Embodiment
Being used for illustrational exemplary application by combining, hereinafter many aspects of the present invention being described.Should be understood that, list many concrete details, relation and method and carry out complete understanding the present invention.But those of ordinary skill in the art easily can recognize that the present invention can implement not in accordance with one or more in detail or can implement by additive method.The present invention by the restriction of illustrational working order or event because the order that certain operations can be different is carried out and/or can be operated with other or event is together carried out simultaneously.
Further, not all illustrational operation or event all need to implement according to method of the present invention.
Term used herein is only used to embodiment is described, and be not intended to limit the present invention.Unless expressly stated otherwise, the article (" a ", " an " and " the ") not indicating concrete number is as used herein intended to comprise plural form.In addition, the term that uses in embodiment part and/or claim " comprises (" including "; " include "), has (" having ", " has ", " with ") or its variant and be intended to comprise and " comprise (comprising) " similar mode with term.
Term " about " or " approximately " refer to that the particular value measured by those of ordinary skill in the art is in acceptable limit of error, and this limit of error depends in part on this value and how to measure or to determine, that is, the restriction of measuring system.Such as, " about " may imply that each operates in 1 Standard deviation-Range or more than a standard deviation in the art.Alternatively, " about " may imply that up to set-point 20% scope, preferably up to set-point 10% scope, more preferably up to set-point 5% scope and more preferably up to set-point 1% scope.Alternatively, especially for biosystem or process, this term may imply that within the scope of certain order of magnitude of certain value, preferably within the scope of 5 times of certain value, more preferably within the scope of 2 times of certain value.Describe the condition of particular value in the application and claims under, except as otherwise noted, should suppose that term " about " means in the acceptable limit of error of particular value.
lexical or textual analysis and abbreviation
Except as otherwise noted, all technical terms used herein and scientific terminology have the implication identical with the implication that general technical staff of the technical field of the invention understands usually usually.Generally speaking, the experimental implementation in naming system used herein and cell cultures, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization is well known in the art and those naming systems generally used and experimental implementation.Standard technique is used for the synthesis of nucleic acid and peptide.Described technology is carried out with the usual ordinary method according to this area of operation and various different routine reference document, and ordinary method and the various different routine reference document of this area provide in this article.Experimental implementation in naming system used herein and analytical chemistry described below and organic synthesis is well known in the art and the naming system generally used and experimental implementation.Standard technique or its improvement are used for chemosynthesis and chemical analysis.
Except as otherwise noted, term " alkyl " himself or refer to containing the carbon atom specified number (that is, C as another substituent part1-C10refer to 1 to 10 carbon atom) straight or branched or cyclic hydrocarbon free radical or its combination, described straight or branched or cyclic hydrocarbon free radical or its combination can be completely saturated, monounsaturated or polyunsaturated and can comprise divalence and multivalence free radical.The example of saturated hydrocarbon free radical includes but not limited to following group, such as, methyl, ethyl, n-propyl group, sec.-propyl, n-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, Cvclopropvlmethvl, their homologue or isomer, such as, n-amyl group, n-hexyl, n-heptyl, n-octyl group etc.Unsaturated alkyl is the alkyl with one or more double bonds or triple bond.The example of unsaturated alkyl includes but not limited to: vinyl, 2-propenyl, crot(on)yl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(Isosorbide-5-Nitrae-pentadienyl), ethynyl, 1-proyl and 3-proyl, 3-butynyl and higher homologue and isomer.Except as otherwise noted, those alkyl derivatives of specific definition more below term " alkyl " is also intended to comprise, such as, " assorted alkyl ".The alkyl group being defined as alkyl is called as " equal alkyl (homoalkyl) ".
Term " alkene " himself or refer to from alkane as another substituent part and derive the biradical obtained, such as, but not limited to-CH2cH2cH2cH2-, and under comprising, look like those groups described by " assorted alkene ".Usually, alkyl (or alkene) group can have 1 to 24 carbon atom, is preferably those groups with 10 or less carbon atom in the present invention." low alkyl group " or " light alkene " is comparatively short-chain alkyl or olefin group, usually has 8 or less carbon atom.
Term " alkoxyl group ", " alkylamino " and " alkylthio " (or thio alkoxy) use with its conventional sense, and refer to those alkyl groups be connected with the remainder of molecule by Sauerstoffatom, amino group or sulphur atom respectively.
Except as otherwise noted, term " assorted alkyl " himself or is combined with another term and refers to that being selected from that the heteroatoms of O, N, Si and S is formed, stable straight or branched or cyclic hydrocarbon free radical or its by the carbon atom of defined amount and at least one combines, and, wherein, nitrogen and sulphur atom can be optionally oxidized and nitrogen heteroatom can by optionally quaternized.Heteroatoms O, N and S and Si can be positioned at any interior location of assorted alkyl or be positioned at the position that alkyl is connected with the remainder of molecule.Example includes but not limited to :-CH2-CH2-O-CH3,-CH2-CH2-NH-CH3,-CH2-CH2-N (CH3)-CH3,-CH2-S-CH2-CH3,-CH2-CH2,-S (O)-CH3,-CH2-CH2-S (O)2-CH3,-CH=CH-O-CH3,-Si (CH3)3,-CH2-CH=N-OCH3, and-CH=CH-N (CH3)-CH3.Maximum two heteroatomss can be continuous, such as, and-CH2-NH-OCH3with-CH2-O-Si (CH3)3.Similarly, term " assorted alkene " himself or refer to as another substituent part the biradical obtained from assorted alkyl derivative, such as but not limited to-CH2-CH2-S-CH2-CH2-He – CH2-S-CH2-CH2-NH-CH2-.For assorted olefin group, heteroatoms also can occupy two ends (such as, alkene oxygen, alkene dioxy, alkene are amino, alkene diamino, etc.) of an end in the end of the chain or the end of the chain.And for the linking group of alkene and assorted alkene, the direction in the molecular formula of the linking group write does not imply the direction of linking group.Such as, molecular formula-C (O)2r '-representative-C (O)2r '-and-R ' C (O)2-.
Generally speaking, " acyl substituent " is also selected from above-named group.Term as used herein " acyl substituent " refers to the group of the many nucleolus heart being connected to compound of the present invention and this group meets the valency of the carbonyl carbon of the many nucleolus heart being connected to compound of the present invention directly or indirectly.
Except as otherwise noted, term " cycloalkyl " and " Heterocyclylalkyl " himself or represent the annular form of " alkyl " and " assorted alkyl " respectively with the combination of other terms.In addition, for Heterocyclylalkyl, heteroatoms can occupy the position that heterocycle is connected with the remainder of molecule.The example of cycloalkyl includes but not limited to: cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl, etc.The example of Heterocyclylalkyl includes but not limited to: 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl, etc.
Except as otherwise noted, term " halo " or " halogen " himself or refer to fluorine, chlorine, bromine or iodine atom as another substituent part.In addition, such as the term of " haloalkyl " and so on is intended to comprise single haloalkyl and multi-haloalkyl.Such as, term " halo (C1-C4) alkyl " be intended to include but not limited to: trifluoromethyl, 2,2,2-trifluoroethyls, 4-chlorobutanol, 3-Bromopropyl, etc.
Term used herein " haloalkyl " refers to the alkyl defined herein replaced by one or more halogen groups defined herein.Haloalkyl preferably can be single haloalkyl, dihalo alkyl or multi-haloalkyl (comprising whole haloalkyl).Single haloalkyl can have iodine, bromine, chlorine or a fluorine in alkyl group.Dihalo alkyl and multi-haloalkyl can have the combination of two or more identical halogen atoms or different halogen groups in alkyl group.Preferably, multi-haloalkyl comprises nearly 12,10 or 8, or 6, or 4, or 3, or 2 halogen groups.The non-limiting example of haloalkyl comprises fluoromethyl, difluoromethyl, trifluoromethy, chloromethyl, dichloro-methyl, three chloromethyl, five fluoroethyl groups, seven fluorinated propyl, two fluoro chloromethyl, dichloro-fluoromethyl, two fluoroethyl groups, two fluorinated propyl, dichloro-ethyl and dichloro-propyl group.Whole haloalkyl refers to all hydrogen atoms all by alkyl that halogen atom replaces.
Term used herein " heteroaryl " refers to have 1 to 8 heteroatomic 5 yuan to 14 yuan monocycle or dicyclo being selected from N, O, S or Se or the multi-loop system condensed.Preferably, heteroaryl be 5 yuan to 10 membered ring system.Typical heteroaryl groups comprises 2-thienyl or 3-thienyl, 2-furyl or 3-furyl, 2-pyrryl or 3-pyrryl, 2-imidazolyl, 4-imidazolyl or 5-imidazolyl, 3-pyrazolyl, 4-pyrazolyl or 5-pyrazolyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl, 3-isothiazolyl, 4-isothiazolyl or 5-isothiazolyl, 2-oxazolyl, 4-oxazolyl or 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl or 5-isoxazolyl, 3-1, 2, 4-triazolyl or 5-1, 2, 4-triazolyl, 4-1, 2, 3-triazolyl or 5-1, 2, 3-triazolyl, tetrazyl, 2-pyridyl, 3-pyridyl or 4-pyridyl, 3-pyridazinyl or 4-pyridazinyl, 3-pyrazinyl, 4-pyrazinyl or 5-pyrazinyl, 2-pyrazinyl, 2-pyrimidyl, 4-pyrimidyl or 5-pyrimidyl.
Term " heteroaryl " also refers to the group that heteroaromatic ring and one or more aryl rings, annular aliphatic ring or heterocycloalkyl ring condense, and wherein, free radical or tie point are positioned on heteroaromatic ring.Non-limiting example includes but not limited to: 1-indolizine base (indolizinyl), 2-indolizine base, 3-indolizine base, 5-indolizine base, 6-indolizine base, 7-indolizine base or 8-indolizine base, 1-pseudoindoyl, 3-pseudoindoyl, 4-pseudoindoyl, 5-pseudoindoyl, 6-pseudoindoyl or 7-pseudoindoyl, 2-indyl, 3-indyl, 4-indyl, 5-indyl, 6-indyl or 7-indyl, 2-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl or 7-indazolyl, 2-purine radicals, 4-purine radicals, 5-purine radicals, 6-purine radicals, 7-purine radicals or 8-purine radicals, 1-quinolizinyl, 2-quinolizinyl, 3-quinolizinyl, 4-quinolizinyl, 6-quinolizinyl, 7-quinolizinyl, 8-quinolizinyl or 9-quinolizinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl or 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl or 8-isoquinolyl, 1-2, 3-phthalazinyl, 4-2, 3-phthalazinyl, 5-2, 3-phthalazinyl, 6-2, 3-phthalazinyl, 7-2, 3-phthalazinyl or 8-2, 3-phthalazinyl, 2-1, 5-phthalazinyl, 3-1, 5-phthalazinyl, 4-1, 5-phthalazinyl, 5-1, 5-phthalazinyl or 6-1, 5-phthalazinyl, 2-quinazolyl, 3-quinazolyl, 5-quinazolyl, 6-quinazolyl, 7-quinazolyl or 8-quinazolyl, 3-cinnolines base, 4-cinnolines base, 5-cinnolines base, 6-cinnolines base, 7-cinnolines base or 8-cinnolines base, 2-pteridyl, 4-pteridyl, 6-pteridyl or 7-pteridyl, 1-4aH carbazyl, 2-4aH carbazyl, 3-4aH carbazyl, 4-4aH carbazyl, 5-4aH carbazyl, 6-4aH carbazyl, 7-4aH carbazyl or 8-4aH carbazyl, 1-carbazyl, 2-carbazyl, 3-carbazyl, 4-carbazyl, 5-carbazyl, 6-carbazyl, 7-carbazyl or 8-carbazyl, 1-carbolinyl, 3-carbolinyl, 4-carbolinyl, 5-carbolinyl, 6-carbolinyl, 7-carbolinyl, 8-carbolinyl or 9-carbolinyl, 1-phenanthridinyl, 2-phenanthridinyl, 3-phenanthridinyl, 4-phenanthridinyl, 6-phenanthridinyl, 7-phenanthridinyl, 8-phenanthridinyl, 9-phenanthridinyl or 10-phenanthridinyl, 1-acridyl, 2-acridyl, 3-acridyl, 4-acridyl, 5-acridyl, 6-acridyl, 7-acridyl, 8-acridyl or 9-acridyl, 1-perimidinyl, 2-perimidinyl, 4-perimidinyl, 5-perimidinyl, 6-perimidinyl, 7-perimidinyl, 8-perimidinyl or 9-perimidinyl (perimidinyl), 2-phenanthroline base, 3-phenanthroline base, 4-phenanthroline base, 5-phenanthroline base, 6-phenanthroline base, 8-phenanthroline base, 9-phenanthroline base or 10-phenanthroline base, 1-phenazinyl, 2-phenazinyl, 3-phenazinyl, 4-phenazinyl, 6-phenazinyl, 7-phenazinyl, 8-phenazinyl or 9-phenazinyl, 1-thiophene phenazinyl, 2-thiophene phenazinyl, 3-thiophene phenazinyl, 4-thiophene phenazinyl, 6-thiophene phenazinyl, 7-thiophene phenazinyl, 8-thiophene phenazinyl, 9-thiophene phenazinyl or 10-thiophene phenazinyl, 1-Phenazoxine base, 2-Phenazoxine base, 3-Phenazoxine base, 4-Phenazoxine base, 6-Phenazoxine base, 7-Phenazoxine base, 8-Phenazoxine base, 9-Phenazoxine base or 10-Phenazoxine base, l-benzisoquinoline base, 3-benzisoquinoline base, 4-benzisoquinoline base, 5-benzisoquinoline base, 6-benzisoquinoline base, 7-benzisoquinoline base, 8-benzisoquinoline base, 9-benzisoquinoline base or 10-benzisoquinoline base, 2-thieno-[2, 3-b] furyl, 3-thieno-[2, 3-b] furyl, 4-thieno-[2, 3-b] furyl or 5-thieno-[2, 3-b] furyl, 2-7H-pyrazine also [2, 3-c] carbazyl, 3-7H-pyrazine also [2, 3-c] carbazyl, 5-7H-pyrazine also [2, 3-c] carbazyl, 6-7H-pyrazine also [2, 3-c] carbazyl, 7-7H-pyrazine also [2, 3-c] carbazyl, 8-7H-pyrazine also [2, 3-c] carbazyl, 9-7H-pyrazine also [2, 3-c] carbazyl, 10-7H-pyrazine also [2, 3-c] carbazyl or 11-7H-pyrazine also [2, 3-c] carbazyl, 2-2H-furo [3, 2-b]-pyranyl, 3-2H-furo [3, 2-b]-pyranyl, 5-2H-furo [3, 2-b]-pyranyl, 6-2H-furo [3, 2-b]-pyranyl or 7-2H-furo [3, 2-b]-pyranyl, 2-5H-pyrido [2, 3-d]-o-oxazines base, 3-5H-pyrido [2, 3-d]-o-oxazines base, 4-5H-pyrido [2, 3-d]-o-oxazines base, 5-5H-pyrido [2, 3-d]-o-oxazines base, 7-5H-pyrido [2, 3-d]-o-oxazines base or 8-5H-pyrido [2, 3-d]-o-oxazines base, 1-1H-pyrazolo [4, 3-d]-oxazolyl, 3-1H-pyrazolo [4, 3-d]-oxazolyl or 5-1H-pyrazolo [4, 3-d]-oxazolyl, 2-4H-imidazo [4, 5-d] thiazolyl, 4-4H-imidazo [4, 5-d] thiazolyl or 5-4H-imidazo [4, 5-d] thiazolyl, 3-pyrazine also [2, 3-d] pyridazinyl, 5-pyrazine also [2, 3-d] pyridazinyl or 8-pyrazine also [2, 3-d] pyridazinyl, 2-imidazo [2, 1-b] thiazolyl, 3-imidazo [2, 1-b] thiazolyl, 5-imidazo [2, 1-b] thiazolyl or 6-imidazo [2, 1-b] thiazolyl, 1-furo [3, 4-c] cinnolines base, 3-furo [3, 4-c] cinnolines base, 6-furo [3, 4-c] cinnolines base, 7-furo [3, 4-c] cinnolines base, 8-furo [3, 4-c] cinnolines base or 9-furo [3, 4-c] cinnolines base, 1-4H-pyrido [2, 3-c] carbazyl, 2-4H-pyrido [2, 3-c] carbazyl, 3-4H-pyrido [2, 3-c] carbazyl, 4-4H-pyrido [2, 3-c] carbazyl, 5-4H-pyrido [2, 3-c] carbazyl, 6-4H-pyrido [2, 3-c] carbazyl, 8-4H-pyrido [2, 3-c] carbazyl, 9-4H-pyrido [2, 3-c] carbazyl, 10-4H-pyrido [2, 3-c] carbazyl or 11-4H-pyrido [2, 3-c] carbazyl, 2-imidazo [1, 2-b] [1, 2, 4] triazinyl, 3-imidazo [1, 2-b] [1, 2, 4] triazinyl, 6-imidazo [1, 2-b] [1, 2, 4] triazinyl or 7-imidazo [1, 2-b] [1, 2, 4] triazinyl, 7-benzo [b] thienyl, 2-benzoxazolyl, 4-benzoxazolyl, 5-benzoxazolyl, 6-benzoxazolyl or 7-benzoxazolyl, 2-benzimidazolyl-, 4-benzimidazolyl-, 5-benzimidazolyl-, 6-benzimidazolyl-or 7-benzimidazolyl-, 2-[4-morpholinodithio base, 4-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl or 7-benzothiazolyl, 1-benzo oxa-leather base, 2-benzo oxa-leather base, 4-benzo oxa-leather base, 5-benzo oxa-leather base, 6-benzo oxa-leather base, 7-benzo oxa-leather base, 8-benzo oxa-leather base or 9-benzo oxa-leather base (benzoxapinyl), 2-benzoxazinyl-, 4-benzoxazinyl-, 5-benzoxazinyl-, 6-benzoxazinyl-, 7-benzoxazinyl-or 8-benzoxazinyl-, 1-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 2-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 3-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 5-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 6-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 7-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 8-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 9-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base, 10-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base or 11-1H-pyrrolo-[1, 2-b] [2] benzo-aza leather base (benzazapinyl).Typical fused heteroaryl group includes but not limited to: 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl or 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl or 8-isoquinolyl, 2-indyl, 3-indyl, 4-indyl, 5-indyl, 6-indyl or 7-indyl, 2-benzo [b] thienyl, 3-benzo [b] thienyl, 4-benzo [b] thienyl, 5-benzo [b] thienyl, 6-benzo [b] thienyl or 7-benzo [b] thienyl, 2-benzoxazolyl, 4-benzoxazolyl, 5-benzoxazolyl, 6-benzoxazolyl or 7-benzoxazolyl, 2-benzimidazolyl-, 4-benzimidazolyl-, 5-benzimidazolyl-, 6-benzimidazolyl-or 7-benzimidazolyl-, 2-[4-morpholinodithio base, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl or 7-benzothiazolyl.
