CN105837608B - Proteasome inhibitor and combinations thereof and purposes - Google Patents

Abstract

This application involves proteasome inhibitors and combinations thereof and purposes.The present invention provides the compounds for being suitable for proteasome inhibitor.The method that various diseases are treated the present invention also provides the Pharmaceutical composition containing the compounds of this invention and using the composition.

Description

Proteasome inhibitor and combinations thereof and purposes

The application is the applying date for August in 2007 6 days, and application No. is 201210398741.0, entitled " protease The divisional application of the application for a patent for invention of body inhibitor ".

Technical field

The present invention relates to the boric acid and boric acid ester compound for being suitable for proteasome inhibitor.The present invention also provides containing The Pharmaceutical composition of the compounds of this invention and the method that various diseases are treated using the composition.

Background technology

Boric acid and boric acid ester compound show various pharmaceutically useful bioactivity.God winged (Shenvi) et al. is in the U.S. It is disclosed in patent the 4,499,082nd (1985)：Peptide boric acid (peptide boronic acid) is certain proteolytic enzymes Inhibitor.Carter is received (Kettner) and god to fly (Shenvi) special in U.S. Patent No. No. 5,187,157 (1993), the U.S. A kind of inhibition tryptose is described in sharp No. 5,242,904 (1993) and U.S. Patent No. 5,250,720 (1993) The peptide boric acid of enzyme sample protease.Ke Liman (Kleeman) et al. is disclosed in U.S. Patent No. No. 5,169,841 (1992) Inhibit the end modified peptide boric acids of N- of the effect of feritin.Ken Da (Kinder) et al. is in U.S. Patent No. No. 5,106,948 (1992 Year) in disclose inhibit growth of cancer cells certain boronic acid compounds.Ba Qiaofuqin (Bachovchin) et al. is in WO 07/ The boronic acid compounds for inhibiting fibroblast activation protein are disclosed in 0005991.

Boric acid and boric acid ester compound especially promise to be proteasome (i.e. a kind of responsible most cells internal protein Turnover more catalytic protein enzymes) inhibitor.Adams (Adams) et al. are in U.S. Patent No. No. 5,780,454 (1998) In describe the peptide boric acid ester and boronic acid compounds for being suitable for proteasome inhibitor.This document also describes borate and boric acid Compound reduces muscle protein degradation rate, reduces the activity of NF- κ B in cell, reduce the degradation rate of p53 albumen in cell, inhibit Cyclin (cyclin) degradation, inhibition growth of cancer cells and the use for inhibiting the adherency of NF- κ B dependent cells in cell On the way.The WO 05/016859 and cloth for inspiring confidence in WO 02/096933, the Cha Teji (Chatterjee) of thunder (Furet) et al. et al. take Being disclosed in the WO 05/021558 of pasture Buddhist nun (Bernadini) et al. and WO 06/08660 according to reports there is proteasome to inhibit The other borates and boronic acid compounds of activity.

Nova (Ciechanover) is looked into west, cell (Cell), and 79:13-21 (1994) is disclosed：Proteasome is ubiquitin-egg The proteolysis component of white enzyme body approach, protein is targeted degradation by being combined with multiple ubiquitin molecules in this approach. West is looked into Nova and is also disclosed：Ubiquitin-Proteasome Pathway plays a crucial role in various important physiology courses.Li Weite (Rivett) Et al., journal of biological chemistry (Biochem.J.) 291:It discloses proteasome and shows trypsase, chymotrypsin protein in 1 (1993) Enzyme and peptidyl glutamyl peptidase activity.Form 26S proteasome catalytic cores is 20S proteasomes.Mike mark (McCormack) et al., biochemistry (Biochemistry) 37:7792 (1998) have taught various peptide substrates by 20S albumen Enzyme body is decomposed, and the substrate includes Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC and Z-Leu-Leu- Glu-2NA, wherein Suc are N- succinyl groups, and AMC is 7- amino -4- methylcoumarins and 2NA is 2- naphthylamines.

Proteasome inhibits to embody a kind of important new strategy for the treatment of of cancer.Golden (King) et al., science (Science) 274:1652-1659 (1996) describes Ubiquitin-Proteasome Pathway in terms of cell cycle regulation, growth and metastasis of tumours Important function.Author teaches：Many key regulatory protein are (including cyclin and cell cycle during the cell cycle Protein dependent kinase p21 and p27^KIP1) be temporarily degraded by Ubiquitin-Proteasome Pathway.Cell needs these The orderly of protein degrades to accelerate the cell cycle and carry out mitosis.

In addition, transcriptional control is also required to Ubiquitin-Proteasome Pathway.Paro shellfish draws (Palombella) et al., cell (Cell),78:773 (1994) are taught：The activation of transcription factor NF-KB is inhibited protein I κ B to degrade by proteasome mediation Regulation and control.The gene that NF- κ B are again related to regulation and control immune response and inflammatory reaction plays an important role.Rui De (Read) et al., exempts from Epidemic disease (Immunity) 2:493-506 (1995) is taught：Cell adhesion molecule (such as e-selectin, ICAM-1 and VCAM-1) Expression needs Ubiquitin-Proteasome Pathway.Ze Teer (Zetter), cancer biology research text volume (Seminars in Cancer Biology)4:219-229 (1993) is taught：Cell adhesion molecule is by guiding tumour cell to be glued to internal distant organs site It echos extravasation and is adhered to from vascular system to internal distant organs site and exosmose and be related to turning in internal (in vivo) tumour Shifting and angiogenesis.In addition, shellfish song (Beg) and Baltimore (Baltimore), science (Science) 274:It teaches 782 (1996) Show：NF- κ B are a kind of anti-apoptotic controlling elements, and make cell to environmental pressure and cell to the inhibition of NF- kB activations Toxic agents are more sensitive.

Proteasome inhibitor(bortezomib (bortezomib)；N-2- pyrazinecarbonyl-L- phenylpropyl alcohol ammonia Acid-L-Leu boric acid) it is the first proteasome inhibitor for obtaining regulatory agency and ratifying.Meath Ya De (Mitsiades) etc. People, contemporary drug target (Current Drug Targets), 7:1341 (2006) review so that bortezomib is approved to be used for Treatment has received the clinical research of the multiple myeloma patients of at least one previous therapies.Fei Sheer (Fisher) et al., faces Bed oncology magazine (J.Clin.Oncol.), 30:4867 describe a kind of confirmation bortezomib in relapsed or stubborn jacket cell The multicenter, international II phases of activity in Lymphoma are studied.Shi Jing (Ishii) et al., the anticancer agent in pharmaceutical chemistry (Anti-Cancer Agents in Medicinal Chemistry),7:359 (2007) and Luo Kaluo (Roccaro) et al., Contemporary Pharmaceutical Biotechnology (Curr.Pharm.Biotech.), 7:1341 (2006), which discuss, a variety of facilitates bortezomib to resist The molecular mechanism of tumor promotion.

By being proved above with reference to document, proteasome is the important target spot of therapeutic intervention.Therefore, it is constantly needed to novelty And/or improved proteasome inhibitor.

Invention content

The present invention provides the compound as effective proteasome inhibitor.These compounds are suitable for external (in Vitro) and in vivo (in vivo) protease inhibition body is active, and is particularly suitable for treating various cell proliferation disorders.

