CN103619356A - Peptide oligonucleotide conjugates - Google Patents

Peptide oligonucleotide conjugates Download PDF

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CN103619356A
CN103619356A CN201180071918.XA CN201180071918A CN103619356A CN 103619356 A CN103619356 A CN 103619356A CN 201180071918 A CN201180071918 A CN 201180071918A CN 103619356 A CN103619356 A CN 103619356A
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CN103619356B (en
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贡纳·J·汉森
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SA Leputa Medical Co
Sarepta Therapeutics Inc
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SA Leputa Medical Co
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Abstract

Oligonucleotide analogues conjugated to carrier peptides are provided. The disclosed compounds are useful for the treatment of various diseases, for example diseases where inhibition of protein expression or correction of aberrant mRNA splice products produces beneficial therapeutic effects.

Description

Peptide oligonucleotide conjugate
The cross reference of related application
The application requires the rights and interests of No. 13/101942 U.S. Patent application of application on May 5th, 2011 and No. 13/107528 U.S. Patent application of application on May 13rd, 2011 according to the 120th, United States code the 35th chapter, these applications are all incorporated herein by reference.
Background
Technical field
The present invention relates in general to the oligonucleotide compound (oligomer) that can be used as antisense compounds, relates more specifically to be attached to the oligomer compound on cell permeability peptide, and the purposes of this type of oligomer compound in antisense application.
Description of Related Art
The actual utility with the bioactive many medicines of potentially useful, is usually obstructed this type of drug delivery owing to being difficult to its target.Conventionally must be by be delivered to intracellular compound from containing delivering the extracellular environment of large water gaging, and penetrate subsequently lipotropy cell membrane and enter cell.By specific transport mechanism, carry out Active transport except immaterial, many molecules, especially macromole, or lipophilic and can not actually dissolving too, otherwise too many hydrophilic and can not penetrate film.
By amino acid residue 49-57 (Tat49-57, thering is sequence RKKRRQRRR) the HIV Tat protein fragments that forms is used to be delivered in cell (Barsoum et al. for example by having bioactive peptides and proteins, 1994, PCT Pubn.No.WO94/04686).Tat (49-60) is used to promote (Astriab-Fisher, the Sergueev et al.2000 of sending of phosphonothiolic acid oligonucleotide; Astriab-Fisher, Sergueev et al.2002).What oppositely Tat or rTat (57-49) (RRRQRRKKR) were compared by report that Tat (49-57) has an improvement sends fluorescein to intracellular effectiveness (Wender, Mitchell et al.2000; Rothbard, Kreider et al.2002).Rothbard and Wender also disclose other and have been rich in arginic transport polymer (WO01/62297 PCT is open; No. 6306993 United States Patent (USP); No. 2003/0032593 U.S. Patent Application Publication).
Oligonucleotide is the medical compounds of the potentially useful of a class, and it is sent is usually the obstacle that treatment is used.With regard to this this, have been found that the morpholino oligomer (PMOs that is connected with di(2-ethylhexyl)phosphate amide; See for example Summerton and Weller, 1997) than the charged oligonucleotide analogs such as D2EHDTPA, have more prospect.PMOs is water solublity, neutral or uncharged antisense molecule substantially, and it splices by prevention or combination or the progress of body translation element are carried out inhibition of gene expression.Also show that PMOs can suppress or stop virus replication (Stein, Skilling et al.2001; McCaffrey, Meuse et al.2003).They have highly resistant (Hudziak, Barofsky et al.1996) to enzymic digestion.(Stein, Foster et al.1997 in acellular and cell culture model in vitro; Summerton and Weller1997), and (Heasman, Kofron et al.2000 in Brachydanio rerio in vivo, Rana nigromaculata and Hemicentrotus seu Strongylocentrotus embryo; Nasevicius and Ekker2000), and in adults model, as rat, mice, rabbit, Canis familiaris L. and pig (see for example Arora and Iversen2000; Qin, Taylor et al.2000; Iversen2001; Kipshidze, Keane et al.2001; Devi2002; Devi, Oldenkamp et al.2002; Kipshidze, Kim et al.2002; Ricker, Mata et al.2002), PMOs has shown high antisense specificity and effect.
Also show that antisense PMO oligomer can be ingested in cell, and compare other widely used antisense oligonucleotides, have more in vivo consistent effectiveness and nonspecific effect still less and (see for example P.Iversen, " Phosphoramidite Morpholino Oligomers ", in Antisense Drug Technology, S.T.Crooke, ed., Marcel Dekker, Inc., New York, 2001).Having shown that PMOs is attached on arginine enrichment peptide can increase their cellular uptake (for example seeing No. 7468418 United States Patent (USP)); Yet the toxicity of this conjugate has slowed down them as the development of feasible drug candidates.
Although obtained obvious progress, this area still exists having the needs of the antisense of raising or the oligonucleotide conjugate of anti-gene performance.The antisense of this type of raising or anti-gene performance comprise: hypotoxicity, DNA and RNA do not endangered to sequence selective compared with strong affinity; The pharmacokinetics and the tissue distribution that improve; The cell improving is sent and reliable and controlled distribution in vivo.
Summary of the invention
Compound of the present invention can address these problems, and the improvement of the antisense molecule existing over this area is provided.By cell permeability peptide being connected to substantially on uncharged nucleic acid analog by glycine or amino proline acid, inventor has solved the toxicity problem relevant to other peptide oligomer conjugates.In addition, between subunit, connect and/or end portion to the modification of the combination of 5 ' and/or 3 ' end of oligonucleotide analogs, for example, morpholino oligonucleotide, can also improve the performance of this conjugate.For example, in certain embodiments, compare other oligonucleotide analogs, conjugate of the present disclosure has the toxicity of minimizing and/or the cell of raising is sent, tired and/or tissue distribution, and/or can be more effectively delivered to target organ.These superior performances can produce the clinical dosage of favourable treatment index, minimizing and lower merchandise cost.
Therefore, in one embodiment, the conjugate that the disclosure provides comprises:
(a) carrier peptides, it comprises aminoacid subunit; With
(b) nucleic acid analog, it comprises substantially uncharged skeleton and is attached to the targeting base sequence on target nucleic acid for sequence-specific;
Wherein:
Two or more described aminoacid subunit is positively charged aminoacid, the glycine that described carrier peptides comprises the c-terminus that is positioned at carrier peptides (G) or proline (P), and described carrier peptides is covalently bound to described nucleic acid analog.The compositions that comprises above conjugate and pharmaceutically acceptable carrier is also provided.
In another embodiment, the method that the disclosure provides Profilin to produce, the method comprises the nucleic acid of this albumen of coding is exposed to conjugate of the present disclosure.
Another aspect of the present disclosure comprises that promotion is transported to intracellular method by nucleic acid analog, the method comprises carrier peptides claimed in claim 1 is attached on nucleic acid analog, and wherein with respect to the non-nucleic acid analog of puting together form, promoted described nucleic acid analog to be transported in cell.
In another embodiment, the disclosure is for the method for disease in treatment target body, and the method comprises the disclosed conjugate of pharmacy effective dose is administered to object.Also provide and prepared method, their using method of this conjugate and can be used for being attached to the carrier peptides on nucleic acid analog.
By reference to the following detailed description, these and other aspect of the present invention can become apparent.For this object, stated different lists of references herein, they have described some background information, program, compound and/or compositions in further detail, and each in them is incorporated to by complete quoting at this.
Accompanying drawing explanation
Figure 1A has shown the exemplary morpholino oligomer structure that comprises the connection of di(2-ethylhexyl)phosphate amide.
Figure 1B has shown the morpholino oligomer that is combined in carrier peptides 5 ' end.
Fig. 1 C has shown the morpholino oligomer that is attached to carrier peptides 3 ' end.
Fig. 1 D-G has shown the repetition subunit fragments of exemplary morpholino oligonucleotide, is appointed as 1D to 1G.
Fig. 2 has described to be connected to morpholino---between the exemplary subunit in T part, connects.
Fig. 3 is for showing the reaction scheme for the preparation of the linking arm of solid phase synthesis.
Fig. 4 has shown the preparation for the synthetic solid support of oligomer (support).
Fig. 5 A, 5B and 5C have shown respectively, compare known conjugate in mice musculus quadriceps, barrier film and heart, the exon skipping data of exemplary conjugate.
Fig. 6 A, 6B and 6C are respectively, and compare known conjugate in mice musculus quadriceps, barrier film and heart, the selective representative of the exon skipping data of exemplary conjugate.
Fig. 7 A has described respectively with 7B Mouse Blood blood urea nitrogen (BUN) level and the survival rate with different peptide-oligomer conjugates, processed.
Fig. 8 A and 8B have shown respectively injury of kidney mark (KIM) data and clusterin (Clu) data of the mice of processing with different peptide-oligomer conjugates.
Fig. 9 A, 9B, 9C and 9D are respectively, and compare known conjugate, the figure of exon skipping, BUN level, % survival rate and KIM level in the Mice Body of relatively processing with exemplary conjugate.
Figure 10 has shown the KIM data of the mice of processing with different conjugates.
Figure 11 has shown the BUN analysis result of the mice of processing with different conjugates.
Figure 12 is for showing the figure of the concentration of different oligomers in kidney of mouse.