Term as used herein " heterocyclic radical " or " heterocycle " refer to the cyclic group of completely saturated or unsaturated, the aromatic or non-aromatic be optionally substituted, such as, it is 4 yuan to 7 yuan single-loop systems, 7 yuan to 12 yuan bicyclic systems or 10 yuan to 15 yuan three-loop systems, it contains in the ring of carbon atom at least one and has at least one heteroatoms.Can have 1 that is selected from nitrogen-atoms, Sauerstoffatom and sulphur atom containing each ring in heteroatomic heterocyclic group, 2 or 3 heteroatomss, wherein, nitrogen-atoms and sulphur atom also can be optionally oxidized.Heterocyclic group can connect at heteroatoms or carbon atom place.
Exemplary monocyclic heterocyclic ring radical comprises: pyrrolidyl, pyrryl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidyl, triazolyl, oxazolyl, oxazolidinyl, isoxazoline base, isoxazolyl, thiazolyl, thiadiazolyl group, thiazolidyl, isothiazolyl, isothiazole alkyl, furyl, tetrahydrofuran base, thienyl, oxadiazoles base, piperidyl, piperazinyl, 2-oxopiperazinyl, 2-oxo-piperidine base, 2-oxo-pyrrolidine base, 2-oxo azatropylidene base (2-oxoazepinyl), azatropylidene base (azepinyl), 4-piperidone base, pyridyl, pyrazinyl, pyrimidyl, pyridazinyl, THP trtrahydropyranyl, morpholinyl, thiomorpholine base, thiomorpholine base sulfoxide, thiomorpholine base sulfone, 1, 3-dioxolane and tetrahydrochysene-1, 1-dioxythiophene base, 1, 1, 4-trioxy--1, 2, 5-thiadiazolidine-2-base, etc..
Exemplary bicyclic heterocyclic group comprises: indyl, indolinyl, benzothiazolyl, benzoxazinyl-, benzoxazolyl, benzothienyl, benzothiazine base, quinuclidinyl, quinolyl, tetrahydric quinoline group, decahydroquinolyl, isoquinolyl, tetrahydro isoquinolyl, Decahydroisoquinolinpreparation base, benzimidazolyl-, benzopyranyl, indolizine base, benzofuryl, chromone base (chromonyl), tonka bean camphor base, benzopyranyl, cinnolines base, quinoxalinyl, indazolyl, pyrrolopyridinyl, furopyridyl (such as, furo [2, 3-c] pyridyl, furo [3, 2-b] pyridyl or furo [2, 3-b] pyridyl), dihydro-iso indolyl, 1, 3-dioxy-1, 3-xylylenimine-2-base, dihydroquinazoline base (such as, 3, 4-dihydro-4-oxo-quinazolinyl), 2, 3-phthalazinyl, etc..
Exemplary tricyclic heterocyclic groups comprises: carbazyl, dibenzazepine Zhuo Ji, two thieno-azatropylidene bases, benzindole base, phenanthroline base, acridyl, phenanthridinyl, Phenazoxine base, phenothiazinyl, xanthenyl, carbolinyl, etc.
Term " heterocyclic radical " refers to the heterocyclic group as defined above replaced by the substituting group that 1,2 or 3 are selected from following groups further:
(a) alkyl;
(b) hydroxyl (or protected hydroxyl);
(c) halogen;
(d) oxygen, that is ,=O;
E () is amino, alkylamino or dialkyl amido;
(f) alkoxyl group;
(g) cycloalkyl;
(h) carboxyl;
(i) heterocyclic oxy group, wherein, heterocyclic oxy group refers to the heterocyclic group connected by oxo bridge key;
(j) alkyl-O-C (O)--;
(k) sulfydryl;
(l) nitro;
(m) cyano group;
(n) sulfamyl or sulfonamido;
(o) aryl;
(p) alkyl-C (O)-O--;
(q) aryl-C (O)-O--;
(r) aryl-S--;
(s) aryloxy;
(t) alkyl-S--;
(u) formyl radical, that is, HC (O)--;
(v) formamyl;
(w) aryl-alkyl--; And
X () is by the aryl of alkyl, cycloalkyl, alkoxyl group, hydroxyl, amino, alkyl-C (O)-NH--, alkylamino, dialkyl amido or halogen substiuted.
Term used herein " thiazolinyl " refers to have 2 to 20 carbon atoms and the straight or branched alkyl containing at least one double bond.Described thiazolinyl preferably has about 2 to 8 carbon atoms.
Except as otherwise noted, term " aryl " refers to how unsaturated aromatic hydrocarbons substituting group, and it can be monocycle or many rings (being preferably 1 ring to 3 ring), and it can be that condense or covalently bound.Term " heteroaryl " refers to the heteroatomic aromatic yl group (or aromatic ring) being selected from N, O and S containing 1 to 4, and wherein, nitrogen-atoms and sulphur atom can be optionally oxidized and nitrogen-atoms can by optionally quaternized.Heteroaryl groups is connected to the remainder of molecule by heteroatoms.The non-limiting example of aryl and heteroaryl comprises: phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, 1-pyrryl, 2-pyrryl, 3-pyrryl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purine radicals, 2-benzimidazolyl-, 5-indyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.The substituting group of each of above-mentioned aryl and heteroaryl ring-member is selected from following acceptable substituting group group.
For simplicity, when combinationally using with other terms (such as, aryloxy, fragrant sulphur oxygen base, arylalkyl), term " aryl " comprises above-mentioned aromatic ring and hetero-aromatic ring.Therefore, term " arylalkyl " is intended to comprise wherein aryl and is connected to those free radicals of alkyl (such as, benzyl, styroyl, picolyl, etc.), described alkyl comprises carbon atom (such as, methylene radical) those alkyl (such as, phenoxymethyl, 2-pyridine yloxymethyl, 3-(1-naphthyl oxygen) propyl group, etc.) of being replaced by such as Sauerstoffatom.
Each of above-mentioned term (such as " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") comprises the replacement form of specified free radical and does not replace both forms.The preferred substituting group of the free radical of every type is hereafter providing.
The substituting group of alkyl and heteroaralkyl radical (comprising those groups being commonly referred to alkene, thiazolinyl, assorted alkene, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) is called as " alkyl substituent " and " assorted alkyl substituent " usually respectively, and they can be selected from following multiple group one or more, but be not limited to following groups :-OR ',=O,=NR ',=N-OR ',-NR ' R " ;-SR ' ;-halogen ,-SiR ' R " R " ' ,-OC (O) R ' ;-C (O) R ' ,-CO2r ' ,-CONR ' R " ,-OC (O) NR ' R " and ,-NR " C (O) R ' ,-NR '-C (O) NR " R " ' ,-NR " C (O)2r ' ,-NR-C (NR ' R " R ' ")=NR " " ,-NR-C (NR ' R ")=NR ' " ,-S (O) R ' ,-S (O)2r ' ,-S (O)2nR ' R " ,-NRSO2r ' ,-CN and-NO2, quantity is 0 to (2m '+1), wherein, and the total number of carbon atoms that m ' is this free radical.R ', R ' ', R ' ' ' and R ' ' ' ' preferably separately refers to hydrogen, substituted or unsubstituted assorted alkyl, substituted or unsubstituted aryl (such as, by 1 aryl to 3 halogen substiuted), substituted or unsubstituted alkyl, alkoxyl group or thio alkoxy, or aromatic yl alkyl group.Such as, when compound of the present invention comprises more than one R group, each in R group is independently selected from each R ', R ' ', R ' ' ' and R ' ' ' ' group (when there being more than in these groups).When R ' and R ' ' is connected to identical nitrogen-atoms, they can with nitrogen-atoms in conjunction with formation 5 ring, 6 rings or 7 rings.Such as ,-NR ' R ' ' is intended to include but not limited to: 1-pyrrolidyl and 4-morpholinyl.By above to substituent discussion, it will be understood by those skilled in the art that, term " alkyl " is intended to comprise following group: this group comprises the carbon atom of the group be connected to except hydrogen group, such as, and haloalkyl (such as ,-CF3with-CH2cF3) and acyl group (such as ,-C (O) CH3,-C (O) CF3,-C (O) CH2oCH3, etc.).
Be similar to the substituent description for alkyl diradical, aryl substituent and heteroaryl substituent are hereinafter referred to as " aryl substituent " and " heteroaryl substituent " usually, and are different, be selected from such as, halogen ,-OR ',=O ,=NR ' ,=N-OR ',-NR ' R " ,-SR ' ,-halogen ;-SiR ' R " R " ' ;-OC (O) R ' ,-C (O) R ' ,-CO2r ' ,-CONR ' R " ,-OC (O) NR ' R " and ,-NR " C (O) R ' ,-NR '-C (O) NR " R " ' ,-NR " C (O)2r ' ,-NR-C (NR ' R ")=NR ' " ,-S (O) R ' ,-S (O)2r ' ,-S (O)2nR ' R " ,-NRSO2r ' ,-CN and-NO2,-R ' ,-N3,-CH (Ph)2, fluoro (C1-C4) alkoxyl group and fluoro (C1-C4) alkyl, quantity is valent sum open on 0 to aromatic ring system, and wherein, R ', R ", R " ' and R " " preferably independently selected from hydrogen, (C1-C8) alkyl and assorted alkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl and (unsubstituted aryl) oxygen-(C1-C4) alkyl.Such as, when compound of the present invention comprises more than one R group, each in R group is independently selected from each R ', R ", R " ' and R " " group (when there being more than in these groups).
Two in aryl substituent on the adjacent atom of aromatic ring or hetero-aromatic ring optionally by Tong Shi – T-C (O)-(CRR ')qthe substituting group of-U-replaces, and wherein, T and U is-NR-,-O-,-CRR independently '-or single chemical bond, and q is the integer of 0 to 3.Alternatively, two in the substituting group on the adjacent atom of aromatic ring or hetero-aromatic ring optionally by Tong Shi – A-(CH2)rthe substituting group of-B-replaces, wherein, A and B independently Wei – CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O)2-,-S (O)2nR '-or single chemical bond, and r is the integer of 1 to 4.One in single chemical bond in the new ring formed thus optionally can be replaced by double bond.Alternatively, two in the substituting group on the adjacent atom of aryl rings or heteroaryl ring optionally by Tong Shi – (CRR ')s-X-(CR " R ' ")d-substituting group replace, wherein, s and d is the integer of 0 to 3 independently, and X is-O-,-NR '-,-S-,-S (O)-,-S (O)2-， Huo – S (O)2nR '-.Substituent R, R ', R ' ' and R ' ' ' preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6) alkyl.
Term used herein " heteroatoms " comprises oxygen (O), nitrogen (N), sulphur (S), phosphorus (P) and silicon (Si).
Term used herein " aryloxy " refers to-O-aryl and both-O-heteroaryls, and wherein, aryl and heteroaryl are defined herein.
Term used herein " pharmacy acceptable salt " refers to and keeps the biological effect of compound of the present invention and the salt of character, its be not biologically undesirable neither other be undesirable.In many cases, compound of the present invention can form acid salt and/or subsalt by the amino that exists and/or carboxyl or similar group (such as, phenol or hydroxamic acid (hydroxyamic acid)).Pharmaceutically acceptable acid salt is formed by mineral acid and organic acid.The mineral acid forming salt can be derived comprise, such as, hydrochloric acid, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid, etc.The organic acid forming salt can be derived comprise, such as, acetic acid, propionic acid, oxyacetic acid, pyruvic acid, oxalic acid, toxilic acid, propanedioic acid, succsinic acid, fumaric acid, tartrate, citric acid, phenylformic acid, styracin, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, tosic acid, Whitfield's ointment, etc.Pharmaceutically acceptable base addition salt is formed by mineral alkali and organic bases.The mineral alkali forming salt can be derived comprise, such as, sodium salt, sylvite, lithium salts, ammonium salt, calcium salt, magnesium salts, molysite, zinc salt, mantoquita, manganese salt, aluminium salt, etc., particularly preferably ammonium salt, sylvite, sodium salt, calcium salt and magnesium salts.The organic bases forming salt can be derived comprise, such as, the amine (comprising the amine of the replacement of natural generation) of primary amine, secondary amine and tertiary amine, replacement, cyclammonium, deacidite, etc., especially such as, isopropylamine, Trimethylamine 99, diethylamine, triethylamine, tripropyl amine and thanomin.Pharmacy acceptable salt of the present invention is by conventional chemical processes by parent compound, and basic group or acidic-group synthesize.Generally speaking, this salt by the free acid form of these compounds and the suitable of stoichiometry alkali (such as, sodium hydroxide, calcium hydroxide, magnesium hydroxide or potassium hydroxide, carbonate, supercarbonate, etc.) reaction to prepare or prepared by reaction by the acid of the free alkali form of these compounds and the suitable of stoichiometry.These reactions are carried out usually in water or organic solvent, or carry out in the mixture of water and organic solvent.Usually, in actual applications, non-aqueous media (such as, ether, ethyl acetate, ethanol, Virahol or acetonitrile) is preferred.The list of other suitable salt can such as, and Remington's Pharmaceutical Sciences, the 20th edition, Mack Publishing Company, Easton, Pa., find in (1985), and this reference is incorporated to herein by reference.
Term used herein " pharmaceutically acceptable carrier/excipient " comprises any and all solvents known to persons of ordinary skill in the art, dispersion medium, dressing, tensio-active agent, antioxidant, sanitas (such as, antiseptic-germicide, anti-mycotic agent), isotonic agent, absorption delay agent, salt, medicine, drug stabilizing agent, bonding agent, vehicle, disintegrating agent, lubricant, sweeting agent, seasonings, dyestuff, etc., and their combination (see, such as, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp.1289-1329, this reference is incorporated to herein by reference).Except hitherto known with the inconsistent any conventional carrier of activeconstituents except, above-mentioned pharmaceutically acceptable carrier/excipient can be used for therapeutic composition or pharmaceutical composition.
Term used herein " patient " refers to animal.Preferably, described animal is Mammals.Patient also refers to such as, primate (such as, the mankind), ox, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc.In a preferred embodiment, described patient is the mankind.
compound and composition
On the one hand, the invention provides the compound that one has the structure of general formula (Ia):
TM-L-AM(Ia)，
Wherein, TM is the targeting moiety with Her2/Neu specific binding, and such as, antibody or its fragment (such as, anti-Her2 antibody), AM is the activated partial of activating dendritic cells, natural killer cell or tumour cell or their combination, and L is linker.
" activated partial " herein refers to the molecule or medicament that can stimulate or strengthen individual immunity system or tumour cell.In general, activated partial acts on toll sample acceptor, Nucleotide-oligomeric structure territory sample acceptor, RIG-I sample acceptor, c type plant lectin receptors or cytosol DNA sensor directly or indirectly, or their combination.
In some embodiments, described activated partial Activation of human immunological cell or tumour cell, or their combination, described people's immunocyte includes but not limited to: the T suppression cell of dendritic cell, scavenger cell, monocyte, bone marrow derived, NK cell, B cell, T cell.
Dendritic cell are the strongest antigen presenting cell.Dendritic cell play Main Function in the reaction of startup innate immunity and the acquired immune response.Dendritic cell also play keying action in induction and maintenance immunological tolerance.
" dendritic cell (DC) " herein refers to foreign cell group, and it comprises two main hypotypes, i.e. marrow sample DC(mDC) and Plasmacytoid DC(pDC) (people such as Steinman, 1979, J.Exp.Med., 149,1-16).These two kinds of blood DC subgroups are at first by their CD11c(integrin complement receptor) and CD123(IL-3R α) expression distinguish.Each in pDC and mDC group forms about 0.2% to about 0.6% of PBMC group in human body.
" pDC " herein refers to plasmacytoid dendritic cells, and which represent the hypotype of the dendritic cell found in blood and peripheral lymphoid organs.These cells expressing surface markers CD123, BDCA-2 (CD303) and BDCA-4 (CD304) and HLA-DR, but do not express CD11c, CD14, CD3, CD20 or CD56, this makes pDC and general dendritic cell, monocyte, T cell, B cell and NK cell be distinguished.As the composition of innate immune system, Toll-like receptor 7 and 9 in these cells express cell, this enable virus and bacterial nucleic acid detected, described virus and bacterial nucleic acid such as, ssRNA or CpG DNA motif.After stimulation and activation subsequently, these cells produce large amounts of type i Interferon, rabbit (being mainly IFN-α and IFN-β) and type iii interferon (such as, IFN-λ), and these two kinds of Interferon, rabbit are important pleiotropy antiviral compounds of the multiple effect of mediation.By producing large amounts of type i Interferon, rabbit, cytokine and chemokine, the reaction of plasmacytoid dendritic cells wide participation human body innate immunity and the acquired immune response.Their adjustable NK cells, T cell, B cell and other relate to the cell of immune response intensity, extended period and reaction pattern; therefore, they play very important effect (Liu YJ.IPC:professional type1interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol.2005 in tumour, infection and autoimmune disorders; 23:275-306.Gilliet M, Cao W, Liu YJ.Plasmacytoid dendritic cells:sensing nucleic acids in viral infection and autoimmune diseases.Nat Rev Immunol.2008Aug; 8 (8): 594-606).
" mDC " herein refers to marrow sample dendritic cell, and they represent the hypotype of the circulation dendritic cell found in blood and peripheral lymphoid organs.These cells expressing surface markers CD11c, any one in CD1a, HLA-DR and BDCA-1 (CD1c) and BDCA-3 (CD141).They do not express BDCA-2 or CD123, and this makes mDC and pDC be distinguished.MDC does not also express CD3, CD20 or CD56.As the composition of innate immune system, mDC expresses Toll-like receptor (TLR), and this receptor comprises TLR2, TLR3, TLR4, TLR5, TLR6 and TLR8, and it enables bacterium and virus composition be detected.After stimulation and activation subsequently, these cells are the most effective antigen presenting cell, thus active antigen specific C D4 and cd8 t cell.In addition, mDC has the ability producing a large amount of IL-12 and IL23, and that this ability mediates for induction Th1 or that Th17 is cell-mediated immunity is extremely important.
Research finds many solid tumors (such as; mammary cancer and head and neck cancer; ovarian cancer) there is pDC infiltration (Treilleux I; Blay JY; the people such as Bendriss-Vermare N, Dendritic cell infiltration and prognosis of early stage breast cancer.Clin Cancer Res2004; 10:7466-7474; Hartmann E; Wollenberg B; the people such as Rothenfusser S, Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer.Cancer Res2003; 63:6478-6487.Zou WP; Machelon V; Coulomb-L'Hermin A; Deng people, Stromal-derived factor-1in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.Nat Med2001; 7:1339-1346) and by the factor of tumor cell secretion suppress DC maturation (Gabrilovich DI; Corak J; the people such as Ciernik IF, Decreased antigen presentation by dendritic cells in patients with breast cancer.Clin Cancer Res1997; 3:483-490.Bell D; Chomarat P; the people such as Broyles D; In breast carcinoma tissue; immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas.J Exp Med1999; 190:1417-1425.Menetrier-Caux C; Montmain G; the people such as Dieu MC, Inhibition of the differentiation of dendritic cells from CD34 (+) progenitors by tumor cells:role of interleukin-6and macrophage colony-stimulating factor.Blood1998; 92:4778-4791).These immature DC cells cannot play a role in raising antineoplastic immune.By contrast, the DC in tumor microenvironment is by suppressing antineoplastic immune and promoting vasculogenesis and promote tumor growth.Evidence suggests that Toll-like receptor 7 agonist Imiquimod and Toll-like receptor 9 agonist CpG medicine can stimulate the pDC in tumor microenvironment; thus Tumor suppression development (Dummer R; Urosevic M; the people such as Kempf W, Imiquimod in basal cell carcinoma:how does it work Br J Dermatol2003; 149:57-58.Miller RL, Gerster JF, the people such as Owens ML, Imiquimod applied topically:a novel immune response modifier and new class of drug.Int J Immunopharmacol1999; 21:1-14.Hofmann MA; Kors C; the people such as Audring H, Phase1evaluation of intralesionally injected TLR9-agonist PF-3512676in patients with basal cell carcinoma or metastatic melanoma.J Immunother2008; 31:520-527).
