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CN103570806A - Polypeptide epoxy ketone compound - Google Patents

Polypeptide epoxy ketone compound Download PDF

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CN103570806A
CN103570806A CN201310317766.8A CN201310317766A CN103570806A CN 103570806 A CN103570806 A CN 103570806A CN 201310317766 A CN201310317766 A CN 201310317766A CN 103570806 A CN103570806 A CN 103570806A
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compound
formula
proteasome
activity
active
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CN103570806B (en
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范晓青
岑坚
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CENTRAX INTERNATIONAL Inc
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CENTRAX INTERNATIONAL Inc
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Abstract

The invention discloses an inhibitor whose new compound and pharmaceutical composition can be used as proteasome. The compound of the invention can prevent three activities CT-L, T-L and PGPH of the proteasome, and can be used for treating various diseases related to proteasome.

Description

Polypeptide epoxy ketone compound
the cross reference of related application
The application requires the right of priority of the U.S. Provisional Application submitted on July 26th, 2012 number 61/675,827, and the right of priority of the Chinese application number 201210352544.5 of submitting on September 20th, 2012.Two parts of applications all quote in full and are incorporated at this.
Invention field
The present invention relates to the compound of many peptide epoxy ketones structure.These compounds can be for the activity of proteasome enzyme inhibition.
Technical background
Proteasome is a proteasome with many catalysiss, in the degradation process of intracellular protein, plays a crucial role.It has been generally acknowledged that proteasome exists with 26S proteasome in vivo, its molecular weight is about 2000kDa, includes a 20S core particle (20S proteasome) and two 19S adjusting particles.20S core particle is hollow structure, and the avtive spot of degrade proteins is trapped among in cavity.Every one end of core particle is all connecting a 19S and is regulating particle, and each regulates particle to contain a plurality of atpase activities site and ubiquitin binding site; Regulate particle can identify the protein of many ubiquitinations, and transfer them in core particle.Except 19S regulates particle, also there is another kind of particle, i.e. the 11S particle of regulating; 11S regulates particle to be combined with core particle to be similar to the mode of 19S particle; 11S particle may play a role on degraded exogenous peptide.Core particle 20S proteasome molecular weight is about 700kDa, has 28 subunits to form 4 rings.In yeast and other eukaryote, each consists of two outer shrouds 7 α subunits, in two ring each by seven β subunits, formed.α ring is the binding site of 19S or 11S REGULATOR, is also the physical barriers of ring in two β.The active site that in two β, ring contains proteolytic enzyme, the β subunit of the degraded of protein in 20S core particle carries out.In vivo, suppressing 20S proteasome is easy to and suppresses the direct correlation of 26S proteasome.The known proteasome that has two kinds of forms: a kind of is structural protein enzyme body, is present in most Eukaryotic cells; Another kind is immunity protease body, is mainly present in hematopoietic cell and is exposed in the cell of inflammatory cytokine.The protein degradation of proteasome mediation is a highly regular governed process, is the necessary program of various biological processes in cell.By using different peptide substrates, defined three major protein enzymic activitys of eukaryotic protein enzyme body: Chymotrypsin sample active (Chymotrypsin-Like is abbreviated as CT-L), its function is the large hydrophobic amino acid residue of hydrolysis; Trypsin-like active (Trypsin-Like is abbreviated as T-L), its function is hydrolysis alkaline amino acid residue; And peptidyl glutamyl peptide hydrolytic activity (Peptidyl Glutamyl Peptide Hydrolyzing, is abbreviated as PGPH), its function is hydrolytic acidity amino-acid residue.For a long time, proteasome is considered to the attractive molecular target of drug development always, and obtained clinical verification (Orlowski and Kuhn in field of antineoplastic medicaments, Clm.Cancer Res. (2008), 14,1649-1657).
Existing a few micromolecular compounds are used to proteasome enzyme inhibition activity, these compounds comprise peptide boric acid, β lactone and many peptide epoxy ketones (Bennett and Kirk, Current Opinion in Drug Discovery & Development (2008), 11,616-625; Borissenko and Groll, Chem.Rev. (2007) 107,687-717).Yet these compounds conventionally lack suitable specificity and biological activity, be difficult to at molecular level abundant development and utilization proteasome in cell levels and body.For example, polypeptide boric acid and β-lactone are all nonspecific 20S proteasome inhibitors, because they also can suppress other proteolytic enzyme (Borissenko and Groll, Chem.Rev. (2007) 107,687-717; Myung et aI., Medicinal Research Reviews (2001), 21,245-273).This just may cause these inhibitor except proteasome enzyme inhibition, also to suppress in vivo other proteolytic enzyme, thereby produces the toxicity of missing the target.On the other hand, at patent US683109981, W02005/105827, CN101044157A, in US2007/0105786A1, disclosed many peptide epoxy ketones have the selectivity of height as the inhibitor of 20S proteasome.It is active that but these many peptide epoxy ketones only suppress the CT-L of 20S proteasome, and can not suppress T-L activity and/or PGPH activity.Existing grinding high showing, the CT-L that simultaneously suppresses 20S proteasome, T-L, with tri-activity of PGPH, can reduce more significantly proteolysis than suppressing wherein one or two activity, thereby growth produces synergy to inhibition tumor cell, improve curative effect (the Chauchan et a1. of oncotherapy, Blood (2008), 111,1654-1664; Kisselev et aI., J.Bio1.Chem. (2006), 281,8582-8590).Therefore, be necessary to develop the CT-L of proteasome enzyme inhibition simultaneously, T-L, and tri-active Novel protease body inhibitor of PGPH.
Summary of the invention
The invention provides the compound with peptide epoxy ketone structure, these compounds are the CT-L of proteasome enzyme inhibition simultaneously, T-L, and PGPH is active.
In some embodiments, compound of the present invention has as shown in the formula the structure shown in (I), and enantiomorph, diastereomer, and tautomer, and the acceptable salt of medicine or solvate or prodrug:
Figure BDA00003571519000031
R wherein 1be selected from
Figure BDA00003571519000032
Figure BDA00003571519000033
Figure BDA00003571519000034
and R 4;
R 4for
Figure BDA00003571519000036
or
Figure BDA00003571519000037
R 2be-(CH 2) mr 5;
R 3independently be selected from hydrogen, hydroxyl, C 1-10alkyl, C 1-10alkoxyl group, C 1-10alkane hydroxyl, C 1-10alkoxyalkyl, amino, NHR 6,-R 7-O (C=O)-R 8,-R 7-(C=O) X-R 8,-R 7-OP0 3m 1m 2,
Figure BDA00003571519000038
Figure BDA00003571519000039
or
Figure BDA000035715190000310
R 5for phenyl, or R y;
R yfor hydroxyl ,-OPO 3m 1m 2,-R 10-O (C=O)-R 11,
Figure BDA00003571519000041
Figure BDA00003571519000042
R 6for C 1-10alkyl, phenyl ,-(C=O) C 1-6alkyl ,-(C=O) phenyl;
Each R 7, R 9and R 10independently be selected from and do not exist, or C 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-); Each R 8and R 11independently be selected from hydrogen, hydroxyl, metal ion (preferred Na +, and K +); C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, or-OP0 3m 1m 2;
Each R 12and R 13independently be selected from hydrogen, C 1-10alkyl (preferred C 1-4alkyl) or the C replacing 1-10alkyl (preferred C 1-4alkyl);
Each M 1, and M 2independently be selected from hydrogen, metal ion (preferred Na +, and K +);
X is not for existing or O;
Y for do not exist or-(C=O)-;
Z is not for existing or O; And
M is 0,1,2,3,4 or 5.
In some embodiments, work as R 5while being phenyl, R 1not R 4.In some embodiments, work as R 1r 4time, R 5it not phenyl.
In some embodiments, formula (I) compound comprises and is selected from Compound I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, the structure in I-16.
In some embodiments, formula (I) compound has the structure of formula (II):
Figure BDA00003571519000051
Each R wherein 1and R 2definition as mentioned above.
In some embodiments, formula (II) compound comprises and is selected from compound 1-5,7-13,20,25,27 and 28 structure, with and enantiomorph, diastereomer, tautomer, and the acceptable salt of medicine or solvate or prodrug.
In some embodiments, formula (I) compound has the structure of formula (III):
R wherein 3definition as mentioned above.
In some embodiments, formula (I) compound has formula (IV) or structure (V):
Figure BDA00003571519000061
Figure BDA00003571519000062
R wherein 3definition as mentioned above.
In some embodiments, formula (I) compound has the structure of formula (VI):
Figure BDA00003571519000063
R wherein 4and R ydefinition as mentioned above.
In some embodiments, the application's compound comprises the structure of following formula (VII):
R wherein 1be selected from following groups:
R wherein 3definition as mentioned above.
In some embodiments, the compound of the application's formula (I) comprises the structure of following formula (VIII):
Figure BDA00003571519000072
R wherein 1be selected from following groups:
Figure BDA00003571519000073
On the one hand, many peptide epoxy ketones provided by the invention can suppress the catalytic activity of 20S proteasome.In some embodiments, many peptide epoxy ketones that the application provides in concentration lower than approximately 5 μ M, 2 μ M, or can suppress 20S proteasome catalytic activity during 1 μ M.In some embodiments, many peptide epoxy ketones provided by the invention not only have stronger restraining effect to the CT-L activity of 20S proteasome, and can effectively suppress in concentration the active and PGPH activity of the T-L of 20S proteasome during lower than approximately 5 μ M.In a preferred embodiment, many peptide epoxy ketones provided by the invention in concentration lower than approximately 5 μ M, 2 μ M, or during 1 μ M, can simultaneously suppress the CT-L of 20S proteasome, the active and PGPH activity of T-L.
On the other hand, the present invention also provides experimentation on animals can suppress in vivo the CT-L of 20S proteasome to prove polypeptide epoxy ketone compound provided by the invention simultaneously, and T-L is active and PGPH is active.Such as, with the polypeptide epoxy ketone compound described in the present invention, for being subject to test product to mouse administration, in mouse blood, the CT-L of proteasome is active, and T-L activity and PGPH activity will be simultaneously suppressed.
On the other hand, the present invention also provides the pharmaceutical composition that can be used for treating mankind's various diseases.These pharmaceutical compositions comprise proteasome inhibitor provided by the invention and the pharmaceutical carrier of effective dose; Human diseases includes but not limited to cancer, inflammation, nerve degenerative diseases (for example senile dementia), muscular dystrophy, chronic infectious disease, fever, the useless use of muscle, denervation, and nerve injury, and Ia disease etc.
In some embodiments, pharmaceutical composition comprises approximately 10 -9gram to approximately 10 grams of compounds provided by the invention.Suitable dosage range is approximately 0.01 milligram to approximately 5 grams for each person every day.
In some embodiments, compound provided by the invention can fill a prescription into suitable preparation with pharmaceutical composition in case by injection administration (as subcutaneous, vein, muscle, artery, sheath, in capsule, in eye frame, in heart, in cortex, intraperitoneal, through tracheae, under epidermis, intraarticular, under capsule, under arachnoid membrane, in backbone, in breastbone, and/or transfusion) and non-injection administration (as oral, enteron aisle, oral cavity, nose, in nose, through mucous membrane, epidermis, emplastrum, skin, medicament for the eyes, lung, hypogloeeis, rectum, vagina or topical).
On the other hand, the invention provides the method for the disease that treatment is relevant to proteasome and the CT-L that simultaneously suppresses body endoproteinase body, the method of T-L activity and PGPH activity, these methods comprise the proteasome inhibitor provided by the invention of using effective dose.
On the other hand, the invention provides the method for the many peptide epoxy ketones of preparation.
Describing in detail and claims below other characteristics and advantages of the present invention is visible.
detailed Description Of The Invention
compound and salt thereof
On the one hand, the structure that compound of the present invention contains formula (I), and enantiomorph, diastereomer, tautomer, and the acceptable salt of medicine or solvate or prodrug:
Figure BDA00003571519000091
R wherein 1be selected from
Figure BDA00003571519000092
Figure BDA00003571519000093
Figure BDA00003571519000094
and R 4;
R 4for
Figure BDA00003571519000095
R 2be-(CH 2) mR 5;
Each R 3independently be selected from hydrogen, hydroxyl, C 1-10alkyl, C 1-10alkoxyl group, C 1-10alkane hydroxyl, C 1-10alkoxyalkyl, amino, NHR 6,-R 7-O (C=O)-R 8,-R 7-(C=O) X-R 8,-R 7-OPO 3m 1m 2,
Figure BDA00003571519000096
Figure BDA00003571519000097
or
Figure BDA00003571519000098
R 5for phenyl, or R y; R yfor-hydroxyl ,-OPO 3m 1m 2,-R 10-O (C=O)-R 11,
Figure BDA00003571519000101
R 6for C 1-10alkyl, phenyl ,-(C=O) C 1-6alkyl ,-(C=O) phenyl;
Each R 7, R 9and R 10independently be selected from and do not exist, or C 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-); Each R 8and R 11independently be selected from hydrogen, hydroxyl, metal ion (preferred Na +, and K +), C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, or-OPO 3m 1m 2;
Each R 12and R 13independently to be selected from hydrogen, C 1-10alkyl (preferred C 1-4alkyl) or replace C 1-10alkyl (preferred C 1-4alkyl);
Each M 1, and M 2independently be selected from hydrogen, metal ion (preferred Na +, and K +);
X is not for existing or O;
Y for do not exist or-(C=O)-;
Z is not for existing or O; And
M is 0,1,2,3,4 or 5.