The compounds of this invention has logical formula (I)：

Or its pharmaceutically acceptable salt or boric anhydride, wherein：

Z¹And Z²It is each independently hydroxyl, alkoxy, aryloxy group or aralkoxy；Or Z¹And Z²It is formed together and is originated from boron The part of sour complexing agent；And

Ring A is selected from by following formed group：

Boronic acid compounds (the wherein Z of formula (I)¹And Z²Respectively hydroxyl) it is referred to following chemical name.

1. proteasome inhibitor of table

The term " alkyl " used alone or as a part for major part refers to 1 to 12 carbon atom Linear chain or branch chain or cyclic aliphatic group.Term " alkoxy " refers to-O- alkyl.

The term " aryl " that is used alone or as a part for major part and " virtue-" (such as " aralkyl ", " aralkoxy " or " aryloxy alkyl ") refer to the C containing 1 to 3 ring₆To C₁₄Aromatic hydrocarbon, the ring is respectively optionally through taking Generation.Preferably, aryl C_6-10Aryl.Aryl includes but is not limited to phenyl, naphthalene and anthryl." aralkyl " or " aryl alkane Base " is optionally substituted each independently containing the aryl being covalently attached with alkyl, the alkyl or the aryl.Preferably, it is fragrant Alkyl is C_6-10Aryl (C_1-6) alkyl, C_6-10Aryl (C_1-4) alkyl or C_6-10Aryl (C_1-3) alkyl, including but not limited to benzene Methyl, phenethyl and menaphthyl.

Term " being substituted " used herein means that the hydrogen-based of specified portions is replaced by the group of specified substituent, premise It is that metalepsis leads to stable or chemically feasible compound.The non-limiting examples of suitable substituent include C_1-6Alkyl, C_3-8Cycloalkyl, C_1-6Alkyl (C_3-8) cycloalkyl, C_2-8Alkenyl, C_2-8Alkynyl, cyano, amino, C_1-6Alkyl amino, two (C_1-6) alkane Base amino, Benzylamino, benzhydryl amino, nitro, carboxyl, carbonyl (C_1-6) alkoxy, trifluoromethyl, halogen, C_1-6Alkane Oxygroup, C_6-10Aryl, C_6-10Aryl (C_1-6) alkyl, C_6-10Aryl (C_1-6) alkoxy, hydroxyl, C_1-6Alkyl sulfenyl, C_1-6Alkyl is sub- Sulfonyl, C_1-6Alkyl sulphonyl, C_6-10Artyl sulfo, C_6-10Aryl sulfonyl kia, C_6-10Aryl sulfonyl, C_6-10Aryl, C_1-6 Alkyl (C_6-10) aryl and halogenated (C_6-10) aryl.

Phrase " one or more substituent groups " used herein refers to the substituent group of certain amount, is equal to based on can Possible one of the number in bonding site utilized is to the maximum number of substituent group, on condition that meeting aforementioned stable and chemistry The condition of feasibility.Unless otherwise stated, optionally substituted group can may replace position tool in each of the group Substituted base, and substituent group may be the same or different.Term " independently selecting " used herein means for individualized Closing in object specifies a variety of situations of code name that can select identical or different value.

Term " about " is used herein to mean that approximate, about, substantially or left and right.When term " about " is combined with numberical range In use, it to the numerical value above and below by envelope extension by modifying that range.In general, term " about " exists Herein for numerical value of the modification higher or lower than described value 10%.

Term "comprising" used herein means " to include but is not limited to ".

Unless otherwise stated, the structure shown in herein is intended to include differing only in the presence of one or more same positions The compound of plain enriched atoms.For example, in addition to deuterium or tritium displacement hydrogen atom or with¹³C or¹⁴Except C enrichment carbon displacement carbon atoms Compound with structure of the present invention is within the scope of the present invention.

Term " boric acid " used herein refers to containing-B (OH)₂Partial compound.In some embodiments, boric acid Compound can form oligomerization acid anhydrides by making boric acid moieties dehydration.For example, Snyder (Snyder) et al., american chemical It can will (J.Am.Chem.Soc.) 80:Oligomerization aryl boric acid is reported in 3611 (1958).

Term " boric anhydride " used herein refers to be combined simultaneously by two or more boronic acid compounds molecules Lose the compound that one or more hydrones are formed.When mixed with water, boric anhydride compound is discharged free by aquation Boronic acid compounds.In various embodiments, boric anhydride can contain there are two, three, four or more boric acid unit, and can With cyclic annular or linear configuration.The oligomerization boric anhydride non-limiting examples of boronic acid compounds of the present invention are as follows.

In formula (1) and (2), integers of the code name n for 0 to about 10, preferably 0,1,2,3 or 4.In some embodiments, Boric anhydride compound contains the cyclic trimer (" boron oxygen hydrocarbon trimer (boroxine) ") of formula (2), and wherein n is 1.Code name W has There is formula (3)：

Its middle ring A has the value described in above for formula (I).

In some embodiments, at least 80% boric acid in the presence of boric anhydride compound is deposited with single oligomerization anhydride form .In some embodiments, the boric acid of at least 85%, 90%, 95% or 99% in the presence of boric anhydride compound is with single oligomerization Anhydride form exists.In certain preferred embodiments, boric anhydride compound by or substantially by the boron oxygen hydrocarbon trimerization with formula (3) Object forms.

It is preferred that (it can including but not limited to be recrystallized, be lyophilized, be exposed to heat by being exposed to dehydration conditions by corresponding boric acid And/or it is exposed to drier) get off to prepare boric anhydride compound.The non-limiting examples of suitable recrystallization solvent include acetic acid second Ester, dichloromethane, hexane, ether, acetonitrile, ethyl alcohol and its mixture.

In some embodiments, Z¹And Z²The part from boric acid complexing agent is formed together.For the present invention, term " boron Sour complexing agent " refers to any compound at least two functional groups, and at least two functional group can respectively be formed with boron Covalent bond.The non-limiting examples of appropriate functional group include amino and hydroxyl.In some embodiments, at least one functional group is Hydroxyl.Term " part for being originated from boric acid complexing agent " refers to by removing hydrogen atom institute shape from the Liang Ge functional groups of boric acid complexing agent Into part.

Term " borate (boronate ester/boronic ester) " used herein refers to contain-B (Z¹)(Z²) part compound, wherein Z¹Or Z²In it is at least one be alkoxy, aralkoxy or aryloxy group；Or Z¹And Z²Together Form the part for being originated from the boric acid complexing agent at least one hydroxyl.

In some embodiments, Z¹And Z²Being formed to be originated from together has at least two to be connected by least two in chain or ring It can be one of N, S or O that the part of the compound for the hydroxyl that atom is separated, the chain or ring, which contain carbon atom and (optionally), Or multiple hetero atoms, wherein the atom being connected in every case with boron is oxygen atom.

Term used herein " compound at least two hydroxyls " refer to any tool there are two or two with The compound of upper hydroxyl.For the present invention, two hydroxyls are preferably connected by least two adjacent atoms, preferably from about 2 to about 5 Atom, more preferable 2 or 3 adjacent atoms are separated.For convenience's sake, term " dihydroxy compounds " is used to refer to above-mentioned The compound at least two hydroxyls of definition.Therefore, term " dihydroxy compounds " used herein is not intended to Being limited to only tool, there are two the compounds of hydroxyl.It part from the compound at least two hydroxyls can be by appointing in its hydroxyl The oxygen atom of two hydroxyls of meaning is connected with boron.Preferably, boron atom, the oxygen atom that is connected with boron and the original for connecting two oxygen atoms Son forms 5- or 6-membered ring together.