Natural killer (NK) cell is a class cytotoxic lymphocyte, and it forms immune main component.NK cell is a hypotype of the peripheral blood lymphocyte defined by the shortage of the expression of CD56 or CD16 and φt cell receptor (CD3).Described natural killer cell identifies and kills and wounds the clone of conversion under the condition not causing MHC non-limiting way.NK cell is at Tumor suppression and prevent cell to be subject to playing a significant role in virus infection.NK cell recognition target cell and send enough signals with trigger target dissolve process determined by a large amount of Inhibitory receptor on cell surface and activated receptor.NK self and the NK self changed are distinguished and relates to the identification of Inhibitory receptor to the non-MHC part of MHC-1 molecule and such as CD48 and Clr-1b and so on.The NK of cell (change self) that is that infect or damage is identified by and (is comprised by various activated receptor, NKG2D, Ly49H and NKp46/Ncr1) part of stress-induced that identifies is (such as, MICA, MICB, Rael, H60, Mult1) or the part of encoding viral (such as, m157, hemagglutinin) regulate.
NK cell represent allosome or autologous stem cell transplantation after main lymphoidocyte in several months peripheral blood, and they play Main Function (people (1989) Blood73:1351-1358 such as Reittie in this time period to pathogenic agent immunity; The people such as Lowdell (1998) Bone Marrow Transplant21:679-686).NK cell acting in as Publication about Document in transplanting, graft versus host disease, anti-leukocythemia liveness and transplanting postoperative infection is looked back: Lowdell (2003) Transfusion Medicine13:399-404.
NK cells of human beings is by the dissolving of natural cytotoxicity and the dissolving of antibody dependent cellular cytotoxicity (ADCC) mediate tumor cell and the cell of virus infection.
NK cells of human beings dissolves signal control by positive and negative cells.Negative (inhibition) signal is by comprising C-phytohemagglutinin structural domain and some fixing condition (KIR) transduction of acceptor CD94/NKG2A.Hypothesis that the adjustment of being dissolved NK by inhibition signal is called as " oneself disappearance ", wherein, the I class allelotrope that specificity HLA(expresses at target cells) Inhibitory receptor on NK cell is combined.The cell of tumour cell and some virus infectiones (such as, CMV) and if on the downward of HLA molecule make this suppression be reduced to below targets threshold target cell also to carry NK and cause and activated molecule, so target cell can become and be subject to the cell-mediated dissolving impact of NK.TLR7, TLR8 or TLR9 agonist can activate both mDC and pDC, thus generates I type IFN and express the costimulatory molecules of such as GITR part and so on, activated NK subsequently, thus generates IFN-g and effectively promote NK cell killing function.
Inhibitory receptor is classified as two groups, one group for being called the Ig-superfamily of fixing condition (KIR), another group is for phytohemagglutinin family (NKG2 forms dimer at cell surface and CD94).KIR has the extracellular structure of 2 structural domains or the extracellular structure of 3 structural domains and is combined with HLA-A, HLA-B or HLA-C.NKG2/CD94 mixture is in conjunction with HLA-E.
Inhibition KIR has nearly 4 intracellular domain, and described structural domain comprises ITIM and the inhibition KIR being characterized best is known KIR2DL1, KIR2DL2 and KIR2DL3 of being combined with HLA-C molecule.KIR2DL2 and KIR2DL3 is in conjunction with first group of HLA-C allelotrope, and KIR2DL1 is in conjunction with second group of allelotrope.Some leukemia/lymphoma cells express both first group of HLA-C allelotrope and second group of HLA-C allelotrope and these leukemia/lymphoma cells known are the cytolytic of anti-NK mediation.
About positive activated signal, ADCC is considered to be mediated by CD16, and has identified the trigger receptor causing natural cytotoxicity in a large number, comprises CD2, CD38, CD69, NKRP-I, CD40, B7-2, NK-TR, NKp46, NKp30 and NKp44.In addition, several KIR molecules with afterbody in short endochylema are also irritating.Known these KIR(KIR2DS1, KIR2DS2 and KIR2DS4) be combined with HLA-C, their extracellular domain is identical with their associated inhibitory KIR.Activation KIR lacks ITIM, is combined on the contrary with causing the DAP12 of NK cell activation.The mechanism controlling the expression of inhibition KIR and activation KIR is still not clear.
Some reports have described the expression of TLR in mouse or human cancer or cancer cell system.Such as; TLR1 to TLR6 expresses (people such as Huang B, Toll-like receptors on tumor cells facilitate evasion of immune surveillance.Cancer Res.2005 by colon, lung, prostate gland and melanoma mouse tumor cell system; 65 (12): 5009 – 5014); TLR3 expresses in human breast cancer cell (Salaun B, Coste I, Rissoan MC; Lebecque SJ, Renno T.TLR3can directly trigger apoptosis in human cancer cells.J Immunol.2006; 176 (8): 4894 – 4901); liver cancer and stomach cancer cell express the people such as TLR2 and TLR4(Huang B, Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor2signaling.Cancer Res.2007; 67 (9): 4346 – 4352), and the people such as TLR9(Droemann D, Human lung cancer cells express functionally active Toll-like receptor9.Respir Res.2005; 6:1) with TLR4(He W; Liu Q; Wang L; Chen W; Li N, Cao X.TLR4signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance.Mol Immunol.2007; 44 (11): 2850 – 2859) expressed by human lung carcinoma cell.TLR7 and TLR8(Cherfils-Vicini J is found, Platonova S, Gillard M in people's lung cancer tumor cell; Laurans L; Validire P, Caliandro R, Magdeleinat P; Mami-Chouaib F; Dieu-Nosjean MC, Fridman WH, Damotte D; Sautes-Fridman C, Cremer I.J.Clin Invest.2010; 120 (4): 1285 – 1297).
TLR is induction microbial product and/or the protein families starting the acquired immune response.TLR activating dendritic cells (DC).TLR is for containing being rich in TIR(Toll/ interleukin-2-receptor in leucic repeating unit ectodomain, membrane spaning domain and cell) the conservative transmembrane molecule of structural domain.TLR identifies the different structure in microorganism, is commonly referred to " PAMP " (pathogenic agent associated molecular pattern).The part be combined with TLR causes the cascade reaction of intracellular signaling pathway, and the induction of this cascade reaction participates in the generation of the factor of inflammation and immunity.
In some embodiments, described activated partial is TLR7 and/or TLR8 agonist.TLR7 and TLR8 is relevant in phylogeny and structure.TLR7 is by people pDC and B cell selective expression.TLR8 is primarily of mDC, monocyte, scavenger cell and bone marrow depression cell expressing.TLR7 specific agonist activation Plasmacytoid DC(pDC), thus generate a large amount of 1 type IFN and express high-caliber costimulatory molecules, described costimulatory molecules promotes the activation of T cell, NK cell, B cell and mDC.The T suppression cell of TLR8 specific agonist activation marrow sample DC, monocyte, scavenger cell or bone marrow derived, thus generate a large amount of 1 type IFN, IL-12 and IL-23, and express high-caliber MHC I quasi-molecule, MHC II quasi-molecule and costimulatory molecules, described MHC I quasi-molecule, MHC II quasi-molecule and costimulatory molecules promote the activation of antigen-specific CD4 and CD8+T cell.
In some embodiments, activated partial is TLR7 and/or TLR8 agonist or its salt pharmaceutically accepted or its solvate, and it is by the representation of following general formula (I):
Wherein, dotted line represents there is chemical bond or there is not chemical bond,for treating the point be connected with linker;
X is S or-NR1, R1shi – W0-W1-W2-W3-W4;
W0chemical bond, alkyl, thiazolinyl, alkynyl, alkoxyl group or-alkyl-S-alkyl--,
W1chemical bond,--O--， Huo – NR2--, wherein, R2hydrogen, alkyl or alkenyl,
W2chemical bond,--O--,--C (O)--,--C (S)--Huo – S (O)2-,
W3chemical bond,--NR3--, wherein, R3hydrogen, alkyl or alkenyl,
W4hydrogen, alkyl, thiazolinyl, alkynyl, alkoxyl group, cycloalkyl, aryl, aryloxy, heteroaryl or heterocyclic radical, each in them is optionally replaced by one or more substituting groups being selected from following groups: hydroxyl, alkoxyl group, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic radical,--NH2, nitro,--alkyl-hydroxyl,--alkyl-aryl-group,--alkyl-heteroaryl,--alkyl-heterocyclyl groups,--O-R4,--O-alkyl-R4,--alkyl-O-R4,--C (O)-R4,--alkyl-C (O)-R4,--alkyl-C (O)-O-R4,--C (O)-O-R4,--S-R4,--S (O)2-R4,--NH-S (O)2-R4,--alkyl-S-R4,--alkyl-S (O)2-R4,--NHR4,--NR4r4,--NH-alkyl-R4, halogen,--CN,--NO2he – SH, wherein, R4be hydrogen independently, alkyl, thiazolinyl,--alkyl-hydroxyl, aryl, heteroaryl, heterocyclic radical or haloalkyl;
Z is hydrogen, alkyl, thiazolinyl, alkynyl, alkoxyl group, aryl, haloalkyl, heteroaryl, heterocyclic radical, each in them optionally can be replaced by one or more substituting groups being selected from following groups: hydroxyl, alkoxyl group, alkyl, thiazolinyl, alkynyl, aryl, heteroaryl, heterocyclic radical, halogen, cyano group, nitro,--N (R5)2,--alkoxy-alkyl,--alkoxyl group-thiazolinyl,--C (O)-alkyl,--C (O)-O-alkyl,--O-C (O)-alkyl,--C (O)-N (R5)2, aryl, heteroaryl,--CO-aryl with – CO-heteroaryl, wherein, R5be separately hydrogen, alkyl, haloalkyl,--alkyl-aryl-group or-alkyl-heteroaryl;
R is hydrogen, alkyl, alkoxyl group, haloalkyl, halogen, aryl, heteroaryl, heterocyclic radical, each in them is optionally replaced by one or more substituting groups being selected from following groups: hydroxyl, alkoxyl group, alkyl, thiazolinyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic radical,--NH2, nitro,--alkyl-hydroxyl,--alkyl-aryl-group,--alkyl-heteroaryl,--alkyl-heterocyclyl groups,--O-R4,--O-alkyl-R4,--alkyl-O-R4,--C (O)-R4,--C (O)-NH-R4,--C (O)-NR4r4,--alkyl-C (O)-R4,--alkyl-C (O)-O-R4,--C (O)-O-R4,--O-C (O)-R4,--S-R4,--C (O)-S-R4,--S-C (O)-R4,--S (O)2-R4,--NH-S (O)2-R4,--alkyl-S-R4,--alkyl-S (O)2-R4,--NHR4,--NR4r4,--NH-alkyl-R4, halogen,--CN He – SH, wherein, R4be hydrogen independently, alkyl, thiazolinyl, alkoxyl group,--alkyl-hydroxyl, aryl, heteroaryl, heterocyclic radical, or haloalkyl;
N is 0,1,2,3 or 4;
Y Wei – NR6r7， – CR6r7r8huo – alkyl-NH2, each in them optionally can be replaced by one or more substituting groups being selected from following groups: hydroxyl, alkoxyl group, alkyl, thiazolinyl, alkynyl,--NH2, halogen,--N (R5)2,--alkoxy-alkyl,--alkoxyl group-thiazolinyl,--C (O)-alkyl,--C (O)-O-alkyl,--C (O)-N (R5)2, aryl, heteroaryl,--CO-aryl with – CO-heteroaryl,
Wherein, R6, R7and R8be hydrogen independently, alkyl, thiazolinyl, alkoxyl group, alkylamino, dialkyl amido, alkylthio, arylthio,--alkyl-hydroxyl,--alkyl-C (O)-O-R9,--alkyl-C (O)-R9huo – alkyl-O-C (O)-R9, wherein, R5be separately hydrogen, alkyl, haloalkyl,--alkyl-aryl-group or-alkyl-heteroaryl, wherein, R9for hydrogen, alkyl, thiazolinyl, halogen or haloalkyl;
Optionally, X and Z together can form 5 to 9 rings.
In some embodiments, the X in general formula (I) is S.
In some embodiments, the X Wei – NR in general formula (I)1, R1for alkyl,--alkyl-W4,--alkyl-O-W4,--alkyl-NH-C (O)-W4,--alkoxyl group-NH-C (O)-W4,--alkyl-NH-C (O)-NH-W4,--alkoxyl group-NH-C (O)-NH-W4,--alkyl-S (O)2-W4, or--alkyl-NH-C (S)-W4, wherein, W4for as defined above.
In some embodiments, Z in general formula (I) is hydrogen, alkyl, alkoxyl group, aryl, heteroaryl, haloalkyl, and each in them is optionally replaced by one to three substituting group being selected from following groups: hydroxyl, alkyl, aryl, heteroaryl, heterocyclic radical, cyano group,--alkoxy-alkyl, nitro and-N (R5)2, wherein, R5be separately hydrogen, alkyl, haloalkyl,--alkyl-aryl-group or-alkyl-heteroaryl.
In some embodiments, the Y in general formula (I) is-NH2,--alkyl-NH2, each in them is optionally replaced by one to three substituting group being selected from alkyl, alkoxyl group, thiazolinyl and alkynyl.
In some embodiments, the n in general formula (I) is 1 or 2.
In some embodiments, the R in general formula (I) is aryl or heteroaryl, and each in them is optionally replaced by one to three substituting group being selected from following groups: hydroxyl, alkoxyl group,--alkyl-hydroxyl,--O-R4,--O-alkyl-R4,--alkyl-O-R4,--C (O)-R4,--C (O)-NH-R4,--C (O)-NR4r4,--alkyl-C (O)-R4,--alkyl-C (O)-O-R4,--C (O)-O-R4,--O-C (O)-R4,--S-R4,--C (O)-S-R4,--S-C (O)-R4,--S (O)2-R4,--NH-S (O)2-R4,--alkyl-S-R4,--alkyl-S (O)2-R4,--NHR4,--NR4r4,--NH-alkyl-R4, halogen,--CN He – SH, wherein, R4be hydrogen independently, alkyl, thiazolinyl, alkoxyl group,--alkyl-hydroxyl, aryl, heteroaryl, heterocyclic radical or haloalkyl.
In some embodiments, activated partial is TLR7 and/or the TLR8 agonist being selected from table 1.Compound in table 1 describes in further detail and characterizes in following patent literature: US4,689,338, US5,389,640, US5,226,575, US6,110,929, US6,194,425, US5,352,784, US6,331,539, US5,482,936, US6,451810, WO2002/46192, WO2002/46193, WO2002/46194, US2004/0014779 and US2004/0162309.
Table 1: representational TLR7 and/or TLR8 agonist
Preferably, AM is resiquimod or Imiquimod.
targeting moiety
In general, compound of the present invention comprises targeting moiety.
Molecule, mixture or aggregate that " targeting moiety (TM) " or " target agent " herein refers to specifically or be optionally combined with target molecule, cell, particle, tissue or aggregate, described target molecule, cell, particle, tissue or aggregate are commonly referred to " target " or " marker " and will discuss these target molecules, cell, particle, tissue or aggregate further in detail herein.
In some embodiments, targeting moiety comprises immunoglobulin (Ig), protein, peptide, small molecules, nano particle or nucleic acid.
The exemplary target agent of the substrate of the part of such as antibody (such as, chimeric antibody, humanized antibody and people's antibody), acceptor, phytohemagglutinin and carbohydrate and some enzymes and so on is identified and in the art without restriction for implementing the present invention.Other target agent comprise the following compound of a class: this compound does not comprise specific molecular identification motif, this compound comprises macromole, the polysaccharide of nano particle, the such as PEG and so on molecular weight being added to activated partial, and polyamino acid.The pharmacokinetics of extra molecular weight effects activated partial, such as serum half-life.
In some embodiments, targeting moiety is antibody, antibody fragment, bi-specific antibody or other molecules based on antibody or compound.Such as, but that other examples of targeting moiety are known in the art and can use other examples of targeting moiety, fit, avimer, receptor-binding ligands, nucleic acid, biotin-avidin combine, binding peptide or protein, etc.Term " targeting moiety " and " bound fraction " in this article synonym use.
" target " or " marker " herein refers to can any entity of the specific targeting moiety of specific binding, such as, and Her2/Neu.
In some embodiments, targeting moiety can specific binding Her2/Neu or can preferentially in conjunction with Her2/Neu relative to non-targeted part.
The combination that " specific binding " herein or " preferentially combine " refers between two basic change partner (such as, at targeting moiety and combine between partner) has selectivity to two basic change partner and can distinguish from undesired or non-specific interaction.Such as, the technology that the ability of antigen-binding portion thereof binding specificity antigenic determinant is familiar with by enzyme linked immunosorbent assay analysis method (ELISA) or other those skilled in the art (such as, surface plasma resonance technology (analyzing on BIAcore the instrument) (people such as Liljeblad, Glyco J17,323-329 (2000)) and traditional binding analysis (Heeley, Endocr Res28,217-229 (2002))) measure.Term " anti-[antigen] antibody " and " antibody combined with [antigen] " refer to can by the antibody of enough avidities in conjunction with respective antigen, and like this, described antibody is used as diagnostic reagent and/or the therapeutical agent of targeting antigen.In some embodiments, the degree of the incoherent protein of anti-[antigen] antibodies is less than about 10% of the antibody antigen combination degree of measured (such as, by radioimmunoassay (RIA)).In some embodiments, the dissociation constant (KD) in conjunction with the antibody of [antigen] is less than 1 μM, is less than 100nM, is less than 10nM, is less than 1nM, is less than 0.1nM, is less than 0.01nM or is less than 0.001nM(such as, 10-8m or less, such as, 10-8m to 10-13m, such as, 10-9m to 10-13m).Should be understood that, above-mentioned definition also can be applicable to the antigen-binding portion thereof be combined with antigen.
In some embodiments, targeting moiety comprises antibody or its function fragment.
" immunoglobulin (Ig) " used herein or " antibody " refer to that total length (namely, natural generation or to be formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecules (such as, IgG antibody) or immunoglobulin molecules immunocompetence (namely, specific binding) part, such as antibody fragment.In the scope of protection of present invention, antibody or antibody fragment can be coupled or derivative.These antibody comprise IgGl, lgG2a, IgG3, IgG4(and IgG4 hypotype) and IgA isotype.
Term " antibody " herein in its broad use and comprise various different antibody structure, include but not limited to: monoclonal antibody, polyclonal antibody, multi-specificity antibody are (such as, bi-specific antibody) and antibody fragment, as long as they show the antigen-binding activity of expectation and comprise the Fc region of immunoglobulin (Ig) or be equal to the region in this Fc region.The term " full length antibody " be used interchangeably herein, " complete antibody " and " whole antibody " refer to the antibody with the structure substantially similar with native antibody structure or the antibody with the heavy chain containing Fc region defined herein.