Detailed Description Of The Invention
I. definition
In the following description, for fully understanding of different embodiments is provided, stated some detail.Yet, it will be appreciated by those skilled in the art that and can in the situation that there is no these details, implement the present invention.In other example, do not have to show or describe the structure of knowing in detail, to avoid the unnecessarily description of fuzzy described embodiment.Unless context separately has requirement, this description and subsequently claim in the whole text in, word " comprises (comprise) " and its distortion, as, " comprise (comprises) " and " comprising (comprising) " will be interpreted as open, comprising property implication, be interpreted as " including, but are not limited to ".And the title providing is herein only for convenient, and does not explain the claimed scope of the invention or implication.
This description in the whole text in, mention that " embodiment " or " embodiment " refer to that specific features, structure or the characteristic relevant with described embodiment of description are included at least one embodiment.Therefore, this description in the whole text in, phrase " in one embodiment " or " in embodiments " appearance at diverse location might not all refer to identical embodiment.In addition, can be in any suitable manner by specific features, structure or property combination in one or more embodiments.In addition, as used in this description and claims, singulative " (a) ", " one (an) " and " described (the) " comprise plural reference object, unless context separately clearly states.Also it should be noted that term "or" is conventionally to comprise that the implication of "and/or" is used, unless context separately clearly states.
As used herein, following term has following implication, except as otherwise noted:
" amino " refer to-NH 2base.
" cyano group " or " nitrile " refer to-CN base.
" hydroxyl (Hydroxy) " or " hydroxyl (Hydroxyl) " refer to-OH base.
" imino group " refer to=NH substituent group.
" guanidine radicals " refers to – NHC (=NH) NH 2substituent group.
" amidino groups " refers to – C (=NH) NH 2substituent group.
" nitro " refer to-NO 2base.
" oxo " refer to=O substituent group.
" sulfo-" refer to=S substituent group.
" cholate " refers to following structure:
Figure BDA0000447333530000051
" dexycholate " refers to following structure:
Figure BDA0000447333530000061
" alkyl " refers to the hydrocarbon chain base of straight or branched, its be saturated or unsaturated (that is, containing one or more pairs of keys and/or triple bond), there is 1 to 30 carbon atom, and it is connected on the remainder of molecule by singly-bound.The alkyl that comprises any number carbon atom that comprises 1 to 30.Comprise the nearly alkyl of 30 carbon atoms and be called as C 1-C 30alkyl, similarly, for example, comprising the nearly alkyl of 12 carbon atoms is C 1-C 12alkyl.Represent similarly the alkyl (with other parts that define) that comprises other number carbon atoms herein.Alkyl includes, but not limited to C 1-C 30alkyl, C 1-C 20alkyl, C 1-C 15alkyl, C 1-C 10alkyl, C 1-C 8alkyl, C 1-C 6alkyl, C 1-C 4alkyl, C 1-C 3alkyl, C 1-C 2alkyl, C 2-C 8alkyl, C 3-C 8alkyl and C 4-C 8alkyl.Representational alkyl comprises, but be not limited to, methyl, ethyl, n-pro-pyl, 1-Methylethyl (isopropyl), normal-butyl, isobutyl group, sec-butyl, n-pentyl, 1,1-dimethyl ethyl (tert-butyl group), 3-methyl hexyl, 2-methyl hexyl, vinyl, third-1-thiazolinyl, but-1-ene base, penta-1-thiazolinyl, penta-Isosorbide-5-Nitrae-dialkylene, acetenyl, propinyl, fourth-2-alkynyl, fourth-3-alkynyl, pentynyl, hexin base etc.Unless separately clearly state in this manual, can be by describing below optionally substituted alkyl.
" alkylidene " or " alkylidene chain " refers to the bivalent hydrocarbon chain that the remainder of molecule is connected to the straight or branched on group.Alkylidene can be saturated or unsaturated (that is, containing one or more pairs of keys and/or triple bond).Representational alkylidene includes, but not limited to C 1-C 12alkylidene, C 1-C 8alkylidene, C 1-C 6alkylidene, C 1-C 4alkylidene, C 1-C 3alkylidene, C 1-C 2alkylidene, C 1alkylidene.Representational alkylidene includes, but not limited to methylene, ethylidene, propylidene, sub-normal-butyl, ethenylidene, allylidene, sub-n-butene base, sub-propinyl, sub-positive butynyl etc.Alkylidene chain is connected on the remainder of molecule and is connected on group by singly-bound or two key by singly-bound or two key.Alkylidene chain is connected on the remainder of molecule and can be for by a carbon or any two carbon in this chain to the connection site on group.Unless separately clearly state in this manual, can be by describing below optionally substituted alkylene chain.
" alkoxyl " refers to formula-OR abase, wherein R afor alkyl as defined.Unless separately clearly state in this manual, can be by describing below optionally substituted alkoxy.
" alkoxyalkyl " refers to formula-R boR abase, wherein R afor alkyl as defined, and R wherein bfor alkylidene as defined.Unless separately clearly state in this manual, can be by describing below optionally substituted alkoxy alkyl.
" alkyl-carbonyl " refers to Shi – C (=O) R abase, wherein R afor as alkyl defined above.Unless separately clearly state in this manual, can be by describing below optionally substituted alkyl carbonyl.
" alkoxy carbonyl " refers to Shi – C (=O) OR abase, wherein R afor alkyl as defined.Unless separately clearly state in this manual, can be by describing below optionally substituted alkoxy carbonyl.
" alkyl amino " refers to formula-NHR abase or-NR ar abase, wherein each R abe independently all as alkyl defined above.Unless separately clearly state in this manual, can be by describing below optionally substituted alkyl amino.
" amide groups " refers to formula-N (H) C (=O) R abase, wherein R afor alkyl or aryl as defined herein.Unless separately clearly state in this manual, can be by describing below optionally substituted amide group.
" amidino groups alkyl " refers to formula-R b-C (=NH) NH 2base, wherein R bfor as alkylidene defined above.Unless separately clearly stated in this manual, can optionally replace amidino groups alkyl by following description.
" amidino groups alkyl-carbonyl " refers to formula-C (=O) R b-C (=NH) NH 2base, wherein R bfor as alkylidene defined above.Unless separately clearly stated in this manual, can optionally replace amidino groups alkyl-carbonyl by following description.
" aminoalkyl " refers to formula-R b-NR ar abase, wherein R bfor as alkylidene defined above, and each R abe all hydrogen or alkyl independently.
" alkylthio " refers to formula-SR abase, wherein R afor as alkyl defined above.Unless separately clearly stated in this manual, can optionally replace alkylthio.
" aryl " refers to the group of the hydrocarbon ring system of self-contained hydrogen, 6 to 30 carbon atoms and at least 1 aromatic ring.Aryl can be monocycle, bicyclo-, three ring or Fourth Ring ring systems, and it can comprise ring system that condense or bridge joint.Aryl comprises, but be not limited to, from the aryl of following hydrocarbon ring system: aceanthrylene, acenaphthylene, the luxuriant and rich with fragrance alkene of vinegar, anthracene, azulene (az μ lene), benzene, bend (chrysene), fluoranthene, fluorenes, asymmetric indacene (as-indacene), symmetrical indacene (s-indacene), indane, indenes, naphthalene, non-that alkene (phenalene), phenanthrene, seven days of the week alkene (pleiadene), pyrene and benzophenanthrene.Unless separately clearly stated in this manual, term " aryl " or prefix " virtue (ar) " (as in " aralkyl (aralkyl) ") are intended to comprise optionally substituted aryl.
" aralkyl " refers to formula-R b-R cbase, wherein R bfor as alkylidene chain defined above, and R cfor as one or more aryl defined above, for example, benzyl, diphenyl methyl, trityl etc.Unless separately clearly stated in this manual, aralkyl is optionally substituted.
" aryl carbonyl " refers to formula-C (=O) R cbase, wherein R cfor as one or more aryl defined above, for example, phenyl.Unless separately clearly stated in this manual, aryl carbonyl is optionally substituted.
" aryloxycarbonyl " refers to formula-C (=O) OR cbase, wherein R cfor as one or more aryl defined above, for example, phenyl.Unless separately clearly stated in this manual, aryloxycarbonyl is optionally substituted.
" aromatic alkyl carbonyl " refers to formula-C (=O) R b-R cbase, wherein R bfor as alkylidene chain defined above, and R cfor as one or more aryl defined above, for example, phenyl.Unless separately clearly stated in this manual, aromatic alkyl carbonyl is optionally substituted.
" aromatic alkoxy carbonyl " refers to Ji Shi-C (=O) OR b-R c, R wherein bfor as alkylidene chain defined above, and R cfor as one or more aryl defined above, for example, phenyl.Unless separately clearly stated in this manual, aromatic alkoxy carbonyl is optionally substituted.
" aryloxy group " refers to formula-OR cbase, wherein R cfor as one or more aryl defined above, for example, phenyl.Unless separately clearly stated in this manual, aryl carbonyl is optionally substituted.
" cycloalkyl " refers to stable, non-aromatic, monocycle or multi-ring carbocyclic ring, and it can comprise ring system that condense or bridge joint, and it is saturated or unsaturated, and by singly-bound, is connected on the remainder of molecule.Representational cycloalkyl includes, but not limited to have the cycloalkanes of 3 to 15 carbon atoms and 3 to 8 carbon atoms.Monocyclic cycloalkyl comprises, for example, and cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl and ring octyl group.Multi-ring base comprises, for example, and adamantyl, norborny, decahydro naphthyl and 7,7-dimethyl-bicyclo-[2.2.1] heptane base.Unless separately clearly stated in this manual, cycloalkyl is optionally substituted.