For the present invention, boric acid complexing agent is preferably pharmaceutically acceptable, that is, is suitble to the administration mankind.At some preferably In embodiment, boric acid complexing agent is sugar.Term " sugar " includes any polyhydroxy carbohydrate portions, including monosaccharide and disaccharide, more Sugar, sugar alcohol and amino sugar.In some embodiments, sugar is monosaccharide and disaccharide, sugar alcohol or amino sugar.The nonrestrictive reality of suitable sugar Example includes glucose, sucrose, fructose, trehalose, mannitol, sorbierite, aminoglucose and N-METHYL-ALPHA-L-GLUCOSAMINE.In some embodiments In, sugar is mannitol or sorbierite.Therefore, in sugar is the embodiment of mannitol or sorbierite, Z¹And Z²Formula is formed together C₆H₁₂O₆Part, the oxygen atom of two of which deprotonated hydroxyl group is formed with boron to be covalently attached and forms boric acid ester compound. In certain specific embodiments, Z¹And Z²The part from PEARLITOL 25C is formed together.

In some embodiments, formula (I) compound such as pula Meng Dong (Plamondon) et al. institutes in WO 02/059131 Description is prepared into freeze-dried powder, and the full text of this case is incorporated herein by reference accordingly.In some embodiments, freeze-dried powder is also Include free dihydroxy compounds.Preferably, free dihydroxy compounds and formula (I) compound are with about 0.5:1 to about 100: 1st, 5 are even more preferably about:1 to about 100:Molar ratio in the range of 1 is present in mixture.It is mannitol in dihydroxy compounds In various embodiments, freeze-dried powder includes molar ratio about 10:1 to about 100:1st, about 20:1 to about 100:1 or about 40:1 to about 100:Free mannitol and mannitol borate in the range of 1.

In some embodiments, freeze-dried powder includes mannitol and formula (I) compound, generally without other components.However, Composition can further contain one or more other pharmaceutically acceptable excipient, supporting agent, diluent, filler, salt, delay Electuary, stabilizer, solubilizer and other materials known to industry.The preparation of pharmaceutically acceptable preparation containing these substances is retouched It is set forth in the theory and practice (Remington of (for example) Lei Mingdengshi pharmacy:The Science and Practice of ), Pharmacy the 20th edition, Zhan Naluo (A.Gennaro) is compiled, Donald Lippincott Williams Louis Wilkins publishing company (Lippincott Williams＆Wilkins), 2000 or latest edition in.

It is preferred that it is prepared according to programs of the pula Meng Dong (Plamondon) et al. described in WO 02/059131 comprising formula (I) freeze-dried powder of compound.Therefore, in some embodiments, included to prepare the method for freeze-dried powder：(a) it prepares comprising peptide The aqueous mixture of boric acid and dihydroxy compounds；(b) mixture is lyophilized.

General synthetic method

Formula (I) compound can be prepared by method known to those skilled in the art.See, e.g. Adams (Adams) et al. U.S. Patent No. 5,780,454；The International Patent Publication of skin Gus gill (Pickersgill) et al. Case WO 2005/097809.Shown in the exemplary following surface current journey 1 of route of synthesis.

Flow 1：

Compound i is coupled with the protected glycine of N (ii), N- end deprotections is then carried out, so as to provide compound iii. The example of appropriate protection base (PG) includes but is not limited to acyl protecting groups, such as formoxyl, acetyl group (Ac), succinyl group (Suc) and methoxysuccinyl；And carbamate protective group, such as tertbutyloxycarbonyl (Boc), benzyloxycarbonyl group (Cbz) and Fluorenylmethyloxycarbonyl (Fmoc).Peptide coupling reaction can by make in advance the carboxylic moiety of compound ii be converted to Acibenzolar (for example, O- (n-hydroxysuccinimide) ester), then carried out with compound i processing.It alternatively, can be by making carboxylic acid and peptide coupling reagent It is in contact and generates Acibenzolar on the spot.The example of suitable peptide coupling reagent includes but is not limited to carbodiimide reagent, such as Dicyclohexyl carbodiimide (DCC) or 1- (3- dimethylamino-propyls) -3- ethylcarbodiimines (EDC)；Phosphorus reagent, example Such as three (dimethylamino) phosphorus hexafluorophosphate (BOP) of benzotriazole -1- bases oxygroup；With urea reagent, for example, O- (1H- benzos Triazol-1-yl)-N, N, N', N'- tetramethylurea tetrafluoro boric acid esters (TBTU).

Then, make compound iii and the benzoic acid (ArCO being substituted₂H) coupling provides compound iv.It is above-mentioned to be used for chemical combination The peptide coupling condition of object i and ii coupling is also suitable for making compound iii and ArCO₂H is coupled.Then, boric acid moieties are deprotected Obtain compound v.It is preferred that including boric acid ester compound iv, organic boronic receptor (acceptor), low-carbon alkanol, C_5-8Hydrocarbon is molten Deprotection step is realized by ester interchange (transesterification) in the two-phase mixture of agent and aqueous inorganic acid Suddenly.

Flow 2：

Alternatively, as shown in flow 2, the sequence of coupling reaction can be overturned.Thus, make first the protected glycine of O (vi) with Benzoic acid (the ArCO being substituted₂H it) is coupled, ester hydrolysis reaction then occurs, so as to form compound vii.Then, as closed above In described by flow 1, realization obtains compound v with compound i couplings and boric acid deprotection.

Purposes, allotment and dispensing

The present invention provides the compound as effective proteasome inhibitor.Compound inhibition can be measured in vitro or in vivo The hydrolase polypeptide of proteasome mediation or the ability of protein degradation.

Therefore, in another aspect, the present invention provides a kind of one or more peptide enzyme activity for inhibiting proteasome in cell Property method, it includes make to need the cell that proteasome inhibits and compound as described herein or its is pharmaceutically acceptable Salt, borate or boric anhydride are in contact.

The present invention also provides a kind of methods for inhibiting cell Proliferation, and it includes make the cell of the needs inhibition and this paper institutes The compound stated is in contact.Phrase " inhibit cell Proliferation " is for representing the chemical combination of the present invention compared with the cell not in contact with inhibitor Object inhibits the ability of cell number or cell growth in the cell contacted.Can cell be counted by using cell counter Or (for example, MTT or WST is measured) is measured to assess cell Proliferation by cell viability.Cell in entity growth (for example, Entity tumor or organ) when, it can be measured by using caliper and grow and compare by exposing cell and not in contact with cell The size of growth assesses cell Proliferation.

Preferably, compared with the growth not in contact with cell, the growth for the cell being in contact with inhibitor is delayed by least about 50%.In various embodiments, compared with not in contact with cell, at least about 75% is inhibited by by the cell Proliferation of exposing cell, At least about 90% or at least about 95%.In some embodiments, phrase " inhibiting cell Proliferation " is including compared with not in contact with cell It is reduced by the number of exposing cell.Therefore, the proteasome inhibitor of cell Proliferation is inhibited to can induce quilt in by exposing cell Exposing cell experience growth delay, growth retardation, programmed cell death (i.e. apoptosis) or meronecrosis.

In another aspect, the present invention provides a kind of Pharmaceutical composition, it includes formula (I) compound or its can pharmaceutically connect The salt or boric anhydride and pharmaceutically acceptable supporting agent received.

