度。化合物均采用利用自身重力的灌流***进行灌流。每个浓度至少测试两个细胞。在电流稳定(或5分钟)后，再比较化合物使用前后的电流大小变化来计算化合物的阻断作用。阳性对照Cisapride以1nM,3nM,10nM，30nM,100nM的浓度梯度进行IC50测试。方法和测试化合物一样。测试电极用PC-10(Narishige，Japan)拉制。全细胞膜片钳记录，噪音用采样频率的五分之一进行过滤。将细胞钳制在–80mV，然后用持续4秒方波去极化到40mV，再用持续2秒方波超极化到-40mV，以得到hERG尾电流。这一程序每20秒重复一次。hERG尾电流是纯hERG电流。检测第二个方波引发的最大电流，待其稳定后，灌流测试化合物，当反应稳定后，计算阻断的强度。待测化合物对稳态表达于HEK293细胞的人类hERG离子通道阻断效应IC50值记录如下表12：The hERG ion channel steady-state expression HEK293 cells were transferred to a perfusion tank and perfused with extracellular fluid at room temperature, each cell being self-control. Test compounds were all dissolved in DMSO and configured to a concentration gradient of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM. The compounds were all perfused using a perfusion system using their own gravity. Test at least two cells per concentration. After the current is stable (or 5 minutes), the change in current magnitude before and after the compound is compared to calculate the blocking effect of the compound. The positive control Cisapride was subjected to an IC50 test at a concentration gradient of 1 nM, 3 nM, 10 nM, 30 nM, 100 nM. The method is the same as the test compound. The test electrodes were drawn with PC-10 (Narishige, Japan). Whole-cell patch clamp recording, noise is filtered using one-fifth of the sampling frequency. The cells were clamped at –80 mV, then depolarized to 40 mV with a square wave lasting 4 seconds, and then hyperpolarized to -40 mV with a square wave lasting 2 seconds to obtain the hERG tail current. This procedure is repeated every 20 seconds. The hERG tail current is a pure hERG current. The maximum current induced by the second square wave is detected. After it is stabilized, the test compound is perfused, and when the reaction is stable, the blocking strength is calculated. The IC50 values of the test compounds for human hERG ion channel blockade expression in HEK293 cells are shown in Table 12 below:
表12.本发明化合物和Ixazomib，Ixazomib-DEA对稳态表达于HEK293细胞的人类hERG离子通道的作用(IC50，μM)Table 12. Effect of compounds of the invention and Ixazomib, Ixazomib-DEA on human hERG ion channels stably expressed in HEK293 cells (IC50, μM)
待测化合物Test compound IC50(μM)IC50 (μM) 待测化合物Test compound IC50(μM)IC50 (μM)
I-1-1I-1-1 >100>100 I-1-3I-1-3 >100>100
I-2-1I-2-1 >100>100 I-2-3I-2-3 >100>100
IxazomibIxazomib 59.9459.94 Ixazomib-DEAIxazomib-DEA 56.4356.43
Cisapride(阳性对照)Cisapride (positive control) 0.0110.011    
结果显示，药物Ixazomib和Ixazomib-DEA对稳态表达于HEK293细胞的人类hERG离子通道具有一定程度的阻断效应。而本发明创造的化合物对于该离子通道阻断效应更弱。The results showed that the drugs Ixazomib and Ixazomib-DEA had a certain blocking effect on the human hERG ion channel stably expressed in HEK293 cells. The compounds created by the present invention have a weaker blocking effect on the ion channel.
本实施例证明，本发明创造的化合物I-1-1、I-1-3、I-2-1、I-2-3在心脏毒性方面表现 明显弱于Ixazomib和Ixazomib-DEA，成药后具有更好的安全性。This example demonstrates that the compounds I-1-1, I-1-3, I-2-1, and I-2-3 created by the present invention are manifested in cardiotoxicity. Significantly weaker than Ixazomib and Ixazomib-DEA, it has better safety after drug administration.