In some embodiments, work as R 5while being phenyl, R 1not R 4.In some embodiments, work as R 1r 4time, R 5it not phenyl.
In some embodiments, R 3be-R 7-O (C=O)-R 8, R 7and R 8definition as mentioned above.In some embodiments, R 7for not existing, R 8be selected from hydrogen, C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, R wherein 12and R 13definition as mentioned above.In some embodiments, R 7c 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-), R 8for hydrogen, C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, R wherein 12and R 13definition as mentioned above.
In some embodiments, R 3for-R 7-(C=O) X-R 8, R 7and R 8definition as mentioned above.In some embodiments, R 7for not existing, X is O, R 8be selected from hydrogen, metal ion (preferred Na +and K ten), NH 4, C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, R wherein 12and R 13definition as mentioned above.In some embodiments, R 7for not existing, X is for not existing, R 8be selected from-NR 12r 13, R wherein 12and R 13definition as mentioned above.
In some embodiments, R 3for amino, NHCOMe, NHCOEt, NHCOC 3h 7, or NHBoc.
In some embodiments,
Figure BDA00003571519000111
Figure BDA00003571519000112
and R 7, R 9, X, Y, and the definition of Z is as mentioned above.In some embodiments, R 7, R 9, X, Y, and in Z, have an existence at least.In some embodiments, X, Y, Z and R 7all not exist, and R 9for C 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-).In some embodiments, R 7, R 9not existing with X, Y is-(C=O)-, Z is O.In some embodiments, R 7while not existing with X, R 9c 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-), Y is-(C=O)-, Z is O.In some embodiments, R 9while not existing with X, R 7c 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-), Y is-(C=O)-, Z is O.
In some embodiments, X does not exist, R 7and R 9all C independently 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-), Y is-(C=O)-, Z is O.In some embodiments, work as R 7, R 9, X and Z are while not existing, Y is-(C=O)-.
In some embodiments, R 1r 4, R 4be
Figure BDA00003571519000113
r 5r y, R ybe-hydroxyl;-OPO 3m 1m 2, M wherein 1and M 2definition as mentioned above;
-R 10-O (C=O)-R 11, R wherein 10for not existing, R 11be selected from hydrogen, C 1-10alkyl (preferred C 1-4one of alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, R wherein 12and R 13definition as mentioned above; Or
Figure BDA00003571519000114
Figure BDA00003571519000115
r wherein 9do not exist with X, Y is (C=O), and Z is O, R 7can be not exist or C alternatively 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-etc.).
In some embodiments, each R 3independently be selected from hydrogen, methyl, ethyl, propyl group,
Figure BDA00003571519000121
or
Figure BDA00003571519000122
In some embodiments, each R 3independently be selected from hydroxyl, methoxyl group, oxyethyl group, propoxy-,-OPO 3na 2,-OC (=O) CH 3,-OC (=O) C 2h 5,-OC (=O) C 3h 7,-OC (=O) C 4h 9,-OC (=O) CH 2nH 2,-OC (=O) CH 2n (CH 3) 2,-OC (=O) CH 2n(C 2h 5) 2,-OC (=O) NH 2,-OC (=O) N (CH 3) 2,-OC (=O) N (C 2h 5) 2,
Figure BDA00003571519000123
In some embodiments, each R 3independently be selected from methylol, hydroxyethyl, hydroxypropyl ,-CH 2oC (=O) CH 3,-CH 2oC (=O) C 2h 5,-CH 2oC (=O) C 3h 7,-CH 2oC (=O) C 4h 9,-CH 2oPO 3na 2,-CH 2oC (=O) CH 2nH 2,-CH 2oC (=O) CH 2n (CH 3) 2,-CH 2oC (=O) CH 2n(C 2h 5) 2,
Figure BDA00003571519000124
-CH 20C (=O) NH 2,-CH 20C (=O) N (CH 3) 2,-CH 20C (=O) N (C 2h 5) 2:
Figure BDA00003571519000126
In some embodiments, each R 3independently be selected from-(C=O) OH-(C=O) ONa ,-(C=O) ONH 4the OCH of ,-(C=O) 3the OC of ,-(C=O) 2h 5the OC of ,-(C=O) 3h 7, or-(C=O) OC 4h 9.
In some embodiments, each R 3independently be selected from-CONH 2,-CON (CH 3) 2,-CON (C 2h 5) 2,
Figure BDA00003571519000127
or
Figure BDA00003571519000128
In some embodiments, R 5for phenyl or R y.R yfor hydroxyl ,-OPO 3h 2,-OPO 3na 2,-OC (=O) H ,-OC (=O) CH 3,-OC (=O) C 2h 5,-OC (=O) C 3h 7,-OC (=0) C 4h 9,-OC (=O) CH 2n (CH 3) 2,-OC (=O) CH 2n(C 2h 5) 2,
Figure BDA00003571519000131
-OC (=O) NH 2,-OC (=O) N (CH 3) 2,-OC (=O) N (C 2h 5) 2,
Figure BDA00003571519000132
or
Figure BDA00003571519000133
In some embodiments, R 6for C 1-10alkyl (preferred C 1-4alkyl) or the C replacing 1-10alkyl (preferred C 1-4alkyl), phenyl ,-(C=O) C 1-6alkyl ,-(C=O) phenyl.
The compound embodiment of formula (I) is including, but not limited to following listed Compound I-1 to I-16:
Figure BDA00003571519000134
Figure BDA00003571519000141
Arbitrary chiral carbon atom in formula (I) is independently R configuration or S configuration.
In some embodiments, formula (I) compound has the structure of formula (II), wherein R 1and R 2definition as mentioned above.
Figure BDA00003571519000142
In some embodiments, the compound relating to contains following structural formula and comprises compound 1-5,7-13, and 20,25,27-28 and enantiomorph thereof, diastereomer, tautomer, and the acceptable salt of medicine or solvate and possible prodrug:
Figure BDA00003571519000151
Figure BDA00003571519000152
Figure BDA00003571519000153
Figure BDA00003571519000154
Figure BDA00003571519000156
Figure BDA00003571519000161
Figure BDA00003571519000162
Figure BDA00003571519000163
Figure BDA00003571519000164
Figure BDA00003571519000165
Figure BDA00003571519000173
Figure BDA00003571519000174
Figure BDA00003571519000175
In some embodiments, formula (I) compound has the structure of formula (III), wherein R 3definition as mentioned above.
Figure BDA00003571519000181
In some embodiments, formula (111) comprises following 53 compounds (compound III-1 is to 111-53) and compound Isosorbide-5-Nitrae, 5 and 7.The enantiomorph that equally also comprises these compounds, diastereomer, tautomer, and the acceptable salt of medicine or solvate and prodrug.
Figure BDA00003571519000191
Figure BDA00003571519000201
Figure BDA00003571519000211
Figure BDA00003571519000221
In some embodiments, formula (I) compound has formula (IV) or structure (V), wherein R 3the definition of group as mentioned above.
Figure BDA00003571519000231
In some embodiments, formula (IV) or (V) comprise following 54 compounds (compound IV-1 is to IV-27, V-1 to V-27) and compound 2,8-11,13 and 28.The enantiomorph that equally also comprises these compounds, diastereomer, tautomer, and the acceptable salt of medicine or solvate and prodrug.
Figure BDA00003571519000241
Figure BDA00003571519000251
Figure BDA00003571519000261
Figure BDA00003571519000271
Figure BDA00003571519000281
In some embodiments, formula (I) compound has the structure of formula (VI).
Figure BDA00003571519000282
R wherein 4and R ydefinition as mentioned above.
In some embodiments, structural formula (VI) comprises following 20 compounds (compound VI-1 is to VI-20) and compound 3 and 12.The enantiomorph that equally also comprises these compounds, diastereomer, tautomer, and the acceptable salt of medicine or solvate and prodrug.
Figure BDA00003571519000291
Figure BDA00003571519000301
In some embodiments, formula (I) comprises the have formula structure of (VII).
Figure BDA00003571519000311
R wherein 1be selected from lower group:
Figure BDA00003571519000312
R 3definition as mentioned above.
In some embodiments, formula (VII) comprises following 43 compounds (compound VI I-1 to VII-43 and compound 20,25 and 27.The enantiomorph that equally also comprises these compounds, diastereomer, tautomer, and the acceptable salt of medicine or solvate and prodrug.
Figure BDA00003571519000313
Figure BDA00003571519000321
In some embodiments, the compound with formula (I) providing in the application has suc as formula the structure shown in (VIII):
Figure BDA00003571519000342
R wherein 1be selected from lower group:
Figure BDA00003571519000351
In some embodiments, the compound shown in formula (VIII) comprises the structure of selecting albefaction compound I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15 and I-16.In some embodiments, the compound shown in formula (VIII) comprises and is selected from compound 1,2,4,5,7-11,13,20,25,27 and 28 structure.The enantiomorph that equally also comprises these compounds, diastereomer, tautomer, and the acceptable salt of medicine or solvate and prodrug.
So that in the compound shown in above formula (VIII), each can both suppress that the CT-L of 20S proteasome is active, T-L is active and PGPH is active simultaneously.In some embodiments, so that in the compound shown in above formula (VIII), each can both suppress that the CT-L of 20S proteasome is active, T-L is active and PGPH is active when the concentration lower than approximately 5 μ M, approximately 2 μ M or approximately 1 μ M simultaneously.In some embodiments, these compounds are to the CT-L activity of 20S proteasome, the IC that T-L is active and PGPH is active 50value is all lower than approximately 5 μ M, approximately 2 μ M or approximately 1 μ M.This simultaneously inhibition that compound shown in the above-mentioned formula of listing (VIII) has is beyond thought, because work as the R that certain compound has other 1group is (for example, in following compound 21
Figure BDA00003571519000352
group) time, it is active significantly reduces, and the CT-L that is difficult to simultaneously to suppress 20S proteasome is active, T-L is active and PGPH is active, so effect is not good enough, and the CT-L that is not suitable for simultaneously suppressing 20S proteasome is active, T-L is active and PGPH is active:
Figure BDA00003571519000353
Pharmacy acceptable salt can be any salt or the ester of physiologically acceptable and applicable administration.For example, pharmacy acceptable salt comprises acid salt (example hydrochloric acid salt, hydrobromate, hydriodate, nitrate, vitriol, hydrosulfate, phosphoric acid salt, superphosphate, γ-picolinic acid salt, acetate, lactic acid salt, salicylate, Citrate trianion, tartrate, pantothenate, choline salt, ascorbate salt, succinate, maleic acid salt, fumarate, gluconate, glucuronate, sucrose hydrochlorate, formate, benzoate, glutaminate, mesylate, esilate, benzene sulfonate, tosilate and pamoate) and base addition salt (as aluminium salt, calcium salt, lithium salts, magnesium salts, sylvite, sodium salt, zinc salt, and diethanolamine salt).
preparation method
The compou nd synthesis route the present invention relates to is as follows.This process is illustrations but not places restrictions on the method that other may prepare this compound.In addition, the step in flow process is only better explanation, depends on the circumstances and can change.
Flow process 1
Figure BDA00003571519000361
Step 1: compound 1001 reacts with isobutyl chlorocarbonate and obtains compound 1002.
Step 2: compound 1002 reacts with bromopropylene and obtains compound 1003.
Step 3: compound 1003 reacts and obtains epoxy compounds with DIEA and superoxol.
Step 4: compound 1004 reacts and obtains compound 1005 with TFA dichloromethane solution.
Flow process 2
Figure BDA00003571519000362
Compound 1006 reacts with NaCN and obtains compound 1007, more further reacts with KOH, obtains product compound 1008 after acidifying.
Flow process 3
Figure BDA00003571519000371
Chlorine trityl chloride resin successively with Fmoc-Phe-OH, Fmoc-Leu-OH, and Fmoc-HomoPhe-OH reaction, synthesize and extended peptide main chain.The product obtaining reacts with compound 1008, and resin obtains compound 1013 after decomposing, and compound 1013 reacts with 1005 and obtains compound 1.
Biological activity and selectivity
Compound provided by the invention has the biological property of proteasome enzyme inhibition catalytic activity.The activity of proteasome can determine by experimental technique known in the art, and these methods are at Stein et al., Biochemistry (1996), 35,3899-3908, Lightcap et a1., Clinical Chemistry, 2000,46,673-683, Kisselev et a1., Journal of Biological Chemistry, (2006), in 281,8582-8590 and U.S. Patent application 09/569748, all have openly.The CT-L of 20S proteasome, PGPH and T-L activity are by determining by fluorogenic substrate detection method.The method is used succinyl-Leu-Leu-Val-Tyr-AMC, Z-leu-Leu-Glu-AMC and Boc-Leu-Arg-Arg-AMC respectively as CT-L, the substrate of PGPH and T-L, reaction completes in damping fluid, the free fluorophore 7-amido-4-methylcoumarin (AMC) decompositing detects with fluorophotometer, can record the CT-L of 20S proteasome, PGPH and T-L are active.
The part purposes of compound provided by the invention is catalytic activity that can proteasome enzyme inhibition.