" cycloalkyl-alkyl " refers to formula-R br dbase, wherein R bfor as alkylidene chain defined above, and R dfor as cycloalkyl defined above.Unless separately clearly stated in this manual, cycloalkyl-alkyl is optionally substituted.
" naphthene base carbonyl " refers to formula-C (=O) R dbase, wherein R dfor as cycloalkyl defined above.Unless separately clearly stated in this manual, naphthene base carbonyl is optionally substituted.
" cyclo alkoxy carbonyl " refers to formula-C (=O) OR dbase, wherein R dfor as cycloalkyl defined above.Unless separately clearly stated in this manual, cyclo alkoxy carbonyl is optionally substituted.
" condense " and refer to any circulus being fused in existing circulus described herein.When the ring condensing is heterocyclic ring or heteroaryl ring, available nitrogen-atoms replaces any carbon atom that becomes the heterocyclic ring condensing or the heteroaryl ring condensing part in existing circulus.
" guanidine alkylation " refers to formula-R b-NHC (=NH) NH 2base, wherein R bfor as alkylidene defined above.Unless separately clearly stated in this manual, be optionally substituted by following description guanidine alkylation.
" guanidine alkylation carbonyl " refers to formula-C (=O) R b-NHC (=NH) NH 2base, wherein R bfor as alkylidene defined above.Unless separately clearly stated in this manual, be optionally substituted by following description guanidine alkylation carbonyl.
" halo " or " halogen " refers to bromo, chloro, fluoro or iodo.
" haloalkyl " refer to by as one or more halogens defined above replace as alkyl defined above, for example, trifluoromethyl, difluoromethyl, methyl fluoride, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-bis-fluoro ethyls, the bromo-2-fluoropropyl of 3-, 1,2-bis-bromoethyls etc.Unless separately clearly stated in this manual, haloalkyl is optionally substituted.
" perhalogeno " or " perfluor " refers to respectively the part that each hydrogen atom is wherein replaced by halogen atom or fluorine atom.
" heterocyclic radical ", " heterocycle " or " ring of heterocycle " refer to 3 yuan to 24 yuan stable non-aromatic cyclic groups, and it comprises 2 to 23 carbon atoms and is selected from 1 to 8 following hetero atom: nitrogen, oxygen, p and s.Unless separately clearly stated in this manual, heterocyclic radical can be monocycle, bicyclo-, three ring systems ring or Fourth Ring, and it can comprise ring system that condense or bridge joint; And the nitrogen in heterocyclic radical, carbon or sulphur atom are optionally oxidized; Described nitrogen-atoms is optionally quaternized; And heterocyclic radical can be for partially or completely saturated.The example of this type of heterocyclic radical comprises, but be not limited to, dioxolanyl (dioxolanyl), thienyl [1, 3] dithiane base, Decahydroisoquinolinpreparation base, imidazolinyl, imidazolidinyl, isothiazole alkyl (isothiazolidinyl), isoxazole alkyl, morpholinyl, octahydro indyl, octahydro isoindolyl, 2-oxygen piperazinyl, 2-Oxypertine base, 2-oxygen pyrrolidinyl, oxazolidinyl, piperidyl, piperazinyl, 4-piperidone base, pyrrolidinyl, pyrazolidinyl, quinoline cyclic group, thiazolidinyl (thiazolidinyl), tetrahydrofuran base, trithiane base (trithianyl), THP trtrahydropyranyl, thio-morpholinyl, thia morpholinyl (thiamorpholinyl), 1-oxygen-thio-morpholinyl, 1, 1-dioxy-thio-morpholinyl, 12-crown-4, 15-hat-5, 18-hat-6, 21-hat-7, azepine-18-hat-6, diaza-18-hat-6, azepine-21-hat-7 and diaza-21-hat-7.Unless separately clearly stated in this manual, heterocyclic group is optionally substituted.
" heteroaryl " refers to 5 to 14 yuan of ring system bases that comprise hydrogen atom, 1 to 13 carbon atom, 1 to 6 hetero atom that is selected from nitrogen, oxygen, p and s and at least 1 aromatic ring.For the purposes of the present invention, heteroaryl can be monocycle, bicyclo-, three ring systems ring or Fourth Ring, and it can comprise ring system that condense or bridge joint; And the nitrogen in heteroaryl, carbon or sulphur atom are optionally oxidized; Described nitrogen-atoms is optionally quaternized.Example includes but not limited to, azepines base, acridinyl, benzimidazolyl, benzothiazolyl, benzindole base, benzo dioxolyl (benzodioxolyl), benzofuranyl, benzoxazolyl, benzothiazolyl, diazosulfide, benzo [b] [Isosorbide-5-Nitrae] Dioxepane base (benzo[b] [Isosorbide-5-Nitrae] dioxepinyl), Isosorbide-5-Nitrae-benzodioxan base (Isosorbide-5-Nitrae-benzodioxanyl), benzo naphtho-furan base, benzoxazolyl, benzo dioxolyl (benzodioxolyl), benzodioxan base (benzodioxinyl), benzopyranyl, .alpha.-5:6-benzopyran ketone group, benzofuranyl, benzofuran ketone group, benzothienyl (benzo thio-phenyl), benzotriazole base, benzo [4,6] imidazo [1,2-a] pyridine radicals, carbazyl, cinnolines base (cinnolinyl), dibenzofuran group, dibenzo thio-phenyl, furyl, furanonyl, isothiazolyl, imidazole radicals, indazolyl, indyl, indazolyl, isoindolyl, indolinyl, iso-dihydro-indole-group, isoquinolyl, indolizine base (indolizinyl), isoxazolyl, naphthyridinyl (naphthyridinyl), oxadiazolyl (oxadiazolyl), 2-oxygen azepines base, oxazolyl, epoxy ethyl, 1-pyridine oxide base, 1-is oxidized pyrimidine radicals, 1-is oxidized pyrazinyl, 1-is oxidized pyridazinyl, 1-phenyl-1H-pyrrole radicals, phenazinyl, phenothiazinyl, phenoxazine group (phenoxazinyl), phthalazinyl, pteridyl, purine radicals, pyrrole radicals, pyrazolyl, pyridine radicals, pyrazinyl, pyrimidine radicals, pyridazinyl, quinazolyl, quinoxalinyl, quinolyl, quinoline cyclic group, isoquinolyl, tetrahydric quinoline group, thiazolyl, thiadiazoles, triazolyl, tetrazole radical, triazine radical and thio-phenyl (that is, thienyl).Unless separately clearly stated in this manual, heteroaryl is optionally substituted.
All groups can be replacement or unsubstituted above.Refer to can be by above any group (for term " replacement " as used herein, alkyl, alkylidene, alkoxyl, alkoxyalkyl, alkyl-carbonyl, alkoxy carbonyl, alkyl amino, amide groups, amidino groups alkyl, amidino groups alkyl-carbonyl, aminoalkyl, aryl, aralkyl, aryl carbonyl, aryloxycarbonyl, aromatic alkyl carbonyl, aromatic alkoxy carbonyl, aryloxy group, cycloalkyl, cycloalkyl-alkyl, naphthene base carbonyl, cycloalkyl alkyl carbonyl, cyclo alkoxy carbonyl, guanidine alkylation, guanidine alkylation carbonyl, haloalkyl, heterocyclic radical and/or heteroaryl) further functionalization, the key that wherein at least 1 hydrogen atom is connected in non-hydrogen atom substituent group replaces.Unless there is in this manual clearly statement, substituent group can comprise and is selected from following one or more substituent groups: oxo (oxo) ,-CO 2h, nitrile, nitro ,-CONH 2, hydroxyl, sulfur oxygen (thiooxy), alkyl, alkylidene, alkoxyl, alkoxyalkyl, alkyl-carbonyl, alkoxy carbonyl, aryl, aralkyl, aryl carbonyl, aryloxycarbonyl, aromatic alkyl carbonyl, aromatic alkoxy carbonyl, aryloxy group, cycloalkyl, cycloalkyl-alkyl, naphthene base carbonyl, cycloalkyl alkyl carbonyl, cyclo alkoxy carbonyl, heterocyclic radical, heteroaryl, dialkylamine, arylamine, alkylarylamine, diaryl amine, N-oxide, acid imide and enamine; The group of silicon atoms, as trialkylsilkl, di alkylaryl silicyl, alkyl diaryl silicyl, diarye silyl, perfluoroalkyl or perfluoro alkoxy, for example, trifluoromethyl or trifluoromethoxy." replacement " also refers to above any group, wherein one or more hydrogen atoms are connected to such as the oxygen in oxo, carbonyl, carboxyl and ester group and for example, such as the higher-order key on the hetero atom of the nitrogen in imines, oxime, hydrazone and nitrile group (, two keys or triple bond) and replace.For example, " replacement " comprises above any group, and wherein one or more hydrogen atoms are replaced by following radicals :-NR gc (=O) NR gr h,-NR gc (=O) OR h,-NR gsO 2r h,-OC (=O) NR gr h,-OR g,-SR g,-SOR g,-SO 2r g,-OSO 2r g,-SO 2oR g,=NSO 2r gwith-SO 2nR gr h." replacement " also refers to above any group, and wherein one or more hydrogen atoms are replaced by following radicals :-C (=O) R g,-C (=O) OR g,-CH 2sO 2r g,-CH 2sO 2nR gr h,-SH ,-SR gor-SSR g.In aforementioned group, R gand R hfor identical or different, and be independently: hydrogen, alkyl, alkoxyl, alkyl amino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkyl-alkyl, haloalkyl, heterocyclic radical, N-heterocyclic radical, heterocyclic radical alkyl, heteroaryl, N-heteroaryl and/or heteroaryl alkyl.In addition, each aforementioned substituent group also can optionally be replaced by one or more above-mentioned substituent groups.In addition, above any group can be substituted to comprise one or more inner oxygen or sulphur atom.For example, alkyl can be replaced to form ether or polyether-based by one or more inner oxygen atoms.Similarly, alkyl can be replaced to form thioether, disulphide etc. by one or more inner sulphur atoms.Amide groups part can be reached 2 halogen atoms and be replaced, and other above-mentioned groups can be replaced by one or more halo atoms.Any group also can be replaced by amino, alkyl monosubstituted amino, guanidine radicals or amidino groups (amidynyl) above.The optional substituent group of any group also comprises aryl phosphoryl above, for example-R ap (Ar) 3, R wherein afor alkylidene, and A rfor aryl moiety, phenyl for example.