综上，本发明化合物具有良好的蛋白酶体抑制活性和抗肿瘤活性，尤其是I-1-1、I-1-3、I-2-1、I-2-3、I-5-1的抗肿瘤活性优良，优于现有化合物Ixazomib和Ixazomib-DEA，而且I-1-1、I-1-3、I-2-1、I-2-3的心脏毒性也极低，明显低于现有化合物Ixazomib和Ixazomib-DEA，临床应用前景优良。 In conclusion, the compounds of the present invention have good proteasome inhibitory activity and antitumor activity, especially I-1-1, I-1-3, I-2-1, I-2-3, and I-5-1. Excellent anti-tumor activity, superior to the existing compounds Ixazomib and Ixazomib-DEA, and the cardiotoxicity of I-1-1, I-1-3, I-2-1, I-2-3 is also extremely low, significantly lower than The existing compounds Ixazomib and Ixazomib-DEA have excellent clinical application prospects.

Claims (67)

  1. 式(Ⅰ)所示的化合物或其晶型、或其药学上可接受的盐、或其溶剂合物或硼酸酐：a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
    Figure PCTCN2017084924-appb-100001
    Figure PCTCN2017084924-appb-100001
    其中，环A表示如下结构之一：Among them, ring A represents one of the following structures:
    Figure PCTCN2017084924-appb-100002
    Figure PCTCN2017084924-appb-100002
    R1选自H或C1-6烷基；R 1 is selected from H or C 1-6 alkyl;
    R2选自C1-6烷基；R 2 is selected from C 1-6 alkyl;
    R3和R4选自氢，或R3和R4与氧和硼原子共同形成一个任选取代的5-20元环，该环包括0-2个选自氮、氧或硫的环杂原子。R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with an oxygen and a boron atom form an optionally substituted 5-20 membered ring comprising 0-2 ring molybdenes selected from nitrogen, oxygen or sulfur. atom.
  2. 权利要求1所述的化合物，其特征在于：所述化合物如式(Ⅰa)所示：The compound of claim 1 wherein said compound is as shown in formula (Ia):
    Figure PCTCN2017084924-appb-100003
    Figure PCTCN2017084924-appb-100003
  3. 根据权利要求1或2所述的化合物，其特征在于：R1选自H。 The compound according to claim 1 or 2, wherein R 1 is selected from H.
  4. 根据权利要求1-3任一项所述的化合物，其特征在于：R2选自异丙基。The compound according to any one of claims 1 to 3, wherein R 2 is selected from isopropyl.
  5. 根据权利要求2-4任一项所述的化合物，其特征在于：X1和X2选自羟基，或X1和X2共同形成与硼酸酯化剂的官能团脱去氢原子后的部分。The compound according to any one of claims 2 to 4, wherein X 1 and X 2 are selected from a hydroxyl group, or X 1 and X 2 together form a portion after deactivating a hydrogen atom with a functional group of a borate esterifying agent .
  6. 根据权利要求5所述的化合物，其特征在于：X1和X2选自羟基。The compound according to claim 5, wherein X 1 and X 2 are selected from a hydroxyl group.
  7. 根据权利要求5所述的化合物，其特征在于：所述硼酸酯化剂选自含有至少一个羟基或至少一个羧基的化合物。The compound according to claim 5, wherein the borating agent is selected from the group consisting of compounds containing at least one hydroxyl group or at least one carboxyl group.
  8. 根据权利要求7所述的化合物，其特征在于：所述硼酸酯化剂为包含有N原子的硼酸酯化剂。The compound according to claim 7, wherein the borating agent is a borating agent containing N atoms.
  9. 根据权利要求5-8任一项所述的化合物，其特征在于：当X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分时，所形成的环为5-12元环。The compound according to any one of claims 5-8, wherein when X 1 and X 2 together form a portion after dehydrogenating the two functional groups of the borate esterifying agent, the ring formed is 5-12 yuan ring.
  10. 根据权利要求7所述的化合物，其特征在于：所述硼酸酯化剂选自单糖或多元醇。The compound according to claim 7, wherein the borating agent is selected from the group consisting of monosaccharides or polyhydric alcohols.