If using the pharmaceutically acceptable salt of the compounds of this invention in these compositions, the salt preferably originates from Inorganic or organic acid or alkali.Summary about acceptable acid addition salts can be found in (for example) Burger (Berge) et al., Journal of Pharmaceutical Sciences (J.Pharm.Sci.)66:Theory and practice (the Remington of 1-19 (1977) and Lei Mingdengshi pharmacy:The Science And Practice of Pharmacy), the 20th edition, Zhan Naluo (A.Gennaro) is compiled, Donald Lippincott William Swail gold This publishing company (Lippincott Williams＆Wilkins), 2000.

The non-limiting examples of Suitable acid addition salts include following：Acetate, adipate, alginate, asparagine Hydrochlorate, benzoate, benzene sulfonate, disulfate, butyrate, citrate, camphor hydrochlorate, camsilate, pentamethylene third Hydrochlorate, digluconate, lauryl sulfate, ethane sulfonate, fumarate, gluceptate (lucoheptanoate), glycerophosphate, Hemisulphate, enanthate, caproate, hydrochloride, hydrobromate, hydriodate, 2- hydroxyethanesulfonic acids salt, lactate, maleate, methane sulfonates, 2- naphthalene sulfonates, nicotinate, oxalates, pamoic acid Salt, pectate, persulfate, 3- phenyl-propionates, picrate, pivalate, propionate, succinate, tartaric acid Salt, rhodanate, toluene fulfonate and undecylate.

Suitable base addition salts include but is not limited to：Ammonium salt；Alkali metal salt, such as lithium salts, sodium salt and sylvite；Alkaline-earth metal Salt, such as calcium salt and magnesium salts；Other multivalent metal salts, such as zinc salt；The salt formed with organic base, the organic bases are if any two Cyclohexylamine, N- methyl-D-glucosamines, tert-butylamine, ethylenediamine, ethanol amine and choline；And with amino acids formed salt, the ammonia Base acid for example has arginine, lysine and such.In some embodiments, pharmaceutically acceptable salt is formula (I) boric acid The base addition salts of compound, wherein Z¹And Z²It is hydroxyl.

Term " pharmaceutically acceptable supporting agent " herein for refer to and receive individual (preferably mammal, it is more excellent Be selected as the mankind) it is compatible and be suitable for target location delivering active agents without make agent of activity terminate substance.To reach work The desired use of property agent, toxicity relevant with supporting agent or ill effect (if present) preferably compare phase with rational risk/benefit When.

Term " supporting agent ", " adjuvant " or " mediator " is used interchangeably herein and including being suitable for wanted particular dosage form It is arbitrary and all solvents, diluent and other liquid vehicles, dispersion or suspension aids, surfactant, pH adjusting agent, isotonic Agent, thickener or emulsifier, preservative, solid binder, lubricant and such.The theory and practice of Lei Mingdengshi pharmacy (Remington:The Science and Practice of Pharmacy), the 20th edition, Zhan Naluo (A.Gennaro) is compiled, Donald Lippincott Williams Louis Wilkins publishing company (Lippincott Williams＆Wilkins), 2000 disclose use In the various supporting agents and its known technology of preparing of allocating pharmaceutically acceptable composition.Unless any conventional carrier medium and this hair Bright compound is incompatible (such as due to generating any undesirable biological effect or not so with harmful way and pharmaceutically acceptable group Any other component for closing object interacts), otherwise its use is encompassed by the scope of the present invention.It may act as pharmaceutically Some examples of the substance of acceptable supporting agent include but is not limited to ion-exchanger, aluminium oxide, aluminum stearate, lecithin, blood Albumin (such as human serum albumins)；Buffer substance, such as phosphate, carbonate, magnesium hydroxide and aluminium hydroxide, sweet ammonia Acid, sorbic acid or potassium sorbate；The partial glyceride mixture of saturated vegetable fatty acid；Water, apyrogeneity matter water, salt or electrolyte, Such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salt；Silica gel, magnesium trisilicate, polyvinylpyrrolidine Ketone, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, lanolin；Sugar, such as lactose, glucose, sucrose and sweet Reveal alcohol；Starch, such as cornstarch and potato starch；Cellulose and its derivative, such as sodium carboxymethylcellulose, ethyl are fine Dimension element and cellulose acetate；Tragacanth gum powder, malt, gelatin, talcum, excipient, such as cocoa butter and suppository wax；Oils, such as Peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil；Glycols, such as propylene glycol and poly- second two Alcohol；Esters, such as ethyl oleate and ethyl laurate；Agar, alginic acid, isotonic saline solution, Ringer's solution (Ringer's solution)；Alcohols, such as ethyl alcohol, isopropanol, hexadecanol and glycerine；Cyclodextrin, such as hydroxypropylβ-cyclodextrin and sulphur butyl Ether beta-cyclodextrin；Lubricant, such as NaLS and magnesium stearate；Petroleum hydrocarbon, such as mineral oil and vaseline (petrolatum).According to the judgement of fitter, colorant, releasing agent, coating agent, sweetener, flavoring agent and aromatizing agent, anti-corrosion Agent and antioxidant also are present in composition.

The Pharmaceutical composition of the present invention can be manufactured by method known to industry, such as conventional granulation, mixing, dissolving, packet Capsule, freeze-drying or emulsifying process etc..Composition can be made as various forms, including granule, sediment or particle, powder (including cold Freeze dried powder, rotary drying powder or spray-dried powders, amorphous powder), tablet, capsule, syrup, suppository, injection, Lotion, elixir, suspension or solution.

It is that the composition of the present invention is adjusted to mammal (the preferably mankind) dispensing according to a preferred embodiment Match.The present invention these Pharmaceutical compositions can take orally, in a manner of not enteral, by nebulizer, with part, per rectum, Intranasal, buccal, Via vagina mode or via implanted reservoir administration.Term " not enteral " used herein is including skin Under, intravenous, intramuscular, intra-articular, intrasynovial, breastbone is interior, intrathecal, liver is interior, intralesional and intracranial injection or infusion techniques.It is excellent Selection of land, intravenously or subcutaneously composition oral, administration.The preparation of the present invention is designed to short-acting, quick-release or long-acting.More into One step, compound can part rather than systemic manner administration, such as at tumor sites (such as passing through injection) administration.

Liquid dosage forms for oral administration includes but is not limited to pharmaceutically acceptable lotion, microemulsion, solution, suspension Liquid, syrup and elixir.In addition to the active compound, liquid dosage form is also containing the usually used inert diluent of industry, such as Water or other solvents, solubilizer and emulsifier, such as ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl Benzoate Ester, propylene glycol, 1,3 butylene glycol, cyclodextrin, dimethylformamide, oils (in particular cottonseed oil, arachis oil, corn oil, embryo Bud oil, olive oil, castor oil and sesame oil), glycerine, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan carboxylic esters and it is mixed Close object.Other than inert diluent, Orally administered composition may also comprise adjuvant, for example, wetting agent, emulsifier and suspending agent, sweet taste Agent, flavoring agent and aromatizing agent.

Can injectable formulation, such as nothing be allocated using suitable dispersant or wetting agent and suspending agent according to known technology The aqueous or oleagenous suspension of bacterium injectable.Sterile injectable preparation can also be in nontoxic, not enteral acceptable diluent or Sterile injectable solution, suspension or lotion in solvent, such as the solution in 1,3-BDO.In acceptable mediator and It is workable to have water, Ringer's solution, U.S.P. and isotonic sodium chlorrde solution in solvent.In addition, it is not waved conventionally with sterile Hair oil is as solvent or suspension media.For this purpose, any mild expressed oi can be used, including synthesis monoglyceride and two glycerine Ester.In addition, aliphatic acid, such as oleic acid are used in the preparation of injection.It can sterilize to injectable formulation, for example, passing through It is filtered via bacteria filter or by being incorporated to fungicide in aseptic solid composite form, the composition can be molten before use Solution in or be scattered in sterile water or other sterile injectable mediums.The formulated composition for not enteral dispensing can pass through group Note is injected or can be by continuous infusion come administration by periodically injecting.