Term " antisense scant polymer " or " antisense compounds " are used interchangeably, and refer to subunit sequence, they have the base being carried on the skeleton subunit being comprised of ribose or other pentoses or morpholino group separately, and wherein said skeleton group connects by the connection between subunit, it allows the base in described compound to form nucleic acid by the target sequence hybridization in Watson-Crick base pairing and nucleic acid (being generally RNA): the oligomer heteroduplex in described target sequence.Oligomer can form accurate complementation or be similar to complementary sequence with target sequence.The mRNA that this type of antisense scant polymer is designed to stop or suppress to contain target sequence translates, and can be said to be the sequence that " sensing " hybridizes with it.
" morpholino oligomer " or " PMO " refer to have support can be with hydrogen bonded the polymerizable molecular to the skeleton of the base on typical polynucleotide, wherein said polymer lacks pentose skeleton part, and more specifically, the ribose skeleton of skeleton for connecting by phosphodiester bond (it typically is the phosphodiester bond of nucleotide and nucleoside, but contain by the ring nitrogen of the combination of described ring nitrogen) described in it.Exemplary " morpholino " oligomer comprises the morpholino subunit structure linking together by (sulfo-) phosphoramidate or (sulfo-) di(2-ethylhexyl)phosphate amido link, its morpholino nitrogen by a subunit is attached on the outer carbon of 5' ring of contiguous subunit, and each subunit comprises and can effectively be attached to the purine in base or pyrimidine bases mating section in polynucleotide by base specific hydrogen bonded.For example, No. 5698685, No. 5217866, No. 5142047, No. 5034506, No. 5166315, No. 5185444, No. 5521063, No. 5506337 United States Patent (USP) and pending trial U.S. Patent application 12/271036,12/271040 and WO/2009/064471 PCT open in, describe morpholino oligomer (comprising antisense scant polymer) in detail, all these files are all incorporated to herein by reference.Representational PMOs comprises the PMOs that is wherein connected to connection (A1) between subunit.
" PMO+ " refers to (1-piperazine) the phosphinylidene oxygen ((1-piperazino) phosphinylideneoxy) that comprises any number being previously described, the di(2-ethylhexyl)phosphate amide morpholino oligomer that (1-(4-(ω-guanidine radicals-silane alcohol base))-piperazine) phosphinylidene oxygen connects (A2 and A3) (is for example shown in, the open WO/2008/036127 of PCT, it is all incorporated to herein by reference).
" PMO-X " refers at least 1 (B) connection or at least one disclosed end modified di(2-ethylhexyl)phosphate amide morpholino oligomer of comprising disclosed herein.
" phosphamide " base comprises the phosphorus with the oxygen atom of 3 connections and the nitrogen-atoms of 1 connection, and the phosphorus of " di(2-ethylhexyl)phosphate amide " base (for example seeing Fig. 1 D-E) oxygen atom that comprises 2 connections of tool and the nitrogen-atoms of 2 connections.The in this article with No. 61/349783 with the neutral of the oligomer of describing in No. 11/801885 pending trial U.S. Patent application or through in being connected between the subunit of modifying, 1 nitrogen always side joint to (pendant to) skeletal chain.In di(2-ethylhexyl)phosphate amide connects, second nitrogen is generally the ring nitrogen in morpholino circulus.
" thioate " or " D2EHDTPA diamides ester " connects and to be respectively phosphoramidate or di(2-ethylhexyl)phosphate carboxylic acid amide esters connects, and 1 oxygen atom is wherein generally side joint to the oxygen on skeleton, by sulfur, is replaced.
" between subunit, connect " and refer to the connection that connects 2 morpholino subunits, for example structure (I).
As used herein, " charged ", " uncharged ", " cation " and " anion " refer in approximate neutral pH, for example the main state of approximately 6 to 8 times chemical parts.For example, this term can refer in physiological pH, i.e. the main state of approximately 7.4 times chemical parts.
" low alkyl group " refers to the alkyl of 1 to 6 carbon atom, as take methyl, ethyl, normal-butyl, isobutyl group, the tert-butyl group, isopentyl, n-pentyl and isopentyl as example.In certain embodiments, " low alkyl group " has 1 to 4 carbon atom.In other embodiments, " low alkyl group " has 1 to 2 carbon atom; Be methyl or ethyl.Similarly, " low-grade alkenyl " refers to 2 to 6, the preferred thiazolinyl of 3 or 4 carbon atoms, as to take acrylic and cyclobutenyl be example.
" non-interference " substituent group produces the adversely substituent group of impact for not being attached to the ability that its pre-determined target puts on to antisense scant polymer as described in this article.This type of substituent group comprises little and/or relatively non-polar group, as methyl, ethyl, methoxyl group, ethyoxyl or fluoro base.
If oligomer to be greater than 37 ℃, to be greater than 45 ℃, preferably at least 50 ℃, and is generally 60 ℃-80 ℃ or higher T under physiological condition mwith target hybridization, oligonucleotide or antisense scant polymer and target polynucleotide " specific hybrid "." the T of term oligomer m" be oligomer 50% with the complementary polynucleotide temperature in when hybridization.T munder standard conditions, in normal saline, record, as for example, Miyada et al., described in Methods Enzymol.154:94-107 (1987).Antisense scant polymer can occur in this type of hybridization and target sequence " is similar to " or " substantially " complementation, and accurately complementary.
When there is hybridization with anti-parallel arrangement between two strand polynucleotide, polynucleotide are described as each other " complementary ".According to the base pairing rules of common acceptance, based on expection in relative chain, form each other the base ratio of hydrogen bond, can quantize complementary (1 degree of polynucleotide and another polynucleotide complementation).
If its sequence of polynucleotide be can specific binding to or can be under physiological condition and the First ray of the second polynucleotide sequence specific hybrid, First ray is " antisense sequences " for the second sequence.
Term " targeting sequence " be in oligonucleotide analogs with rna gene group in the sequence of target complement sequence (looking like for substantially complementary in addition).The full sequence of analog compounds or only its part can with target complement sequence.For example, in having the analog of 20 bases, only 12-14 may be targeting sequence.Conventionally, the continuous base composition of targeting sequence in analog, but alternatively by discrete sequence, formed,, when these discrete sequences are put together, for example, from the other end of analog, formed the sequence of crossing over target sequence.
" skeleton " of oligonucleotide analogs (for example, uncharged oligonucleotide analogs) refers to the structure that supports base pairing part; For example, for morpholino oligomer, as described in this article, " skeleton " for example comprises, by connecting the morpholino circulus that (, phosphorous connection) connects between subunit." uncharged skeleton substantially " refers to the skeleton of oligonucleotide analogs, is wherein less than between 50% subunit and is connected to and approaches under neutral pH as charged.For example, uncharged skeleton can comprise and be less than 50%, is less than 40%, is less than 30%, is less than 20%, is less than 10%, is less than between 5% or even 0% subunit and connects substantially, and it is approaching under neutral pH as charged.In some embodiments, substantially uncharged skeleton, every 4 uncharged (under physiological pH) connect comprise at the most between 1 charged (under physiological pH) subunit, connect, every 8 comprise at the most 1 or every 16 and comprise 1 uncharged connection at the most.In some embodiments, nucleic acid analog described herein is entirely uncharged.
When hybridization occurs with anti-parallel arrangement, by target and targeting sequence description, be " complementation " each other.Targeting sequence can have target sequence " approximate " or " substantially " complementarity, and still with the object of the method for current description, works, and is still " complementary ".Preferably, the oligonucleotide analogs compound and the target sequence that in the method for current description, adopt, every 10 nucleotide have 1 mispairing at the most, and preferably have 1 mispairing at the most in 20.Or, antisense scant polymer used with as the exemplary targeting sequence of appointment herein there is at least 80%, at least 90% sequence homology or at least 95% sequence homology.For complementation is attached on RNA target, and as described below, guanine base can with cytosine or uracil RNA base complementrity.
" heteroduplex " refers to the two strands forming between the complementary portion of oligonucleotide analogs and target RNA." heteroduplex of resistance to nuclease " refers to by antisense scant polymer being attached to the heteroduplex that its complementary target is put on formation, thereby this heteroduplex tolerates substantially such as in the cell of RNA enzyme H and the vivo degradation of extracellular nuclease, and wherein nuclease can cut double-stranded RNA/RNA or RNA/DNA complex.