In some embodiments, compound provided by the invention concentration during lower than 5 μ M (such as compound 1-5,7-13,20,25,27 and 28) can suppress CT-L in 20S proteasome, arbitrary activity of T-L and PGPH (such as have 50% or higher restraining effect).In some embodiments, compound provided by the invention in concentration lower than 5 μ M, 2 μ M, during 1 μ M (such as compound l-5,7-13,20,25,27 and 28) during or lower than 0.01 μ M (such as compound 1-2,4-5,7-13,20,25,27 and 28) can suppress the CT-L activity of 20S proteasome.In some embodiments, compound provided by the invention concentration during lower than 5 μ M (such as compound 1-5,7-13,20,25,27 and 28) during, lower than 2 μ M (such as compound 1-5,8-13,20,25,27 and 28) during, lower than 1 μ M (such as compound 1-5,8-13,20,25,27 and 281, or during lower than 0.5 μ M (such as compound 1,2,8-11,13,25 and 27) can suppress the T-L of 20S proteasome active.In some embodiments, compound provided by the invention concentration during lower than 5 μ M (such as compound 1-5,7-13,20,25,27 and 28) during, lower than 2 μ M (such as compound 1,2,4,5,7-11,13,25,27 and 28), during lower than 1 μ M (such as compound 1,2,4,5,7-11,13,25,27 and 28) during, or lower than 0.5 μ M (such as compound 2,5,8-11,13,25 and 27) can suppress the PGPH of 20S proteasome active.
Compound provided by the invention can suppress the CT-L of 20S proteasome simultaneously, and T-L and PGPH are active.In this application, " suppress " to refer to that compound can significantly suppress the CT-L of 20S proteasome, each in T-L and PGPH activity simultaneously.In embodiment preferably, compound provided by the invention lower than 5 μ M, can suppress the CT-L of 20S proteasome in concentration simultaneously when 2 μ M or approximately 1 μ M, and T-L and PGPH are active.In better embodiment, compound provided by the invention in concentration lower than 5 μ M (such as compound 1-5,7-13,20,25,27 and 28), lower than 2 μ M (such as compound 1,2,4,5,8-11,13,25,27 and 28), lower than 1 μ M (such as compound 1,2,4,5,8-11,13,25,27 and 281, or during lower than 0.5 μ M (such as compound 2,8-11,13,25 and 27) CT-L that can simultaneously suppress 20S proteasome time, T-L and PGPH are active.
In some embodiments, compound provided by the invention can suppress the CT-L of 20S proteasome in vivo simultaneously, T-L and PGPH are active, and in the biological tissue taking out in vivo, (in vitro) suppresses CT-L, T-L and the PGPH activity of 20S proteasome simultaneously.
In the in vitro embodiment of an example, in body, (such as mouse) gets blood, then use compound treatment blood sample provided by the invention, the compound that the application provides can suppress at such in vitro the CT-L of 20S proteasome in blood, tri-kinds of activity of T-L and PGPH.In some in vitro embodiment, compound provided by the invention can suppress the CT-L of 20S proteasome during lower than 5 μ M, 2 μ M, 1 μ M or 0.5 μ M simultaneously in concentration, and T-L and PGPH are active (such as compound 1-2,4-5,7-13,20,25,27 and 28).
Compound provided by the invention can suppress the CT-L of 20S proteasome in body simultaneously, and T-L and PGPH are active.Such as, with the polypeptide epoxy ketone compound described in the present invention, for being subject to test product to live body (as mouse) administration, gathering this biological blood the CT-L to 20S proteasome in blood preparation, T-L and PGPH activity are measured.In some embodiments, compound provided by the invention can suppress the CT-L of this 20S proteasome in vivo simultaneously, T-L and PGPH active (such as compound 1-5,7-13,20,25,27 and 28).
the purposes of compound
The invention provides proteasome enzyme inhibition CT-L simultaneously, T-L, and the method for PGPH activity, comprise the compounds of this invention for the treatment of effective dose." treatment effective dose " refer in giving the individuality of drug compound, is enough to provide the amount of required result for the treatment of or active described compound.This effective dose may change according to different factors, such as the age of curer, and sex, healthy state, the formulation of compound, seriousness of curer disease etc.Treatment effective dose can be determined according to factor above-mentioned or other factors by doctor.
On the other hand, the invention provides the method for the disease that treatment is relevant to proteasome.The method comprises the compound in the present invention who treats effective dose.These compounds can be used for treatment various situations or the disease relevant with proteasome, these that include but not limited to enumerate below.
Known have various diseases or situation relevant with the adjusting of proteasome catalysis.Proteasome inhibitor has been proposed for prevention or treatment various diseases, comprises cancer, neurotoxic/degenerative disease, alzheimer's disease, ischemic disease, inflammation, Ia disease, HIV infects, organ-graft refection, septic shock, antigen presentation suppresses, parasitic infection, the disease that oxypathy is relevant, macular degeneration, pulmonary disorder, muscular dystrophy, fibrotic disease, bone and hair growth disease.Therefore, polypeptide epoxy ketone protease body inhibitor provided by the invention provides a kind of approach for the treatment of for above-mentioned disease.
Proteasome inhibitor is had treatment malignant tumour function by clinical proof.Therefore, compound provided in this article can be used for treating cancer.The cancer being treated of example comprises leukemia, lymphoma, myelomatosis, hepatocellular carcinoma etc.Compound of the present invention also may be treated following cancer: adrenocortical carcinoma, the cancer that acquired immune deficiency syndrome (AIDS) is relevant, astrocytoma, osteocarcinoma, osteosarcoma, polymorphism neuroblastoma, malignant fibrous histiocytoma, melanoma, malignant mesothelioma, pheochromocytoma, pineocytoma and primitive neuroectodermal tumor, neuroblastoma, sarcoma of uterus, cholangiocarcinoma, bladder cancer, mastocarcinoma, stomach cancer, cervical cancer, colorectal carcinoma, the rectum cancer, esophagus cancer, cancer eye, ovarian cancer, incidence cancer, kidney, lip and oral carcinoma, lung cancer, nasal cavity and paranasal sinus cancer, nasal sinus cancer, penile cancer, prostate cancer, transitional cell carcinoma, glandula cancer, soft tissue cancer, skin carcinoma, Tiroidina and Parathyroid cancer, carcinoma of vagina etc.
Proteasome inhibitor suppresses relevant to NF-kB activity.NF-κ B is a kind of efficient transcription factor, can transcribing of regulatory gene comprise that inflammation molecule is as tumour necrosis factor, interleukin 1, cyclooxygenase, ICAM-1 etc.Therefore, the compound in the present invention can be used for the treatment of diseases associated with inflammation as a kind of immunosuppressor, as allergy, and asthma, organ-/ tissue graft-rejection, autoimmunity lupus erythematosus, rheumatic arthritis, psoriasis, multiple sclerosis and inflammatory bowel.Can give the compound providing in the present invention of effective dose to suffering from the patient of above-mentioned disease, this compound can be included in pharmaceutical composition, to treat these diseases.
Found that proteasome inhibitor can reduce the degraded of muscle protein, thus in suppressing amyotrophy and muscle fiber atrophy of great use.Therefore the compound relating in the present invention can be used for treating emaciation and muscular dystrophy, and for example chronic infectious disease, has a fever, and muscle is useless to be used and denervation, and nerve injury, because of the renal failure that acidismus causes, liver failure.Can give the compound providing in the present invention of effective dose to suffering from the patient of above-mentioned disease, this compound can be included in pharmaceutical composition, thereby reduces or reduce the degraded of mytolin, ICBP or p53 albumen.
The compound relating in the present invention can be used for treating nerve degenerative diseases equally, comprise apoplexy, neural system ischemia injury, traumatic nerve injury (for example knocks Brain Injury After, Spinal injury, neural system is damaged outward), multiple sclerosis and other immune-mediated property neuropathy are (as Guillain-Barre syndrome and its varient, acute exercise axon type neuropathy, Guillain Barre syndrome, Fisher syndrome), hiv virus/aids dementia complex, diabetic neuropathy, parkinsonism, Heng Dingdun chorea, multiple sclerosis, bacterial speck, parasite, fungi and viral meningitis, encephalitis, vascular dementia, multi infarct dementia, dementia with Lewy body, frontal lobe is dull-witted as Pick's disease, under cortex dull-witted (as Heng Dingdun or stein-leventhal syndrome), burnt cortical atrophy syndrome (as primary progressive aphasia), the poisonous dementia of metabolism, because infecting the dementia (as syphilis or chronic meningitis) causing.
The compound relating in the present invention can also deposit relevant albumen processing for regulating and controlling to the amyloid beta of extracellular matrix, and amyloid beta is the major cause of Alzheimer's disease.Therefore, the compound relating in the present invention to treatment Alzheimer's disease of great use, for example, by reducing the speed of amyloid beta regulation process, reduce the speed of the plaque test of amyloid beta, reduce the formation speed and the clinical symptom that reduces Alzheimer's disease of amyloid beta.
Proteasome inhibitor is also helpful to reducing cystic fibrosis.Therefore, the compound relating in the present invention can be used for treating the disease that fibrosis is relevant, diabetic nephropathy for example, glomerulosclerosis, IgA nephropathy, liver cirrhosis, Biliary atresia, congestive heart failure, scleroderma, radioactive fibrosis, pulmonary fibrosis, myocardial fibrosis.
pharmaceutical composition and administering mode
On the other hand, pharmaceutical composition provided by the invention comprises compound provided by the invention and pharmaceutically acceptable carrier.
Term " pharmaceutically acceptable carrier " refers to a kind of pharmaceutically acceptable material as mentioned herein, and composition or medium, such as liquid or solid weighting agent, thinner, vehicle, solvent or Embedding Material, it participates in the compound the present invention relates to from a certain position, body fluid, tissue, organ (inner or outside), or another location is loaded or be delivered to body portion, body fluid, organ (inner or outside), or body portion.Pharmaceutically acceptable carrier can be medium, thinner, and vehicle or other do not have excessive toxicity or side effect can be for contacting the material of animal tissues.Typical pharmaceutically acceptable carrier comprises carbohydrate, starch, cellulose family, maltose, tragacanth gum, gelatin, Ringer's solution, Lalgine, physiological saline, buffer reagent etc.
Every kind of pharmaceutically acceptable carrier should be compatible with other moiety, for example with the present invention in the compound formation preparation that provides, biological vital tissue or organ are not had to excessive toxicity, stimulation, anaphylaxis, immunogenicity or other problem or complication, and have more rational benefit risk ratio.
The material of some pharmaceutically acceptable carriers comprises: (1) carbohydrate, and such as lactose, dextrose plus saccharose; (2) starch, such as W-Gum and yam starch; (3) Mierocrystalline cellulose and its derivative, such as Xylo-Mucine, ethyl cellulose, cellulose acetate; (4) powdered tragacanth; (5) maltose; (6) gelatin; (7) talcum powder; (8) vehicle, such as theobroma oil and suppository wax; (9) oils, such as peanut oil, Oleum Gossypii semen, Thistle oil, sesame oil, sweet oil, Semen Maydis oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyalcohols, such as glycerine, sorbyl alcohol, N.F,USP MANNITOL and polyoxyethylene glycol; (12) lipid, such as ethyl oleate, Laurate ethyl; (13) agaropectin; (14) buffer reagent, such as magnesium hydroxide and aluminium hydroxide; (15) Lalgine; (16) sterile pyrogen-free water; (17) physiological saline; (18) Ringer's solution; (19) alcohols, such as ethanol and propyl alcohol; (20) phosphoric acid buffer; (21) the compatible material of other nontoxicity in pharmaceutical dosage form, such as acetone.
Pharmaceutical composition may comprise pharmaceutically acceptable auxiliary material, to simulate physiological condition, and such as pH regulator and buffer reagent, toxicity conditioning agent etc., as sodium acetate, sodium-chlor, Repone K, calcium chloride, Sodium.alpha.-hydroxypropionate etc.
Pharmaceutical cpd can be made into any suitable formulation, for example, for example, as solid dosage (tablet, capsule, powder, particle etc.) and liquid dosage form (aqueous solution, emulsion, elixir, syrup etc.).Preparation method's technique of pharmaceutical composition is well known, can be prepared according to common process, such as at Remington, The Science and Practice of Pharmacy (Gennaro ed.20th edition, Williams & Wilkins PA, USA) provide in (2000).
In some embodiments, pharmaceutical composition comprises approximately 10 -9g to the compound providing in the present invention of about 10g (such as about 0.01mg is to about 10g, about 0.1mg is to about 10g, about 1mg is to about 10g, about 5mg is to about 10g, about 10mg is to about 10g, about 20mg is to about 10g, about 30mg is to about 10g, about 40mg is to about 10g, about 50mg is to about 10g, about 80mg is to about 10g, about 100mg is to about 10g, about 150mg is to about 10g, about 200mg is to about 10g, about 300mg is to about 10g, about 400mg is to about 10g, about 500mg is to about 10g, about 600mg is to about 10g, about 700mg is to about 10g, about 800mg is to about 10g, about 900mg is to about 10g, about 1g is to about 10g, about 10mg is to about 5g, about 10mg is to about 3g, about 10mg is to about 1g, about 10mg is to about 900mg, about 10mg is to about 700mg, about 10mg is to about 500mg, or about 10mg is to about 300mg).Reasonably dosage is the about 5g extremely of about 0.01mg for each person every day.