" natural antibody " herein refers to the immunoglobulin molecules of the natural generation with different structure.Such as, native IgG antibodies is about 150,000 daltonian allos tetramer glycoprotein, and its heavy chain identical with two by two that are connected by disulfide linkage identical light chains is formed.From N end to C end, each heavy chain has variable region (VH), also referred to as variable heavy chain domain or heavy-chain variable domains, is three constant domain (CH1, CH2 and CH3), also referred to as CH after heavy chain.Similarly, from N end to C end, each light chain has variable region (VL), also referred to as variable light chain domain or light variable domains, is constant light structural domain (CL), also referred to as constant region of light chain after light chain.Based on the aminoacid sequence of antibody constant structural domain, the light chain of antibody can be appointed as the one in two types (being called κ and λ).
" antibody fragment " herein refers to the molecule being different from complete antibody, and described molecule comprises a part for the complete antibody of conjugated antigen, and described antigen is combined with complete antibody.The example of antibody fragment includes but not limited to: Fv, Fab, Fab', Fab'-SH, F (ab') 2, Diabody, linear antibodies, single-chain antibody molecules (such as, scFv), single domain antibody and the multi-specificity antibody formed by antibody fragment.Summary about some antibody fragments refers to, the people such as Hudson, Nat Med9,129-134 (2003).Summary about scFv fragment refers to such as, Pliickthun, in The Pharmacology of Monoclonal Antibodies; vol.113, Rosenburg and Moore edit, Springer-Verlag; New York, pp.269-315 (1994); And WO93/16185; With United States Patent (USP) the 5th, 571, No. 894 and the 5th, 587, No. 458.About containing saving receptor binding domain residue and there is the discussion of Fab and F (ab ') 2 fragments of the Half-life in vivo of raising, refer to United States Patent (USP) the 5th, 869, No. 046.Diabody is the antibody fragment with two antigen binding sites that is that can be divalence or dual specific.Refer to such as, EP404,097; WO1993/01161; The people such as Hudson, Nat Med9,129-134 (2003); With people such as Hollinger, Proc Natl Acad Sci USA90,6444-6448 (1993).Three body antibody and limbs antibody, also people such as Hudson, describe in Nat Med9,129-134 (2003).Single domain antibody is the antibody fragment containing all of antibody or a part of heavy-chain variable domains or all or a part of light variable domains containing antibody.In some embodiments, single domain antibody behaviour single domain antibody (Domantis, Inc., Waltham, MA; See such as, U.S. Patent No. 6,248,516Bl).Antibody fragment is prepared by various different technology, and described technology includes but not limited to: as described herein, the proteolytic digestion of complete antibody and being generated by recombinant host cell (such as, intestinal bacteria or phage).
" antigen-binding domains " herein refers to and comprises with all or part of specific binding of antigen and a part for the antibody in the region of complementation.Antigen-binding domains is by such as, and one or more antibody variable territories (also referred to as antibody variable region) provide.Specifically, antigen-binding domains comprises antibody chain variable region (VL) and antibody heavy chain variable region (VH).
" variable region " or " variable domains " herein refers to the heavy chain of antibody structural domain or light chain domain that relate to and antibody is combined with antigen.The heavy chain of natural antibody and the variable domains (being respectively VH and VL) of light chain have similar structure usually, and wherein, each structural domain comprises four conservative framework regions (FR) and three hypervariable regions (HVR).See such as, the people such as Kindt, Kuby Immunology, sixth version, W.H.Freeman and Co., the 91st page (2007).Single VH or VL structural domain can be enough to bring antigen-binding specificity.
" hypervariable region " or " HVR " herein refers to the regional in the antibody variable territory of the ring (" Gao Bianhuan ") of sequence alterable height and/or formation structure qualification.Generally speaking, natural four chain antibodies comprise six HVR, and three at VH(H1, H2, H3) in, three at VL(L1, L2, L3) in.HVR comprises usually from the amino-acid residue of Gao Bianhuan and/or the amino-acid residue from complementary determining region (CDR), and the latter has the highest sequence variability and/or relates to antigen recognition.Except the CDR1 in VH, CDR comprise the amino-acid residue forming Gao Bianhuan usually.Hypervariable region (HVR) is also referred to as " complementary determining region (CDR) " and these terms relevant with the variable region portion forming antigen binding domain exchange use in this article.This specific region is by people such as Kabat, U.S.Dept.of Health and Human Services, the people such as Sequences of Proteins of Immunological Interest (1983) and Chothia, J Mol Biol196:901-917 (1987) describes, wherein, when being compared to each other, definition comprises overlap or the subset of amino-acid residue.But the application about the CDR of antibody or any definition of its variant is intended in the scope of term defined herein and used herein.The definite number of residues comprising specific CDR is different with the sequence of CDR and the difference of size.Under the condition of variable region amino acid sequence providing antibody, those skilled in the art routine can determine which residue comprises specific CDR.
Antibody of the present invention can be chimeric antibody, humanized antibody, people's antibody or antibody fusion protein.
" chimeric antibody " herein refers to the recombinant protein of the variable domains comprising both heavy chain of antibody and light chain, described variable domains comprises the antibody deriving from species and (is preferably rodent animal antibody, be more preferably murine antibodies) complementary determining region (CDR), and the constant domain of antibody molecule derives from the constant domain of people's antibody.For animal doctor application for, the constant domain of chimeric antibody can derive from the constant domain of other species, other species described such as, class human primate, cat or dog.
" humanized antibody " herein refers to following recombinant protein: in this recombinant protein, the CDR coming from the antibody (such as, rodent animal antibody) of species is transferred to people's heavy-chain variable domains and people's light variable domains from the variable heavy chain of rodent animal antibody and variable light.The constant domain of antibody molecule derives from the constant domain of people's antibody.In some embodiments, the specific residue of the framework region of humanized antibody, especially contact or near those specific residues of CDR sequence, can be modified, such as, can be come from original rodent, the corresponding residue of class human primate or other antibody and replace.
" people's antibody " herein refers to the antibody such as obtained from transgenic mice, and described transgenic mice is generated specific people's antibody by " transformation " for responding antigenic stimulation.In the art, the element of people's heavy chain gene seat and people's light chain gene seat is introduced into and derives from the mouse species of embryonic stem cell line, and described embryonic stem cell line comprises the targeted disruption of endogenous heavy chain locus and light chain gene seat.Transgenic mice can synthesize and has specific people's antibody to human antigen, and mouse can be used for the hybridoma generating secretion people antibody.The method of people's antibody is obtained by people such as Green from transgenic mice, Nature Genet.7:13 (1994), the people such as Lonberg, Nature368:856 (1994), the people such as Taylor, Int.Immun.6:579 (1994) describes.Fully human antibodies also builds by gene transfection method or chromosomal transfection method and display technique of bacteriophage, and all these methods are known in the art.Referring to such as, the people such as McCafferty, Nature348:552-553 (1990), which describing the external raw human antibodies of immunoglobulin variable domain domain gene pedigree by coming from non-immune donor and fragment thereof.In the art, be cloned in antibody variable domain gene frame in the main of filobactivirus or secondary coat protein gene, and be function antibody fragment at the displaying on surface of phage particle.Because filamentous particle comprises the single-stranded DNA copy of phage genome, the selection based on the functional property of antibody also causes the selection of gene coding schedule being revealed to the antibody of those character.By which, phage imitates the properties of B cell.Phage display can carry out in a variety of forms, and the summary about phage display refers to such as, Johnson and Chiswell, Current Opinion in Structural Biology3:5564-571 (1993).People's antibody also produces by Activated in Vitro B cell.Refer to United States Patent (USP) the 5th, 567, No. 610 and the 5th, 229, No. 275, the full content of this United States Patent (USP) is incorporated to herein by reference.
" antibody fusion protein " herein refers to the antigen binding molecules produced by restructuring, wherein, connect identical or different natural antibody, have in identical or different specific single-chain antibody or antibody fragment two or more.Fusion rotein comprises at least one specific binding site.The valency of fusion rotein represents the sum of the brachium conjunctivum be combined with antigen or epi-position that fusion rotein has or binding site, that is, unit price, divalence, trivalent or multivalence.The antibody fusion protein of multivalence refers to that this antibody fusion protein can utilize the multiple interaction be combined with antigen, therefore increases and antigen or the not avidity that is combined of synantigen.Specificity represents that antibody fusion protein can the antigen dissimilar with how many kinds of or epi-position be combined, that is, monospecific, dual specific, tri-specific, polyspecific.Use these to define, natural antibody (such as, IgG) is divalence, because it has two basic change arm, but it is monospecific, because its antigen in conjunction with a type or epi-position.Monospecific multivalent fusion proteins has more than one binding site for same antigen or epi-position.Such as, Monospecific diabodies antibody be have two with the fusion rotein of identical antigen reactive binding site.Fusion rotein can comprise the multivalence of different antibodies composition or multiple copies of multispecific combination or same antibody component.Fusion rotein also can comprise therapeutical agent.
In some embodiments, TM is monoclonal anti-HER2 antibody.HER2 is by the transmembrane tyrosine kinase acceptor of neu proto-oncogene encodes.There is heterozygosis dimerization (heterodimerize) in HER2 and HER1, HER3 or HER4, and causes the signal of cell proliferation and survival.The process LAN of cell surface HER2 is relevant to tumour or cancer.Therefore, the treatment for HER2 is useful for suffering from the patient of HER2 positive cancer or tumour.These therapeutic anti-HER2 antibody by the combination of following mechanism of action mediate these antibody to HER2 positive tumor/cancer antitumor/antitumour activity, described mechanism of action comprises: promote the adjustment (internalization and/or " coming off ") of acceptor, cell death inducing, antibody-mediated cytotoxicity (ADCC) and conditioning the antigenic determinant of cell in being handed to antigen presenting cell.
In some embodiments, anti-HER2 antibody is Herceptin (Trastuzumab), handkerchief trastuzumab or margetuximab(MGAH22).Herceptin and handkerchief trastuzumab are monoclonal antibody, and they have anti-tumor activity to HER2 positive tumor cell or HER2 positive cancer cell.Margetuximab(or MGAH22) be the monoclonal antibody (mAb) that Fc-of new generation optimizes, its target HER2.Clinical trial has illustrated that margetuximab is effective to the tumour cell of HER2 or cancer cells of expressing medium level.
Trastuzumab (Herceptin) for acting on the Humanized monoclonal antibodies of Her2 Human epidermal growth factor receptor extracellular domain, Her2 25% to 30% breast carcinoma.Herceptin is considered to have the antitumor action of following three aspects: (1) lowers Her2 acceptor, suppresses Her2 intracellular signal transduction path and cell death inducing; (2) immunologic mechanism that antibody dependent ADCC and CDC is associated with killing tumor cell is made; (3) chemotherapy effect is improved.
In some embodiments, targeting moiety comprises Fab, and Fab ', F (ab ') 2, single domain antibody, T and Abs dipolymer, Fv, scFv, dsFv, ds-scFv, Fd, linear antibodies, mini-antibody, Diabody, bispecific antibody fragment, bibody, tribody, sc-Diabody, κ (λ) body, BiTE, DVD-Ig, SIP, SMIP, DART, or the antibody analog containing one or more CDR.
In some embodiments, targeting moiety comprises extracellular domain (ECD) or PD-1, CTLA4, CD47, BTLA, KIR, the soluble form of TIM3,4-1BB and LAG3, the part surface ligand amphiregulin of total length, β tunicin, EGF, liver joins albumen, Epigen, epiregulin, IGF, neuregulin, TGF, TRAIL or VEGF.
In some embodiments, targeting moiety comprises particle (target particle), is preferably nano particle, and optionally, for being connected to the targeted nano particle of targeted molecular, described targeted molecular can specific binding or preferentially in conjunction with target.In some embodiments, target particle himself guides compound of the present invention (such as, the enrichment by tumour cell or tumor tissues), and without the need to being connected extra targeted molecular with it.
" nano particle " herein refers to that diameter is less than any particle of 1000nm.In some embodiments, therapeutical agent and/or targeted molecular can be combined with polymeric matrix.In some embodiments, targeted molecular can with the surperficial covalent attachment of polymeric matrix.In some embodiments, covalent attachment is mediated by linker.In some embodiments, therapeutical agent can be combined with polymeric body surface, is encapsulated in polymeric matrix, is surrounded by polymeric matrix, and/or is scattered in whole polymeric matrix.United States Patent (USP) the 8th, 246, No. 968, the full content of this United States Patent (USP) is incorporated to herein by reference at this.
In general, nano particle of the present invention comprises the particle of any type.Any particle can be used according to the present invention.In some embodiments, particle is biodegradable and biocompatible.In general, biocompatible substance is to cytotoxic.In some embodiments, if certain material to be added to the result producing in cell and be less than a certain threshold value of necrocytosis, so think that this material is biocompatible.In some embodiments, do not induce side effect if added in cell by certain material, so think that this material is biocompatible.In general, biodegradable material is the material issuing solution estranged through treatment section correlation time (such as, several weeks, several months or several years) at physiological conditions.In some embodiments, biodegradable material is carry out by cell mechanism the material that decomposes.In some embodiments, biodegradable material is the material decomposed by chemical process.In some embodiments, particle is physiologically acceptable and biodegradable material.In some embodiments, particle is biocompatible substance, but is not biodegradable material.In some embodiments, particle is biodegradable material, but is not biocompatible substance.
In some embodiments, the granularity of particle is greater than the renal excretion limit (such as, diameter is greater than the particle of 6nm).In some embodiments, particle size is the size (such as, diameter is less than the particle of 1000nm) being enough to avoid being removed from blood flow by liver.In general, the physiochemical properties of particle should allow target particle to be circulated for a long time in blood plasma by reduction renal excretion and hepatic clearance.
Usually, it is desirable to the particle swarm using size, shape and/or composition relatively uniform, like this, each particle has similar character.Such as, the particle of at least 80%, the diameter of the particle of at least 90% or the particle of at least 95% or overall dimension are mean diameter or overall dimension plus-minus 5%, 10% or 20%.In some embodiments, the size of particle swarm, shape and/or composition can be inhomogenous.
Multiple different particle can be used according to the present invention.In some embodiments, particle is spherical or class is spherical.In some embodiments, particle is spherical or class is spherical.In some embodiments, particle is flat or tabular.In some embodiments, particle is cubes or class cubes.In some embodiments, particle is avette or oval.In some embodiments, particle is cylindrical, conical or pyramid.
In some embodiments, particle is microparticle (such as, microballoon).In general, " microparticle " refers to that diameter is less than any particle of 1000 μm.In some embodiments, particle is micro-minitype particle (picoparticle) (such as, slight spheroid).In general, " micro-minitype particle " refers to that diameter is less than any particle of 1nm.In some embodiments, particle is liposome.In some embodiments, particle is micella.
Particle can be solid or hollow and can comprise one or more layers (such as, nanoshell, nano-rings).In some embodiments, every layer has unique composition and unique character relative to other each layers.Such as, particle can have core/shell structure, and wherein, core is one deck, and shell is another layer.Particle can comprise multiple different layer.In some embodiments, one deck can be full cross-linked, and another layer is insufficient crosslinked, etc.In some embodiments, the one deck in different layers, which floor or all layers can comprise one or more therapeutical agent to be delivered or diagnostic reagents.In some embodiments, one deck comprises medicament to be delivered, and another layer does not contain medicament to be delivered, etc.In some embodiments, each layer separately comprises different medicaments to be delivered or the set of medicament.
In some embodiments, particle is porous, and it refers to that particle comprises hole or passage, described hole or passage are usually little than the size of particle.Such as, particle can be porous silica silicon grain, such as, mesoporous silica nano-particle, or particle can have meso-porous titanium dioxide silicon coating (people such as Lin, 2005, J.Am.Chem.Soc., 17:4570).Particle can have the hole that diameter is about 1nm to about 50nm, and such as, diameter is the hole of about 1nm to 20nm.About 10% to 95% of particle volume can be made up of the space in hole or passage.
Particle can have coating.Such as, as fruit granule comprises the virose material of cell tool, so the use of biocompatible coating can be and has superiority.Suitable coated substance includes but not limited to: the carbohydrate of the biocompatible hydrophilic polymkeric substance of natural protein, the such as polyoxyethylene glycol (PEG) or PEG derivative and so on of such as bovine serum albumin (BSA) and so on, phosphatide-(PEG), silicon-dioxide, lipid, polymkeric substance, such as glucosan and so on, other nano particles that can be combined with nano particle of the present invention, etc.Coating is by such as dipping, using various ways coating or the assembling of layer-layer technology, self-assembly, conjugation etc.Self-assembly refers to the process being spontaneously assembled into higher structure, and this process depends on composition (such as, molecule) the natural sucking action each other of higher structure.This process is occurred by the random motion of molecule and the formation of key based on size, shape, composition or chemical property usually.
The example of polymkeric substance comprises polyolefine (such as, polyethylene), polycarbonate (such as, poly-(1, 3-dioxane-2 ketone)), polyanhydride (such as, poly-(sebacic anhydride)), polyhydroxy acid (such as, poly-(beta-hydroxy alkanoates)), poly-fumaric acid esters, polycaprolactone, polymeric amide (such as, polycaprolactam), polyacetal resin, polyethers, polyester (such as, poly(lactic acid), polyglycolide), poly-(ortho ester), polyvinyl alcohol, urethane, polyphosphonitrile, polyacrylic ester, polymethacrylate, polybutylcyanoacrylate, polyureas, polystyrene and polyamine.In some embodiments, polymkeric substance according to the present invention comprises the polymkeric substance being ratified to be used for human body by FDA (Food and Drug Adminstration) (FDA) according to 21C.F.R. § 177.2600, include but not limited to: polyester (such as, poly(lactic acid), polyglycolic acid, poly-(lactic-co-glycolic acid), polycaprolactone, poly-valerolactone, poly-(1,3-dioxane-2 ketone)), polyanhydride is (such as, poly-(sebacic anhydride)), polyethers (such as, polyoxyethylene glycol), urethane, polymethacrylate, polyacrylic ester and polybutylcyanoacrylate.
In some embodiments, particle can be non-polymeric particle (such as, metallic particles, quantum dot, ceramic particle, containing the polymkeric substance of organic and/or inorganic materials, the material that bone is derivative, bone substitute, virion, etc.).In some embodiments, therapeutical agent to be delivered or diagnostic reagent can with the surface bonding of so non-polymeric particle.In some embodiments, non-polymeric particle is the aggregate of non-polymeric constituents, such as, and the aggregate of atoms metal (such as, gold atom).In some embodiments, therapeutical agent to be delivered or diagnostic reagent with the surface bonding of the aggregate of non-polymeric constituents and/or can be encapsulated in the aggregate of non-polymeric constituents, by the aggregate of non-polymeric constituents around and/or be scattered in the aggregate of whole non-polymeric constituents.