When medicament can be when entering cell except the passive mechanism diffusing through cell membrane, this medicament " is initiatively absorbed by mammalian cell ".This medicament for example, can transport by " Active transport ", refer to by for example ATP dependency transport mechanism and transport medicament through mammalian cell membrane, or by " facilitation transportation ", transport, refer to by medicament being attached to the medicament transportation that can promote subsequently institute's combination and transport antisense agents through cell membrane through the transport mechanism on the transport protein of film.
Term " regulates and expresses " and/or " antisense is active " refers to antisense scant polymer by the expression of RNA interfering or translate the ability that increases or more generally reduce the expression of given albumen.In the situation of the protein expression reducing, antisense scant polymer can directly stop the expression of given gene, or promotes from the accelerated decomposition of the next RNA of that genetic transcription.Morpholino oligomer it is believed that by last (check in space) mechanism and works as described in this article.The preferred antisense target that oligomer is checked in space comprises the region of ATG initiation codon subregion, splicing site, next-door neighbour's splicing site and the 5'-untranslated region of mRNA, although use morpholino oligomer successfully using other region as target.
" aminoacid subunit " is generally a-amino acid residue (CO-CHR-NH-); But can be also β-or other amino acid residues (for example ,-CO-CH 2cHR-NH-), wherein R is amino acid side chain.
Term " naturally occurring aminoacid " refers to the aminoacid in the protein that is present in occurring in nature discovery.Term " alpha-non-natural amino acid " refers to those aminoacid in the protein that is not present in occurring in nature discovery; Example comprises Beta-alanine (β-Ala) and 6-aminocaprolc acid (Ahx).
" effective dose " or " treatment effective dose " refers to the amount of the antisense scant polymer that is administered to mammalian object, no matter be single dose or as the parts of a series of dosage, it effectively produces the therapeutic effect of expectation by suppressing the translation of selected target nucleic acid sequence conventionally.
" treatment " of individual (for example mammal, as people) or cell is for attempting to change any type intervention of the natural process of this individuality or cell.Treatment includes, but not limited to give pharmaceutical composition, and can be used as prevention, or carries out after contacting in pathology event or with cause of disease medicament.
II. carrier peptides
A. the performance of carrier peptides
As mentioned above, the disclosure relates to the conjugate of carrier peptides and nucleic acid analog.This carrier peptides can effectively increase the Premeabilisation of cells of nucleic acid analog conventionally.In addition, applicant is surprised to find glycine (G) or proline (P) subunit (is for example included between nucleic acid analog and the residue of carrier peptides, c-terminus or aminoterminal in carrier peptides) can reduce the toxicity of this conjugate, the conjugate simultaneously connecting with respect to the difference having between carrier peptides and nucleic acid analog, effect remains unchanged or is improved.Therefore, when conjugate of the present disclosure has better treatment window than other peptide-oligomer conjugates and be more promising drug candidates.
Except the toxicity reducing, the existence of the glycine between nucleic acid analog and carrier peptides or proline subunit also it is believed that other advantage can be provided.For example, glycine is cheap, and it is upper to be easily incorporated into nucleic acid analog (or optional linking arm), and does not have any probability of racemization.Similarly, proline is easily combined and racemization does not occur, and also to provide be not the carrier peptides of spiralization thing.The hydrophobicity of proline can also be given about carrier peptides and interactional some advantage of cytolipin bilayer, and the carrier peptides that comprises a plurality of proline (for example in certain embodiments) can resist G-tetra-chain formation.Finally, in certain embodiments, when proline is partly close to arginine subunit, this proline is partly given conjugate metabolic, because this Arg-Pro amido link can not be by conventional endopeptidase enzymatic lysis.
As mentioned above, compare other known conjugates, comprise the conjugate that is connected to the carrier peptides on nucleic acid analog by glycine or proline subunit and there is lower toxicity and similar effect.Compare other conjugates, the support the application's who carries out experiment shows, use the toxicity much lower (for example seeing injury of kidney mark (KIM) and blood urea nitrogen (BUN) data described in embodiment 30) of the marker for nephrotoxicity of conjugate of the present disclosure.Although do not wish to be subject to theoretical constraint, inventor believes that the toxicity of the minimizing of disclosure conjugate may relate in being connected to the peptide moiety of nucleic acid analog (for example, c-terminus) and not exist alpha-non-natural amino acid as aminocaproic acid or Beta-alanine.Because these alpha-non-natural amino acids are cut in vivo, it is believed that the TC that does not cut peptide can accumulate and cause poisonous effect.
Glycine or proline part can be positioned at aminoterminal or the c-terminus of carrier peptides, and in some cases, carrier peptides can be directly connected on nucleic acid analog by glycine or proline subunit, or carrier peptides can be connected on nucleic acid analog by optional linking arm.
In one embodiment, the disclosure relates to conjugate, and it comprises:
(a) carrier peptides, comprises aminoacid subunit; With
(b) nucleic acid analog, comprises uncharged skeleton substantially and for sequence-specific, is attached to the targeting base sequence of target nucleic acid;
Wherein:
Two or more aminoacid subunits are positively charged aminoacid, the glycine that described carrier peptides comprises the c-terminus that is positioned at described carrier peptides (G) or proline (P) subunit, and described carrier peptides is covalently bound to nucleic acid analog.In some embodiments, no more than 7 continuous amino acid subunits are arginine, and for example 6 or continuous amino acid subunit are still less arginine.In some embodiments, described carrier peptides comprises the glycine subunit that is positioned at c-terminus.In other embodiments, described carrier peptides comprises the proline subunit that is positioned at c-terminus.Further in other embodiments, described carrier peptides comprises single glycine or the proline (that is, not comprising glycine or proline dimer or the trimer etc. that are positioned at c-terminus) that is positioned at c-terminus.
In certain embodiments, in the time of on being attached to the antisense scant polymer with uncharged skeleton substantially, with respect to the non-antisense scant polymer of puting together form, carrier peptides can effectively promote that antisense scant polymer is attached to its target sequence, as proved by following:
(i), when antisense scant polymer being attached to the translation initiation codon of the albumen that can effectively stop coding on its target sequence, with respect to the expression being provided by the non-oligomer of puting together, the expression of the albumen of coding declines, or
(ii), when antisense scant polymer is attached to its target sequence and can effectively stops the aberrant splicing site in Pre-mRNA (encoding said proteins when it is correctly spliced), with respect to the expression being provided by the non-oligomer of puting together, the expression of the albumen of coding increases.Below further described the analysis of applicable these effects of measurement.In one embodiment, provide the puting together of peptide cell free translation this activity in analyzing, as described in this article.In some embodiments, activity has been enhanced at least 2 times, at least 5 times or at least 10 times.
Alternatively or in addition, with respect to the non-analog of puting together form, carrier peptides can effectively promote nucleic acid analog to be transported in cell.In certain embodiments, transportation has been increased at least 2 times, at least 2 times, at least 5 times or at least 10 times.
In other embodiments, with respect to the conjugate that comprises the carrier peptides that lacks end glycine or proline subunit, carrier peptides can effectively reduce the toxicity (that is, increasing maximum tolerated dose) of conjugate.In certain embodiments, toxicity has been reduced at least 2 times, at least 2 times, at least 5 times or at least 10 times.
Other advantages of peptide transport section can be stablized the double-stranded ability between antisense scant polymer and its target nucleic acid sequence for its expection.Although do not wish to be subject to theoretical constraint, this is stablized double-stranded ability and may be caused by the electrostatic interaction between positively charged transport section and electronegative nucleic acid.
The length of carrier peptides is not subject to specific limited, and there are differences in different embodiments.In some embodiments, carrier peptides comprises 4 to 40 aminoacid subunits.In other embodiments, carrier peptides comprises 6 to 30,6 to 20,8 to 25 or 10 to 20 aminoacid subunits.In some embodiments, carrier peptides is straight chain, and in other embodiments, it is to have side chain.
In some embodiments, carrier peptides is rich in positively charged aminoacid subunit, for example arginine subunit.If at least 10% aminoacid subunit is with positive electricity, carrier peptides " is rich in " positively charged aminoacid.For example, in some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% aminoacid subunit is with positive electricity.Even in other embodiments, all aminoacid subunits except glycine or proline subunit are all with positive electricity.In other embodiments, all positively charged aminoacid subunits are all arginine.
In other embodiments, in carrier peptides, the number of positively charged aminoacid subunit is 1 to 20, for example 1 to 10 or 1 to 6.In certain embodiments, in carrier peptides, positively charged amino acid whose number is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20.
Positively charged aminoacid can be naturally occurring, that non-natural exists, synthetic, that modify or naturally occurring amino acid whose analog.For example, can specificity design with the aminoacid of the modification of clean positive charge for the present invention, as described in more detail below.Well known to being modified to of amino acid whose number of different types.In certain embodiments, positively charged aminoacid is histidine (H), lysine (K) or arginine (R).In other embodiments, carrier peptides only comprises natural amino acid subunit (that is, not comprising alpha-non-natural amino acid).In other embodiments, can for example by acetyl group, benzoyl or stearyl part, to end amino acid, add cap, for example, at N-end.