In some embodiments, the compound providing in the present invention or pharmaceutical composition can be made into be suitable for the formulation of drug release, by injection administration (as subcutaneous, vein, muscle, artery, sheath, in capsule, in frame, in heart, intradermal, intraperitoneal, through tracheae, epidermis, intraarticular, under capsule, under arachnoid membrane, in backbone, in breastbone, and/or transfusion) and non-injection administration (as oral, enteron aisle, oral cavity, nose, in nose, mucous membrane, epidermis, emplastrum, corium, medicament for the eyes, lung, hypogloeeis, rectum, vagina or epidermis topical).
The formulation that suitable formulation includes but is not limited to inject purposes is such as milk sap, solution and suspension, and the formulation of oral use is as tablet, capsule, pill, drageeing, powder and particle, local application or the formulation that absorbs through skin be as spray, ointment, paste, breast frost, lotion, gel, solution, patche and inhalation, the formulation of vagina or rectal administration is as suppository.These formulations can be prepared according to compound and suitable vehicle under conditions suitable, preparation method and technique are well-known, such as by Remington: at The Science and Practice ofPharmacy (Gennaro ed.20th edition, Williams & Wilkins PA, USA) (2000) provide.
Pharmaceutical cpd can enter in organism by any suitable approach, oral such as passing through, intravenous injection, and in nose, external application, intramuscular injection, intradermal injection, percutaneous dosing or subcutaneous route.
In some embodiments, the compound the present invention relates to or pharmaceutical composition can be used with the second active substance simultaneously, can reach in vivo the even collaborative effect of stack like this.For example, the compound the present invention relates to can become with the second active substance combination a pharmaceutical composition, or uses with independent composition simultaneously, or uses successively with independent composition.Can use with the compounds of this invention simultaneously, the second active substance of being used for the treatment of cancer is including, but not limited to Fluracil, Zorubicin, daunorubicin, Tamoxifen, Leuprolide, goserelin, Drogenil, Nilutamide, finasteride, dexamethasone, aminoglutethimide, amsacrine, Anastrozole, asparaginase, bacille Calmette-Guerin vaccine, bicalutamide, bleomycin, clinical, busulfan, camptothecine, capecitabine, carboplatin, carmustine, Chlorambucil, cis-platinum, CldAdo, colchicine, endoxan, medicine, cyproterone, cytosine arabinoside, Dacarbazine, actinomycin d, zhengdingmeisu, Retalon, stilboestrol, Docetaxel, Zorubicin, adriamycin, epirubicin, estradiol, Emcyt, Etoposide, Exemestane, filgrastim, fludarabine, fluohydrocortisone, Fluracil, Fluoxymesterone, Drogenil, gemcitabine, genistein, goserelin, tamoxifen, teniposide, testosterone, titanocene dichloride, opens up Pu Taikang, trastuzumab, vitamin A acid, vincaleucoblastine, hydroxyurea, idarubicin, ifosfamide, imatinib, Interferon, rabbit, irinotecan, irinotecan, letrozole, formyl tetrahydrofolic acid, pentostatin, mithramycin, procarbazine, Raltitrexed porphin Fei Er, Rituximab streptozotocin, Suramine, Leuprolide, LEVAMISOLE HCL, lomustine, mustargen, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, Nilutamide, R 17934, Sostatin, platinum, taxol, Pamidronic Acid, Tioguanine, tespamin, methyl chloride, the luxuriant titanium of topotecan two, trastuzumab, vitamin A acid, vincaleucoblastine, vincristine(VCR), vindesine, Changchun is auspicious refined.
In some embodiments, compound provided by the invention can be used simultaneously and carry out cancer therapy with non-chemical method.In some embodiments, compound provided by the invention can carry out with radiotherapy simultaneously.In some embodiments, compound provided by the invention can with surgical operation, tumour heat cure, focus ultrasonic therapy, psychrotherapy or above several therapy are combined with.
In some embodiments, compound provided by the invention can be used with steroid simultaneously.Suitable steroid is including, but not limited to amcinonide, beclometasone, Betamethasone Valerate, budesonide, Chloroprednisonum, clobetasol, corticosterone, cortisone, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, two pregna-fluoride butyl esters, glycyrrhetinic acid, L-6400, fluorine compound, flunisolide, flucloronide, lidex, fluocinonide, fluocortin butyl, fluocortolone, flurandrenolide, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurrenolone, propionic acid fluorine, formocortal, clobetasol propionate, halcinonide, halometasone, hydrocortisone, P-5604, depersolon, Zpoflogin, meprednisone, 6-first Prednisolone Acetate, furoate in office, paramethasone, prednisolone, dexamethasone, with 25-diethylamine vinegar Ultracortene-H.
In some embodiments, compound provided by the invention can be used with immunotherapeutic agent simultaneously.Suitable immunotherapeutic agent comprises: tumor multidrug-resistance reversal agent (such as verapamil), rapamycin, mycophenlate mofetil, Thalidomide, endoxan, ciclosporin, and monoclonal antibodies.
embodiment
Embodiment 1: external 20S proteasome CT-L, T-L, and the mensuration of PGPH activity inhibition.
With the people source 20S proteasome of purifying, carry out CT-L; the external test of T-L and PGPH activity; the concentration of 20S proteasome is respectively 2; 4 and 8nmol/L; use respectively succinyl-Leu-Leu-Val-Tyr-AMC (10 μ mol/L); Z-leu-Leu-Glu-AMC (10 μ mol/L) and Boc-Leu-Arg-Arg-AMC (50 μ mol/L) are as substrate, and reaction is carried out in the 20mM triisopropyl second sulphonyl buffered soln (pH8.0) that comprises 0.5mM ethylenediamine tetraacetic acid (EDTA), 0.001% sodium lauryl sulphate (SDS) and 0.05%NP-40.The stoste of proteasome inhibitor is prepared in dimethyl sulfoxide (DMSO) (DMSO), and in test mixing thing, the ultimate density of DMSO is 1%.Reaction is carried out at 27 ℃ of temperature.With fluorophotometer, detect cracking 7-amino-4-methylcoumarin fluorophore out, thereby measure proteasome activity.IC 50the speed of response of the detection of (503nhibiting concentration) numerical value based between 60 to 75 minutes.IC 50value is the concentration of inhibitor when the inhibition of 20S proteasome activity is reached to 50%.
Embodiment 2: the CT-L of 20S proteasome in blood, the mensuration of T-L and PGPH activity inhibition.
By cardiac puncture, from CD-1 mouse, get 800 microlitre fresh whole blood samples and inject the test tube contain heparin sodium, at 4 ℃ in 150xg centrifugal 5 minutes, ice-cold phosphate buffered saline cleaning three times for centrifugal throw out out.At every turn by throw out back dissolving in the cold phosphate buffered saline of 1mL, and at 4 ℃ in 6000xg centrifugal 10 minutes.After last cleaning, (phosphate buffered saline that contains 5mM EDTA, pH8.0), keeps cell after 1 hour to be decomposed, then at 4 ℃ in 6000xg centrifugal 10 minutes to add 200 μ L decomposed solution.Protein concentration in the lysate of above-mentioned blood is measured by two quinolinic acid methods (BCA).Approximately 100 μ g protein are added in HEPES buffered soln (pH7.5); Add again compound in the present invention of 0.2,1 or 5 μ M to said mixture; Add again Succinyl-Leu-Leu-Val-Tyr-AMC (25 μ mol/L) Z-leu-Leu-Glu-AMC (10 μ mol/L) or Boc-Leu-Arg-Arg-AMC (10 μ mol/L) respectively as CT-L, the substrate of PGPH or T-L, it is 50 μ L that final reaction liquid amasss, contain 0.5mM EDTA, 0.05%NP-40 and 0.002%SDS.Said mixture is hatched and is placed in fluorophotometer for 30 minutes at 37 ℃, with 360/460nm filtering, three 7-amino-4-methylcoumarin fluorophores are carried out quantitatively, and then measure the CT-L of 20S proteasome, T-L or PGPH are active.By relatively calculating inhibition percentage with reference substance.
Embodiment 3: the CT-L of 20S proteasome in body, the mensuration of T-L and PGPH activity inhibition.
Proteasome activity in body is determined in BALB/c nude mice and carries out.Take the polypeptide epoxy ketone compound described in the present invention as be subject to test product with the dosage that can tolerate by intravenous injection to mouse (every group of 5-6 mouse) administration, in the carrier (pH3.0-3.5) that be formulated in by test product to contain 10-20% (w/v) hydroxypropyl-B-cyclodextrin, 2.5%DMSO and 10mM Trisodium Citrate.Inject latter one hour, by cardiac puncture, gather whole blood sample and inject the test tube contain heparin sodium, at 4 ℃ in 150xg centrifugal 5 minutes.Centrifugal throw out out cleans three times with ice-cold phosphate buffered saline.At every turn by throw out back dissolving in the cold phosphate buffered saline of 1mL, at 4 ℃ in 6000xg centrifugal 10 minutes.After last cleaning, (phosphate buffered saline that contains 5mM EDTA, pH8.0) keeps 1 hour, and cell is decomposed, then at 4 ℃ in 6000xg centrifugal 10 minutes to add 200 μ L decomposed solution.Protein concn in the lysate of blood is measured by two quinolinic acid methods (BCA).Approximately 100 μ g protein are added to and contain 0.5mM EDTA, in the HEPES damping fluid of 0.05%NP-40 and 0.002%SDS (pH7.5), add again Succinyl-Leu-Leu-Val-Tyr-AMC (25 μ mol/L), Z-leu-Leu-Glu-AMC (10 μ mol/L) or Boc-Leu-Arg-Arg-AMC (10 μ mol/L) be respectively as CT-L, the substrate of PGPH or T-L determination of activity.Then mixture is placed at 37 ℃ and hatches 30 minutes.Free fluorophore with fluorophotometer at the quantitative 7-amino-4-in 360/460nm wavelength place methylcoumarin, and then the CT-L of mensuration 20S proteasome, the activity of T-L or PGPH.By with vehicle group comparison, calculate and to be subject to the inhibition percentage of test product to body endoproteinase body activity.
Embodiment 4: compound 1, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 1 structural formula is as follows
Figure BDA00003571519000451
The restraining effect of 1 pair of 20S structural protein enzyme body of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L to 20S structural protein enzyme body and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 5ovalue.Compound 1 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 1/IC 50ka Feizuo meter) respectively 0.18 and 0.26.
Figure BDA00003571519000461
In mouse blood lysate, the restraining effect of 1 pair of proteasome of compound completes by the measuring method providing in embodiment 2.When compound 1 concentration is 0.2 μ m, CT-L activity 96.7% is suppressed, and T-L activity 60.5% is suppressed, and PGPH activity 40.2% is suppressed.When compound 1 concentration is 1 μ m, CT-L activity 99.2% is suppressed, and T-L activity 82.5% is suppressed, and PGPH activity 61.5% is suppressed.When compound 1 concentration is 5 μ m, CT-L activity 98.9% is suppressed, and T-L activity 87.3% is suppressed, and PGPH activity 74.3% is suppressed.
1 pair of proteasome activity restraining effect of compound is measured by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 1 can suppress CT-L in mouse blood simultaneously, and T-L and PGPH are active, and inhibiting rate surpasses 40%.
Synthesizing of compound 1
Flow process 1
Figure BDA00003571519000462
Synthetic compound 1002.Free dimethyl hydroxylamine solution preparation: add N in reaction flask 1, the methylene dichloride of O-dimethyl azanol hydrochloride (8.2g, 84mmo1) (100 milliliters) solution, drips (11.7 milliliters of TEA, methylene dichloride 84mmo1) (50 milliliters) solution, drips off for approximately 20 minutes.To reaction flask 2, add methylene dichloride (200 milliliters) solution of compound 1001 (18.5g, 80mmo1), then add isobutyl chlorocarbonate (10.9g, 80mmo1), be chilled to-10 ℃ add below NMM (8.1 grams, 80mmo1).Again the dimethyl hydroxylamine solution in reaction flask 1 is slowly added.Then the solution obtaining is at room temperature stirred and spent the night, then dilute with water, layering, water is with DCM extraction once.Merge organic phase salt water washing, anhydrous magnesium sulfate drying, removes by filter sal epsom.Evaporated under reduced pressure solvent obtains compound 1002 (17.5g).
Synthetic compound 1003.Under argon shield, compound 1002, (8.2 grams, tetrahydrofuran (THF) 30mmol) (100 milliliters) solution is chilled to-78 ℃, adds t-BuLi solution (46mL1.3M).Under-78 ℃ of stirrings, add pseudoallyl bromine (3.63g, 30mmo1).Add again saturated aqueous ammonium chloride (100 milliliters), in-78 ℃ of stirring reactions 1 hour.Then by ethyl acetate, extract.Organic layer after merging, uses salt water washing.Organic phase anhydrous sodium sulfate drying, filters, and after underpressure distillation is concentrated, crosses post and obtains compound 1003.
Synthetic compound 1004.To compound 1003 (5.1 grams, add DIEA and H in methyl alcohol 20mmo1) (50 milliliters) solution 2o 2solution (4.6 milliliter 30%, 80mmo1).Stir after 2 hours, inject aqueous solution of sodium bisulfite (100 milliliters).Then by ethyl acetate, extract.Merge after organic layer, use salt water washing.Organic phase anhydrous sodium sulfate drying, filters, and after concentrating under reduced pressure, crosses post and obtains required compound 1004.