Particle (such as, nano particle, microparticle) can use any method preparation known in the art.Such as, the additive method that bead dosage form is known by following method and those of ordinary skill in the art is formed: such as nanoprecipitation, flow focusing fluid channel, spraying dry, single emulsion and two emulsion solvent evaporation, solvent extraction, is separated, grinding, microemulsion operates, micro-manufacture, nanometer manufacture, sacrifice layer, simple and complex coacervation.Alternatively or extraly, described for single dispersing semiconductor nanoparticle, conductive nanoparticle; magnetic nanoparticle, the water-based of organic nanometer granule and other nano particles and organic solvent synthetic method (people such as Pellegrino, 2005; Small, 1:48; The people such as Murray, 2000, Ann.Rev.Mat.Sci., 30:545; And the people such as Trindade, 2001, Chem.Mat., 13:3843).
The method of microparticle for the preparation of the medicament sending encapsulation describes in the literature (see, such as, Doubrow; editor, " Microcapsules and Nanoparticles in Medicine and Pharmacy, " CRC Press; Boca Raton, 1992; The people such as Mathiowitz, 1987, J.Control.Release, 5:13; The people such as Mathiowitz, 1987, Reactive Polymers, δ: 275; And the people such as Mathiowitz, 1988, J.Appl.Polymer Sci., 35:755).
In some embodiments, targeting moiety comprises nucleic acid targeting moiety.
In general, nucleic acid targeting moiety is any polynucleotide combining the composition (target) relevant with organ, tissue, cell, extracellular matrix components and/or intracellular compartments.
In some embodiments, nucleic acid targeting moiety is fit.
Fitly be generally the polynucleotide be combined with specific objective structure, described specific objective structure is relevant with certain organs, tissue, cell, extracellular matrix components and/or intracellular compartments.In general, fit target function is based on fit three-dimensional structure.In some embodiments, the combination of fit and target is mediated by the interaction between the two dimension of both fit and target and/or three-dimensional structure usually.In some embodiments, the fit combination with target, not only based on fit basic sequence, also depends on three-dimensional structure that is fit and/or target.In some embodiments, fit pairing by Watson-Crick complementary base is combined with its target, and the structure (such as, hairpin loop) that described Watson-Crick base pairing is destroyed base pairing hinders.
In some embodiments, nucleic acid targeting moiety is that spiegelmers(PCT announces WO98/08856, WO02/100442 and WO06/117217).In general, spiegelmers is the mirror image nucleic acid of synthesis, and it can specific binding target (that is, mirror image is fit).Spiegelmers is characterized by following constitutional features: the impact of described constitutional features makes them not be subject to circumscribed-nuclease and endo-nuclease.
One skilled in the art would recognize that, according to the present invention can use any can the nucleic acid targeting moiety (such as, fit or spiegelmers) of specific binding target.In some embodiments, nucleic acid targeting moiety to be used according to the present invention can determine the marker relevant with disease, imbalance and/or illness by target.In some embodiments, nucleic acid targeting moiety to be used according to the present invention can determine cancer associated target by target.In some embodiments, nucleic acid targeting moiety to be used according to the present invention can determine tumor marker by target.Use and can determine the cancer markers of any type and/or any tumor marker by target according to nucleic acid targeting moiety of the present invention.For example, nucleic acid targeting moiety can determine the marker relevant with prostate cancer, lung cancer, mammary cancer, carcinoma of the colon and rectum, bladder cancer, carcinoma of the pancreas, carcinoma of endometrium, ovarian cancer, osteocarcinoma, the esophageal carcinoma, liver cancer, cancer of the stomach, cerebral tumor, cutaneous melanoma and/or leukemia by target.
Nucleic acid of the present invention (comprises nucleic acid targeting moiety and/or functional r NA to be delivered, such as, RNAi-inducing entity, ribozyme, tRNA, etc., describe in further detail below) can according to any obtainable technology preparation, include but not limited to: the enzymatic lysis of chemosynthesis, enzymic synthesis, longer precursor or chemical cracking, etc.The method of synthesis RNA be known in the art (see such as, Gait, M.J. (editor) Oligonucleotide synthesis:a practical approach; Oxford [Oxfordshire]; Washington, D.C.:IRL Press, 1984; And Herdewijn, P. (editor) Oligonucleotide synthesis:methods and applications, Methods in molecular biology, v.288 (Clifton, N.J.) Totowa, N.J.:Humana Press, 2005).
The nucleic acid forming nucleic acid targeting moiety can comprise the nucleosides of natural generation, the nucleosides modified, there is the hydrocarbon linker that inserts between one or more nucleosides (such as, alkene) or polyethers linker is (such as, PEG linker) the nucleosides of natural generation, there is the nucleosides of the modification of hydrocarbon linker or the PEG linker inserted between one or more nucleosides, or their combination.In some embodiments, the Nucleotide of nucleic acid targeting moiety or the Nucleotide of modification can be replaced by hydrocarbon linker or polyethers linker, as long as the binding affinity of nucleic acid targeting moiety and selectivity substantially can not due to replace (such as, nucleic acid targeting moiety should not be greater than about 1 × 10 to the dissociation constant of target-3m) reduce.
Those of ordinary skill in the art are known, can comprise the Nucleotide of all types found in the nucleic acid of natural generation or can comprise one or more nucleotide analogs or have the structure different from the structure of the nucleic acid of natural generation according to nucleic acid of the present invention.United States Patent (USP) the 6th, 403, No. 779, the 6th, 399, No. 754, the 6th, 225, No. 460, the 6th, 127, No. 533, the 6th, 031, No. 086, the 6th, 005, No. 087, the 5th, 977, No. 089, the reference in these United States Patent (USP)s discloses multiple different concrete nucleoside analog and spendable modification.See Crooke, S. (editor) Antisense Drug Technology:Principles, Strategies, and Applications (first version), Marcel Dekker; ISBN:0824705661; First version (2001) and reference wherein.Such as, 2 '-modification comprises halo, alkoxyl group and allyloxy.In some embodiments, 2 '-OH group is selected from following group replacement: H, OR, R, halogen, SH, SR, NH2, NHR, NR2 or CN, wherein, R is C1-C6 alkyl, thiazolinyl, or alkynyl, and halogen is F, Cl, Br or I.The example of the connecting key modified comprises thiophosphatephosphorothioate and 5 '-N-phosphoramidite connecting key.
The nucleic acid comprising multiple different nucleotide analog, the skeleton of modification or the internucleoside bridges of non-natural generation can be used according to the present invention.The nucleosides that nucleic acid of the present invention can comprise natural nucleus glycoside (that is, adenosine, thymidine, guanosine, cytidine, uridine, Desoxyadenosine, deoxythymidine, pancreatic desoxyribonuclease, Deoxyribose cytidine) or modify.The example of the Nucleotide modified comprises nucleosides (such as, the cytosine arabinoside (aracytidine) of base modification, flesh nucleosides, isoguanine riboside, pigment meat and vegetables (nebularine), pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-sulfo-thymidine, 3-denitrogenation-5-azacytidine, 2 '-deoxyuridine, 3-nitro-pyrrole, 4-skatole, 4-thiourdine, 4-sulfo-thymidine, 2-amino adenosine, 2-sulfo-thymidine, 2-thio uridine, 5-bromo cytidine, 5-iodo uridine, flesh nucleosides, 6-azauridine, 6-chloro-purine, 7-denitrogenation adenosine, 7-denitrogenation guanosine, 8-azepine adenosine, 8-nitrine adenosine, benzoglyoxaline, M1-methyladenosine, pyrrolopyrimidine, 2-amino-6-chloro-purine, 3-methyladenosine, 5-proyl cytidine, 5-proyl uridine, 5-bromo uridine, 5-fluoro uridine, 5-methylcytidine, 7-denitrogenation adenosine, 7-denitrogenation guanosine, 8-oxygen adenosine, 8-oxygen guanosine, O (6)-methyl guanine and 2-thiacydidine), the base (such as, methylated base) of chemistry or bio-modification, carbohydrate (such as, the 2 '-fluororibose of modification, 2 '-amino ribose, 2 '-nitrine ribose, 2 '-O-methylribose, L-enantiomer nucleosides arabinose and hexose), the bound phosphate groups (such as, thiophosphatephosphorothioate and 5 '-N-phosphoramidite connecting key) of modification and their combination.Be easy to obtain for the natural nucleus glycoside acid mono of the chemosynthesis of nucleic acid and the nucleotide monomer of modification.In some cases, show the character of improvement relative to the nucleic acid be only made up of the Nucleotide of natural generation containing these nucleic acid modified.In some embodiments, nucleic acid as herein described modify be used to reduce and/or prevent nuclease (such as, exonuclease, endonuclease, etc.) digestion.Such as, the structure of nucleic acid is stablized to reduce digestion by comprising nucleotide analog at 3 ' end of a chain or two chains.
The nucleic acid modified does not need to carry out consistence modification along the total length of molecule.Different nucleotide modifications and/or skeleton structure can be present in each different positions of nucleic acid.Those of ordinary skill in the art will be understood that, nucleotide analog or other modifications can be positioned at any position making the substantially impregnable nucleic acid of the function of nucleic acid.For example, any position that can be positioned at the substantially impregnable nucleic acid targeting moiety of ability making nucleic acid targeting moiety specific binding target is modified.The region modified can be positioned at 5 ' end and/or the 3 ' end of a chain or two chains.Such as, the following nucleic acid targeting moiety modified has been used: about 1 to 5 residue of the 5 ' end and/or 3 ' end place that are arranged in arbitrary chain of two chains of the nucleic acid targeting moiety of this modification is nucleotide analog and/or has backbone modification.Described modification can be 5 ' or 3 ' end modified.Article one, or two nucleic acid chains can comprise Nucleotide, the Nucleotide of at least 80% unmodified, the Nucleotide of at least 90% unmodified or the Nucleotide of 100% unmodified of at least 50% unmodified.
Such as, the modification to carbohydrate, nucleosides or internucleoside bridges can be comprised according to nucleic acid of the present invention, such as, No. 2003/0175950th, U.S. Patent Application Publication, No. 2004/0192626, No. 2004/0092470, in No. 2005/0020525 and No. 2005/0032733 describe those.The present invention includes the application with any one in modification as herein described or more than one any nucleic acid.Such as, report multiple end conjugate (such as, lipid (such as, cholesterol), lithocholic acid, lauric acid (aluric acid), long-chain branch alkyl) and improve cellular uptake.Such as, any suitable testing method known in the art can be used to detect analogue and modification, thus select to make therapeutical agent or diagnostic reagent send those analogues and modification that be improved, the make targeting moiety of nucleic acid and the specific binding of target be improved etc.In some embodiments, one or more non-natural nucleoside connecting keys can be comprised according to nucleic acid of the present invention.In some embodiments, the connecting key of one or more are positioned at 3 ' end of nucleic acid targeting moiety, 5 ' end or 3 ' end and 5 ' hold the inherent Nucleotide at these two ends to be reversed generation such as 3 '-3 ' connecting key or 5 '-5 ' connecting key and so on.
In some embodiments, be not synthesis according to nucleic acid of the present invention, it is the entity of the natural generation of separating from its natural surroundings.
Any method can be used (to refer to such as following United States Patent (USP): 6,716,583 to design new nucleic acid targeting moiety; 6,465,189; 6,482,594; 6,458,543; 6,458,539; 6,376,190; 6,344,318; 6,242,246; 6,184,364; 6,001,577; 5,958,691; 5,874,218; 5,853,984; 5,843,732; 5,843,653; 5,817,785; 5,789,163; 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588 and 5,270,163 and following U.S. Patent Application Publication: 2005/0069910,2004/0072234,2004/0043923,2003/0087301,2003/0054360 and 2002/0064780).The invention provides a kind of method for designing new nucleic acid targeting moiety.The present invention also provides a kind of method for being separated or identifying new nucleic acid targeting moiety in the mixture from candidate nucleic acid targeting moiety.
Can design and/or identify the nucleic acid targeting moiety be combined with protein, carbohydrate, lipid and/or nucleic acid.In some embodiments, nucleic acid targeting moiety can be designed and/or be identified as and use in the mixture of the present invention be combined with protein and/or its characteristic, described protein and/or its characteristic are such as, tumor marker, integrin, cell surface receptor, transmembrane protein, intercellular protein matter, ionic channel, protein called membrane transporters, enzyme, antibody, chimeric protein, etc.In some embodiments, nucleic acid targeting moiety can be designed and/or be identified as and use in the mixture of the present invention be combined with carbohydrate and/or its characteristic, described carbohydrate and/or its characteristic are such as, glycoprotein, carbohydrate are (such as, monose, disaccharides and polysaccharide), glycocalyx (namely, the outer region of the carbohydrate enrichment on most of eukaryotic outside surface), etc.In some embodiments, nucleic acid targeting moiety can be designed and/or be identified as and use in the mixture of the present invention be combined with lipid and/or its characteristic, described lipid and/or its characteristic are such as, oil, saturated fatty acid, unsaturated fatty acids, glyceryl ester, hormone, steroid are (such as, cholesterol, bile acide), VITAMIN (such as, vitamin-E), phosphatide, sphingolipid, lipoprotein, etc.In some embodiments, nucleic acid targeting moiety can be designed and/or be identified as and use in the mixture of the present invention be combined with nucleic acid and/or its characteristic, described nucleic acid and/or its characteristic are such as, the DNA nucleic acid of DNA nucleic acid, RNA nucleic acid, modification, the RNA nucleic acid of modification and comprise the nucleic acid of any combination of DNA, RNA, the DNA of modification and the RNA of modification, etc.
Any obtainable method design and/identification nucleic acid targeting moiety (such as, fit or spiegelmer) can be used.In some embodiments, nucleic acid targeting moiety by identifying nucleic acid targeting moiety to design and/or identifying from the nucleic acid mixture of candidate.Index concentration Fas lignand system is evolved (SELEX) or its modification method is the common method identifying the nucleic acid targeting moiety be combined with target from the nucleic acid mixture of candidate.
The nucleic acid targeting moiety of any target of selective binding is separated by SELEX method or its modification method, and condition is the target that described target can be used as in SELEX method.
linker
In general, compound of the present invention comprises linker, and targeting moiety is connected with activated partial by described linker.But some compounds are not containing linker, and activated partial is directly connected with targeting moiety.
" linker " is herein the part of instigating the first molecule to be connected by chemical bond with the second molecule.In linker of the present invention, can connection be cut off, thus release first and/or dimolecular biologically active form.The preferred embodiment of linker is comprise stable under conditions of neutral ph but under lower pH condition, be easy to the part of the chemical bond that cracking occurs.Specifically, the example of preferred linker is that to be included in pH be stable under the condition of 7 to 8 but is the part being easy to the chemical bond of cracking under the condition of 4 to 6 at pH.Another example of linker be included in there is enzyme condition under be easy to the part of the chemical bond of cracking.These are peptide to the preferred embodiment of the linker of enzyme sensitivity, and described peptide comprises the recognition sequence of endosome peptase.Another example of linker is the linker to redox potential sensitivity, this linker at low reduction potential (such as, the mercaptan of lower concentration or gsh) stable but cracking under high reduction potential (such as, the mercaptan of high density or gsh) condition under condition.These comprise disulphide and sulphenamide to the preferred embodiment of the linker of redox potential sensitivity.Specifically, preferred example comprises the aryl-alkyl disulphide of replacement, wherein, aryl by the demand of having living space and electrophilic or replace to the substituting group of electronics, thus control to be tending towards the susceptibility with the disulfide linkage of thiol reactant.Another example of linker is the part comprising the chemical bond being easy to cracking after exposure to radiation.The preferred embodiment of these radiosensitive linkers is 2-nitrobenzyl ether, and it is cracking after being exposed to light.Specifically, the preferred embodiment of these linkers is as lower part: covered the biological activity of in two molecules connected at connecting key by this part before cutting off.
In some embodiments, compound of the present invention comprises the linker being selected from following group: diazanyl group, polypeptide, disulphide group and sulfide group.
" diazanyl group " or " hydrazine linker " or " from cyclisation hydrazine linker " herein refers to and cyclization can occur and the linker part forming one or more rings after change condition (such as, pH change).When attached, diazanyl group is converted into hydrazone.This connection occurs by such as reacting with ketone groups in L4 part.Therefore, term hydrazine linker also can be used for describing linker of the present invention, because this conversion to hydrazone occurs after connection.
" five yuan of hydrazine linkers " herein refers to the molecular moiety containing hydrazine, and this part can be carried out cyclization and form one or more five-rings after condition changes (such as, pH changes).Alternatively, these five yuan of linkers can be described as five yuan of hydrazine linkers similarly.
" hexa-atomic hydrazine linker " herein refers to the molecular moiety containing hydrazine, and this part can be carried out cyclization and form one or more six-rings after condition changes (such as, pH changes).This hexa-atomic linker can be described as hexa-atomic hydrazine linker similarly.
" cyclization " herein refers to the cyclisation of peptide, hydrazine or disulfide linkers, " cyclization " should represent that linker cyclisation formed ring and starts the separation of Drug Ligand mixture.Cyclisation speed can be measured and complete cyclisation when the product of at least 90%, 95% or 100% is formed in strange land.
In some embodiments, compound of the present invention comprises the linker region between targeting moiety and activated partial, and described linker can be present in the cracking agent cracking in intracellular environment (such as, lysosome or endosome or little recessed interior).Linker can be such as, peptidyl linkers, and this peptidyl linkers is by intracellular peptidases or proteolytic enzyme (including but not limited to: lysosomal protein enzyme or endosome proteolytic enzyme) cracking.Usually, the length of peptidyl linkers is at least two amino acid whose length or at least three amino acid whose length.Cracking agent can comprise cathepsin B, cathepsin D and plasmin, known tissue Cathepsin B, cathepsin D and plasmin equal hydrolyzable dipeptide medicament derivative, the active medicine of target cell interior is caused to discharge (see such as, Dubowchik and Walker, 1999, Pharm.Therapeutics83:67-123).Most typical linker is peptidyl linkers, and it can by the enzymatic lysis be present in target cell or tissue.Such as, can use can by the peptidyl linkers of mercaptan dependence protein enzyme Cathepsin B cracking (such as, Phe-Leu or (Gly-Phe-Leu-Gly) linker), described mercaptan dependence protein enzyme Cathepsin B high expression level in cancerous tissue.Other this kind of linkers, such as at United States Patent (USP) the 6th, describe in 214, No. 345.In some embodiments, can by the peptidyl linkers of intracellular protein enzymatic lysis be Val-Cit linker or Phe-Lys linker (see such as, United States Patent (USP) the 6th, 214, No. 345, this us patent describes the synthesis of the Zorubicin with val-cit linker).An advantage of intracellular proteolysis release therapeutical agent is used to be that described therapeutical agent is weakened when coupling usually and the serum stability of conjugate is usually higher.
In some embodiments, the linker of cleavable is pH sensitivity, namely responsive to a certain pH value condition and be hydrolyzed.Usually, the linker of pH sensitivity hydrolyzable in acid condition.Such as, (such as, hydrazone, semicarbazone, thiosemicarbazone, Immuno toxin amine, ortho ester, acetal, the ketal of the unstable linker of hydrolyzable acid in lysosome can be used in; etc.), see such as, United States Patent (USP) the 5th; 122, No. 368, the 5th; 824, No. 805, the 5th; 622, No. 929, Dubowchik and Walker; 1999, Pharm.Therapeutics83:67-123; The people such as Neville, 1989, Biol.Chem.264:14653-14661.These linkers are relatively stable under conditions of neutral ph, such as, and those linkers in blood, but unstable under lower than the pH condition (being approximately lysosomal pH) of 5.5 or 5.0.In some embodiments, hydrolyzable linker is thioether linker (such as, the thioether be connected with therapeutical agent by acyl group hydrazone chemical bond, see such as, United States Patent (USP) the 5th, 622, No. 929).