  11. 根据权利要求7所述的化合物，其特征在于：所述硼酸酯化剂选自C4-10的饱和硼酸酯化剂，其中，羟基和羧基的数量之和为2～4。The compound according to claim 7, wherein the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the amounts of the hydroxyl group and the carboxyl group is 2 to 4.
  12. 根据权利要求7所述的化合物，其特征在于：所述硼酸酯化剂选自甘露醇、柠檬酸、苹果酸、酒石酸、葡萄糖、二乙醇胺、二丙醇胺、三乙醇胺、三丙醇胺、3-((2-羟基乙基)氨基)-1-丙醇、N-甲基二乙醇胺、N-丁基二乙醇胺、2-((2-羟基丙基)氨基)-1-丙醇、二异丙醇胺和N，N-双(2-羟乙基)甘氨酸中的任一种；或，所述硼酸酯化剂任选被C1-6的烷基、C3-C6的环烷基、C2-C6的羧烷基、C1-C6的羟烷基取代。The compound according to claim 7, wherein the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine. , 3-((2-hydroxyethyl)amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol Any one of diisopropanolamine and N,N-bis(2-hydroxyethyl)glycine; or the borate esterifying agent is optionally C1-6 alkyl, C3-C6 ring Alkyl, C2-C6 carboxyalkyl, C1-C6 hydroxyalkyl substituted.
  13. 根据权利要求1～12任一项所述的化合物，其特征在于：所述环A选自下述结构：The compound according to any one of claims 1 to 12, wherein the ring A is selected from the following structures:
    Figure PCTCN2017084924-appb-100004
    Figure PCTCN2017084924-appb-100004
  14. 根据权利要求12或13所述的化合物，其特征在于：所述硼酸酯化剂选自二丙醇胺。The compound according to claim 12 or 13, wherein the borating agent is selected from the group consisting of dipropanolamine.
  15. 根据权利要求12或13所述的化合物，其特征在于：所述硼酸酯化剂选自二乙醇胺。The compound according to claim 12 or 13, wherein the borating agent is selected from the group consisting of diethanolamine.
  16. 根据权利要求6任一项所述的化合物或其甘露醇酯，其特征在于：所述化合物为如下化合物之一： The compound according to any one of claims 6 or a mannitol ester thereof, wherein the compound is one of the following compounds:
    Figure PCTCN2017084924-appb-100005
    Figure PCTCN2017084924-appb-100005
  17. 根据权利要求1-12任意一项所述的化合物，其特征在于：所述化合物为如下化合物之一：The compound according to any one of claims 1 to 12, wherein the compound is one of the following compounds:
    Figure PCTCN2017084924-appb-100006
    Figure PCTCN2017084924-appb-100006
    Figure PCTCN2017084924-appb-100007
    Figure PCTCN2017084924-appb-100007
    Figure PCTCN2017084924-appb-100008
    Figure PCTCN2017084924-appb-100008
  18. 根据权利要求17所述的化合物，其特征在于：所述化合物为如下化合物之一：The compound according to claim 17, wherein the compound is one of the following compounds:
    Figure PCTCN2017084924-appb-100009
    Figure PCTCN2017084924-appb-100009
  19. 一种制备权利要求1所述化合物的方法，其特征在于：所述化合物如式(Ⅰaa)所示，它包括以下步骤：A process for the preparation of a compound according to claim 1, characterized in that the compound is represented by the formula (Iaa) and comprises the steps of:
    Figure PCTCN2017084924-appb-100010
    Figure PCTCN2017084924-appb-100010
    (1)在碱的存在下，将S1所示化合物与Z-X所示的羧基活化剂反应，制备得到M1所示的活化酯；其中，X表示羧基活化剂在反应中离去的部分，Z表示羧基活化剂在反应中进行取代的部分； (1) reacting a compound represented by S1 with a carboxyl activating agent represented by ZX in the presence of a base to prepare an activated ester represented by M1; wherein X represents a portion of the carboxyl activator which leaves in the reaction, and Z represents a moiety in which a carboxyl activator is substituted in the reaction;
    Figure PCTCN2017084924-appb-100011
    Figure PCTCN2017084924-appb-100011
    (2)以M1所示的活化酯和S2所示的化合物或其盐为原料，制备得到式S3所示化合物；(2) using the activated ester represented by M1 and the compound represented by S2 or a salt thereof as a raw material to prepare a compound represented by the formula S3;
    Figure PCTCN2017084924-appb-100012
    Figure PCTCN2017084924-appb-100012
    (3)将S3所示化合物水解，得到式(Ⅰaa)所示化合物。(3) Hydrolysis of the compound represented by S3 to give a compound of the formula (Iaa).