Any number, combination and/or the sequence of H, K and/or R can be present in carrier peptides.In some embodiments, all aminoacid subunits except carboxyl terminal glycine or proline are all positively charged aminoacid.In other embodiments, at least 1 positively charged aminoacid is arginine.For example, in some embodiments, all positively charged aminoacid is all arginine, and even in other embodiments, and carrier peptides is comprised of arginine and carboxyl terminal glycine or proline.Further, in other embodiments, carrier peptides comprises no more than 7 continuous arginine, for example no more than 6 continuous arginine.
The positively charged aminoacid of other types is also considered.For example, in certain embodiments, at least 1 positively charged aminoacid is arginine analog.For example, this arginine analog can be and comprises R an=C (NH 2) R bthe cationic a-amino acid of side-chain structure, wherein R afor H or R c; R bfor R c, NH 2, NHR or N (R c) 2, R wherein cfor low alkyl group or low-grade alkenyl, and optionally comprise oxygen or nitrogen, or R aand R bcan form together ring; And wherein said side chain passes through R aor R bbe connected on aminoacid.Carrier peptides can comprise these arginine analogs of any number.
Positively charged aminoacid can occur in any sequence in carrier peptides.For example, in some embodiments, positively charged aminoacid can be that replace or continuous.For example, carrier peptides can comprise sequence (R d) m, R wherein dwhen occurring, be all positively charged aminoacid independently, and m is 2 to 12,2 to 10,2 to 8 or 2 to 6 integer at every turn.For example, in certain embodiments, R dfor arginine, and carrier peptides comprises and is selected from following sequence: (R) 4, (R) 5, (R) 6, (R) 7(R) 8, or be selected from: (R) 4, (R) 5, (R) 6(R) 7, for example, in specific embodiment, carrier peptides comprises sequence (R) 6, for example (R) 6g or (R) 6p.
In other embodiments, carrier peptides is by sequence (R d) mform with carboxyl terminal glycine or proline, wherein R dwhen occurring, be all positively charged aminoacid independently, and m is 2 to 12,2 to 10,2 to 8 or 2 to 6 integer at every turn.In certain embodiments, R dwhen occurring, be all arginine, histidine or lysine independently at every turn.For example, in certain embodiments, R dfor arginine, and carrier peptides forms by being selected from following sequence: (R) 4, (R) 5, (R) 6, (R) 7(R) 8, and carboxyl terminal glycine or proline.For example, in specific embodiment, carrier peptides is by sequence (R) 6g or (R) 6p forms.
In some other embodiments, carrier peptides can comprise one or more hydrophobic amino acid subunits, described hydrophobic amino acid subunit comprises alkyl replacement or unsubstituted, thiazolinyl, alkynyl, aryl or aralkyl side chain, and every 6 carbon atoms of wherein said alkyl, thiazolinyl and alkynyl side chain comprise 1 hetero atom at the most.In some embodiments, hydrophobic amino acid is phenylalanine (F).For example, carrier peptides can comprise two or more continuous hydrophobic amino acids as phenylalanine (F), for example 2 continuous phenylalanine parts.Hydrophobic amino acid can be arranged in any point of carrier peptides sequence.
In other embodiments, carrier peptides comprises sequence [(R dy br d) x(R dr dy b) y] zor [(R dr dy b) y(R dy br d) x] z, R wherein dwhen occurring, be all positively charged aminoacid independently, x and y are 0 or 1 independently at every turn when occurring at every turn, and condition is that x+y is 1 or 2, and z is 1,2,3,4,5 or 6, and Y bfor:
-C(O)-(CHR e) n-NH-
(Yb)
Wherein n is 2 to 7, and each R ewhen occurring, be all hydrogen or methyl independently at every turn.In some of these embodiments, R dwhen occurring, be all arginine, histidine or lysine independently at every turn.In other embodiments, each R dit is all arginine.In other embodiments, n is 5, and Y bfor aminocaproic acid part.In other embodiments, n is 2, and Y bfor Beta-alanine part.In other embodiments, R efor hydrogen.
In aforementioned some embodiment, x is that 1, y is 0, and carrier peptides comprises sequence (R dy br d) z.In other embodiments, n is 5, and Y bfor aminocaproic acid part.In other embodiments, n is 2, and Y bfor Beta-alanine part.Further in other embodiments, Re is hydrogen.
Further, in aforementioned other embodiments, x is that 0, y is 1, and carrier peptides comprises sequence (R dr dy b) z.In other embodiments, n is 5, and Y bfor aminocaproic acid part.In other embodiments, n is 2, and Y bfor Beta-alanine part.Further in other embodiments, R efor hydrogen.
In other embodiments, carrier peptides comprises sequence (R dy b) p, R wherein dand Y bas defined above, and p be 2 to 8 integer.In other embodiments, each R dit is all arginine.In other embodiments, n is 5, and Y bfor aminocaproic acid part.In other embodiments, n is 2, and Y bfor Beta-alanine part.Further in other embodiments, Re is hydrogen.
In other embodiments, carrier peptides comprises sequence ILFQY.Except any other sequence disclosed herein, described peptide can also comprise ILFQY sequence.For example carrier peptides can comprise ILFQY and [(R dy br d) x(R dr dy b) y] z, [(R dr dy b) y(R dy br d) x] z, (R dy b) por their combination, wherein R d, x, y and Y bas defined above.Described [(R dy br d) x(R dr dy b) y] z, [(R dr dy b) y(R dy br d) x] zor (R dy b) psequence can be positioned at aminoterminal, c-terminus or this two ends of ILFQY sequence.In certain embodiments, x is that 1, y is 0, and carrier peptides comprises by optional Z linking arm and is connected to (the R in ILFQY sequence dy br d) z.
In other relevant embodiments, carrier peptides comprises sequence ILFQ, IWFQ or ILIQ.Other embodiments comprise the carrier peptides that comprises sequence PPMWS, PPMWT, PPMFS or PPMYS.Except any other sequence described herein, for example, except sequence [(R dy br d) x(R dr dy b) y] z, [(R dr dy b) y(R dy br d) x] zor (R dy b) p, carrier peptides can also comprise these sequences, wherein R d, x, y and Y bas defined above.
Some embodiments of carrier peptides comprise the modification to naturally occurring aminoacid subunit, for example, can modify amino terminal or carboxyl terminal aminoacid subunit.This type of modification comprises with hydrophobic group and adds cap to free amine group or free carboxy.For example, can to aminoterminal, add cap by acetyl group, benzoyl or stearyl part.For example, in table 1, any peptide sequence can have this type of modification, even clearly describe in table.In these embodiments, the aminoterminal of carrier peptides can be as described below:
Figure BDA0000447333530000211
Further in other embodiments, carrier peptides comprises at least 1 in alanine, aspartic acid, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.
In embodiments more disclosed herein, carrier peptides is comprised of the sequence annotating and carboxyl terminal glycine or proline subunit.
In some embodiments, carrier peptides be can't help following sequence and is formed (amino terminal is to carboxyl terminal): R 6g, R 7g, R 8g, R 5gR 4g, R 5f 2r 4g, Tat-G, rTat-G, (RXR 2g 2) 2or (RXR 3x) 2g.In other embodiments, carrier peptides be can't help R 8g, R 9g or R 9f 2g forms.Further in other embodiments, carrier peptides be can't help following sequence and is formed: Tat-G, rTat-G, R 9f 2g, R 5f 2r 4, R 4g, R 5g, R 6g, R 7g, R 8g, R 9g, (RXR) 4g, (RXR) 5g, (RXRRBR) 2g, (RAR) 4f 2or (RGR) 4f 2.In other embodiments, carrier peptides be can't help " wearing film peptide Penetratin " or " R 6pen " form.
In yet another aspect, the disclosure provides peptide-nucleic acid analog conjugate, and it comprises:
Nucleic acid analog, it has uncharged skeleton and targeting base sequence substantially, and
Covalently bound peptide to nucleic acid analog, it comprises carboxyl terminal glycine or proline subunit, and forms by being selected from 8 to 16 following other other subunits: R dsubunit, Y subunit and optional Z subunit, comprise at least 8 R dsubunit, at least 2 Y subunits and at the most 3 Z subunits, wherein the described subunit of >50% is R dsubunit, and wherein:
(a) each R dsubunit all represents arginine or arginine analog independently, and described arginine analog is for comprising R an=C (NH 2) R bthe cationic a-amino acid of side-chain structure, wherein R afor H or R c; R bfor R c, NH 2, NHR or N (R c) 2, R wherein cfor low alkyl group or low-grade alkenyl, and optionally comprise oxygen or nitrogen, or R aand R bcan form together ring; And wherein said side chain passes through R aor R bbe connected on aminoacid;
(b) at least 2 Y subunits are Y aor Y b, wherein:
(i) each Y aall independently for thering is the neutral a-amino acid subunit independently selected from the side chain of following group: alkyl replacement or unsubstituted, thiazolinyl, alkynyl, aryl and aralkyl, wherein said side chain, be elected to when the alkyl replacing, thiazolinyl and alkynyl, every 2, preferably every 4, and more preferably every 6 carbon atoms comprise 1 hetero atom at the most, and wherein said subunit is continuous or is positioned at the side of linking arm part, and
(ii) Y bfor:
-C(O)-(CHR e) n-NH-
(Yb)
Wherein n is 2 to 7, and each R ewhen occurring, be all hydrogen or methyl independently at every turn; And
(c) Z represents to be selected from following aminoacid subunit: alanine, aspartic acid, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, threonine and have the aminoacid of side chain, described side is linked as natural 1 carbon or the 2 carbon homologues that have side chain, is not included in electronegative side chain under physiological pH (for example carboxylate side chain).In some embodiments, side chain is neutral.In other embodiments, Z side chain is the natural amino acid whose side chain that exists.Z subunit optional in some embodiments is selected from: alanine, glycine, methionine, serine and threonine.Carrier peptides can comprise 0,1,2 or 3 Z subunits, and comprises in some embodiments 2 Z subunits at the most.