In other embodiments, linker is (such as, the disulfide linkers) of cleavable under reductive condition.Multiple disulfide linkers known in the art; comprise such as; SATA(N-succinimido-5-acetylthioacetate can be used); SPDP(N-succinimido-3-(2-pyridyl dithio) propionic ester); SPDB(N-succinimido-3-(2-pyridyl dithio) butyric ester) and SMPT(N-succinimido-oxygen carbonyl-Alpha-Methyl-α-(2-pyridyl dithio) toluene); SPDB and SMPT(is see such as; the people such as Thorpe; 1987, Cancer Res.47:5924-5931; The people such as Wawrzynczak, In Immunoconjugates:Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W.Vogel edits), Oxford U. publishes, 1987. also see United States Patent (USP) the 4th, 880, No. 935) those linkers of being formed.
In other embodiments; linker is the malonic ester linker (people such as Johnson; 1995; Anticancer Res.15:1387-93); maleimidobencoyl linker (people such as Lau, 1995, Bioorg-Med-Chem.3 (10): 1299-1304) or the 3 '-N-amide analogue (people such as Lau; 1995, Bioorg-Med-Chem.3 (10): 1305-12).
Usually, linker is substantially insensitive to extracellular environment." substantially insensitive to extracellular environment " that use in the part of linker herein refers in compound sample of the present invention, when compound of the present invention is present in extracellular environment (such as, in blood plasma) time, be no more than the linker cracking of about 20%, typically be no more than the linker cracking of about 15%, more typically be no more than about 10% linker cracking, even more typically be no more than the linker cracking of about 5%, be no more than the linker cracking of about 3%, or be no more than the linker cracking of about 1%.Such as, by the antibody of the non-coupling by compound of the present invention to (a) (" compound sample ") and (b) equimolar amount or therapeutical agent (" control sample ") together hatch with blood plasma independently one section predetermined time section (such as, 2 hours, 4 hours, 8 hours, 16 hours or 24 hours) and the amount (such as by high-efficient liquid phase color spectrometry) comparing the antibody or therapeutical agent being present in the antibody of non-coupling in compound sample or the amount of therapeutical agent and be present in the non-coupling in control sample subsequently whether measure linker substantially insensitive to extracellular environment.
In the embodiment that other do not repel mutually, linker promotes cell internalization.In some embodiments, when linker is coupled to activated partial, linker promotes cell internalization.In other embodiments, when linker is coupled to both targeting moiety and activated partial, linker promotes cell internalization.
The multiple linker that can use in the compositions and methods of the invention is called " Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease " in WO2004010957(name) and US20120141509A1 and US20120288512A1(content disclosed in it be incorporated to by reference herein) in describe.
In some embodiments, linker unit has following general formula:
-Ta-Ww-Yy-
Wherein ,-T-is carrier unit, and a is 0 or 1 ,-W-is separately Amino Acid Unit, and w is the integer of 2 to 12 independently, and-Y-is spacer units, and y is 0,1 or 2.
carrier unit
When there is carrier unit (-T-), targeting moiety is connected to Amino Acid Unit (-W-) by this carrier unit.Can include but not limited to natively or by the useful functional group that chemical operation is present on targeting moiety (such as, antibody): the different head hydroxyl of sulfydryl, amino, hydroxyl, carbohydrate and carboxyl.Suitable functional group is sulfydryl and amino.Sulfydryl generates by the intramolecular disulfide bond going back original antibody.Alternatively, the reaction that sulfydryl generates reagent by the amino of the sine group of antibody and 2-iminothiolane (Traut reagent) or other sulfydryls generates.In some embodiments, antibody is recombinant antibodies and is designed to one or more Methionin.In other embodiments, recombinant antibodies is designed to extra mercapto groups, such as, and extra halfcystine.
In some embodiments, the sulphur atom of carrier unit and antibody forms chemical bond.Described sulphur atom can derive from the mercapto groups (-SH) of the antibody (A) of reduction.The representational carrier unit of these embodiments describes in the square brackets of general formula (IIa) and (IIb), and wherein, A-,-W-,-Y-,-D, w and y are as defined above, and R1be selected from :-C1-C10alkene-,-C3-C8-carbocyclic ring-,-O-(C1-C8alkyl)-,-arylidene-,-C1-C10alkene-arylidene-,-arylidene-C1-C10-alkene-,-C1-C10-alkene-(C3-C8carbocyclic ring)-,-(C3-C8carbocyclic ring)-C1-C10alkene-,-C3-C8-heterocycle-,-C1-C10-alkene-(C3-C8heterocycle)-,-(C3-C8heterocycle)-C1-C10alkene-,-(CH2cH2o) r-and-(CH2cH2o) r-CH2-, and r is the integer of 1 to 10.
Exemplary carrier unit is R1for-(CH2)5the carrier unit of the general formula (IIa) in-time:
Another exemplary carrier unit is R1for-(CH2cH2o) r-CH2-and r is 2 time the carrier unit of general formula (IIa):
Another exemplary carrier unit is R1for-(CH2)5the carrier unit of the general formula (IIb) in-time:
In some other embodiments, carrier unit is connected to antibody units (A) by the disulfide linkage between the sulphur atom of antibody units and the sulphur atom of carrier unit.The representational carrier unit of present embodiment describes in the square brackets of general formula (III), wherein, and R1, A-,-W-,-Y-,-D, w and y be as defined above.
In other embodiments, the reactive group of support comprises the reaction site can reacted with the amino group of antibody.Amino group can be arginine or Methionin.Suitable amine reaction site includes but not limited to: the ester (such as, succinimide ester, 4-nitrophenyl ester, phenyl-pentafluoride base ester) of activation, acid anhydrides, acyl chlorides, SULPHURYL CHLORIDE, isocyanic ester and lsothiocyanates.The representational carrier unit of these embodiments describes in the square brackets of general formula (IVa) and (IVb), wherein, and R1, A-,-W-,-Y-,-D, w and y be as above.
On the other hand, the reactive functional groups of support comprises the reaction site of reacting with the carbohydrate group of the modification that can be present on antibody.In some embodiments, antibody is glycosylated by the mode of enzyme, thus generates carbohydrate group.Carbohydrate can by the reagent mildly oxidising of such as sodium periodate and so on, the carbonyl unit of the carbohydrate of the oxidation obtained can with containing such as hydrazides, oxime, reactive amine, hydrazine, thiosemicarbazide, hydrazinecarboxylate and the aryl hydrazide (people such as such as Kaneko, those described in 1991, Bioconjugate Chem2:133-41) and so on the support condensation of functional group.The representational carrier unit of present embodiment describes in the square brackets of general formula (Va) to (Vc), wherein, and R1, A-,-W-,-Y-,-D, w and y be as above.
amino Acid Unit
If spacer units exists, carrier unit (-T-) is connected to spacer units (-Y-) by Amino Acid Unit (-W-), if the non-existent words of spacer units, carrier unit is connected to cytotoxic agent or cytostatics (activated partial, D) by Amino Acid Unit.-Ww-is dipeptides, tripeptides, tetrapeptide, pentapeptide, six peptides, seven peptides, octapeptide, nonapeptide, decapeptide, 11 peptides or dodecapeptide unit.Each-W-unit has the general formula described in following square brackets independently, and w is the integer of 2 to 12.
Wherein, R2for hydrogen, methyl, sec.-propyl, isobutyl-, sec-butyl, benzyl, to hydroxybenzyl ,-CH2oH ,-CH (OH) CH3,-CH2cH2sCH3,-CH2cONH2,-CH2cOOH ,-CH2cH2cONH2,-CH2cH2cOOH ,-(CH2)3nHC (═ NH) NH2,-(CH2)3nH2,-(CH2)3nHCOCH3,-(CH2)3nHCHO ,-(CH2)4nHC (═ NH) NH2,-(CH2)4nH2,-(CH2)4nHCOCH3,-(CH2)4nHCHO ,-(CH2)3nHCONH2,-(CH2)4nHCONH2,-CH2cH2cH (OH) CH2nH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
The Amino Acid Unit of linker unit by enzymatic lysis, thus discharges activated partial (-D) by the mode of enzyme (including but not limited to: tumor correlated albumen enzyme), and after release, this activated partial is protonated in vivo, thus produces activated molecule (D).
Exemplary Wwunit is represented by general formula (VI) to (VIII):
Wherein, R3and R4as shown in the table:
Wherein, R3, R4and R5as shown in the table:
Wherein, R3, R4, R5and R6as shown in the table:
Suitable Amino Acid Unit includes but not limited to: general formula (VI) unit, wherein, and R3for benzyl, R4for-(CH2)4nH2, R3for sec.-propyl and R4for-(CH2)4nH2, or R3for sec.-propyl and R4for-(CH2)3nHCONH2.Another suitable Amino Acid Unit is general formula (VII) unit, wherein, and R3for benzyl, R4for benzyl and R5for-(CH2)4nH2.-Ww-unit can be designed and be optimized for carry out enzymatic lysis by specific tumor correlated albumen enzyme in its selectivity.Suitable-Ww-unit passes through the unit of proteolytic enzyme (cathepsin B, cathepsin C and cathepsin D) and its cracking of plasmin catalysis for those.
In some embodiments ,-Ww-is dipeptides, tripeptides or four peptide units.
At R2, R3, R4, R5or R6be not under the condition of hydrogen, R2, R3, R4, R5or R6the carbon atom connected is chirality.With R2, R3, R4, R5or R6the each carbon atom connected is (S) configuration or (R) configuration independently.
In some embodiments, Amino Acid Unit is phe-lysine dipeptides (Phe-Lys or FK linker).In some embodiments, Amino Acid Unit is valine-citrulline dipeptides (Val-Cit or VC linker).In some embodiments, Amino Acid Unit is 5-aminovaleric acid, all phenylalanine Methionin, four 1-isoquinolinecarboxylic acid's ester Methionins, Cyclohexylalanine Methionin, isonipecotic acid (isonepecotic acid) Methionin, Beta-alanine Methionin, glycine Serine α-amino-isovaleric acid glutamine or isonipecotic acid.
Amino Acid Unit can comprise natural amino acid.In other embodiments, Amino Acid Unit can comprise alpha-non-natural amino acid.
spacer units
When there is spacer units (-Y-), Amino Acid Unit is connected to drug unit by this spacer units.Spacer units is two large classes: excise from (self-immolative) that excise and non-self.The spacer units of non-self excision be Amino Acid Unit enzymatic lysis in TM-linker-AM conjugate or drug-linker compound after part or all of spacer units be still connected to the spacer units of activated partial unit.The example of non-self excision spacer units includes but not limited to: (Gly-Gly) spacer units and glycine spacer unit.When the TM-linker-AM conjugate containing Gly-Gly spacer units or glycine spacer unit is by tumour cell associated protein enzyme, cancer cells associated protein enzyme or lymphocyte associated protein enzyme generation enzymatic lysis, cracking is out from A-T-Ww-for Gly-Gly-drug moiety or glycine-drug moiety.In order to discharge AM, independently hydrolysis reaction should be carried out in target cell, thus make glycine-drug unit chemical bond cracking.
In typical embodiment ,-Yy-is the PAB ether that can be replaced by Qm, and wherein, Q is-C1-C8alkyl ,-C1-C8alkoxyl group ,-halogen ,-nitro or-cyano group, and m is the integer of 0 to 4.
In some embodiments, non-self excision spacer units (-Y-) is-Gly-Gly-.
In some embodiments, non-self excision spacer units (-Y-) is-Gly-.
In one embodiment, AM-linker compound or TM-linker-AM conjugate lack spacer units (y=0).
Alternatively, can AM(D be discharged containing the TM-linker-AM conjugate from excision spacer units), without the need to independent hydrolysing step.In these embodiments ,-Y-is PAB alcohol (PAB) unit, and this PAB alcohol (PAB) unit to be connected with-Ww-by the nitrogen-atoms of PAB group and to be directly connected in-D by carbonate group, carbamate groups or ether group.
Include but not limited to from other examples of excision spacer units: with the aromatics of PAB group electronic equivalence, such as, 2-aminooimidazole base-5-carbinol derivatives is (see such as, the people such as Hay, 1999, Bioorg.Med.Chem.Lett.9:2237) and ortho position or contraposition-aminobenzyl acetal.The spacer units that cyclisation occurs is easy to after amido linkage can be used to be hydrolyzed, such as, replace and the unsubstituted 4-Aminobutanoicacid acid amides (people such as Rodrigues, 1995, Chemistry Biology2:223), the dicyclo [2.2.1] suitably replaced and dicyclo [2.2.2] loop systems (people such as Storm, 1972, and 2-aminophenyl propionic acid (people such as Amsberry, 1990, J.Org.Chem.55:5867) J.Amer.Chem.Soc.94:5815).The elimination containing drug amine that the alpha-position of glycine is substituted people such as (, 1984, J.Med.Chem.27:1447) Kingsbury is also the example of the strategy from excision spacer units, and this strategy can be used for TM-linker-AM conjugate.
In alternative embodiments, spacer units is two (methylol) vinylbenzene (BHMS) unit of side chain, and it can be used for merging multiple part.
Typical spacer units (-Yy-) is represented by general formula (IX)-(XI):
Wherein, Q is C1-C8alkyl, C1-C8alkoxyl group, halogen, nitro or cyano group, and m is the integer of 0 to 4.
In some embodiments, linker is enzyme cleavable.In some embodiments, linker is not enzyme cleavable.
In some embodiments, described linker is by the representation of following general formula (III):
Wherein, L is by the representation of general formula (II):
M is 1,2,3,4,5 or 6, b is separately 0 or 1, and D is represented independently by the structure of following general formula (III):
Wherein, i is separately 0 or 1;
J is separately 0,1,2,3,4,5 or 6;
A is separately S, O or N-Ra, and wherein, Ra is hydrogen, alkyl, thiazolinyl or alkoxyl group;
B is separately alkyl, thiazolinyl,--O-alkyl--,--alkyl-O--,--S-alkyl--,--alkyl-S--, aryl, heteroaryl, heterocyclic radical or peptide, each in them is optionally replaced by one or more substituting groups being selected from following groups: hydroxyl, alkoxyl group, alkyl, thiazolinyl, alkynyl, cycloalkyl,--alkyl-aryl-group,--alkyl-heteroaryl,--alkyl-heterocyclyl groups,--O-R4,--O-alkyl-R4,--C (O)-R4,--C (O)-O-R4,--S-R4,--S (O)2-R4,--NHR4,--NH-alkyl-R4, halogen,--CN,--NO2， He – SH, wherein, R4for alkyl, thiazolinyl,--alkyl-hydroxyl, aryl, heteroaryl, heterocyclic radical or haloalkyl.
In some embodiments, described linker by following logical formula V to the representation of general formula (VII):
A, B, i and j are as defined above.
In some embodiments, linker is selected from: S1, S2, S3, S4, S5, S6, S7 ， – Gly-Phe-Leu-Gly-,-Ala-Leu-Ala-Leu-,-Phe-Arg-,-Phe-Lys-,-Val-Lys-,-Val-Ala-, or Val-Cit-, wherein, S1 to S7 is by following representation:
Wherein, m is separately 1 to 20.Preferably, m is 1 to 3,1 to 5,1 to 10 or 2 to 5.
Therefore, the invention provides the compound of a kind of general formula (Ia), wherein, TM is anti-HER2 antibody; L is selected from: S1, S2, S3, S4, S5, S6, S7 ， – Gly-Phe-Leu-Gly-,-Ala-Leu-Ala-Leu-,-Phe-Arg-,-Phe-Lys-,-Val-Lys-,-Val-Ala-, or Val-Cit-; AM is the compound of general formula (I).In one embodiment, TM is Herceptin (Trastuzumab), handkerchief trastuzumab or margetuximab(MGAH22); AM is the compound being selected from table 1, and wherein, the amine groups on quinoline ring is the point be connected with linker.In another embodiment, TM is Herceptin (Trastuzumab), handkerchief trastuzumab or margetuximab(MGAH22); L is S1, S2 or S3; AM is the compound being selected from table 1, and wherein, the amine groups on quinoline ring is the point be connected with linker.In yet, TM is Herceptin (Trastuzumab); L is S1, S2 or S3; AM is resiquimod or Imiquimod, and wherein, the amine groups on quinoline ring is the point be connected with linker.
In another embodiment, compound of the present invention is by the representation of following general formula A to general formula C:
In a kind of embodiment of general formula A to general formula C, anti-HER2 antibody is Herceptin (Trastuzumab), handkerchief trastuzumab or margetuximab(MGAH22); The compound of general formula (I) is selected from table 1, and wherein, the amine groups on quinoline ring is the point be connected with linker.In general formula A another embodiment to general formula C, anti-HER2 antibody is Herceptin (Trastuzumab), handkerchief trastuzumab or margetuximab(MGAH22); The compound of general formula (I) is resiquimod or Imiquimod, and wherein, the amine groups on quinoline ring is the point be connected with linker.In the another embodiment of general formula A to general formula C, anti-HER2 antibody is Herceptin (Trastuzumab); AM is resiquimod or Imiquimod, and wherein, the amine groups on quinoline ring is the point be connected with linker.
the preparation of compound
In general, the activated partial of the representation of general formula (I) can use the following synthesis step preparation listed.In step (1), the 4-chloro-3-nitroquinoline of general formula A and general formula R1nH2amine reaction generate the 3-nitroquinoline-4-amine of Formula B.In step 2, the 3-nitroquinoline-4-amine of Formula B is reduced, thus generates the quinoline-3-4-diamines of general formula C.In step 3, the quinoline-3-4-diamines of general formula C and carboxylic acid or its equivalent react, and generate 1H-imidazo [4, the 5c] quinoline of general formula D.
Alternatively, the compound of general formula (I) can according to US6,331,539B1, US6,451,810B1, US7,157,452 and described in US7,301027B2 synthetic method preparation.
On the other hand, the compound of general formula (Ia) is by using linker to connect both targeting moiety and activated partial to prepare.Described linker uses its reaction site to be combined with targeting moiety and activated partial.In some embodiments, described in combination with at linker and form covalent linkage between targeting moiety and activated partial and carry out.In some embodiments, described reaction site is nucleophilic group.In some embodiments, described reaction site is electrophilic group.Nucleophilic group useful in linker includes but not limited to: hydrazides group, oximido group, amino group, diazanyl group, thiosemicarbazone group, hydrazinecarboxylate group and aryl hydrazide group.Useful electrophilic group includes but not limited to: maleimide base group, carbonate group and haloacetyl amine groups.
pharmaceutical dosage form and administering mode
The invention still further relates to pharmaceutical composition, described pharmaceutical composition comprises compound of the present invention or its pharmacy acceptable salt, and one or more pharmaceutically acceptable carriers.
Compound as herein described (such as, its additive salt or hydrate) and pharmaceutically acceptable carrier can use multiple different mode of administration or approach to be delivered to patient.Suitable route of administration includes but not limited to: inhalation, transdermal administration, oral administration, rectal administration, through mucosa delivery, enterally administering and administered parenterally (comprising intramuscular, subcutaneous and intravenous injection).Preferably, comprise as the antibody of targeting moiety or the compound of the present invention of antibody fragment with parenteral mode administration, more preferably, with intravenous administration.