  20. 根据权利要求19所述的方法，其特征在于：步骤(1)中，所述羧基活化剂选自氯甲酸乙酯、氯甲酸丙酯和氯甲酸异丁酯中的任一种或多种。The method according to claim 19, wherein in the step (1), the carboxyl activating agent is selected from any one or more selected from the group consisting of ethyl chloroformate, propyl chloroformate and isobutyl chloroformate.
  21. 根据权利要求19或20所述的方法，其特征在于：步骤(1)中，所述碱选自N-甲基吗啉、三乙胺和N-乙基二异丙胺中的任一种或多种。The method according to claim 19 or 20, wherein in the step (1), the base is selected from any one of N-methylmorpholine, triethylamine and N-ethyldiisopropylamine or A variety.
  22. 根据权利要求19-21任一项所述的方法，其特征在于：步骤(2)中，S2所示化合物的盐为三氟醋酸盐或盐酸盐。The method according to any one of claims 19 to 21, wherein in the step (2), the salt of the compound represented by S2 is a trifluoroacetate or a hydrochloride.
  23. 权利要求1～18任一项所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐在制备抗肿瘤药物或蛋白酶体抑制剂类药物中的用途。Use of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof or a boric anhydride, for the preparation of an antitumor drug or a proteasome inhibitor.
  24. 根据权利要求23所述的用途，其特征在于：所述蛋白酶体抑制剂类药物是蛋白酶体胰凝乳蛋白酶样蛋白酶抑制剂类药物或者蛋白酶体胱天蛋白酶样蛋白酶抑制剂类药物。The use according to claim 23, wherein the proteasome inhibitor drug is a proteasome chymotrypsin-like protease inhibitor drug or a proteasome caspase-like protease inhibitor drug.
  25. 根据权利要求23所述的用途，其特征在于：所述抗肿瘤药物是预防和/或治疗浆细胞瘤、淋巴瘤、乳腺癌、结肠癌、肺癌、肾癌、***、鼻咽癌的药物。The use according to claim 23, wherein the antitumor drug is a drug for preventing and/or treating plasmacytoma, lymphoma, breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer, nasopharyngeal cancer .
  26. 根据权利要求25所述的用途，其特征在于：所述浆细胞瘤是多发性骨髓瘤；所述淋巴瘤为非霍奇金淋巴瘤，包括套细胞淋巴瘤和滤泡性淋巴瘤。 The use according to claim 25, wherein the plasmacytoma is multiple myeloma; the lymphoma is a non-Hodgkin's lymphoma, including mantle cell lymphoma and follicular lymphoma.
  27. 式(Ⅰ)所示的化合物或其晶型、或其药学上可接受的盐、或其溶剂合物或硼酸酐：a compound of the formula (I) or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof or boric anhydride:
    Figure PCTCN2017084924-appb-100013
    Figure PCTCN2017084924-appb-100013
    其中，环A表示如下结构之一：Among them, ring A represents one of the following structures:
    Figure PCTCN2017084924-appb-100014
    Figure PCTCN2017084924-appb-100014
    其中，R5、R6、R7选自H、F、Cl、Br、I、C1-6烷基、C1-6烷氧基，三氟甲基；Wherein R 5 , R 6 , and R 7 are selected from the group consisting of H, F, Cl, Br, I, C 1-6 alkyl, C 1-6 alkoxy, trifluoromethyl;
    R1选自H、C1-6烷基、C1-6环烷基、烷氧基、苄基；所述C1-6烷基、C1-6环烷基、烷氧基、苄基可以被进一步取代；R 1 is selected from the group consisting of H, C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl; the C 1-6 alkyl, C 1-6 cycloalkyl, alkoxy, benzyl The base can be further substituted;
    R2选自C1-6烷基；R 2 is selected from C 1-6 alkyl;
    R3和R4选自氢，或R3和R4与氧和硼原子共同形成一个任选取代的5-20元环，该环包括0-2个选自氮、氧或硫的环杂原子。R 3 and R 4 are selected from hydrogen, or R 3 and R 4 together with an oxygen and a boron atom form an optionally substituted 5-20 membered ring comprising 0-2 ring molybdenes selected from nitrogen, oxygen or sulfur. atom.