In the embodiment of selecting, carrier peptides has type Y ajust in time 2 Y subunits, it is side continuous or that be positioned at cysteine subunit.In some embodiments, 2 Y asubunit is continuous.In other embodiments, Y athe side chain of subunit comprises natural exist amino acid whose side chain and its 1 carbon or 2 carbon homologues, is not included in side chain charged under physiological pH.Other possible side chains are the natural amino acid whose side chain that exists.In other embodiments, side chain is aryl or aralkyl side chain; For example, each Y acan be independently selected from: phenylalanine, tyrosine, tryptophan, leucine, isoleucine and valine.
In the embodiment of selecting, each Y aall independently be selected from phenylalanine and tyrosine; In other embodiments, each Y ait is all phenylalanine.This comprises, for example, and the conjugate being formed by arginine subunit, phenylalanine subunit, glycine or proline subunit, optional linking arm part and nucleic acid analog.A kind of this type of conjugate comprises having formula Arg 9phe 2the peptide of aa, wherein aa is glycine or proline.
Aforementioned bearer peptide can also comprise ILFQY, ILFQ, IWFQ or ILIQ.Other embodiments comprise the aforementioned bearer peptide that comprises sequence PPMWS, PPMWT, PPMFS or PPMYS.
Peptide-oligomer conjugate of the present invention is more more effective than the non-oligomer of puting together in difference in functionality, comprising: in protein expression system, suppress the expression of said target mrna, comprise cell free translation system; Suppress the splicing of target Pre-mRNA; With by controlling viral nucleic acid copies or mRNA transcribes cis acting element as target, to suppress copying of virus.
The conjugate that also comprises within the scope of the present invention other pharmacological agent (that is, not being nucleic acid analog) and carrier peptides.Specifically, some embodiments provide conjugate, and it comprises:
(a) carrier peptides that comprises aminoacid subunit; With
(b) pharmacological agent;
Wherein:
Two or more aminoacid subunit is positively charged aminoacid, the glycine that described carrier peptides comprises the c-terminus that is positioned at described carrier peptides (G) or proline (P) subunit, and described carrier peptides is covalently bound to pharmacological agent.Carrier peptides in these embodiments can be any carrier peptides described herein.Also provide by pharmacological agent is conjugated to and in carrier peptides, sent its method.
Pharmacological agent to be delivered can be bioactivator, for example therapeutic agent or diagnostic agent, although its can for for detection of compound, as fluorescent chemicals.Bioactivator comprises and is selected from following medicine: biomolecule, for example peptide, protein, saccharide or nucleic acid, especially antisense oligonucleotide, or " micromolecule " organic or inorganic compound." micromolecule " compound can be defined as to organic and inorganic or organo-metallic compound more widely, it is not biomolecule as above.Conventionally, this compounds has the molecular weight that is less than 1000, or, in one embodiment, be less than 500.
In one embodiment, pharmacological agent to be delivered does not comprise single amino acids, dipeptides or tripeptides.In another embodiment, it does not comprise short oligopeptide; That is, there is the oligopeptide that is less than 6 aminoacid subunits.In other embodiments, it does not comprise longer oligopeptide; That is the oligopeptide, with 7 to 20 aminoacid subunits.Further in other embodiments, it does not comprise having oligopeptide or the protein that is greater than 20 aminoacid subunits.
With respect to the non-pharmacological agent of puting together form and/or having less toxicity, with respect to the pharmacological agent being conjugated on the corresponding peptide that lacks glycine or proline subunit, described carrier peptides can effectively promote pharmacological agent to be transported in cell.In some embodiments, transportation has been provided at least 2 times, at least 5 times or at least 10 times.In other embodiments, toxicity has been reduced (that is, maximum tolerated dose has been reduced) at least 2 times, at least 5 times or at least 10 times.
B. peptide linking arm
Can use several different methods that carrier peptides is for example connected to, on medicament to be delivered (, nucleic acid analog, pharmacological agent etc.) by those skilled in the art.In some embodiments, carrier peptides is directly connected on nucleic acid analog, and linking arm in the middle of not using.Thus, between the end amino acid on nucleic acid analog and the unhindered amina of free carboxy, forming amido link may be useful to forming conjugate.In certain embodiments, carboxyl terminal glycine or proline subunit are directly connected on 3 ' end of nucleic acid analog, for example can carry out connection carrier peptide (for example seeing Fig. 1 C) by form amido link between carboxyl terminal glycine or proline part and 3 ' morpholino ring nitrogen.
In some embodiments, by being selected from following linking arm part, nucleic acid analog is conjugated in carrier peptides: Y aor Y bsubunit, cysteine subunit and uncharged non-aminoacid linking arm part.In other embodiments, by being positioned at glycine or the proline part of 5 ' or 3 ' end of nucleic acid analog, directly nucleic acid analog is connected in carrier peptides.In some embodiments, by glycine or proline subunit, directly carrier peptides is connected to 3 ' end of nucleic acid analog, for example, by amido link, is directly connected on 3 ' morpholino nitrogen.
In other embodiments, conjugate comprises the coupling part between end glycine or proline subunit.In some of these embodiments, linking arm is 18 atomic lengths nearly, and it comprises and is selected from following connection (bonds): alkyl, hydroxyl, alkoxyl, alkyl amino, amide, ester, carbonyl, carbamate, di(2-ethylhexyl)phosphate amide, phosphoamide, phosphonothiolic acid and di-phosphate ester.In certain embodiments, linking arm comprises di(2-ethylhexyl)phosphate amide and piperazine.For example, in some embodiments, linking arm has following structure (XXIX):
Figure BDA0000447333530000251
R wherein 24for not existing, H or C 1-C 6alkyl.In certain embodiments, R 24for not existing, and in other embodiments, structure (XXIX) is connected to 5 ' end of nucleic acid analog (for example, morpholino oligomer) in carrier peptides and (for example sees, Figure 1B).
In some embodiments, R dthe pendant moiety of subunit is independently selected from guanidine radicals (HN=C (NH 2) NH-), amidino groups (HN=C (NH 2) C<), the amino dihydro-pyrimidin base of 2-, the amino tetrahydro-pyrimidine base of 2-, PA base and 2-aminopyrimidine base.
If desired, a plurality of carrier peptides can be connected on individualized compound; Or, a plurality of compounds can be attached on single transportation thing.Linking arm between carrier peptides and nucleic acid analog can also for example, be comprised of natural or alpha-non-natural amino acid (, 6-aminocaprolc acid or Beta-alanine).Linking arm can also comprise and transports the direct key that (for example,, at 3 ' morpholino nitrogen or 5 ' OH place) between the c-terminus of peptide and the amino of nucleic acid analog or hydroxyl forms by the condensation by for example carbodiimides promotes.
In general, linking arm can comprise any non-reacted part, and it does not disturb transportation or the function of conjugate.Linking arm can be selected from the linking arm for cutting under the normal condition of using, and for example, contains ether, thioether, amide or amino-formate bond.In other embodiments, comprise that the carrier peptides that can cut in vivo and for example, connection between compound (, oligonucleotide analogs, pharmacological agent etc.) are desirable.In vivo for being connected to of can cutting is known in the art, and for example comprise carboxylate (it is by enzymatic hydrolysis) and disulphide (it leaves cut at glutathion).By the radiation of application suitable wavelength, also can cut in vivo the connection that photodissociation can be cut, as O-Nitrophenylfluorone ether.The exemplary Heterobifunctional that further contains the disulphide group that can cut is rolled into a ball bridging agent, comprise 3-[(4-azido phenyl) dithio] propanoic acid-N-hydroxy-succinamide ester and Vanin, E.F. and Ji, T.H., other materials of describing in Biochemistry20:6754-6760 (1981).
C. exemplary carrier peptide
The table of exemplary carrier peptide sequence and oligonucleotide sequence is provided in following table 1.In some embodiments, the disclosure provides peptide oligomer conjugate, and wherein said peptide comprises or is comprised of any peptide sequence in table 1.In another embodiment, nucleic acid analog comprises or is comprised of any oligonucleotide sequence in table 1.Further in other embodiments, the disclosure provides peptide oligomer conjugate, and wherein said peptide comprises or is comprised of any peptide sequence in table 1, and nucleic acid analog comprises or be comprised of any oligonucleotide sequence in table 1.In other embodiments, the disclosure provides the peptide that comprises or be comprised of any sequence in table 1.