Term used herein " administration " is intended to all modes comprising action site that is direct and that indirectly extremely expected by compound delivery.
Compound as herein described or its pharmacy acceptable salt and/or its hydrate can be individually dosed, with other compound Combined Preparation of the present invention and/or combine the form administration with mixture with other treatment agent.Certainly, to the illness that can depend in part on the selection of the therapeutical agent of compound Combined Preparation of the present invention in treatment.
Such as, when to when suffering from patient's administration of the disease states caused by the organism depending on Autoinducer, compound of the present invention can the form administration of mixture, and described mixture contains the medicament being used for the treatment of pain, infection and other symptoms and usually relevant with disease side effect.These medicaments comprise such as, analgesic agent, microbiotic, etc.
When to when carrying out patient's administration of cancer therapy, compound can the form administration of mixture, and described mixture comprises carcinostatic agent and/or supplementary synergistic agent.Described compound can also the form administration of mixture of medicament of side effect containing treatment radiotherapy, the medicament of the side effect of described treatment radiotherapy such as, antiemetic, antiradiation agent, etc.
Can comprise such as with the supplementary synergistic agent of compound Combined Preparation of the present invention, tricyclic antidepressant (such as, meter Pa Ming (imipramine), Desipramine (desipramine), amitriptyline (amitriptyline), clomipramine (clomipramine), Trimipramine (trimipramine), doxepin (doxepin), nortriptyline (nortriptyline), protriptyline (protriptyline), amoxapine (amoxapine) and maprotiline (maprotiline)), non-tricyclic antidepressant (such as, Sertraline (sertraline), trazodone (trazodone) and citalopram (citalopram)), Ca2+ antagonist (such as, verapamil (verapamil), nifedipine (nifedipine), nitrendipine (nitrendipine) and caroverine (caroverine)), amphotericin, triparanol analogue (such as, tamoxifen (tamoxifen)), antiarrhythmic drug (such as, Quinidine (quinidine)), antihypertensive drug (such as, serpentine (reserpine)), mercaptan consumes thing (such as, fourth methyllanthionine and sulphoxide imine) and calcium leucovorin.
Active compound of the present invention with the form administration of himself or with the form administration of pharmaceutical composition, in described pharmaceutical composition, described active compound and one or more pharmaceutically acceptable carrier, vehicle or mixing diluents.Pharmaceutical composition used according to the invention uses one or more physiology acceptable carriers to prepare in a usual manner usually, described physiology acceptable carrier comprises vehicle and adjuvant, and described physiology acceptable carrier is conducive to active compound being processed into the preparation that can pharmaceutically use.Suitable formulation depends on selected route of administration.
For for mucosa delivery, in formulation, use the permeate agent being suitable for barrier to be penetrated.These permeate agents are generally known in the art.
For oral administration, compound is prepared easily through being combined with pharmaceutically acceptable carrier known in the art by active compound.These carriers can make compound of the present invention be mixed with by oral tablet, pill, dragee, capsule, liquid, gel, syrup, soup compound and the suspension of patient to be treated.Oral pharmaceutical preparation is by such as under type acquisition: mixed with compound active agent by solid excipient; optionally grind the mixture obtained; and granular mixture is processed, if want to obtain tablet or sugar-coat label, need to add suitable adjuvant before this procedure.Specifically, suitable vehicle be weighting agent (such as, comprise the sugar of lactose, sucrose, N.F,USP MANNITOL or sorbyl alcohol), cellulose preparation (such as, W-Gum, wheat starch, Starch rice, potato starch, gelatin, tragacanth gum, methylcellulose gum, Vltra tears, sodium hydroxyethlcellulose) and/or polyvinylpyrrolidone (PVP).If necessary, can disintegrating agent be added, such as, crosslinked polyvinylpyrrolidone, agar or Lalgine or its salt (such as, sodium alginate).
Suitable dressing is provided to sugar-coat label.In order to achieve this end, can use concentrated sugar soln, this solution is optionally containing Sudan Gum-arabic, talcum powder, polyvinylpyrrolidone, carbomer gel, polyoxyethylene glycol and/or titanium dioxide, paint solution and suitable organic solvent or solvent mixture.Dyestuff or pigment can be added in tablet or dragee dressing, for identifying or characterize different active compound doses combinations.
The pharmaceutical preparation that can orally use comprises the capsule of the push style capsule be made up of gelatin and the soft sealing of being made up of gelatin and softening agent (such as, glycerol or Sorbitol Powder).Push style capsule can containing with the tackiness agent of the weighting agent of such as lactose and so on, such as starch and so on and/or the lubricant of such as talcum powder or Magnesium Stearate and so on and the optionally activeconstituents that mixes of stablizer.In soft capsule, active compound is dissolvable in water or is suspended in suitable liquid, such as, and fatty oil, whiteruss or liquid macrogol.In addition, stablizer can be added.Dosage for all formulations of oral administration should be suitable for this administering mode.
For orally administering, the form administration of the tablet that composition can be prepared by conventional methods or lozenge.
For inhalation, compound used according to the invention is convenient to by using suitable propelling agent (such as, Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas) to send with the form of the spraying provided by compression wrap or atomizer.When using pressurized spray, dose unit is by providing the valve of the amount of sending metering to determine.The capsule used in sucker or insufflator and cartridge case (such as, gelatine capsule and cartridge case) can be configured to the formulation of the powdered mixture containing compound and suitable powder matrix (such as, lactose or starch).
Compound can be configured to the formulation by injecting (such as, fast injection or continuous infusion) mode administered parenterally.Injection is the preferred medication of composition of the present invention.Formulation for injecting can provide with the unit dosage form of the sanitas added, and such as, provides with the form of ampulla or multi-dose container.Composition can adopt suspension in such as oiliness carrier or aqueous carrier, solution or emulsion and so on form and can blender (formulatory) containing such as suspension agent, stablizer and/or dispersion agent and so on, and such as crosslinked polyvinylpyrrolidone, agar or Lalgine or its salt (such as, sodium alginate) can be added.
Pharmaceutical dosage form for administered parenterally comprises the aqueous solution of the active compound of water-soluble form.In addition, the suspension of active compound can be prepared into suitable oily injection suspensions.Suitable lipophilic solvent or carrier comprise fatty acid ester (such as, ethyl oleate or triglyceride level) or the liposome of fatty oil (such as, sesame oil) or synthesis.Water injection suspension liquid can comprise following material: this material increases the viscosity of suspension, such as, and Xylo-Mucine, Sorbitol Powder or dextran.Optionally, suspension also can contain suitable stablizer or medicament, and this stablizer or medicament increase the solubleness of compound, thus prepare the solution of high enrichment.For injection, medicament of the present invention can be formulated into aqueous solution, is preferably mixed with physiological compatibility damping fluid (such as, Hanks solution, Ringer's solution or normal saline buffer solution).
Alternatively, activeconstituents is dissolved in the powder type in suitable carrier before can be use, described suitable carrier such as, aseptic apirogen water.
Compound also can be formulated into rectal compositions, and such as, suppository or retention enema formulation, such as, containing conventional suppository matrix (such as theobroma oil or other glyceryl ester).
Except aforementioned formulation, compound also can be formulated into prolonged action preparation.The formulation of this long-term onset is by implanting or dermal delivery (such as, subcutaneous delivery or intramuscular delivery), intramuscular injection or transdermal patch delivery.Therefore, such as, compound maybe can be mixed with sparing soluble derivative by suitable polymeric material or hydrophobic material (such as, the emulsion in acceptable oil) or ion exchange resin preparation by compound, such as, is mixed with sl. sol. salt.
Pharmaceutical composition also can comprise suitable solid or gel phase carriers or vehicle.The example of these carriers or vehicle comprises the polymkeric substance of calcium carbonate, calcium phosphate, various carbohydrate, starch, derivatived cellulose, gelatin and such as polyoxyethylene glycol and so on.
Preferred pharmaceutical composition is the composition being mixed with injection type, such as, intravenous form, and this preferred pharmaceutical composition comprise based on total 100 % by weight pharmaceutical composition about 0.01 % by weight to about 100 % by weight compound of the present invention.Drug-ligand conjugate can be antibody-cytotoxin conjugate, wherein, selects the antibody of target particular cancers.
In some embodiments, pharmaceutical composition of the present invention also comprises other treatment agent.
In some embodiments, described other treatment agent is carcinostatic agent.
In some embodiments, other carcinostatic agents are selected from: the inhibitor of antimetabolite, topoisomerase I and topoisomerase II, alkylating agent, microtubule inhibitors, antiandrogen agent, GNRh conditioning agent or their mixture.
In some embodiments, described other treatment agent is chemotherapeutics.
" chemotherapeutics " herein refers to chemical compound useful in Therapeutic cancer.Example includes but not limited to: gemcitabine (Gemcitabine), irinotecan (Irinotecan), Zorubicin (Doxorubicin), 5 FU 5 fluorouracil (5-Fluorouracil), cytosine arabinoside (Cytosine arabinoside, " Ara-C "), endoxan (Cyclophosphamide), phosphinothioylidynetrisaziridine (Thiotepa), busulfan (Busulfan), cytotoxin, taxol, methotrexate (Methotrexate), cis-platinum (Cisplatin), melphalan (Melphalan), vinealeucoblastine(VLB) (Vinblastine) and carboplatin (Carboplatin).
In some embodiments, second chemotherapeutics is selected from: tamoxifen (tamoxifen), raloxifene (raloxifene), Anastrozole (anastrozole), Exemestane (exemestane), letrozole (letrozole), imatinib (imatanib), taxol (paclitaxel), endoxan (cyclophosphamide), lovastatin (lovastatin), mimosine (minosine), gemcitabine (gemcitabine), cytosine arabinoside (cytarabine), 5 FU 5 fluorouracil, methotrexate, docetaxel (docetaxel), goserelin (goserelin), vincristine(VCR) (vincristine), vinealeucoblastine(VLB) (vinblastine), R 17934 (nocodazole), teniposide (teniposide), Etoposide (etoposide), gemcitabine, ebormycine (epothilone), vinorelbine (vinorelbine), camptothecine (camptothecin), daunorubicin (daunorubicin), dactinomycin (actinomycin D), mitoxantrone (mitoxantrone), acridine (acridine), Zorubicin (doxorubicin), epirubicin (epirubicin) or idarubicin (idarubicin).
test kit
On the other hand, the invention provides a kind of test kit, described test kit comprise in compound of the present invention or composition one or more and use the specification sheets of described compound or composition.In an exemplary embodiment, the invention provides for the test kit by linker arm of the present invention and another molecule coupling.Described test kit comprises linker and the specification sheets for making linker be connected with specific functional group.Described test kit also comprise in cytotoxic drug, target agent, detectable mark, pharmacy acceptable salt or buffer reagent one or more.Described test kit also can comprise container and optionally one or more bottles, testing tube, flask, bottle or syringe.Other forms of test kit is apparent and within the scope of the invention to those skilled in the art.
medical use
Compound of the present invention comprises anti-HER2 antibody, linker and as TLR7/8 agonist and the cytotoxic agent represented by general formula (I).In the prior art, the cytokine reagent of several major chemical groups is only had to be developed for antibody drug conjugates.They are divided into two classes: DNA damage agent and microtubule disrupting agent.The invention provides the element of the Chemical cell for antibody drug conjugates reagent-TLR7/8 agonist that a class is new, its immunity moderation reacts, subsequently activating dendritic cells and natural killer cell and promote anti-tumor activity.For expectation, the present inventor finds that compounds exhibit of the present invention goes out extraordinary anti-tumor activity.Carrying out in the process of typical treatment with compound of the present invention, cytotoxic agent partial delivery is extremely expressed the tumour cell/cancer cells of HER2 by the antibody moiety worked as guided missile, in this tumour cell/cancer cells, described cytotoxic agent part is directly or indirectly to antitumor cell/cancer cells.This methods for the treatment of has benefited from the reduction of toxic side effects and the improvement of pharmacokinetic properties.And cytotoxic agent (medicine) slow releasing from carrier antibody makes the intratumoral concentrations of active cytotoxins reagent maintain higher level and makes the Plasma Concentration of active cytotoxins reagent maintain lower level.
Therefore, on the other hand, the invention provides a kind of method suppressing HER2 positive tumor cell/cancer cell multiplication, described method comprises compound administration of the present invention in described tumour cell.In some embodiments, tumour can be transfer or non-diverting.
" cancer " or " tumour " herein refers to the pathological state in human body, it is characterized by uncontrolled cell proliferation.Example includes but not limited to: cancer, lymphoma, blastoma and leukemia.The more concrete example of cancer includes but not limited to: lung (minicell and non-small cell) cancer, mammary cancer, prostate cancer, the carcinous cancer of class, bladder cancer, cancer of the stomach, carcinoma of the pancreas, liver cancer (hepatocellular carcinoma), hepatoblastoma, colorectal carcinoma, squamous cell carcinoma of the head and neck, the esophageal carcinoma, ovarian cancer, cervical cancer, carcinoma of endometrium, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma, thyroid carcinoma, fibroma durum, acute myelocytic leukemia (AML) and chronic myelocytic leukemia (CML).
" suppression " or " treatment " herein refers to reduction, therapeutic and prophylactic treatment, and wherein, object is the pathologic disorders or illness that reduce or prevent to specify.In one embodiment, after administration compound of the present invention, cancer patients can experience tumor size and reduce." treatment " comprises (1) suppression to be suffered from or shows the disease in the pathology of disease or the subject of symptom; (2) alleviation suffers from or shows the disease in the pathology of disease or the subject of symptom; And/or (3) impact suffers from or shows any measurable reduction of the disease in the pathology of disease or the patient of symptom or patient body.Compound of the present invention can prevent growth of cancer cells to a certain extent and/or kill cancer cells, and the compound of the present invention in this degree can be cytostatic and/or Cytotoxic.
" treatment significant quantity " herein refers to the amount of the compound provided herein of the imbalance in effective " treatment " patient or mammalian body.When Therapeutic cancer, the medicine for the treatment of significant quantity can reduce the number of cancer cells, tumor size is reduced, anticancer infiltrates and enters peripheral organ, Tumor suppression shifts, Tumor suppression grows to a certain degree, and/or alleviates one or more in the symptom relevant to cancer to a certain extent.
On the other hand, the invention provides a kind of method of HER2 positive tumor/cancer for the treatment of in subject, described method comprises the compound administration of the present invention for the treatment of significant quantity in described patient.In some embodiments, tumour or cancer can be the tumour or the cancer that are in any stage, such as, and early stage or late tumor or cancer, such as, I phase, II phase, III phase, IV phase or V phase tumour or cancer.In some embodiments, tumour or cancer can be transfer or non-diverting.When metastatic tumo(u)r or cancer, method of the present invention can slow down or suppress primary tumor or cancer to shift to other sites, or slows down or suppress formed in other sites away from primary tumor or cancer therapy or produce metastatic tumo(u)r or cancer.Therefore, method of the present invention also comprises: 1) slow down or suppress may occur to shift or occurred the growth of tumour cell or the cancer cells (such as, disseminated tumor cell, DTC) shifted, propagation, migration or invasion; 2) slow down or suppress primary tumor or cancer are different from other sites, position or the region formation transfer of primary tumor or cancer to one or more or set up transfer; 3) after transfer has been formed or set up, slow down or suppress one or more other sites being different from primary tumor or cancer, the growth of transfer in position or region or propagation; And 4) after transfer has been formed or set up, slow down or suppressed formation or the foundation of extra transfer.
In some embodiments, tumour or cancer are entity or liquid cell agglomerate." entity " knurl refers to and usually flocks together and form the cancer of agglomerate, knurl or transfer.Concrete non-limiting example comprises: breast tumor/mammary cancer, ovarian tumor/ovarian cancer, uterus tumor/uterus carcinoma, tumor of cervix/cervical cancer, gastric tumor/cancer of the stomach, lung tumor/lung cancer, gastric tumor/cancer of the stomach, colon tumor/colorectal carcinoma, tumor of bladder/bladder cancer, glia tumour/colloid carcinoma and endometrial tumors/carcinoma of endometrium etc." liquid tumors " refers to the knurl of nature dispersion or diffusion, and it can not form solid mass usually.Concrete example comprises: reticuloendothelial system knurl or hemopoietic system knurl, such as, and lymphoma, myelomatosis and leukemia.Leukemic non-limiting example comprises: acute and chronic lymphoblastic leukemia, myeloblastic leukemia and multiple myeloma.Usually, these diseases are caused by low differentiation acute leukemia (such as, EBL and acute megakaryocytic leukemia).Concrete myeloide disease includes, but are not limited to: acute promyelocytic leukemia (APML), acute myeloblastic leukemia (AML) and chronic myelocytic leukemia (CML).Lymphoid malignancies comprises, but be not limited to: acute lymphoblastic leukemia (ALL), it comprises B cell system ALL(B-ALL) and T cell system ALL(T-ALL), lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstroem macroglobulinemia (WM).Concrete malignant lymphoma comprises: non-Hodgkin lymphoma and variant thereof, lymphoma peripheral T cell, adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocyte leukemia (LGF), Huo Qijin disease and Reed-Sternberg disease.
In some embodiments, method of the present invention together can be implemented with other treatment method or therapy (such as, surgical operation, radiotherapy, ion or chemical radiation therapy, chemotherapy, immunotherapy, local or region thermotherapy (hyperthermia) or vaccination).These other treatment methods or therapy can before the administration of compound of the present invention, basic (separately or as a mixture) simultaneously or give afterwards.
In some embodiments, method of the present invention comprises compound of the present invention and the other treatment agent Combined Preparation for the treatment of significant quantity.In some embodiments, described other treatment agent is carcinostatic agent/antineoplastic agent.In some embodiments, described other treatment agent is the inhibitor of metabolic antagonist, topoisomerase I and topoisomerase II, alkylating agent, microtubule inhibitors, antiandrogen agent, GNRh conditioning agent or their mixture.In some embodiments, described other treatment agent is selected from: Tamoxifen (tamoxifen), raloxifene (raloxifene), Anastrozole (anastrozole), Exemestane (exemestane), letrozole (letrozole), imatanib, taxol, endoxan, lovastatin (lovastatin), minosine, gemcitabine (gemcitabine), cytosine arabinoside (cytarabine), 5 FU 5 fluorouracil, methotrexate, docetaxel (docetaxel), goserelin (goserelin), vincristine(VCR), vinealeucoblastine(VLB), nocodazole (nocodazole), teniposide (teniposide), Etoposide (etoposide), gemcitabine, ebormycine, vinorelbine (vinorelbine), camptothecine, daunomycin (daunorubicin), dactinomycin, mitoxantrone, acridine, Zorubicin, epirubicin or idarubicin.