  28. 权利要求27所述的化合物，其特征在于：所述化合物如式(Ⅰa)所示：The compound of claim 27, wherein the compound is as shown in formula (Ia):
    Figure PCTCN2017084924-appb-100015
    Figure PCTCN2017084924-appb-100015
  29. 根据权利要求27或28所述的化合物，其特征在于：R1选自H或苄基。The compound according to claim 27 or 28, wherein R 1 is selected from H or benzyl.
  30. 根据权利要求27-29任一项所述的化合物，其特征在于：R2选自异丙基。The compound according to any one of claims 27-29, wherein R 2 is selected from isopropyl.
  31. 根据权利要求27-30任一项所述的化合物，其特征在于：X1和X2选自羟基，或X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分。The compound according to any one of claims 27 to 30, wherein X 1 and X 2 are selected from a hydroxyl group, or X 1 and X 2 together form a dehydrogenation atom with two functional groups of the borate esterifying agent. part.
  32. 根据权利要求30所述的化合物，其特征在于：X1和X2选自羟基。The compound according to claim 30, wherein X 1 and X 2 are selected from a hydroxyl group.
  33. 根据权利要求30所述的化合物，其特征在于：所述硼酸酯化剂选自含有至少一 个羟基或至少一个羧基的化合物。The compound according to claim 30, wherein said borating agent is selected from the group consisting of at least one a compound having a hydroxyl group or at least one carboxyl group.
  34. 根据权利要求30所述的化合物，其特征在于：所述硼酸酯化剂为包含有N原子的硼酸酯化剂。The compound according to claim 30, wherein the borate esterifying agent is a borate esterifying agent containing N atoms.
  35. 根据权利要求30-33任一项所述的化合物，其特征在于：当X1和X2共同形成与硼酸酯化剂的两个官能团脱去氢原子后的部分时，所形成的环为5-12元环。The compound according to any one of claims 30 to 33, wherein when X 1 and X 2 together form a portion after deactivating a hydrogen atom from two functional groups of the borate esterifying agent, the ring formed is 5-12 yuan ring.
  36. 根据权利要求33所述的化合物，其特征在于：所述硼酸酯化剂选自单糖或多元醇。The compound according to claim 33, wherein the borating agent is selected from the group consisting of monosaccharides or polyhydric alcohols.
  37. 根据权利要求33所述的化合物，其特征在于：所述硼酸酯化剂选自C4-10的饱和硼酸酯化剂，其中，羟基和羧基的数量之和为2～8。The compound according to claim 33, wherein the borating agent is selected from the group consisting of C 4-10 saturated borating agents, wherein the sum of the amounts of the hydroxyl group and the carboxyl group is 2-8.
  38. 根据权利要求33所述的化合物，其特征在于：所述硼酸酯化剂选自甘露醇、柠檬酸、苹果酸、酒石酸、葡萄糖、二乙醇胺、二丙醇胺、三乙醇胺、三丙醇胺、3-((2-羟基乙基)氨基)-1-丙醇、N-甲基二乙醇胺、N-丁基二乙醇胺、2-((2-羟基丙基)氨基)-1-丙醇、二异丙醇胺和N，N-双(2-羟乙基)甘氨酸中的任一种；或，所述硼酸酯化剂任选被C1-6的烷基、C3-C6的环烷基、C2-C6的羧烷基、C1-C6的羟烷基取代。The compound according to claim 33, wherein the borating agent is selected from the group consisting of mannitol, citric acid, malic acid, tartaric acid, glucose, diethanolamine, dipropanolamine, triethanolamine, tripropanolamine. , 3-((2-hydroxyethyl)amino)-1-propanol, N-methyldiethanolamine, N-butyldiethanolamine, 2-((2-hydroxypropyl)amino)-1-propanol Any one of diisopropanolamine and N,N-bis(2-hydroxyethyl)glycine; or the borate esterifying agent is optionally C1-6 alkyl, C3-C6 ring Alkyl, C2-C6 carboxyalkyl, C1-C6 hydroxyalkyl substituted.