Include capsule, tablet, pill, powder and granule for oral solid dosage forms.It, will in these solid dosage forms Reactive compound and at least one inertia, pharmaceutically acceptable excipient or supporting agent (such as sodium citrate or Dicalcium Phosphate) And/or a) filler or incremental agent (such as starch, lactose, sucrose, glucose, mannitol and silicic acid), b) adhesive (such as carboxylic Methylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum), c) moisturizer (such as glycerine), d) Disintegrant (such as aga agar, calcium carbonate, potato or tapioca, alginic acid, certain silicates and sodium carbonate), e) resistance Solvent (such as paraffin), f) sorbefacient (such as quaternary ammonium compound), g) wetting agent (such as cetanol and glycerol monostearate Ester), h) absorbent (such as kaolin and bentonite) and i) lubricant (such as gather by talcum, calcium stearate, magnesium stearate, solid-state Ethylene glycol, NaLS and the mixing of its mixture.In the case of capsule, tablet and pill, the dosage form also may include Buffer, such as phosphate or carbonate.

Excipient (for example, lactose or toffee and high molecular weight polyethylene glycol and such) also can be used by Similarity Class The solid composite of type is used as the filler in soft filling and hard-filled gelatin capsule.Tablet, dragee, capsule, pill and The solid dosage forms of granula can be prepared as having well known other packets in coating and shell, such as enteric coating and modification of drug technology Clothing.It optionally contains opacifier, and can also be only or preferentially in enteron aisle specific part optionally with delayed mode The composition of discharge active component.The example of workable embedding composition includes polymeric material and wax.Excipient also can be used The solid composite of similar type is used as soft fill out by (for example, lactose or toffee and high molecular weight polyethylene glycol and such) It fills and the filler in hard-filled gelatin capsule.

Reactive compound can be in also microencapsulation form together with one or more above-mentioned excipient.Tablet, dragee, glue The solid dosage forms of capsule, pill and granule can be prepared as having coating and shell, such as enteric coating, controlled release coat and modification of drug Well known other coatings in technology.In these solid dosage forms, can by reactive compound and at least one inert diluent (such as Sucrose, lactose or starch) mixing.Such as in normal operation, these dosage forms also may include other objects besides inert diluents Matter, such as tableting lubricant and other compression aids, such as magnesium stearate and microcrystalline cellulose.In capsule, tablet and pill In the case of, the dosage form also may include buffer.It optionally contains opacifier, and can also be only or preferentially in enteron aisle Certain specific part is optionally with the composition of delayed mode discharge active component.The example of workable embedding composition includes poly- Close substance and wax.

Dosage form for part or percutaneous administration the compounds of this invention includes ointment, paste, emulsifiable paste, lotion, gel, powder End, solution, spray, inhalant or patch.Aseptically, by active component and pharmaceutically acceptable supporting agent and any Required preservative or buffer mixes as required.Eye-drops preparations, auristilla and eye drops be also covered by the scope of the present invention it It is interior.In addition, present invention also contemplates that using transdermal patch, have and provide compound in the additional benefit of controlled delivery in body. These dosage forms can be made by the way that compound is made to be dissolved or dispersed in suitable media.Absorption enhancer can also be used come increase Close the flux that object passes through skin.It can be by providing rate controlling membranes or by the way that compound is scattered in polymer substrate or gel Carry out speed control.

In some embodiments, the intravenous administration of formula (I) compound.In these embodiments, formula (I) compound is (wherein Z¹And Z²The part from boric acid complexing agent is formed together) form of above-mentioned freeze-dried powder can be prepared into.It is preferred that it is suitable for by addition The aqueous solvent of drug administration compounds freeze-dried powder.The example of suitable complex solvent includes but is not limited to water, brine and phosphoric acid Salt buffer brine (PBS).Preferably, freeze-dried powder is compounded using standard (0.9%) brine.After compounding, in boric acid ester compound It establishes and balances between corresponding free boronic acid compounds.In some embodiments, balance is quickly reached after aqueous medium is added, Such as within 10-15 minutes.The relative concentration of existing borate and boric acid depends on all multi-parameters during balance, such as molten The ratio of boric acid complexing agent and boric acid ester compound present in liquid pH value, temperature, the property of boric acid complexing agent and freeze-dried powder.

The Pharmaceutical composition of the present invention is preferred to administration with proteasome mediated illness or in suffering from protease Patient among body mediated conditions or the risk of experience proteasome mediated illness recurrence and allocate.It is used herein Term " patient " means animal, preferably mammal, the more preferably mankind.The preferred Pharmaceutical composition of the present invention is formulated For those compositions of oral administration medicine supplying, Intravenous administration or subcutaneous administration.However, the chemical combination of the present invention containing therapeutically effective amount Any of the above-described kind of dosage form of object is all completely within the scope of routine test, and therefore, completely within the scope of the present invention. In some embodiments, Pharmaceutical composition of the invention can further contain another therapeutic agent.In some embodiments, it is described another Therapeutic agent is usually to the therapeutic agent with treated disease or patient's administration of symptom.

" therapeutically effective amount " means to be enough to cause proteasome activity or proteasome mediated disorder severity that can examine The amount of the reduction measured.The amount of required proteasome inhibitor should depend on inhibitor to give cell category validity and Treat the time span needed for the illness.It should also be clear that for any particular patient, specific dosage and therapeutic scheme should Depending on various factors, age of activity, patient including used particular compound, weight, general health, gender and Diet, time of administration, excretion rate, pharmaceutical composition, the judgement for the treatment of physician and treated specified disease severity.This hair The amount of other therapeutic agents in the presence of bright composition should usually be no more than and just contain this therapeutic agent as sole active Usually by the amount of administration for composition.Preferably, the range of the amount of other therapeutic agents can be to contain this therapeutic agent as only About the 50% to about 100% of usually existing amount in the composition of one therapeutically active agent.

In another aspect, the present invention provides a kind for the treatment of with proteasome mediated illness or in suffering from protease The method of patient among body mediated conditions or the risk of the proteasome mediated illness recurrence of experience.It is used herein Term " proteasome mediated illness " is including as caused by proteasome expression or the increase of activity or being characterized in that protease Body surface reaches or the increase of activity or needs any illness, disease or the symptom of proteasome activity." proteasome mediates term Venereal disease disease " further includes helpful any illness, disease or the symptom of the inhibition to proteasome activity.

For example, the compounds of this invention and Pharmaceutical composition are suitable for treatment via the egg regulated and controled by proteasome activity White matter is (for example, NF κ B, p27^Kip、p21^WAF/CIP1, p53) illness that is mediated.Associated disease includes inflammatory conditions (for example, class wind Wet arthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, skin disease are (for example, atopy skin Scorching, psoriasis)), blood vessel proliferative illness (for example, atherosclerosis, restenosis), eye proliferative disorders are (for example, sugar Urinate characteristic of disease retinopathy), benign proliferative illness (for example, hemangioma), autoimmune disease is (for example, multiple sclerosis, group Knit and organ rejection response) and with infecting relevant inflammation (for example, immune response), Neurodegenerative conditions (for example, Ah Er Cihai Mo's diseases (Alzheimer's disease), Parkinson's disease (Parkinson's disease), motor neuron Disease, neurogenic pain, triplet repeat illness (triplet repeat disorder), astrocytoma and by wine Nerve degeneration caused by essence hepatopathy), ischemia injury (for example, apoplexy) and cachexia be (for example, with various physiological and pathological shapes State the degradation of acceleration muscle protein (for example, neurotrosis, go on a hunger strike, have a fever, acid poisoning, HIV infection, cancer and certain endocrines Disease)).