" combine " administration with one or more other treatment agent and comprise simultaneously administration and with sequentially any.Term used herein " drug regimen " refers to the product obtained by mixed active composition or combined activity composition, and both the fixed Combination comprising activeconstituents and non-fixed combinations.Term " fixed Combination " refers to that activeconstituents (such as the compound of general formula (1)) and associating medicament deliver medicine to patient with single entity or dosage simultaneously.Term " non-fixed combinations " refers to that activeconstituents (such as the compound of general formula (1)) and associating medicament are as the entity that separates simultaneously or (do not have specific time limitation) in order and deliver medicine to patient, and this administration provides the activeconstituents for the treatment of significant quantity in patient body.The latter also treats for mixture, such as, and administration three kinds or more activeconstituents.
effective dose
Be applicable to the composition that pharmaceutical composition of the present invention comprises the activeconstituents comprising treatment significant quantity, described treatment significant quantity is the amount effectively realizing expecting object.By (especially), illness in treatment is depended on to the effective actual amount of application-specific.The determination of significant quantity just in the limit of power of those skilled in the art (especially according to the concrete disclosed content of this paper).
For any compound as herein described, treatment significant quantity can be determined by cell cultures detection method at first.Target Plasma Concentration is can the concentration of active compound of cell growth inhibiting or division.In a preferred embodiment, at least 25% of cytoactive is suppressed.The target Plasma Concentration of the active compound suppressed at least about 30%, 50%, 75% or even 90% or higher cytoactive can be induced for preferred at present.The percentage ratio that the cytoactive in patient body suppresses can be monitored, thus assess the appropriateness of the Plasma Concentration reached, and can raise or lower dosage to realize the suppression percentage ratio expected.
As known in the art, the treatment significant quantity for human body is also determined by animal model.Such as, can be formulated into for the dosage of human body the Efficient Cycle concentration that realization finds in animal body.As mentioned above, the dosage in human body regulates by monitoring Carbazole alkaloid and rise or lowering dosage.
Treatment effective dose is also determined by the known somatic data showing the compound of similar pharmacological activity.The dosage used can regulate based on the relative bioavailability of the compound of institute's administration of comparing with known compound and effect.
Be realize maximum effectiveness in human body to carry out regulating just in the limit of power of those of ordinary skill in the art to dosage based on aforesaid method and additive method well known in the art.
When topical, the systemic circulation concentration of the compound of institute's administration is not particularly important.In this case, effectively realize at regional area the concentration of result expected to reach to drug compound.
For preventing and/or treating the application of the disease aspect relevant with abnormal cell proliferation, preferably, the circulation composition of the compound of institute's administration is about 0.001 μM to 20 μMs, preferably about 0.01 μM to 5 μMs.
The patient dose of the oral administration of compound as herein described typically is about 1mg/ days to about 10,000mg/ days, typically more is about 10mg/ days to about 1,000mg/ day, typically is about 50mg/ days most to about 500mg/ days.According to weight in patients, typical dosage is about 0.01mg/kg/ days extremely about 150mg/kg/ days, typically more is about 0.1mg/kg/ days to about 15mg/kg/ days, typically is about 1mg/kg/ days most to about 10mg/kg/ days, such as, 5mg/kg/ days or 3mg/kg/ days.
In at least some embodiment, postpone or the patient dose of Tumor suppression growth can be 1 μm ol/kg/ days or less.Such as, patient dose can be 0.9 μm ol/kg/ days, 0.6 μm ol/kg/ days, 0.5 μm ol/kg/ days, 0.45 μm ol/kg/ days, 0.3 μm ol/kg/ days, 0.2 μm ol/kg/ days, 0.15 μm ol/kg/ days or 0.1 μm ol/kg/ days or less (mole number of reference drug).Preferably, when continuing the time period of at least five days with dosed administration every day, antibody drug conjugates postpones tumor growth.
For other mode of administration, dosage and interval can separate regulation, thus provide the Plasma Concentration of the compound for the effective administration of specific clinical indication in treatment.Such as, in one embodiment, concentration multiple dosing every day that can be relatively high according to compound of the present invention.Alternatively, more preferably, the dosage regimen of lower frequency is used with minimum effective concentration administration compound of the present invention.This can provide the treatment plan matched with the severity of the disease of individuality.
Use instruction herein can arrange effective treatment plan, and do not cause larger toxicity, and treat the clinical symptom that particular patient shows completely effectively.This arrangement should comprise by considering that many factors carefully selects active compound, described many factors such as, the toxic characteristic of the adverse side effect of compound potencies, relative bioavailability, weight in patients, existence and seriousness thereof, preferred mode of administration and selected medicament.
Although describe and illustrate preferred embodiment of the present invention herein, it is obvious to the skilled person that these embodiments only illustrate.Those skilled in the art can make multiple change, amendment and replacement to these embodiments, and do not deviate from the present invention.Should be understood that, Alternate embodiments of the present invention as herein described can be used for putting into practice the present invention.Limit scope of the present invention by appended claim, and method and structure in these right and equivalent thereof are also contained by appended claim.
embodiment
The present invention illustrates further by following embodiment, but the present invention is not limited thereto, and following embodiment illustrates the preparation method of compound of the present invention.
embodiment 1
the generation of the L clone of her2 transfection
Reagent: from ATCC(Manassas, VA; Cat No.CRL2648) L cell, purchased from GeneCopoeia, the Her2/pEZ-Lv105cDNA of (Cat No.Z2866), glucose DMEM, L-glutamine, Lipofectamine2000 (Invitrogen; Carlsbad, CA).
In order to the clone of the Herceptin for the preparation of screening coupling, generate the L cell of the Her2 of presentation markup.Her2/pEZ-Lv105 construct passes through the transfection of standard Lipofectamine2000 working specification to L cell (this cell grows in high concentration glucose DMEM+10%FBS+2mM L-glutamine).
binding analysis (facs analysis)
In order to determine the binding ability of the Herceptin of coupling, the L cell of his-and-hers watches intelligent her2 carries out facs analysis.In brief, by 100 μ l about 106the L cell of the individual her2 with transient transfection is together hatched from the antibody of the Herceptin coupling of different amount, uses the anti-or uncorrelated huIgG of PBS or independent two as negative control.After washing, cell to be resuspended in FACS damping fluid and 20 μ L mouse anti human IgG at room temperature anti-with being coupled to phycoerythrin (the anti-human PE of Mu) two in 100 μ L reaction volumes together hatch 30 minutes.After washing, cell is fixed in 200 μ L2% paraformaldehyde/PBS, carries out flow cytometry analysis subsequently.Identical step is used for uncorrelated human IgG antibody as isotype controls to set baseline PMT/ clone.At BDon carry out flow cytometry analysis and record the geometric mean fluorescence intensity of each sample.Use the data of FlowJo software analysis record.Result shows in FIG.
the generation of antibody toll sample receptors ligand conjugate
Reagent: Herceptin (Roche/Genentech Corporation; South San Francisco; CA) MC-val-cit-PAB(MC-vc-PAB-TLRL, be connected with resiquimod) or the MC-(MC-TLRL that is connected with resiquimod) (at Contract Research Organization; China synthesizes), Sodium Tetraborate, sodium-chlor, dithiothreitol (DTT) (DTT), Sephadex G25, DTPA, DTNB and maleimidocaproyl-monomethyl (Sigma-Aldrich; Milwaukee, Wisconsin).
Medicine for generating antibody TLR ligand conjugates comprises Herceptin and resiquimod (TLRL).Be the linker MC-vc-PAB of cleavable or the linker MC of non-cleavable for generating the linker of TLAC.
the preparation of Herceptin MC-TLRL
Herceptin by the buffer-exchanged under 20mg/mL concentration fromin be purified, be that the excessive 100mM dithiothreitol (DTT) (DTT) of antibody being dissolved in 500mM Sodium Tetraborate and 500mM sodium-chlor under the condition of 8.0 processes at pH.After 37 DEG C hatch about 30 minutes, also carry out wash-out with the PBS containing 1mM DTPA by the wash-out exchange buffering liquid on Sephadex G25 resin.Mercaptan/Ab value is detected by the concentration and concentrations of mercaptans measuring the antibody of reduction, the concentration of the antibody of described reduction is detected by the absorbancy of solution at 280nm, described concentrations of mercaptans by with DTNB(Sigma-Aldrich, Milwaukee, Wisconsin) reaction and absorbancy at 412nm place determine.The antibody being dissolved in the reduction in PBS is freezing on ice.Under concentration known, be dissolved in the drug-linker reagent (Mc-be connected with resiquimod) of DMSO at acetonitrile and dilution with water, and added to freezing the going back in original antibody in PBS.After about 1 hour, add excessive maleimide cancellation and react and hide any unreacted antibody thiol group.In some cases, coupling is carried out by the Methionin of standard step and immunoglobulin (Ig).Reaction mixture is concentrated by centrifugal ultrafiltration, and the antibody of coupling carries out purifying and desalination by the wash-out of the G25 resin in PBS, aseptically by the metre filter of 0.2 μm, and refrigerated storage.
the preparation of Herceptin MC-vc-TLRL
Antibody is connected to TLRL by halfcystine by maleimidocaproyl-valine-citrulline (vc)-p-aminobenzyloxycarbonyl (MC-vc-PAB).MC-vc-PAB linker is by the intercellular protein enzymatic lysis of such as cathepsin B and so on, when cracking, release free drug (people such as Doronina, Nat.Biotechnol., 21:778-784 (2003)), and MC linker is protease cracking between anti-cell.The Herceptin of purifying is be dissolved in 500mM Sodium Tetraborate and 500mM sodium-chlor under the condition of 8.0 at pH, and processes by excessive 100mM dithiothreitol (DTT) (DTT) further.Hatch about 30 minutes at 37 DEG C after, by the wash-out exchange buffering liquid on Sephadex G25 resin and with the PBS wash-out containing 1mM DTPA.Mercaptan/Ab value is detected by the concentration and concentrations of mercaptans measuring the antibody of reduction, the concentration of the antibody of described reduction is detected by the absorbancy of solution at 280nm place, described concentrations of mercaptans by with DTNB(Sigma-Aldrich, Milwaukee, Wisconsin) reaction and absorbancy (optical extinction coefficient=13600cm at 412nm place-1m-1) detect.The antibody being dissolved in the reduction in PBS is freezing on ice.The MC-val-cit-PAB-PNP be connected with resiquimod in DMSO is dissolved in acetonitrile and water, and is added in the antibody of the freezing reduction in PBS.After hatching 1 hour, add excessive maleimide cancellation and react and hide any unreacted antibody thiol group.In some cases, coupling is carried out by the Methionin of standard step and immunoglobulin (Ig).By centrifugal ultrafiltration concentrated reaction mixture, antibody drug conjugates carries out purifying and desalination by the wash-out of the G25 resin in PBS, aseptically by the metre filter of 0.2 μm, and refrigerated storage.
Usually, the linked reaction of antibody and MC-TLRL or MC-vc-TLRL generates nonuniform mixture, and this mixture comprises antibody that be connected with the TLRL medicine of different quantities, coupling, that is, medicament distribution is the medicine loading of 1 to about 8.Therefore, antibody MC-TLRL or antibody MC-vc-TLRL comprises the mixture that the species molecule of the purifying of separation and average drug are loaded as 1 to 8.By controlled working process, medicine is loaded as 3 to 5.In the compound formulation obtained by linked reaction, in each antibody, the average number of TLRL drug moiety can be characterized by usual manner, such as mass spectroscopy, elisa assay method, electrophoretic separation method and HPLC.Also can determine to distribute according to the antibody MC-TLRL of medicine or the amount of antibody MC-vc-TLRL.By ELISA, mean value (people (2004) the Clinical Cancer Res.10:7063-7070 such as Hamblett of the medicine efficient loading number in the particular formulations of antibody and TLRL coupling can be determined; The people such as Sanderson (2005) Clinical Cancer Res.11:843-852).But the apportioning cost of medicine does not combine by antibody-antigene and ELISA detectability distinguishes.And the elisa assay for detecting antibody drug conjugates can not determine which position drug moiety is connected in antibody, such as, heavy chain or light chain segments or specific amino-acid residue.In some cases, homogeneous Herceptin MC-TLRL or the separation of Herceptin MC-vc-TLRL, purifying and sign (medicine wherein, had in the Herceptin MC-TLRL or Herceptin MC-vc-TLRL that other drug loads is particular value) realize by the mode of such as reversed-phase HPLC or electrophoretic separation and so on.
the cytotoxicity analysis (ADCC) of antibody dependent cellular mediation
Reagent: SKBR3 cell (ATCC, Cat No.HTB-30), McCoy ' s5A(Invitrogen; Cat No.22400, Lot No.747809), RPMI-1640(Invitrogen; Cat No.11835; Lot No.764956), FCS(Hyclone, Cat No.SH30084.03; Lot No.GRH0054); Ficoll-Hypaque(Amersham Biosciences, Piscataway, NJ; Cat No.17-1440-02), 96 orifice plates (Costar, Cat No.3599, Cat No.3916), trypan blue (Invitrogen Cat No15250-061), LDH test kit (Promega, Cat No.G7891), ELISA plate reading MD5(Molecule device).Humanized antibody(Genentech Corporation, South San Francisco, CA; Trade(brand)name Herceptin) multiple soluble in water to make 10mg/ml stoste before use.
In order to determine whether still there is effector function with the Herceptin of the TLRL coupling of high-efficient carrier, the cytotoxicity (ADCC) detecting the antibody dependent cellular mediation of several different unconjugated form active and with demonstrate there is the ADCC activity significantly improved Herceptin reference material compared with.Use come from peripheral blood lymphocytes (PBMC) the action effect cell of healthy donors, end user SKBR3 clone carries out ADCC analysis as target cell.First day, by 1 × 104/ 100 μ L SKBR3 cells are inoculated on 96 hole flat boards, subsequently 37 DEG C, under 5%CO2 condition, hatch 48 hours.
At the 3rd day, prepare fresh human PBMC by the human blood obtained from the voluntary donor of health.People's venous blood sample is collected in ammonium citrate (ACD-A) pipe.Pipe is put upside down for several times, by whole blood transfer to 50ml conical tube.With the PBS in 2%FBS with 1:3 dilute blood.Ficoll-Hypaque is slowly dispensed to bottom blood/PBS mixture.At room temperature with the speed of 2400rpm, centrifugation is carried out to sample and continue 30 minutes, subsequently by suction removing upper strata (blood plasma/PBS).Erythrocyte sedimentation rate layer (Buffy coat) is collected with Sterile pipette and collects in 50ml conical tube.If still visible WBC block under erythrocyte sedimentation rate layer, so collects all WBC materials, does not carefully remove too much Ficoll-Hypaque.Add aseptic PBS-2%FBS and mix by being inverted.Under the condition of room temperature, 250 × g, centrifugation is carried out to the PBMC suspension of dilution and continue 20 minutes, collecting cell granule.PBMC granule is suspended in the RPMI-1640 substratum with 2% heat-inactivated FBS, and washing also checks viable count by trypanblue exclusion method.Preparation 1.2 × 107the density of cell/mL is stand-by.Meanwhile, in RPMI-1640, the ultimate density of Dispersal risk is from 12 μ g/mL, 4 μ g/mL, 1.2 μ g/mL, 0.4 μ g/mL, 0.12 μ g/mL, 0.04 μ g/mL, 0.012 μ g/mL, 0.004 μ g/mL to 0 μ g/mL.
Subsequently the diluent of a series of test antibody and control antibodies is added in the hole containing target cell, PBMC effector cell in 50 μ L RPMI-1640 substratum is added to (ratio of effector cell and target cell is 60:1) in each hole, and hatch 17 hours again.At the end of hatching, centrifugal treating carried out to flat board and use the lactate dehydrogenase (LDH) in LDH measurement kit assay supernatant liquor active.Cytolysis uses microplate reader quantitative by the absorbancy at 490nm place.The absorbancy in hole only containing target cell contrasts as a setting, and the hole of the target cell of dissolving containing useful Triton-X100 provides maximum and obtains signal.Antibody independent cell toxicity is measured in the hole not adding antibody containing target cell and effector cell.Cytotoxicity calculates according to following equations:
% cytotoxicity=100%x [A490nm (sample) – A490 (target cell) – A490 (effector cell]/[A490nm (target cell) – A490nm (target cell) of dissolving]
The average A DCC value in duplicate sample diluting liquid is drawn relative to antibody concentration, and by matching to Prism5(GraphPad) obtain EC50 value and ADCC(%) at utmost.
enrichment people dendritic cell (DC) from PBMC
Centrifugal by Ficoll, prepare human PBMC by the erythrocyte sedimentation rate layer obtained from volunteer's donor of health.By using negative sense abatement, by magnetic bead (Miltenyi Biotec) the enrichment dendritic cell of the mixture had from the anti-cd 3 antibodies of human PBMC, anti-CD 19 antibodies, anti-CD20 antibody, anti-CD 14 antibody and anti-CD16 antibody.By goat anti-mouse FITC(pedigree), HLA-DR-APCCy7, CD123-BV421 and CD11C-APC DC to enrichment dyes.The cell of dyeing is analyzed on BD LSR Fortessa.CD 3-resisting monoclonal antibody, anti-CD4monoclonal antibody, anti-CD11C monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD14 monoclonal antibody, anti-CD16 monoclonal antibody, anti-CD123 monoclonal antibody are purchased from BD Biosciences or Biolgend.
Fig. 3 A represents the percentage ratio of DC before and after enrichment.The DC(HLA-DR+Lin-of total cell before and after numeral pedigree abatement in two figure on top) percentage ratio.The mDC(CD11C+CD123-of total DC before and after numeral pedigree abatement in bottom graph) and percentage ratio pDC(CD123+CD11C-).
to stimulation and the cytokine-expressing of the people DC of enrichment
By 1-2 × 105the DC of enrichment is inoculated in 96 hole flat boards in 100 μ l substratum, to be added in flat board by the stimulant that 100 μ l dilute and cultivates 20 to 22 hours in the couveuse of 37 DEG C.Collect supernatant liquor and pass through ELISA(Mabtech AB) analyze humanIFN-α, IL-6, IL-12 (p70) and TNF-α.
statistical analysis
The significance of all comparisons uses the two tail t detection computations of Student, and the variance between hypothetical simulation group and sample sets is unequal, thinks that result is significant as p<0.05.Dependency between parameter uses Spearman rank correlation test evaluation, and P value <0.05 is considered to statistically significant.
herceptin TLRL conjugate is used to carry out interior tumor cell fragmentation test
For be derived from patient cancer of the stomach heterograft (PDX) mouse model research for, by 6 to 8 week ages female Balb/c nude mice (available from SLAC, Shanghai, China) transplant for tumor fragment.According to Laboratory Animal Care and instruction manual and the code of animal care and the council of use (Institutional Animal Care and Use Committee), with normal nude mice diets animal, animal is supported in SPF animal facility.About 15mm will be of a size of3to 30mm3patient STO#69 gastric tumor fragment transplant (hypodermically) to right side of Balb/c nude mice.
Medicine or the reference agent (QWKx3) of 5mg/kg to 20mg/kg antibody is contained by Intravenous administration route administration.A tumour is measured weekly to determine its subcutaneous growth by slide calliper rule.The two-dimensional of twice tumour is measured weekly with slide calliper rule.Following equation is used to calculate gross tumor volume: gross tumor volume=(length x width2) × 0.5.Mean tumour volume or body weight use paint program Prism5 (GraphPad) to draw.The terminal of effect research is arranged on first time and treats latter 30 days to 45 days or when tumor size reaches 2000mm3time above (whichever first reaches).If mouse loses body weight more than 20% or seriously ill and cannot obtain enough food or water, so it removed from research and implement euthanasia.The tumour of mouse is collected, at LN at terminal2in half freezing and semifixed in formalin, organize for the preparation of FFPE.