  39. 根据权利要求27～38任意一项所述的化合物，其特征在于：所述化合物为如下化合物之一：The compound according to any one of claims 27 to 38, wherein the compound is one of the following compounds:
    Figure PCTCN2017084924-appb-100016
    Figure PCTCN2017084924-appb-100016
    Figure PCTCN2017084924-appb-100017
    Figure PCTCN2017084924-appb-100017
    Figure PCTCN2017084924-appb-100018
    Figure PCTCN2017084924-appb-100018
    Figure PCTCN2017084924-appb-100019
    Figure PCTCN2017084924-appb-100019
    Figure PCTCN2017084924-appb-100020
    Figure PCTCN2017084924-appb-100020
    Figure PCTCN2017084924-appb-100021
    Figure PCTCN2017084924-appb-100021
    Figure PCTCN2017084924-appb-100022
    Figure PCTCN2017084924-appb-100022
    Figure PCTCN2017084924-appb-100023
    Figure PCTCN2017084924-appb-100023
    Figure PCTCN2017084924-appb-100024
    Figure PCTCN2017084924-appb-100024
    Figure PCTCN2017084924-appb-100025
    Figure PCTCN2017084924-appb-100025
    Figure PCTCN2017084924-appb-100026
    Figure PCTCN2017084924-appb-100026
    Figure PCTCN2017084924-appb-100027
    Figure PCTCN2017084924-appb-100027
    Figure PCTCN2017084924-appb-100028
    Figure PCTCN2017084924-appb-100028
    Figure PCTCN2017084924-appb-100029
    Figure PCTCN2017084924-appb-100029
    Figure PCTCN2017084924-appb-100030
    Figure PCTCN2017084924-appb-100030
    Figure PCTCN2017084924-appb-100031
    Figure PCTCN2017084924-appb-100031
    Figure PCTCN2017084924-appb-100032
    Figure PCTCN2017084924-appb-100032
    Figure PCTCN2017084924-appb-100033
    Figure PCTCN2017084924-appb-100033
    Figure PCTCN2017084924-appb-100034
    Figure PCTCN2017084924-appb-100034
    Figure PCTCN2017084924-appb-100035
    Figure PCTCN2017084924-appb-100035
    Figure PCTCN2017084924-appb-100036
    Figure PCTCN2017084924-appb-100036
  40. 一种药物组合物，其特征在于：它是以权利要求1～18、27～39任一项所述的化合物或其药学上可接受的盐或硼酸酐为活性成分，加上药学上可接受的辅料制备而成的制剂。A pharmaceutical composition comprising the compound according to any one of claims 1 to 18, 27 to 39, or a pharmaceutically acceptable salt thereof or boric anhydride as an active ingredient, and pharmaceutically acceptable Preparation of excipients.
  41. 根据权利要求39所述的药物组合物，其特征在于：所述药学上可接受的辅料选自稀释剂、填充剂、着色剂、助流剂、润滑剂、粘合剂、稳定剂、助悬剂或缓冲剂的任一种或多种。The pharmaceutical composition according to claim 39, wherein said pharmaceutically acceptable adjuvant is selected from the group consisting of a diluent, a filler, a coloring agent, a glidant, a lubricant, a binder, a stabilizer, and a suspension. Any one or more of a dose or a buffer.