Synthetic compound 1005.To compound 1004 (2.7 grams, add TFA in DCM solution 10mmo1).Fully, after stirring reaction, evaporated under reduced pressure obtains compound 1005.
Flow process 2
Synthetic compound 1007.To compound 1006 (2.6 grams, in DMSO 20mmo1) (10 milliliters) solution, add sodium cyanide (2 grams, 40mmo1).After stirred overnight at room temperature, dilute with water, ethyl acetate is extracted.Organic phase salt water washing.Anhydrous sodium sulfate drying, filters, and volatile matter is removed in decompression, then recrystallization obtains compound 1007.
Synthetic compound 1008.To compound 1007 (1.2 grams, 10mmo1) in dioxane (20 milliliters) solution, add potassium hydroxide solution (1 milliliter, 3M).After stirring is spent the night, reaction solution is slowly added to the water, and filters, and filter cake recrystallization obtains compound 1008.
Flow process 3
Figure BDA00003571519000481
Synthetic resins 1009.In the ethylene dichloride (30 milliliters) of 2-chlorine trityl chloride resin (3.2mmo1) and Fmoc-Phe-OH (6.4mmo1), (1.2 milliliters, 6.6mmo1) stir 1 hour to add DIEA.With DCE, dimethyl formamide, Virahol, ether is washed, then dry air.To the dimethyl formamide solution that adds 20% piperidines in dimethyl formamide (40 milliliters) solution of gained resin, after fully stirring, filter, by DMF, methyl alcohol and dichloromethane rinse, dry air obtains resin 1009.
Synthetic resins 1010.In resin 1009 (3.2mmo1), add DCE (40 milliliters), Fmoc-Leu-OH (2.2g, 6.2mmo1), DIED is (2.3 milliliters, 13.2mmo1), HOBT (0.86g, 6.4mmo1) and BOP (1.78g, 6.4mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air.To the dimethyl formamide solution that adds 20% piperidines in dimethyl formamide (40 milliliters) solution of gained resin, after fully stirring, filter, with DMF and DCM, to rinse, dry air obtains resin 1010.
Synthetic resins 1011.In resin 1010 (3.2mmo1), add DCE (40 milliliters), Fmoc-HFE-OH (2.56g, 6.4mmo1), DIAD (2.3 milliliters, 13.2mmo1), HOBT (0.86g, 6.4mmo1) and BOP (1.78g, 6.4mmo1), after fully stirring, filter, with DCE, dimethyl formamide, Virahol, and ether flushing, dry air.Dimethyl formamide (40 milliliters) solution to gained resin adds the dimethyl formamide solution of 20% piperidines, after fully stirring, filters, and with DMF, methyl alcohol and DCM rinse, and dry air obtains resin 1011.
Synthetic resins 1012.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1008 (0.40g, 2.8mmo1), (1.1 milliliters of DIED, 6.3mmo1), HOBT (0.43g, 3.2mmo1), and BOP (0.89g, 3.2mmo1), after fully stirring, filter, with DCE, dimethyl formamide, Virahol and ether rinse, and dry air obtains resin 1012 for 24 hours.
Synthetic compound 1013.To the mixing solutions that adds 10 milliliters of TFA/DCM=1/4 (V/V) in resin 1012 (1.6mmo1).Stirring at room, filters, and evaporated under reduced pressure obtains compound 1013.
Synthetic compound 1.In reaction flask, add the acetonitrile solution of compound 1005 (0.33mmo1), then add compound 1013 (0.3mmo1), DIEA (1.5mmo1), HOBT (0.6mmo1), and BOP (0.64mmo1), stir under room temperature.Then dilute with water, ethyl acetate is extracted.Organic phase washes with water, anhydrous sodium sulfate drying.Filter, filtrate decompression evaporate to dryness, crosses post and obtains compound 1 (110 milligrams).Compound 1 nuclear magnetic resonance data: 1hNMR (400H z, Methanol-d 4) δ 7.32-7.28 (m, 2H), 7.29-7.19 (m, 3H), 7.14-7.10 (m, 5H), 6.20 (s, 1H), 4.61-4.53 (m, 2H), 4.31-4.27 (m, 2H), 3.70 (dd, 2H), 3.23-3.14 (m, 2H), 2.93-2.82 (m, 2H), 2.74-2.66 (m, 2H), 2.44 (s, 3H), 2.12-2.02 (m, 1H), 2.01-1.89 (m, 1H), 1.75-1.38 (m, 9H), 0.96-0.87 (m, 12H). Low Resolution Mass Spectra: molecular formula C 40h 53n 5o 7, molecular weight calculated value 715.4, observed value (M+H +) 716.3.
Embodiment 5: compound 2, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 2 structures are as follows
Figure BDA00003571519000491
The restraining effect of 2 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 2 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 2 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 2/IC 50being respectively Ka Feizuo meter) is respectively 0.05 and 0.21.
Figure BDA00003571519000501
In mouse blood lysate, the restraining effect of 2 pairs of 20S proteasomes of compound completes by the analytical procedure providing in embodiment 2.When compound 2 concentration are 0.2 μ M, CT-L activity 98.0% is suppressed, and T-L activity 70.1% is suppressed, and PGPH activity 44.7% is suppressed.When compound 2 concentration are 1 μ M, CT-L activity 98.1% is suppressed, and T-L activity 81.8% is suppressed, and PGPH activity 68.4% is suppressed.When compound 2 concentration are 5 μ M, CT-L activity 93.0% is suppressed, and T-L activity 89.3% is suppressed, and PGPH activity 68.5% is suppressed.
2 pairs of proteasome activity restraining effect of compound are assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 2 can suppress CT-L97.1% in mouse blood simultaneously, and T-L57.8% and PGPH45.7% are active.
Synthesizing of compound 2
Flow process 4
Synthetic resins 1015.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1014 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1), and BOP (3.2mmo1), after fully stirring, filter, with DCE, dimethyl formamide, Virahol and ether rinse, and dry air obtains resin 1015 for 24 hours.
Synthetic compound 1016.Compound 1016 is identical with the preparation process of compound 1013.
Synthetic compound 2.To adding in the dichloromethane solution of compound 1016 (0.17mmo1), add DEPBT (0.21mmo1) and DIEA (0.34mo1), stir half an hour, add compound 1005 (0.20mmo1), stirred overnight at room temperature, concentrated post and obtained compound 2.Compound 2 nuclear magnetic resonance datas: 1hNMR (400MHz, DMSO-d6): δ 0.76-0.87 (m, 12H), 1.26-1.39 (m, 7H), 1.45-1.63 (m, 2H), 1.73-1.87 (m, 2H), 2.44-2.54 (t, 2H), 2.57 (s, 3H), 2.9-2.76 (m, 1H), 2.93-2.98 (m, 2H), 3.08-3.10 (d, J=5.2Hz, 1H), 3.65-3.76 (m, 2H), 4.20-4.4 (m, 3H), 4.51-4.55 (t, 1H), 7.06-7.19 (m, 8H), 7.25-7.29 (t, 2H), 7.38 (s, 1H), 7.82-7.84 (d, J=7.6Hz, 1H), 7.99-8.01 (d, J=8.4Hz, 1H), 8.20-8.22 (d, J=7.2Hz, 1H), 8.39-8.41 (d, J=7.6Hz, 1H). Low Resolution Mass Spectra: molecular formula C 40h 53n so 6s, molecular weight calculated value 731.4, observed value (M+H +) 732.5.
Embodiment 6: compound 3, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 3 structural formulas are as follows
The restraining effect of 3 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 3 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 3 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 3/IC 50ka Feizuo meter) respectively 2.40 and 4.08.
Figure BDA00003571519000512
In mouse blood lysate, the restraining effect of 3 pairs of 20S proteasomes of compound completes by the analytical procedure providing in embodiment 2.When compound 3 concentration are 5[tM, the CT-L activity inhibited of >80%, the T-L activity inhibited of >50%, the PGPH activity inhibited of >40%.
3 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 3 can suppress the CT-L of >30% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 3
Flow process 5
Figure BDA00003571519000521
Synthetic resins 1017.In ethylene dichloride (30 milliliters) solution of 2-chlorine trityl chloride resin (3.2mmo1) and Fmoc-Phe-OH (6.4mmo1), add the rear stirring of DIEA (6.6mmo1) 1 hour.Then use DCE, dimethyl formamide, Virahol, ether washing, then dry air.To the dimethyl formamide solution that adds 20% piperidines in dimethyl formamide (40 milliliters) solution of gained resin, after fully stirring, filter, by DMF, methyl alcohol and dichloromethane rinse, dry air obtains resin 1017.
Synthetic resins 1018.Resin 1018 is identical with the preparation process of resin 1010
Synthetic resins 1019.Resin 1019 is identical with the preparation process of resin 1011
Synthetic resins 1021.In resin 1019 (1.6mmo1), add DCE, compound 1020 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), filter after fully stirring, with DCE, DMF and ether rinse, and dry air obtains resin 1021.
Synthetic compound 1022.Compound 1022 is identical with the preparation process of compound 1013.
Synthetic compound 3.To adding in the dichloromethane solution of compound 1022 (0.2mmo1), add DEPBT (0.26mmo1) and DIEA (0.4mol), stir half an hour, add compound 1005 (0.24mmo1), stirred overnight at room temperature, concentrated post and obtained compound 3.Compound 3 nuclear magnetic resonance datas 1hNMR (400MHz, Methanol-d4): δ 0.92-1.00 (m, 12H), 1.30-1.42 (m, 2H), 1.46-1.55 (m, 4H), 1.63-1.75 (m, 4H), 1.99-2.20 (m, 1H), 2.00-2.20 (m, 1H), 2.65-2.79 (m, 6H), 2.92-2.94 (d, J=4.8Hz, 1H), 3.23-3.25 (d, J=5.2Hz, 1H), 3.77-3.80 (m, 6H), 4.40-4.48 (m, 3H), 4.56-4.60 (m, 1H), 4.62-4.80 (br, 1H), 7.19-7.32 (m, 5H). Low Resolution Mass Spectra: molecular formula C 34h 53n 5o 8, molecular weight calculated value 659.4, observed value (M+H ten) 660.3.
Embodiment 7: compound 4, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 4 structural formulas are as follows
Figure BDA00003571519000531
The restraining effect of 4 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 4 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 4 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 4/IC 50ka Feizuo meter) respectively 0.66 and 0.93.
Figure BDA00003571519000532
In mouse blood lysate, the restraining effect of 4 pairs of 20S proteasomes of compound completes by the analytical procedure providing in embodiment 2.When compound 4 concentration are 1 μ M, the CT-L activity inhibited of >90%, the T-L activity inhibited of >60%, the PGPH activity inhibited of >40%.
4 pairs of proteasome activity restraining effect of compound are assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 4 can suppress the CT-L of >30% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 4
Flow process 6
Figure BDA00003571519000541
Synthetic compound 1024.The methanol solution stirred overnight at room temperature of compound 1023 (0.6mo1) and oxammonium hydrochloride (0.718mo1), after steaming desolventizes, adds water, with dichloromethane extraction, organic phase salt water washing, with anhydrous sodium sulfate drying, filter, evaporate to dryness obtains thick compound 1024.
Synthetic compound 1026.In the DMF solution of compound 1024, add NCS (0.5mo1), be heated to 60, stir and within one hour, obtain compound 1025.Reaction system is chilled to after 0 ℃, adds prop-2-yn-1-ol (0.5mol) and triethylamine (0.5mo1), stirs 10 minutes, is added to the water, be extracted with ethyl acetate organic phase salt water washing, dried over sodium sulfate, filter, vacuum concentration, crosses post and obtains compound 1026.
Synthetic compound 1027.In the dichloromethane solution of compound 1026 (40mmo1) and imidazoles (123mo1), add tertiary butyl dimethyl-silicon (TBDPSCl) (45mmo1).Under room temperature, stir 2 hours, add water, with dichloromethane extraction, organic phase salt water washing, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains compound 1027.
Synthetic compound 1028.In the THF solution of compound 1027 (37mmo1), add diisobutyl aluminium hydride (15mmo1), under room temperature, stir 1 hour, drip water, with dichloromethane extraction, organic phase is washed with sodium bicarbonate, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains compound 1028.
Synthetic compound 1029.By compound 1028 (40.87mmo1), the dichloromethane solution of tetrabromomethane (44.96mmo1) and triphenylphosphine (44.96mmo1) stirs 1 hour in 0 ℃.Mixture is concentrated, crosses column purification and obtains compound 1029.
Synthetic compound 1030.By stirring 3 hours under the DMSO solution room temperature of compound 1029 (22.8mmo1) and sodium cyanide (27.3mmo1), add water, be extracted with ethyl acetate organic phase anhydrous sodium sulfate drying, the concentrated compound 1030 that obtains.
Synthetic compound 1031.The hydrochloric acid of compound 1030 (16mmo1)/methanol solution is stirred and spent the night, add water, stir 1 hour, under vacuum, steam and desolventize, cross post and obtain compound 1031.
Synthetic compound 1032.To under the methanol solution room temperature of compound 1031 (4.68mmo1) and NaOH (9.35mmo1), stir and spend the night, under vacuum, steam except methyl alcohol, pH be adjusted to 5, obtains compound 1032.