The compounds of this invention and Pharmaceutical composition are especially suitable for treating cancer.Term " cancer " used herein is Refer to a kind of cell conditions, it is characterized in that uncontrolled or imbalance cell Proliferation, the inadequately cell differentiation reduced, invasion week It encloses the ability of tissue and/or the ability of new growth is formed in ectopic sites.Term " cancer " includes but is not limited to entity and swells Knurl and neoplastic hematologic disorder.Term " cancer " covers the disease of skin, tissue, organ, bone, cartilage, blood and blood vessel.Term " cancer " Further include primary carcinoma and metastatic carcinoma.

It can include cancer of pancreas with the non-limiting examples of entity tumor that revealed proteasome inhibitor is treated；Bladder Cancer；Colorectal cancer；Breast cancer, including metastatic breast cancer；Prostate cancer swashs including androgen-dependent prostate cancer and hero Plain dependent/non-dependent prostate cancer；Kidney, including (for example) metastatic renal cell cancer；Hepatocellular carcinoma；Lung cancer, including (for example) non-small Cell lung cancer (NSCLC), bronchioalveolar carcinoma (BAC) and adenocarcinoma of lung；Oophoroma, including (for example) progressive epithelioma or original Hair property peritoneal cancer；Cervix cancer；Gastric cancer；Cancer of the esophagus；Incidence cancer, including (for example) head and neck squamous cell carcinoma；Melanoma； Neuroendocrine carcinoma, including metastatic neuroendocrine tumor；Brain tumor, including (for example) glioma, tail off branch Spongiocytoma, into becoming astrocytoma between Human glioblastoma and adult；Osteocarcinoma；And soft tissue sarcoma.

It can include acute bone with the non-limiting examples of hematologic malignancies that revealed proteasome inhibitor is treated Marrow leukaemia (AML)；Chronic myelogenous leukemia (CML), including accelerated period CML and rapid change period CML (CML-BP)；Acute lymphoblastic Blast cell leukemia (ALL)；Chronic lymphocytic leukemia (CLL)；Hodgkin's disease (Hodgkin's disease, HD)；It is non-suddenly Strange gold lymthoma (NHL), including follicular lymphoma and lymphoma mantle cell；B cell lymphoma；T cell lymphoma；It is multiple Myeloma (MM)；Macroglobulinemia Waldenstron (Waldenstrom's macroglobulinemia)；Development of bone marrow Bad syndrome (MDS), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), intractable Anaemia is with the RAEB (RAEB-T) in initial cell excessive (RAEB) and transformation；With bone marrow proliferative syndrome.

In some embodiments, the compounds of this invention or composition are for treating with cancer or in suffering from cancer or warp The patient among the risk of cancer return is gone through, the cancer is selected from the group being made of Huppert's disease and lymphoma mantle cell Group.

In some embodiments, proteasome inhibitor of the invention and another therapeutic agent administration.It is described another to control Agent is treated also to can inhibit proteasome or can work with different mechanisms.In some embodiments, another therapeutic agent is usual To the therapeutic agent with treated disease or patient's administration of symptom.The proteasome inhibitor of the present invention can be with another therapeutic agent Together with single formulation or with separate dosage forms administration.When with separate dosage forms administration, another therapeutic agent can be in this hair of administration Administration prior to, concurrently with, or after bright proteasome inhibitor.

In some embodiments, the proteasome inhibitor of formula (I) and anti-cancer agent in conjunction administration.Art used herein Language " anticancer agent " refers to any medicament of the purpose for treating cancer to cancered individual administration.

The non-limiting examples of DNA damage chemotherapeutics include topoisomerase I inhibitor (for example, Irinotecan (irinotecan), topotecan (topotecan), camptothecine (camptothecin) and its analog or metabolin and Ah mould Plain (doxorubicin))；Topoisomerase II inhibitors are (for example, etoposide (etoposide), Teniposide (teniposide) and daunorubicin (daunorubicin))；Alkylating agent is (for example, melphalan (melphalan), benzenebutanoic acid Mustargen (chlorambucil), busulfan (busulfan), thiotepa (thiotepa), ifosfamide (ifosfamide), Carmustine (carmustine), lomustine (lomustine), Semustine (semustine), streptozotocin (streptozocin), dacarbazine (decarbazine), methotrexate (MTX) (methotrexate), mitomycin C (mitomycin C) and cyclophosphamide (cyclophosphamide))；DNA intercalators are (for example, cis-platinum (cisplatin), Austria Husky profit platinum (oxaliplatin) and carboplatin (carboplatin))；DNA intercalators and free-radical generating agent, such as bleomycin (bleomycin)；And nucleoside mimics thing is (for example, 5 FU 5 fluorouracil (5-fluorouracil), capecitabine (capecitibine), gemcitabine (gemcitabine), fludarabine (fludarabine), cytarabine (cytarabine), purinethol (mercaptopurine), thioguanine (thioguanine), Pentostatin (pentostatin) and hydroxycarbamide (hydroxyurea)).

The chemotherapeutics for destroying cellular replication includes：Taxol (paclitaxel), Docetaxel (docetaxel) and phase Close analog；Vincristine (vincristine), vincaleukoblastinum (vinblastin) and related analogs；Thalidomide (thalidomide), lenalidomide (lenalidomide) and related analogs (for example, CC-5013 and CC-4047)；Albumen Tyrosine kinase inhibitor (such as imatinib mesylate (imatinib mesylate) and Gefitinib (gefitinib))； Proteasome inhibitor (for example, bortezomib)；NF- kB inhibitors, including I kappa b kinase inhibitor；With being overexpressed in cancer Protein combine and and then lower cellular replication antibody (for example, Herceptin (trastuzumab), Rituximab (rituximab), Cetuximab (cetuximab) and bevacizumab (bevacizumab))；With it is known in cancer it is upper The protein or other inhibitor of enzyme adjusted, be overexpressed or activated, the inhibition can lower cellular replication.

In order to be more fully understood by the present invention, following preparating example and test case are stated.These embodiments illustrate how It prepares or tests specific compound, but be not construed as limiting the scope of the invention in any way.