  42. 根据权利要求40或41所述的药物组合物，其特征在于：所述制剂是片剂、胶囊剂、口服液、注射剂、透皮剂、气雾剂固体制剂、脂质体制剂或缓控释制剂。The pharmaceutical composition according to Claim 40 or 41, wherein the preparation is a tablet, a capsule, an oral solution, an injection, a transdermal agent, an aerosol solid preparation, a liposome preparation or a controlled release preparation.
  43. 一种联合用药物，其特征在于：它含有相同或不同规格单位制剂的用于同时或者分别给药的权利要求1～18、27～39任一项所述化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐与抗肿瘤药物或辅助***的药物，以及药学上可接受的载体。A combination drug comprising the same or different unit preparations for simultaneous or separate administration of the compound according to any one of claims 1 to 18, 27 to 39, or a pharmaceutically acceptable compound thereof a salt, or a prodrug thereof, or a solvate thereof or a boric anhydride, and an antitumor drug or a drug for adjuvant treatment of a tumor, and a pharmaceutically acceptable carrier.
  44. 根据权利要求43所述的联合用药物，其特征在于：所述抗肿瘤药物为细胞毒类药物、激素类药物或者分子靶向药物。The combination drug according to claim 43, wherein the antitumor drug is a cytotoxic drug, a hormonal drug or a molecular targeted drug.
  45. 根据权利要求44所述的联合用药物，其特征在于：所述细胞毒类药物是卡铂、顺铂、伊立替康、紫杉醇、氟脲嘧啶、阿糖胞苷、来拉度胺或维甲酸；所述激素类药物， 如***、氟维司群或他莫昔芬；所述分子靶向药物是厄洛替尼、拉帕替尼或曲妥珠单抗。The combination drug according to claim 44, wherein the cytotoxic drug is carboplatin, cisplatin, irinotecan, paclitaxel, fluorouracil, cytarabine, lenalidomide or retinoic acid. The hormone drug, Such as dexamethasone, fulvestrant or tamoxifen; the molecular targeted drug is erlotinib, lapatinib or trastuzumab.
  46. 根据权利要求44所述的联合用药物，其特征在于：所述辅助***的药物为重组人粒细胞集落刺激因子、***、帕米膦酸二钠或唑来膦酸。The combination drug according to claim 44, wherein the drug for adjuvant treatment of tumor is recombinant human granulocyte colony stimulating factor, erythropoietin, pamidronate disodium or zoledronic acid.
  47. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在10.56、11.68、12.04、13.4、15.51、16.81、17.83、20.33、21.24度处有特征峰。A crystal form of a borate compound characterized in that X-ray powder diffraction of the crystal form has characteristics of 2θ diffraction angles at 10.56, 11.68, 12.04, 13.4, 15.51, 16.81, 17.83, 20.33, 21.24 degrees. peak.
  48. 根据权利要求47所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 47, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100037
    Figure PCTCN2017084924-appb-100037
  49. 根据权利要求47或48所述的晶型，其特征在于：该晶型具有基本如图4所示的X射线粉末衍射图谱。A crystal form according to claim 47 or 48, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in Fig. 4.
  50. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在8.38、11.4、12.1、13.29、18.27、19.62、20.47、23.32、24.74度处有特征峰。A crystal form of a borate compound characterized in that X-ray powder diffraction of the crystal form has characteristics of 2θ diffraction angles at 8.38, 11.4, 12.1, 13.29, 18.27, 19.62, 20.47, 23.32, 24.74 degrees. peak.
  51. 根据权利要求50所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 50, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100038
    Figure PCTCN2017084924-appb-100038
  52. 根据权利要求50或51所述的晶型，其特征在于：该晶型具有基本如图5所示的X射线粉末衍射图谱。A crystal form according to claim 50 or 51, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in Fig. 5.
  53. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在6.19、7.98、10.05、14.73、14.92、17.16、18.8、20.03、21.05度处有特征峰。A crystal form of a borate compound characterized in that X-ray powder diffraction of the crystal form has characteristics of 2θ diffraction angles at 6.19, 7.98, 10.05, 14.73, 14.92, 17.16, 18.8, 20.03, and 21.05 degrees. peak.