Flow process 7
Figure BDA00003571519000551
Synthetic resins 1033.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1032 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air 24/J, time, must be to 0 resin 1033.
Synthetic compound 1034.Compound 1034 is identical with the preparation process of compound 1013.
Synthetic compound 4.DEPBT (0.31mmo1) is added in the dichloromethane solution of compound 1034 (0.24mmo1).0 ℃ is stirred 2 hours, adds compound 1005 (0.31mmo1), stirred overnight at room temperature, and concentrating under reduced pressure, crosses post and obtains compound 4.Compound 4 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.84-0.94 (m, 12H), 1.36-1.68 (m, 9H), 1.91-2.10 (m, 2H), 2.57-2.68 (m, 2H), 2.78-2.89 (m, 2H), 3.10-3.19 (m, 2H), 3.70 (q, 2H), 4.24-4.30 (m, 2H), 4.49-4.64 (m, 5H), 6.37 (s, 1H), 7.06-7.28 (m, 1OH). Low Resolution Mass Spectra: molecular formula C 40h 53n 5o 8, molecular weight calculated value 731.4, observed value (M+H +) 732.3.
Embodiment 8: compound 5, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 5 structural formulas are as follows
Figure BDA00003571519000561
The restraining effect of 5 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 5 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 5 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 5/IC 50ka Feizuo meter) respectively 0.27 and 0.52.
Figure BDA00003571519000562
In mouse blood lysate, the restraining effect of 5 pairs of 20S proteasomes of compound completes by the analytical procedure providing in embodiment 2.When compound 5 concentration are 1 μ M, the CT-L activity inhibited of >80%, the T-L activity inhibited of >60%, the PGPH activity inhibited of >40%.
5 pairs of proteasome activity restraining effect of compound are assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 5 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 5
Flow process 8
Figure BDA00003571519000571
Synthetic compound 1036.The dichloromethane solution of compound 1035 (7mmo1) and carbonyl dimidazoles (7.7mmo1) is stirred 4 hours, concentrate and obtain compound 1036, be directly used in next step.
Synthetic compound 1037.At 0 ℃, in the dichloromethane solution of compound 1036 (7mmo1) and triethylamine (21mmo1), add dimethylamine hydrochloride (14mmo1), under room temperature, stir 3 hours, concentrated, purifying obtains compound 1037.
Synthetic compound 1038.By the methanol solution stirring at room of compound 1037 (2.07mmo1) and lithium hydroxide (4.14mmo1) 4 hours, under vacuum, steam except methyl alcohol, add water, with acetic acid, regulate pH=6, ethyl acetate extraction, organic phase salt water washing, anhydrous sodium sulfate drying, filter the concentrated compound 1038 that obtains.
Flow process 9
Figure BDA00003571519000572
Synthetic resins 1039.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1038 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air is 24 hours, obtains final resin 1039.
Synthetic compound 1040.Compound 1040 is identical with the preparation process of compound 1013.
Synthetic compound 5.DEPBT (0.26mmo1) and DIEA (0.60mo1) are added in the dichloromethane solution of compound 1040 (0.2mmo1), in 0 ℃, stir 2 hours, add compound 1005 (0.26mmo1), stirred overnight at room temperature, the concentrated and purified compound 5 that obtains.Compound 5 nuclear magnetic resonance datas: 1hNMR (400MHz, CDCl 3): 60.84-0.94 (m, 12H), 1.32-1.72 (m, 9H), 1.91-2.10 (m, 2H), 2.50-2.64 (m, 2H), 2.80-2.93 (m, 7H), 3.10-3.17 (m, 1H), 3.21-3.30 (m, 1H), 3.50-3.70 (m, 2H), 4.49-4.90 (m, 5H), 5.17 (s, 1H), (6.30-6.45 d, J=13.2Hz, 1H), 7.00-7.26 (m, 11H). Low Resolution Mass Spectra: molecular formula C 43h s8n 6o 9, molecular weight calculated value 802.4, observed value (M+H +) 803.4.
Embodiment 9: compound 7, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 7 structural formulas are as follows
Figure BDA00003571519000581
The restraining effect of 7 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 7 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 7 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 7/IC 50ka Feizuo meter) respectively 1.61 and 2.00.
Figure BDA00003571519000582
In mouse blood lysate, the restraining effect of 7 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 7 concentration are 1 μ M, the CT-L activity inhibited of >80%, the T-L activity inhibited of >60%, the PGPH activity inhibited of >40%.
7 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 7 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 7
Flow process 10
Figure BDA00003571519000591
Synthetic compound 1042.Under room temperature, the oxammonium hydrochloride aqueous solution (149mmo1) is added in the methanol solution of compound 1041 (57mmo1), stirred overnight at room temperature, steams except methyl alcohol residuum acetic acid ethyl dissolution, filtrate anhydrous sodium sulfate drying, removes solvent and obtains compound 1042.
Synthetic compound 1043.The methanol solution that the 1.5N sodium hydroxide solution of compound 1042 (53mmo1) and 100ml is mixed stirs 3 hours, steam except methyl alcohol, the solution obtaining carries out acidifying, is then extracted with ethyl acetate, organic phase anhydrous sodium sulfate drying, the concentrated oily compound 1043 that obtains.
Flow process 11
Resin 1044.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1043 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred, filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air is 24 hours, obtains resin 1044.
Synthetic compound 1045.Compound 1045 is identical with the preparation process of compound 1013.
Synthetic compound 7.By in the dichloromethane solution of DEPBT (0.179mmo1) and DIEA (0.411mol) power H55 compound 1045 (0.137mmo1).In 0 ℃ of stirring 2 hours, add compound 1005 (0.179mmo1), stirred overnight at room temperature, concentrated, cross post and obtain compound 7.Compound 7 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.83-0.91 (m, 12H), 1.25-1.61 (m, 9H), 1.85-2.02 (m, 2H), 2.62-2.68 (m, 2H), 2.85-2.88 (m, 2H), 3.07-3.19 (m, 2H), 3.48-3.60 (m, 2H), 4.23-4.31 (m, 2H), 4.47-4.60 (m, 2H), 7.06-7.28 (m, 11H). Low Resolution Mass Spectra: molecular formula C 39h 51n so 8, molecular weight calculated value 717.4, observed value (M+H ten) 718.4.
Embodiment 10: compound 8, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
The structural formula of compound 8 is as follows
Figure BDA00003571519000601
The restraining effect of 8 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 8 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 8 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 8/IC 50ka Feizuo meter) respectively 0.28 and 0.55.
Figure BDA00003571519000602
In mouse blood lysate, the restraining effect of 8 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 8 concentration are 0.2 μ M, CT-L activity 98.0% is suppressed, and T-L activity 65.2% is suppressed, and PGPH activity 50.1% is suppressed.When compound 8 concentration are 1 μ M, CT-L activity 100% is suppressed, and T-L activity 79.5% is suppressed, and PGPH activity 75.1% is suppressed.When compound 8 concentration are 5 μ M, CT-L activity 98.5% is suppressed, and T-L activity 90.2% is suppressed, and PGPH activity 89.4% is suppressed.
8 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 5mg/kg, compound 8 can suppress in mouse blood 92.5% CT-L simultaneously, 50.5% T-L, and 41.0% PGPH is active.
Synthesizing of compound 8
Flow process 12
Figure BDA00003571519000611
Compound 1047.By the DMF solution stirred overnight at room temperature of compound 1046 (0.120mo1) and potassium cyanide (0.156mo1), add successively reaction solution to extract in water and ethyl acetate, then organic phase concentrates, and crosses post and obtains compound 1047.
Synthetic compound 1048.By hydrochloric acid/methanol solution (50ml, 4N) stirring at room of compound 1047 (25.2mmo1) 3 hours, by mixture distillation, the product purification obtaining obtained compound 1048.
Synthetic compound 1049.The methanol solution of sodium methylate (3N, 30m1) of compound 1048 (15.7mmo1) is stirred 3 hours in 40 ℃, add water after being chilled to room temperature, under room temperature, stir 3 hours, with DCM extraction, the concentrated compound 1049 that obtains of organic phase.
Flow process 13
Figure BDA00003571519000612
Synthetic resins 1050.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1038 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air is 24 hours, obtains final resin 1050.
Synthetic compound 1051.Compound 1051 is identical with the preparation process of compound 1013.
Synthetic compound 8.In the dichloromethane solution of DEPBT (0.21mmo1) and DIEA (0.48mo1) power N55 compound 1051 (0.16mmo1).In 0.C stirs 2 hours, adds compound 1005 (0.213mmo1), and stirred overnight at room temperature is concentrated, crosses post and obtains compound 8.Compound 8 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.74-0.91 (m, 12H), 1.32-1.58 (m, 9H), 1.90-2.02 (m, 2H), 2.52-2.65 (m, 2H), 2.85-2.90 (m, 2H), 3.06-3.16 (m, 2H), 3.45 (s, 2H), 4.26-4.33 (m, 2H), 4.49-4.60 (m, 2H), 6.59 (s, 1H), 7.06-7.26 (m, 10H). Low Resolution Mass Spectra: molecular formula C 39h 51n 5o 7s, molecular weight calculated value 733.4, observed value (M+H +) 734.3.
Embodiment: compound 9, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 9 structural formulas are as follows
Figure BDA00003571519000621
The restraining effect of 9 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 9 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 9 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 9/IC 50ka Feizuo meter) respectively 0.35 and 0.54.
In mouse blood lysate, the restraining effect of 9 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 9 concentration are 1 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
9 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 9 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 9
Flow process 14
Figure BDA00003571519000632
Synthetic compound 1054.The DMF solution of compound 1052 (40mmo1) and compound 1053 (20mmo1) is heated to reflux 12 hours, adds water and be extracted with ethyl acetate, organic phase salt water washing, anhydrous sodium sulfate drying, concentrated, cross column purification, obtain compound 1054.
Synthetic compound 1055.The ethanolic soln of compound 1054 (10mmo1) and the vitriol oil (5m1) is heated to reflux 6 hours, under vacuum, distills, add water and be extracted with ethyl acetate, with sodium carbonate, regulate the pH=8 of water.Ethyl acetate extraction, organic phase salt water washing, anhydrous sodium sulfate drying, the concentrated compound 1055 that obtains.
Synthetic compound 1056.To under the ethanolic soln room temperature of compound 1055 (10mmo1) and sodium hydroxide (8mmo1), stir and spend the night.Pressure reducing and steaming ethanol under vacuum, adjusts pH=5, filters the concentrated compound 1056 that obtains of filtrate.
Flow process 15
Figure BDA00003571519000641
Synthetic resins 1057.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1038 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air is 24 hours, obtains resin 1057.
Synthetic compound 1058.Compound 1058 is identical with the preparation process of compound 1013.
Synthetic compound 9.By DEPBT (0.65mmo1) and DIEA (1.5mo1) power [in the dichloromethane solution of 155 compounds 1058 (0.50mmo1).In 0 ℃ of stirring 2 hours, add compound 1005 (0.65mmo1), stirred overnight at room temperature, concentrated, cross column purification and obtain compound 9.Compound 9 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.83-0.90 (m, 12H), 1.32-1.60 (m, 9H), 1.90-2.04 (m, 2H), 2.59-2.63 (m, 2H), 2.81-2.87 (m, 2H), 3.05-3.16 (m, 2H), 3.73 (s, 2H), 4.26-4.32 (m, 2H), 4.48-4.60 (m, 2H), 4.79 (s, 2H), 7.06-7.30 (m, 11H). Low Resolution Mass Spectra: molecular formula C 40h 53n 5o ts, molecular weight calculated value 747.4, observed value (M+H +) 748.3.
Embodiment 12: compound 10, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 10 structural formulas are as follows:
The restraining effect of 10 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 10 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 10 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 10/IC 50ka Feizuo meter) respectively 0.27 and 0.35.
In mouse blood lysate, the restraining effect of 10 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 10 concentration are 1 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
10 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 10 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active
Synthesizing of compound 10
Flow process 16
Figure BDA00003571519000653
Synthetic compound 10.Compound 9 (0.087mmo1) is dissolved in to 1ml pyridine, then adds 1ml diacetyl oxide, under room temperature, stir and spend the night, concentrated, cross post and obtain compound 10.Compound 10 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.83-0.90 (m, 12H), 1.32-1.57 (m, 9H), 1.91-2.07 (m, 2H), 2.06 (s, 3H), 2.60-2.63 (m, 2H), 2.81-2.87 (m, 2H), 3.05-3.16 (m, 2H), (3.76 d, J=5.2Hz, 2H), 4.26-4.32 (m, 2H), 4.48-4.51 (m, 1H), 4.56-4.57 (m, 1H), 5.31 (s, 2H), 7.06-7.27 (m, 10H), 7.40 (s, 1H). Low Resolution Mass Spectra: molecular formula C 42h 55n so 8s, molecular weight calculated value 789.4, observed value (M+H +) 790.3.
Embodiment 13: compound 11, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 11 structural formulas are as follows
Figure BDA00003571519000661
The restraining effect of 11 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 11 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 11 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 11/IC 50ka Feizuo meter) respectively 0.36 and 0.56.