Specific embodiment

Example

Abbreviation

DCM dichloromethane

DIEA diisopropylethylamine

EDCI N- (3- dimethylaminopropyls)-N'- ethyl-carbodiimide hydrochlorides

EtOAc ethyl acetate

H hours

HPLC high performance liquid chromatography

TBTU o- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid esters

HOBt I-hydroxybenzotriazole hydrates

LCMS liquid chromatography mass

Min minutes

The residence time of tr diode array spectrum

Analytic type LC-MS methods

Using following gradient spectral measurement is carried out on Symmetry C18-3.5 μm -4.6 × 50mm columns：

Solvent A：2% isopropanol, 98% water, 10mM NH₄OAc

Solvent B：75% acetonitrile, 25% methanol, 10mM NH₄OAc

Time [min]	Flow velocity [mL/min]	The percentage of solvent B
			0.0	1.0	5.0
3.5	1.0	100.0
			4.9	1.0	100.0
5.0	1.0	5.0

Example 1：[(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boron The synthesis of sour 20D- mannitol (I-1)

Step 1：[(2,3- difluoro benzoyls) amino] methyl acetate

Glycine methyl ester salt is added into tetrahydrofuran (5mL) solution of 2,3- difluoro-benzoic acids (0.190g, 1.2mmol) Hydrochlorate (0.150g, 1.2mmol), HOBt (0.162g, 1.2mmol), DIEA (0.209mL, 1.2mmol) and EDCI (0.252g, 1.3mmol).It is stirred overnight reaction mixture.It is reacted with saturated sodium bicarbonate solution stopped reaction mixture, and makes product point Assigned in DCM.Organic layer is detached, solvent is then removed, obtains [(2,3- difluoro benzoyl) amino] methyl acetate, without Purifying is i.e. for next step.

Step 2：[(2,3- difluoro benzoyls) amino] acetic acid

Add into methanol (7mL) solution of [(2,3- difluoro benzoyls) amino] methyl acetate (0.250g, 1.1mmol) Add lithium hydroxide (0.053g, 2.2mmol) and water (3mL).It is stirred overnight reaction mixture.Mixture is diluted with water (20mL) And it is acidified with 1N HCl (5mL).Make product in DCM/ methanol (4:1) it is distributed in.Organic layer is dried, and remove molten with sodium sulphate Agent obtains [(2,3- difluoro benzoyl) amino] acetic acid, without further purification i.e. for next step.

Step 3：Bis- fluoro- N- of 2,3- [2- ({ (1R) -3- methyl-1s-[(3aR, 4R, 6R, 7aS) -3a, 5,5- trimethyls six Hydrogen -4,6- methylene -1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide

It is molten to the dimethylformamide (10mL) of [(2,3- difluoro benzoyls) amino] acetic acid (0.205g, 0.95mmol) TBTU (0.337g, 1.0mmol) and (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyls six are added in liquid Hydrogen -4,6- methylene -1,3,2- benzo dioxy borine -2- bases] butyl- 1- amine trifluoroacetate (0.362g, 0.95mmol).Make Mixture is cooled to 0 DEG C, and DIEA (0.498mL, 2.9mmol) is added dropwise.Reaction mixture is made to be warming up to room temperature and is stirred overnight. With 100mL water stopped reactions, and product is made to be allocated in DCM.Organic layer is dried, and remove solvent with sodium sulphate, obtain 2,3- Two fluoro- N- [2- ((1R) -3- methyl-1s-[(3aR, 4R, 6R, 7aS) -3a, 5,5- trimethyl hexahydro -4,6- methylene -1,3, 2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide.

Step 4：[(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid

To bis- fluoro- N- of 2,3- [2- ((1R) -3- methyl-1s-[(3aR, 4R, 6R, 7aS) -3a, 5,5- trimethyl hexahydro -4, 6- methylene -1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide (0.536g, Methanol 1.2mmol)/1N HCl (1:1) in (1.5mL) solution add enanthol (1mL) and boric acid isobutyl ester (0.207g, 2.0mmol).It is stirred overnight reaction mixture.By enanthol layer separation and concentrate methanol/HCl layers.It is thick by reversed-phase HPLC purifying Product obtains [(1R) -1- ({ [(2,3- difluoro benzoyl) amino] acetyl group } amino) -3- methyl butyls] boric acid.

Step 5：[(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boron Sour 20D- mannitol (I-1)

To [(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid In the solution of (0.085g, 0.26mmol) in the tert-butyl alcohol (2mL) and water (5mL) add PEARLITOL 25C (0.943g, 5.2mmol).Warm soloution is simultaneously allowed to stirring until whole dissolvings.Then solution is freezed and solvent is removed by desivac, obtained To [(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid 20D- mannitol (I-1) (0.98g, 97%).

Example 2：[(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid The synthesis of 20D- mannitol (I-5)

Step 1：[2- ({ (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4,6- methylenes Base -1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] t-butyl carbamate

Through 15 minutes to (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4,6- methylenes Base -1,3,2- benzo dioxy borine -2- bases] butyl- 1- amine trifluoroacetate (4.9g, 10.8mmol), N-- (tertiary butyloxycarbonyls Base) it is added dropwise in the mixture of glycine (1.98g, 11.3mmol) and TBTU (3.81g, 11.9mmol) in DCM (100mL) DCM (25mL) solution of DIEA (5.64mL, 32.4mmol).Reaction mixture is made to be stirred overnight and concentrate.

By column chromatography purification of crude product, obtain [2- ((1R) -3- methyl-1s-[and (3aS, 4S, 6S, 7aR) -3a, 5,5- Trimethyl hexahydro -4,6- methylene -1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] carbamic acid The tert-butyl ester (2.5g, 55%).

Step 2：2- amino-N- { (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydros -4,6- Methylene -1,3,2- benzo dioxy borine -2- bases] butyl } acetamide

To [2- ((1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4,6- methylene -1, 3,2- benzo dioxy borine -2- bases] butyl amino) -2- oxoethyls] and t-butyl carbamate (2.5g, 5.9mmol) DCM The 4M HCl in dioxane (5.9mL) are added in (15mL) solution.Reaction mixture is made to stir 2 hours and is concentrated, obtains 2- ammonia Base-N- { (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4,6- methylene -1,3,2- benzos Dioxy borine -2- bases] butyl } acetamide, without further purification i.e. for next step.

Step 3：The bromo- N- of 2- [2- ((1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4, 6- methylene -1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide

Into DCM (2.25mL) solution of 2- bromobenzoic acids (0.124g, 0.62mmol) add EDCI (0.119g, 0.62mmol), HOBt (0.084g, 0.62mmol), N-methylmorpholine (0.185mL, 1.68mmol) and 2- amino-N- (1R)- 3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydro -4,6- methylene -1,3,2- benzo dioxy borines -2- Base] butyl } acetamide (0.2g, 0.56mmol).Reaction mixture is made to stir 2 hours and is concentrated.By residue diluted with water simultaneously It is extracted with EtOAc.Organic solution is merged, is washed with brine, through MgSO₄It is dried, filtered and concentrated.It is thick by column chromatography purifying Product obtains the bromo- N- of 2- [2- ({ (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydros -4,6- Asias Methyl-1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide (0.22g, 78%).

Step 4：[(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid

To the bromo- N- of 2- [2- ({ (1R) -3- methyl-1s-[(3aS, 4S, 6S, 7aR) -3a, 5,5- trimethyl hexahydros -4,6- Asias Methyl-1,3,2- benzo dioxy borine -2- bases] butyl } amino) -2- oxoethyls] benzamide (0.220g, 0.44mmol) In methanol/hexane (1:1) in the solution in (2.2mL) add 1N HCl (1mL, 1.0mmol) and boric acid isobutyl ester (0.078g, 0.76mmol).Reaction mixture is stirred overnight.Concentrated reaction mixture is simultaneously purified by reversed-phase HPLC, obtains [(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid (0.119g, 73%).

Step 5：[(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid 20D- mannitol (I-5)

To [(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid (0.103g, PEARLITOL 25C (1.01g, 5.5mmol) 0.28mmol) is added in the solution in the tert-butyl alcohol (9mL) and water (15mL).It warms molten Liquid is simultaneously allowed to stirring until whole dissolvings.Then solution is freezed and solvent is removed by desivac, obtain [(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid 20D- mannitol (I-5) (0.92g, 84%).