  54. 根据权利要求53所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 53, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100039
    Figure PCTCN2017084924-appb-100039
  55. 根据权利要求53或54所述的晶型，其特征在于：该晶型具有基本如图6所示 的X射线粉末衍射图谱。A crystal form according to claim 53 or claim 54 wherein the crystal form has a structure substantially as shown in Fig. 6. X-ray powder diffraction pattern.
  56. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在9.23、12.53、14.18、17.06、20.82、21.46、22.62度处有特征峰。A crystal form of a borate compound, characterized in that in the X-ray powder diffraction of the crystal form, the 2θ diffraction angle has characteristic peaks at 9.23, 12.53, 14.18, 17.06, 20.82, 21.46, 22.62 degrees.
  57. 根据权利要求56所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 56, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100040
    Figure PCTCN2017084924-appb-100040
  58. 根据权利要求56或57所述的晶型，其特征在于：该晶型具有基本如图7所示的X射线粉末衍射图谱。A crystal form according to claim 56 or 57, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in Fig. 7.
  59. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在10.59、11.76、13.19、15.56、17.76、19.5、20.26、21.37、22.2度处有特征峰。A crystal form of a borate compound characterized in that X-ray powder diffraction of the crystal form has characteristics of 2θ diffraction angles at 10.59, 11.76, 13.19, 15.56, 17.76, 19.5, 20.26, 21.37, and 22.2 degrees. peak.
  60. 根据权利要求59所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 59, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100041
    Figure PCTCN2017084924-appb-100041
  61. 根据权利要求59或60所述的晶型，其特征在于：该晶型具有基本如图8所示的X射线粉末衍射图谱。A crystal form according to claim 59 or 60, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in Fig. 8.
  62. 一种硼酸酯类化合物的晶型，其特征在于：所述晶型的X射线粉末衍射中，2θ衍射角度在7.06、10.61、12.24、14.9、17.23、20.21、23.49、26.48度处有特征峰。A crystal form of a borate compound, characterized in that in the X-ray powder diffraction of the crystal form, the 2θ diffraction angle has characteristic peaks at 7.06, 10.61, 12.24, 14.9, 17.23, 20.21, 23.49, and 26.48 degrees.
  63. 根据权利要求62所述的晶型，其特征在于：该晶型X射线粉末衍射中，2θ衍射角度特征峰的相对强度值为：The crystal form according to claim 62, wherein in the X-ray powder diffraction, the relative intensity values of the characteristic peaks of the 2θ diffraction angle are:
    Figure PCTCN2017084924-appb-100042
    Figure PCTCN2017084924-appb-100042
  64. 根据权利要求62或63所述的晶型，其特征在于：该晶型具有基本如图9所示的X射线粉末衍射图谱。A crystal form according to claim 62 or 63, wherein the crystal form has an X-ray powder diffraction pattern substantially as shown in Fig. 9.
  65. 一种肿瘤的预防和/或治疗方法，其特征在于：它是对肿瘤患者给予权利要求1～18、 27～39任一项所述的化合物、或其药学上可接受的盐、或其前药、或其溶剂合物或硼酸酐。A method for preventing and/or treating a tumor, characterized in that it is administered to a tumor patient according to claims 1 to 18, The compound according to any one of items 27 to 39, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof or a boric anhydride.
  66. 根据权利要求65所述的方法，其特征在于：所述肿瘤患者是浆细胞瘤、淋巴瘤、乳腺癌、结肠癌、肺癌、肾癌、***和/或鼻咽癌的患者。The method according to claim 65, wherein the tumor patient is a patient of plasmacytoma, lymphoma, breast cancer, colon cancer, lung cancer, kidney cancer, cervical cancer, and/or nasopharyngeal cancer.
  67. 根据权利要求66所述的方法，其特征在于：所述患者是多发性骨髓瘤患者；所述患者是非霍奇金淋巴瘤患者，进一步地是套细胞淋巴瘤和/或滤泡性淋巴瘤患者。 The method according to claim 66, wherein said patient is a patient with multiple myeloma; said patient is a non-Hodgkin's lymphoma patient, further a patient with mantle cell lymphoma and/or follicular lymphoma .
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