In mouse blood lysate, the restraining effect of 11 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 11 concentration are 1 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
11 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 11 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 11
Flow process 17
Synthetic compound 11.In the dichloromethane solution of compound 9 (0.1mmo1), add DMG ester (0.1mmo1), EDCI (0.1mmo1), HOBt (0.05mmo1) and DIEA (0.3mmo1).Under room temperature, stir and spend the night, concentrated, cross post and obtain compound 11.Compound 11 nuclear magnetic resonance datas: 1hNMR (400MHz, Methanol-d4): δ 0.83-0.90 (m, 12H), 1.32-1.60 (m, 9H), 1.91-2.06 (m, 2H), 2.30 (s, 6H), 2.61-2.66 (m, 2H), 2.80-2.87 (m, 2H), 3.04-3.06 (m, 1H), 3.15 (d, J=4.8Hz, 1H), 3.24 (s, 2H), 3.76 (d, J=7.2Hz, 2H), 4.27-4.33 (m, 2H), 4.47-4.51 (m, 1H), 4.56-4.57 (m, 1H), 5.38 (s, 2H), 7.06-7.27 (m, 10H), 7.41 (s, 1H). Low Resolution Mass Spectra: molecular formula C 44h 60n 6o 8s, molecular weight calculated value 832.4, observed value (M+H +) 833.4.
Embodiment 14: compound 12, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 12 structural formulas are as follows
Figure BDA00003571519000672
The restraining effect of 12 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 12 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 12 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 12/IC 50ka Feizuo meter) respectively 1.16 and 1.71.
Figure BDA00003571519000681
In mouse blood lysate, the restraining effect of 12 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 12 concentration are 5 μ M, the active >80% of CT-L is suppressed, and the active >40% of T-L is suppressed, and the active >30% of PGPH is suppressed.
12 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 12 can suppress the CT-L of >30% in mouse blood simultaneously, and T-L and PGPH are active
Synthesizing of compound 12
Flow process 18
Figure BDA00003571519000682
Synthetic compound 12.In the THF solution of compound 3 (0.11rmno1), add 7mg sodium hydride, under room temperature, stir 30 minutes, then add 31.6mg dimethylaminoethyl chloride, under room temperature, stir 6 hours, reaction product is concentrated, and purifying obtains compound 12.Compound 12 nuclear magnetic resonance datas: 1hNMR (400MHz, CDCl 3): δ 0.87-0.95 (m, 12H), 1.26-1.35 (m, 3H), 1.50-1.72 (m, 7H), 1.96-2.06 (m, 1H), 2.15-2.25 (m, 1H), 2.53 (d, J=2.4Hz, 4H), 2.63-2.70 (m, 2H), 2.82-2.90 (m, 6H), 3.04 (s, 2H), 3.28-3.30 (m, 1H), 3.73 (d, J=2.4Hz, 4H), 4.27-4.35 (m, 2H), 4.40-4.50 (m, 2H), 4.55-4.63 (m, 2H), 7.16-7.32 (m, 5H), 6.71 (d, J=6.0Hz, 1H), 6.91 (d, J=8.0Hz, 1H), 7.54-5.78 (m, 2H). Low Resolution Mass Spectra: molecular formula C 37h 58n 6o 9, molecular weight calculated value 730.4, observed value (M+H +) 731.4.
Embodiment 15: compound 13, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 13 structural formulas are as follows:
Figure BDA00003571519000691
The restraining effect of 13 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 13 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 13 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 13/IC 50ka Feizuo meter) respectively 0.24 and 0.34.
Figure BDA00003571519000692
In mouse blood lysate, the restraining effect of 13 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 13 concentration are 1 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
13 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 13 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 13
Flow process 19
Synthetic compound 1059.To under the DCM solution room temperature of compound 1055 (6mmo1) and CDI (6.6mmo1) mixing, stir 2 hours the not purified crude product compound 1059 that obtains.
Synthetic compound 1060.At 0 ℃, in the dichloromethane solution of compound 1059 (6mmo1) and triethylamine (18mmo1), add dimethylamine hydrochloride (12mmo1), stir 3 hours under room temperature, enriched product is crossed column purification and is obtained compound 1060.
Flow process 20
Figure BDA00003571519000701
Synthetic resins 1061.In resin 1011 (1.6mmo1), add DCE (30 milliliters), compound 1038 (2.8mmo1), DIEA (6.3mmo1), HOBT (3.2mmo1) and BOP (3.2mmo1), mixture is fully stirred.Filter, with DCE, dimethyl formamide, Virahol and ether, rinse, then dry air obtains final resin 1061 for 24 hours.
Synthetic compound 1062.Compound 1062 is identical with the preparation process of compound 1013.
Synthetic compound 13.In the dichloromethane solution of compound 1062 (0.20mmo1), add DEPBT (0.26mmo1) and DIEA (0.60mmol).0。Under C, stir 2 hours, then add compound 1005 (0.26mmo1), under room temperature, stir and spend the night, the concentrated and purified compound 13 that obtains.Compound 13 nuclear magnetic resonance datas: 1hNMR (400MHz, CDCl 3): δ 0.76-0.89 (m, 12H), 1.22-1.60 (m, 9H), 1.85-1.97 (m, 1H), 2.00-2.10 (m, 1H), 2.56-2.62 (m, 2H), 2.83 (d, J=4.8Hz, 1H), 2.91 (d, J=6.8Hz, 6H), 2.98-3.07 (m, 1H), 3.09-3.13 (m, 1H), 3.27 (d, J=4.8Hz, 1H), 3.70 (s, 2H), 4.15-4.27 (m, 2H), 4.45-4.52 (m, 1H), 4.58-4.68 (m, 1H), 5.33 (s, 2H), 6.46 (d, J=6.4Hz, 1H), 6.51 (d, J=7.6Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 7.15-7.30 (m, 11H). Low Resolution Mass Spectra: molecular formula C 43h 58n 6o ss, molecular weight calculated value 818.4, observed value (M+H +) 819.3.
Embodiment 16: compound 20, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 20 structural formulas are as follows
The restraining effect of 20 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 20 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 20 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 20/IC 50ka Feizuo meter) respectively 0.17 and 0.18.
Figure BDA00003571519000712
In mouse blood lysate, the restraining effect of 20 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 20 concentration are 5 μ m, the active >80% of CT-L is suppressed, and the active >50% of T-L is suppressed, and the active >40% of PGPH is suppressed.
20 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 20 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 20
Flow process 21
Synthetic compound 1064.To compound 1001 (70.1mmo1), in the DMF solution of compound 1063 (69.8mmo1) and HATU (69.8mmo1), add DIEA (24ml, then stirred overnight at room temperature, reactant is concentrated, the product dilute with water obtaining, ethyl acetate extraction, organic phase anhydrous sodium sulfate drying, concentrated, the product obtaining carried out column purification, obtained compound 1064.
Synthetic compound 1065.Dichloromethane solution (40m1) to adding TFA in the methylene dichloride of compound 1064, then stirs reaction mixture dilute with water 3 hours, with dichloromethane extraction, the methylene dichloride phase water merging rinses, and anhydrous sodium sulfate drying obtains white solid compound 1065 after concentrating.
Synthetic compound 1066.To compound 1065 (25.7mmo1), Boc-HFE-OH (25.7mmo1), in the DMF solution of HATU (9.8g, 25.7mmo1), add DIEA (9.5rnL), then stirred overnight at room temperature, reaction mixture dilute with water, ethyl acetate extraction, the organic phase dried over mgso of merging, concentrated, by purification by flash chromatography, obtain white solid compound 1066.
Synthetic compound 1067.Methyl alcohol/THF of compound 1066 (3.6mmo1) (10:10m1) solution is cooled to 0 ℃, adds LiOH.H2O (10.8mmo1), then stirring at room is 3 hours.Reaction mixture slowly drips, and is acidified to pH<6, ethyl acetate extraction, and the organic phase water of merging rinses, anhydrous sodium sulfate drying, the concentrated colorless oil compounds 1067 that obtains.
Synthetic compound 1068.To compound 1067 (3.1mmo1), compound 1005 (3.5mmo1), in the DMF solution of HATU (3.2mmo1), add DIEA (0.5mL), then stirred overnight at room temperature, reaction mixture dilute with water, ethyl acetate extraction, the organic phase dried over mgso merging, concentrated, by purification by flash chromatography, obtain white solid compound 1068.
Synthetic compound 1069.In the dichloromethane solution of compound 1068 (4.3mmo1), add TFA (10m1), stir 4 hours under room temperature, concentrated not being further purified of reaction mixture obtains brown oily compound 1069 (500mg).
Flow process 22
Figure BDA00003571519000731
Synthetic compound 20.To compound 1069 (0.15mmo1), compound 1070 (0.2mmo1), in the DMF solution of HATU (0.16mmo1), add DIEA (0.1mL), then stirred overnight at room temperature, reaction mixture dilute with water, dichloromethane extraction, the organic phase dried over mgso merging, concentrated, the product purified by flash chromatography obtaining, obtains white solid compound 20.Compound 20 nuclear magnetic resonance datas: 1hNMR (400MHz, DMSO-d6): δ 0.79-0.88 (m, 12H), 1.28-1.39 (m, 7H), 1.51-1.53 (m, 2H), 1.99-2.03 (t, 2H), 2.50-2.58 (m, 2H), 2.71-2.77 (m, 1H), 2.93-2.97 (m, 2H), 3.10-3.12 (d, J=5.2Hz, 1H), 4.32-4.42 (m, 2H), 4.52-4.59 (m, 2H), 7.04-7.26 (m, 10H), 8.01-8.03 (d, J=8Hz, 1H), 8.22-8.24 (d, J=7.6Hz, 2H), 8.76-8.80 (m, 2H), 8.90-8.91 (d, J=2.4Hz, 1H), 9.19-9.20 (d, J=1.6Hz, 1H). Low Resolution Mass Spectra: molecular formula C 39h 50n 6o 6, molecular weight calculated value 698.4, observed value (M+H +) 699.4.
Embodiment 17: simultaneous test: compound 21, structure, proteasome activity rejection, and synthetic method
Compound 21 structural formulas are as follows
Figure BDA00003571519000732
The restraining effect of 21 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 21 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 21 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 21/IC 50carfilzomib) be respectively 23 and 39.The active IC suppressing of T-L of 21 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body 50value is respectively up to 181 μ M and 39.7 μ M.The active IC suppressing of PGPH of 21 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body 50be worth also highly, be respectively 4.41 μ M and 12.3 μ M.The restraining effect of 21 pairs of proteasome activities of compound is not good enough.Work as R 1group becomes
Figure BDA00003571519000741
time, the CT-L of compound to 20S proteasome, the restraining effect of T-L and PGPH activity significantly reduces, and can not suppress the CT-L of 20S proteasome simultaneously, and T-L and PGPH are active, and this makes compound be unfavorable for the result for the treatment of of expectation.This shows, the R providing in the application 1group can suppress the CT-L of 20S proteasome simultaneously, and T-L and PGPH activity are beyond thought.
Synthesizing of compound 21
Flow process 23
Figure BDA00003571519000743
Synthetic compound 1073.In the THF solution of compound 1071 (27.7mmo1), add TEA (27.7mmo1) at 0 ℃.Add compound 1072 (27.7mmo1) again, stir 5 hours under room temperature, filter, concentrated, purifying obtains yellow oily compound 1073.
Synthetic compound 1074.In the THF solution of compound 1073 (0.38mmo1), add LiOH.H2O (0.58mmo1), then stirred overnight at room temperature.Reaction mixture is concentrated, and the product obtaining slowly drips, and is acidified to pH<6, dichloromethane extraction, and the organic phase water of merging rinses, anhydrous sodium sulfate drying, the concentrated and purified colorless oil compounds 1074 that obtains.
Synthetic compound 1075.To compound 1074 (0.50mmo1), compound 1069 (0.50mmo1), in the DMF solution of HATU (0.55mmo1), add DIEA (0.2mL), stirred overnight at room temperature then, reaction mixture is concentrated, the product dilute with water obtaining, aqueous solution dichloromethane extraction, organic phase dried over mgso, concentrated, the product purification obtaining, obtains white solid compound 1075.
Synthetic compound 21.In the THF solution of compound 1075 (0.08mmo1), add water and 10%pd/C, stir 2 hours under room temperature hydrogen shield, filter, filtrate is processed with sodium carbonate, filters and obtains white solid compound 21.Compound 21 nuclear magnetic resonance datas: 1hNMR (400MHz, DMSO-d6): δ 0.72-0.86 (m, 12H), 1.06-1.11 (m, 1H), 1.21-1.49 (m, 7H), 1.66-1.89 (t, 1H), 1.90-1.97 (m, 2H), 2.49-2.67 (m, 2H), 2.90-2.94 (m, 3H), 2.98-3.19 (m, 1H), 4.10-4.46 (m, 6H), 7.06-7.35 (m, 10H), 8.10-8.11 (d, 1H), 8.46 (s, 1H), 8.78-8.79 (t, 1H), 9.04-9.05 (t, 1H). Low Resolution Mass Spectra: molecular formula C 36h 49n 4na 2o 10p, molecular weight calculated value 774.3, observed value (M+H +) 775.3.
Embodiment 18: compound 25, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 25 structural formulas are as follows:
Figure BDA00003571519000751
The restraining effect of 25 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 25 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 25 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 25/IC 50ka Feizuo meter) respectively 0.26 and 0.30.