In the method similar with example 1 or 2 by the compound in appropriate initial substance preparation following table.

Example 2：20S Proteasome assays

25 μ L are added containing someone in the 1 μ L test compounds that 37 DEG C are dissolved in DMSO into 384 hole black microtiter plates Class PA28 activators (Boston Biochem, final concentration 12nM) and Ac-WLA-AMC (5 selective substrates of β) (15 μM of final concentration) Measure buffer solution, then add 25 μ L again at 37 DEG C and contain the mankind 20S proteasome (Boston Biochem, final concentration Measure buffer solution 0.25nM).It measures buffer solution to be made of 20mM HEPES, 0.5mM EDTA and 0.01%BSA, pH value is 7.4.With BMG Galaxy plate reader (37 DEG C, 380nm excitations, 460nm emits, 20 gains) tracking reaction.Inhibit relative to 0% (DMSO) and 100% inhibits (10 μM of bortezomibs) control group to calculate suppression percentage.

When measuring test with this, compound I-1 to I-21 shows IC₅₀Value is less than 50nM.

Example 3：Antiproliferative measures

100 μ L are supplemented with to the appropriate cell culture medium of 10% fetal calf serum (hero company (Invitrogen)) HCT-116 (1000) or other tumor cell inoculations in (McCoy's 5A for HCT-116, hero company) is thin in 96 holes In the hole of born of the same parents' culture plate, and cultivated overnight at 37 DEG C.Test compound is added in hole and cultivates plate 96 hours at 37 DEG C. MTT or WST reagents (10 μ L, Roche Holding Ag (Roche)) are added into each hole, and are cultivated 4 hours at 37 DEG C as described in manufacturer. For MTT, stay overnight be metabolized dye solubilization according to manufacturer specification (Roche Holding Ag).Spectrophotometric is used for MTT Meter (Molecular Devices (Molecular Devices)) reads each hole under 595nm (dominant wavelength) and 690nm (reference wavelength) Optical density, then read the optical density in each hole at 450 nm for WST.For MTT, ginseng is subtracted from the OD value of dominant wavelength Specific optical density value.Suppression percentage is calculated using the value for the DMSO control groups for being set as 100%.

Example 4：In-vivo tumour efficacy models

Using 26 No. 3/8 needles of 1mL (Bi Di companies (Becton Dickinson) Ref#309625) by 100 μ L RPMI- HCT-116 (2-5 × 10 of fresh separated in 1640 culture mediums (Sigma-Aldrich (Sigma-Aldrich))⁶It is a) or Other tumour cell aseptic injections are to female CD-1 nude mouses (5-8 week old, company of Charles River (Charles River)) the right back of the body In the subcutaneous space of side.Alternatively, some heteroplastic transplantation models need the continuous passage of tumor fragment.In such cases, via No. 13 trochars (Popper's Sen Si companies (Popper＆Sons) 7927) are by small fragment (the about 1mm of tumor tissues³) through subcutaneous It is implanted to the right back of the body through anaesthetizing (3-5% isofluranes/oxygen mixture) C.B-17/SCID mouse (5-8 week old, company of Charles River) Side.Since after inoculation the 7th day, tumour is measured 2 times a week using vernier caliper.Use standardization program (0.5 × (length × wide Degree²)) calculate gross tumor volume.When gross tumor volume reaches about 200mm³When, mouse is randomly divided into several treatment groups and starts to receive Drug therapy.Each experiment is determined based on the result previously obtained from pharmacokinetics/pharmacodynamics and maximum tolerated dose research Dosage and time-histories.Control group will receive the mediator without any drug.In general, with various dose and time-histories by testization Object (100-200 μ L) is closed via vein (No. 27 needles), oral (No. 20 tube feed needles) or subcutaneous (No. 27 needles) approach administration.Weekly two Secondary measurement tumor size and weight, and when control tumor reaches about 2000mm³When terminate research.

Although the present invention has been described in detail for clarity and the purpose understood above, these particular implementations Example should be considered as illustrative rather than be restrictive.One of ordinary skill in the art are after this disclosure is read it will be appreciated that not taking off In the case of from true scope of the present invention can to form and details, various changes can be made, true scope of the invention should be by accompanying right Claim rather than specific embodiment define.

Patent and scientific and technical literature mentioned herein provide available knowledge for one of ordinary skill in the art.Unless Defined otherwise, otherwise all scientific and technical terminologies used herein have is generally understood phase with those skilled in the art of the invention Same meaning.Granted patent, application case and bibliography cited herein is incorporated herein by reference, and is drawn With degree just as specifically and individually instruction by each granted patent, application case and bibliography by reference simultaneously Enter general.In the case of contradiction, it should be subject to this disclosure (including definition).

Claims (9)

1. a kind of formula (I) compound,

Or the purposes of its pharmaceutically acceptable salt or boric anhydride in the drug for protease inhibition body activity is prepared, In：

Z¹And Z²For hydroxyl；Or Z¹And Z²It is formed together by removing what hydrogen atom was formed from the Liang Ge functional groups of boric acid complexing agent Part；And

Ring A is selected from by following formed group：

2. the Z in purposes according to claim 1, wherein formula (I)¹And Z²For hydroxyl.

3. the Z in purposes according to claim 1, wherein formula (I)¹And Z²It is formed together by from the two of boric acid complexing agent The part that a functional group's removal hydrogen atom is formed.

4. purposes according to claim 1, wherein the compound is selected from by following formed group：

[(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 2- fluoro benzoyls of 5-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,5- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,5- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 5- fluoro benzoyls of 2-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(4- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,4- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,5- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,4- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 4- fluoro benzoyls of 2-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,3- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,4 difluorobenzene formoxyl) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 2- fluoro benzoyls of 4-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(4- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,4 dichloro benzene formoxyl) amino] acetyl group } amino) -3- methyl butyls] boric acid；With

[(1R) -1- ({ [(3,5- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

Or its pharmaceutically acceptable salt or boric anhydride.

5. purposes according to claim 1, wherein the compound is the boration being selected from by following formed group Close the Nitranitol of object：

[(1R) -1- ({ [(2,3- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 2- fluoro benzoyls of 5-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,5- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,5- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- benzoyl bromides) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 5- fluoro benzoyls of 2-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(4- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,4- difluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,5- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3,4- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(3- fluoro benzoyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 4- fluoro benzoyls of 2-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,3- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,4 difluorobenzene formoxyl) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(the chloro- 2- fluoro benzoyls of 4-) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(4- chlorobenzene formacyls) amino] acetyl group } amino) -3- methyl butyls] boric acid；

[(1R) -1- ({ [(2,4 dichloro benzene formoxyl) amino] acetyl group } amino) -3- methyl butyls] boric acid；With

[(1R) -1- ({ [(3,5- dichloro-benzoyls base) amino] acetyl group } amino) -3- methyl butyls] boric acid.

6. purposes according to claim 1, wherein the compound is [(1R) -1- ({ [(2,5- dichloro-benzoyl base) ammonia Base] acetyl group } amino) -3- methyl butyls] boric acid or its pharmaceutically acceptable salt or boric anhydride.

7. purposes according to claim 1, the wherein purposes are combined with second therapeutic agent.

8. purposes according to claim 7, wherein the second therapeutic agent is melphalan.

9. purposes according to claim 7, wherein the second therapeutic agent is lenalidomide.