Figure BDA00003571519000761
In mouse blood lysate, the restraining effect of 25 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 25 concentration are 1 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
25 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 2mg/kg, compound 25 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 25
Flow process 24
Figure BDA00003571519000762
Synthetic compound 25.To compound 1069 (0.33mmo1), compound 1076 (0.35mmo1), in the DMF solution of BOP (0.35mmo1), add TEA (0.2mL), stirred overnight at room temperature then, reaction mixture is concentrated, the product dilute with water obtaining, dichloromethane extraction, organic phase dried over mgso, the concentrated volatile matter of removing, by purification by flash chromatography, obtain white solid compound 25.Compound 25 nuclear magnetic resonance datas: 1hNMR400MHz, DMSO-d6): δ 0.71-0.87 (m, 12H), 1.23-1.52 (m, 7H), 1.58-1.63 (m, 2H), 1.85-1.95 (m, 2H), 2.44-2.60 (m, 2H), 2.63-2.76 (m, 1H), 2.95-2.98 (t, 2H), 3.08-3.11 (t, 1H), 4.22-4.35 (m, 3H), 4.52-4.53 (d, J=4Hz, 1H), 7.08-7.30 (m, 10H), 7.46-7.49 (d, J=12.4Hz, 2H), 7.77 (s, 1H), 7.85-7.96 (m, 2H), 8.18-8.22 (t, 2H). Low Resolution Mass Spectra: molecular formula C 38h 50n 6o 6s, molecular weight calculated value 718.4, observed value (M+H +) 719.4.
Embodiment 19: compound 27, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 27 structural formulas are as follows
Figure BDA00003571519000771
The restraining effect of 27 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 27 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 27 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 27/IC 50ka Feizuo meter) respectively 0.37 and 0.40.
Figure BDA00003571519000772
In mouse blood lysate, the restraining effect of 27 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 27 concentration are 5 μ M, the active >80% of CT-L is suppressed, and the active >60% of T-L is suppressed, and the active >40% of PGPH is suppressed.
27 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, compound 27 can suppress the CT-L of >40% in mouse blood simultaneously, and T-L and PGPH are active.
Synthesizing of compound 27
Flow process 25
Figure BDA00003571519000781
Synthetic compound 1078.Sodium formiate (7.05mmo1) is dissolved in the formic acid of 2.15ml, then adds compound 1077 (8.54mmo1), under room temperature, stir 4 hours, filter.In filtrate, add 2.92ml diacetyl oxide, under room temperature, stir 4 hours.Solvent evaporated, under remaining product high vacuum, distillation obtains N-formyl sarcosine ethyl.Potassium tert.-butoxide (6.14mmo1) is suspended in diethyl ether and is chilled to 0 ℃.N-formyl sarcosine ethyl (6.2mmo1) and methyl-formiate (4.32mmo1) are dissolved in 5ml diethyl ether, and drop in above-mentioned potassium tert.-butoxide diethyl ether solution, and time for adding is 30 minutes, drip off rear stirring 1 hour.Filter solid product, soluble in water, add 1.8ml concentrated hydrochloric acid, in steam bath, heat 30 minutes, cooling, adjust pH=5.Add 5.95mmol cyanamide, be heated to 100 ℃ and keep 1 hour.Cooling, filter and obtain compound 1078.
Synthetic compound 1079.Compound 1078 (0.52mmo1) is dissolved in to THF, adds dimethyl dicarbonate butyl ester acid anhydride (1.29mmo1) and 4-DMAP (1.03mmo1), under room temperature, stir and spend the night.Ethyl acetate extraction, the concentrated and purified compound 1079 that obtains of organic phase.
Synthetic compound 1080.Compound 1079 (0.43mmo1) is dissolved in to mixture (the 10ml THF: water=2:1), then add LiOH.H of THF and water 2o (4.3mmo1), stirs 4 hours, and evaporate to dryness is adjusted pH=5.Ethyl acetate extraction, the product evaporate to dryness by obtaining, obtains compound 1080.
Synthetic compound 1081.By compound 1080 (0.33mmo1), compound 1069 (0.34mmo1), in the DMF solution of HATU (0.37mmo1), add DIEA (0.2mL), stirred overnight at room temperature then, reaction mixture is concentrated, the product dilute with water obtaining, aqueous solution dichloromethane extraction, organic phase dried over mgso, concentrated, the product purification obtaining, obtains white solid compound 1081.
Synthetic compound 27.In the dichloromethane solution of compound 1081 (0.1mmo1), add TFA (0.2ml), stir 3 hours under room temperature, concentration of reaction solution, by flash chromatography purifying, obtains white solid compound 27.Compound 27 nuclear magnetic resonance datas: 1hNMR (400MHz, DMSO-d6): δ 0.77-0.87 (m, 12H), 1.23-1.39 (m, 7H), 1.46-1.68 (m, 2H), 1.88-1.94 (m, 2H), 2.49-2.50 (t, 1H), 2.72-2.76 (t, 2H), 2.94-2.99 (m, 2H), 3.10-3.11 (d, J=5.2Hz, 1H), 3.5 (s, 3H), 4.23-4.36 (m, 3H), 4.52-4.53 (d, J=4.8Hz, 1H), 5.82 (s, 2H), 7.08-7.33 (m, 11H), 7.82-7.89 (m, 3H), 8.17-8.19 (d, J=8Hz, 1H). Low Resolution Mass Spectra: molecular formula C 39h 53n 7o 6. molecular weight calculated value 715.4, observed value (M+H +) 716.5.
Embodiment 20: compound 28, structure, proteasome activity rejection, in vitro proteasome activity rejection, the active rejection of body endoproteinase body, and synthetic method
Compound 28 structural formulas are as follows:
Figure BDA00003571519000791
The restraining effect of 28 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body activity completes by the inhibition of enzyme activity measuring method providing in embodiment 1.Form has been listed the CT-L of 28 pairs of 20S structural protein enzyme bodies of compound and 20S immunity protease body below, the active IC suppressing of T-L and PGPH 50value.Compound 28 and Ka Feizuo meter (Carfilzomib) are to the active IC suppressing of the CT-L of 20S structural protein enzyme body and 20S immunity protease body 50ratio (IC 50compound 28/IC 50ka Feizuo meter) respectively 0.16 and 0.15.
Figure BDA00003571519000792
In mouse blood lysate, the restraining effect of 28 pairs of 20S proteasomes of compound can complete by the analytical procedure providing in embodiment 2.When compound 28 concentration are 0.2 μ M, CT-L activity 96.1% is suppressed, and T-L activity 57.8% is suppressed, and PGPH activity 36.0% is suppressed.When compound 28 concentration are 1 μ M, CT-L activity 98.4% is suppressed, and T-L activity 73.5% is suppressed, and PGPH activity 48.2% is suppressed.When compound 28 concentration are 5 μ M, CT-L activity 100% is suppressed, and T-L activity 83.2% is suppressed, and PGPH activity 64.9% is suppressed.
28 pairs of proteasome activity restraining effect of compound can be assessed by the method providing in embodiment 3 in vivo.When injected dose is 4mg/kg, it is active that compound 28 can suppress in mouse blood 97.5% CT-L simultaneously, and 72.1% T-L is active, and 68.4% PGPH is active.
Synthesizing of compound 28
Flow process 26
Figure BDA00003571519000801
Synthetic compound 28 is to compound 1082 (0.19mmo1), compound 1069 (0.17mmo1), in the DMF solution of HATU (0.18mmo1), add DIEA (0.1mL), stirred overnight at room temperature then, reaction mixture is concentrated, the product dilute with water obtaining, aqueous solution dichloromethane extraction, organic phase dried over mgso, concentration of reaction solution, by flash chromatography purifying, obtain white solid compound 28.Compound 28 nuclear magnetic resonance datas: 1nMR (400MHz, DMSO-d6): δ 0.73-0.87 (m, 12H), 1.19-1.40 (m, 7H), 1.39-1.50 (m, 1H), 1.52-1.63 (m, 1H), 1.73-1.76 (m, 1H), 1.84-1.88 (m, 1H), 2.33-2.44 (m, 2H), 2.56-2.76 (m, 1H), 2.93-3.22 (m, 5H), 4.22-4.36 (m, 3H), 4.51-4.53 (t, 1H), 6.25 (s, 1H), 6.88 (s, 2H), 7.06-7.29 (m, 10H), 7.88-7.90 (d, J=8.4Hz, 1H), 7.97-7.99 (d, J=8Hz, 1H), 8.13-8.15 (d, J=8Hz, 1H), 8.20-8.22 (d, J=7.2Hz, 1H). Low Resolution Mass Spectra: molecular formula C 39h 52n 6o 6s, molecular weight calculated value 732.4, observed value (M+H +) 733.4.

Claims (16)

1. the compound with structure shown in formula (I), and enantiomorph, diastereomer, tautomer, and the acceptable salt of medicine or solvate or prodrug:
Figure FDA00003571518900011
R wherein 1be selected from
Figure FDA00003571518900013
R 2be-(CH 2) mR 5;
Each R 3independently be selected from hydrogen, hydroxyl, C 1-10alkyl, C 1-10alkoxyl group, C 1-10alkane hydroxyl, C 1-10alkoxyalkyl, amino, NHR 6,-R 7-O (C=O)-R 8,-R 7-(C=O) X-R 8,-R 7-OPO 3m 1m 2,
Figure FDA00003571518900014
R 5for phenyl, or R y,
R yfor-hydroxyl ,-OP0 3m 1m 2,-R 10-O (C=O)-R 11,
Figure FDA00003571518900022
Figure FDA00003571518900023
R 6for C 1-10alkyl, phenyl ,-(C=O) C 1-6alkyl ,-(C=O) phenyl;
Each R 7, R 9and R 10independently be selected from and do not exist, or C 1-10alkylidene group (preferably-CH 2-,-C 2h 4-,-C 3h 7-, etc.); Each R 8and R 11independently be selected from hydrogen, hydroxyl, metal ion (preferred Na +, and K +), C 1-10alkyl (preferred C 1-4alkyl) ,-C 1-10alkylidene group-NR 12r 13,-NR 12r 13, or-OPO 3m 1m 2; Each R 12and R 13independently be selected from hydrogen, C 1-10alkyl is (as C 1-4alkyl) or replace C 1-10alkyl (preferred C 1-4alkyl);
Each M 1, and M 2independently be selected from hydrogen, metal ion (preferred Na +, and K +);
X is not for existing or O;
Y for do not exist or-(C=O)-;
Z is not for existing or O; And
M is 0,1,2,3,4 or 5.
2. the acceptable salt of compound as claimed in claim 1, and enantiomer, diastereomer, tautomer and medicine thereof, solvate or prodrug, wherein said compound comprises and is selected from formula I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, the structure of I-16.
3. compound as claimed in claim 1 or 2, the compound of wherein said formula (I) has the structure shown in formula (II):
Figure FDA00003571518900031
R wherein 1and R 2definition described in claim 1.
4. compound as claimed in claim 3, and enantiomer, diastereomer, tautomer and pharmacy acceptable salt thereof, solvate or prodrug, wherein said compound comprises and is selected from compound 1,2,3,4,5,7,8,9,10,11,12,13,20,25,27 and 28 structure.
5. the compound as described in claim 1 or 3, the compound of described formula (I) has the structure shown in formula (III):
Figure FDA00003571518900032
R wherein 3definition described in claim 1.
6. as the compound in claim 1 or 3, the compound of wherein said formula (I) has the structure shown in formula (1V):
R wherein 3definition described in claim 1.
7. the compound as described in claim 1 or 3, the compound of wherein said formula (I) has the structure shown in formula (v):
Figure FDA00003571518900041
Wherein the definition of R3 is described in claim 1.
8. the compound as described in claim 1 or 3, the compound of wherein said formula (I) has the structure shown in formula (VI):
Figure FDA00003571518900042
R wherein 4and R ydefinition as claimed in claim 1.
9. the compound as described in claim 1 or 3, the compound of wherein said formula (I) has the structure shown in formula (VII)
Figure FDA00003571518900043
R wherein 1be selected from
Figure FDA00003571518900044
Figure FDA00003571518900051
R wherein 3definition described in claim 1.
10. the compound as described in claim 1 or 3, the compound of wherein said formula (I) has the structure shown in formula (VIII):
Figure FDA00003571518900052
R wherein 1be selected from
11. pharmaceutical compositions comprise arbitrary compound and the pharmaceutically acceptable carrier in claim 1-10.
12. 1 species specificity suppress the method for the catalytic activity of 20S proteasome, comprise the compound in the claim 1-10 that treats effective dose.
13. methods as claimed in claim 11, the CT-L of wherein said 20S proteasome is active, and T-L activity and PGPH activity are all suppressed simultaneously.
The method of 14. 1 kinds of treatments and proteasome relative disease, comprises the compound in the claim 1-10 that treats effective dose.
The purposes of compound in the medicine for the preparation of the treatment disease relevant to 20S proteasome in 15. claim 1-10.
Method in 16. claims 14 or the purposes in claim 15, the relevant disease of wherein said 20S proteasome is selected from lower group: cancer, neurotoxic/degenerative disease, alzheimer's disease, ischemic disease, inflammation, Ia disease, HIV infects, organ-graft refection, septic shock, antigen presentation suppresses, and viral gene expression reduces, parasitic infection, the disease that oxypathy is relevant, macular degeneration, pulmonary disorder, muscular dystrophy, fibrotic disease, bone and hair growth disease.
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