Table 1. exemplary carrier peptide and oligonucleotide sequence
Figure BDA0000447333530000261
Figure BDA0000447333530000271
Figure BDA0000447333530000281
Figure BDA0000447333530000301
Figure BDA0000447333530000311
Figure BDA0000447333530000321
Figure BDA0000447333530000331
Figure BDA0000447333530000341
Figure BDA0000447333530000361
Figure BDA0000447333530000371
Figure BDA0000447333530000381
Figure BDA0000447333530000391
Figure BDA0000447333530000401
Figure BDA0000447333530000411
Aa=glycine or proline; B=Beta-alanine; X=6-aminocaproic acid; The aminoterminal of tg=unmodified, or with acetyl group, benzoyl or stearyl, add the amino terminal (that is, acetyl group amide, benzoyl amide or stearyl amide) of cap and Y bfor:
-C(O)-(CHR e) n-NH-
Wherein n is 2-7, and each R ewhen occurring, be all hydrogen or methyl independently at every turn.For the sake of simplicity, not all sequence has all annotated end tg group; Yet each of above sequence can comprise the aminoterminal of unmodified or add the aminoterminal of cap with acetyl group, benzoyl or stearyl.
III. antisense scant polymer
For example be included in nucleic acid analog in conjugate of the present invention, for can base specific being attached to uncharged synthetic oligomer substantially of polynucleotide target sequence, antisense oligonucleotide analog.This type of analog comprises, for example, and methyl phosphonate, peptide nucleic acid(PNA), uncharged N3' → P5' phosphoramidate and morpholino oligomer substantially.
The nucleic acid analog base sequence that the base pairing group who is supported by analog skeleton provides can be any sequence, the base pairing group of wherein said support comprises A, T, C, G and U base standard or that process is modified, or off-gauge creatinine (I) and mix-G base of 7-denitrogenation.
In some embodiments, nucleic acid analog is morpholino oligomer, the oligonucleotide analogs that the morpholino subunit structure of form forms as shown in Figure 1, wherein: (i) this structure is by phosphorous connection (1 to 3 atomic length, preferred 2 atomic lengths) be joined together, the morpholino nitrogen of 1 subunit is attached on the 5' outer shroud carbon of contiguous subunit, and (ii) Pi and Pj for can being effectively attached to the purine in base or pyrimidine bases mating section in polynucleotide by base specific hydrogen bonded.Described purine or pyrimidine bases mating section are generally adenine, cytosine, guanine, uracil or thymus pyrimidine.Below further described synthetic, the structure of morpholino oligomer and in conjunction with feature, and in No. 5698685, No. 5217866, No. 5142047, No. 5034506, No. 5166315, No. 5521063 and No. 5506337 United States Patent (USP), have been described in detail, all these is all incorporated to herein by reference.
The chemical property of the oligomer expectation based on morpholino comprises and has high T mcomplementary base target nucleic acid comprise target RNA, or even be as short as the ability of the oligomer selective cross of 8-14 base, Active transport is to the ability in mammalian cell, and oligomer: the ability of the RNA heteroduplex enzymatic degradation of resistance to RNA.
In preferred embodiments, morpholino oligomer is the length of an about 8-40 subunit.More typically, oligomer is about 8-20, about 8-16 is individual, about 10-30 is individual or the length of an about 12-25 subunit.For some application, as antibacterial, short oligomer, for example the length of an about 8-12 subunit, may be particularly advantageous, especially when being connected to as peptide carrier disclosed herein (peptide transporter) above.
A. there is the oligomer connecting between the subunit through modifying
An embodiment of the present disclosure relates to the peptide-oligomer conjugate that comprises the nucleic acid analog (for example, morpholino oligomer) connecting between the subunit containing through modifying.In some embodiments, described conjugate compare answer through the oligomer of modifying, the affinity of DNA and RNA is not had to higher affinity, and compare the cell that the oligomer connecting between the subunit with other represents raising and send, tire and/or tissue distribution performance.In one embodiment, conjugate comprises between one or more (A) as defined below type subunit and connects.In other embodiments, conjugate comprises at least 1 and between type (B) subunit, connects as defined below.Between the subunit that further in other embodiments, conjugate comprises (A) type and type (B), be connected.Further in other embodiments, conjugate comprises morpholino oligomer as described in more detail below.Architectural feature and the performance of different connection types and oligomer have been described in the following discussion in more detail.
1. connect (A)
Applicant has been found that by preparation to have the oligomer connecting between different subunits, can optimize the raising of the performance of antisense activity, bio distribution and/or other expectations.For example, oligomer can optionally comprise between one or more (A) type subunit and connect, and in certain embodiments, oligomer comprises at least 1 (A) type and connects, and for example each connection can be (A) type.In some other embodiments, each (A) type connection has identical structure.(A) type connects and can comprise disclosed connection in No. 7943762 United States Patent (USP) of owning together, and it is all incorporated to herein by reference at this.Connect salt or isomer that (A) has following structure (I) or this structure, wherein 3 ' and 5 ' indicate respectively morpholino ring (that is, structure discussed below (i)) to the junction point of 3 ' and 5 ' end:
Figure BDA0000447333530000431
Wherein:
W is S or O independently at every turn when occurring;
X is independently-N (CH at every turn when occurring 3) 2,-NR 1r 2,-OR 3or
Figure BDA0000447333530000432
Y when occurring at every turn all independently for O or-NR 2,
R 1when occurring, be all hydrogen or methyl independently at every turn;
R 2when occurring at every turn all independently for hydrogen or-LNR 4r 5r 7;
R 3when occurring, be all hydrogen or C independently at every turn 1-C 6alkyl;
R 4when occurring, be all hydrogen, methyl ,-C (=NH) NH independently at every turn 2,-Z-L-NHC (=NH) NH 2or-[C (=O) CHR'NH] mh, wherein Z is-C (=O)-or direct key, naturally there are amino acid whose side chain or its 1 carbon or 2 carbon homologues in R', and m is 1 to 6;
R 5when occurring, be all hydrogen, methyl or electron pair independently at every turn;
R 6when occurring, be all hydrogen or methyl independently at every turn;
R 7when occurring, be all hydrogen, C independently at every turn 1-C 6alkyl or C 1-C 6alkoxyalkyl; And
L is the optional nearly linking arm of 18 atomic lengths, and it comprises alkyl, alkoxyl or alkylamino, or their combination.
In some instances, oligomer comprises at least 1 (A) type connection.In some other embodiments, the continuous connection that oligomer comprises at least 2 (A) types.In other embodiments, (A) type that is connected to of at least 5% in oligomer; For example in some embodiments, 5% to 95%, 10% to 90%, 10% to 50% or 10% to 35% connection can be for connecting (A) type.In some specific embodiments, at least 1 be connected to-N of (A) type (CH 3) 2.In other embodiments, each (A) type connection is-N (CH 3) 2, and even in other embodiments, each connection in oligomer is-N (CH 3) 2.In other embodiments, at least 1 (A) type is connected to piperazine-1-base, for example unsubstituted piperazine-1-base (for example, A2 or A3).In other embodiments, each (A) type for example connects, for piperazine-1-base, unsubstituted piperazine-1-base.
In some embodiments, W is S or O independently at every turn when occurring, and in certain embodiments, W is O.
In some embodiments, X is independently-N (CH at every turn when occurring 3) 2,-NR 1r 2,-OR 3.In some embodiments, X is-N (CH 3) 2.Aspect other, X is-NR 1r 2, and in other example, X is-OR 3.
In some embodiments, R 1when occurring, be all hydrogen or methyl independently at every turn.In some embodiments, R 1for hydrogen.In other embodiments, X is methyl.
In some embodiments, R 2when occurring, be all hydrogen at every turn.In other embodiment, R 2when occurring, be all-LNR at every turn 4r 5r 7.In some embodiments, R 3when occurring, be all hydrogen or C independently at every turn 1-C 6alkyl.In other embodiments, R 3for methyl.In other embodiments, R 3for ethyl.In some other embodiments, R 3for n-pro-pyl or isopropyl.In some other embodiments, R 3for C 4alkyl.In other embodiments, R 3for C 5alkyl.In some embodiments, R 3for C 6alkyl.
In certain embodiments, R 4when occurring, be all hydrogen independently at every turn.In other embodiments, R 4for methyl.Further in other embodiments, R 4for-C (=NH) NH 2, and in other embodiments, R 4for-Z-L-NHC (=NH) NH 2.Further in other embodiments, R 4for-[C (=O) CHR'NH] mh.In one embodiment, Z is-C (=O)-, and in another embodiment, Z is direct key.R' is the natural amino acid whose side chain that exists.In some embodiments, R ' is natural 1 carbon or the 2 carbon homologues that have amino acid whose side chain.
M is 1 to 6 integer.M can be 1.M can be 2.M can be 3.M can be 4.M can be 5.M can be 6.
In some embodiments, R 5when occurring, be all hydrogen, methyl or electron pair independently at every turn.In some embodiments, R 5for hydrogen.In other embodiments, R 5for methyl.In other embodiments, R 5for electron pair.
In some embodiments, R 6when occurring, be all hydrogen or methyl independently at every turn.In some embodiments, R 6for hydrogen.In other embodiments, R 6for methyl.
In other embodiments, R 7when occurring, be all hydrogen, C independently at every turn 1-C 6alkyl or C 2-C 6alkoxyalkyl.In some embodiments, R 7for hydrogen.In other embodiments, R 7for C 1-C 6alkyl.Further in other embodiments, R 7for C 2-C 6alkoxyalkyl.In some embodiments, R 7for methyl.In other embodiments, R 7for ethyl.Further in other embodiment, R 7for n-pro-pyl or isopropyl.In some other embodiments, R 7for C 4alkyl.In some embodiments, R 7for C 5alkyl.In some embodiments, R 7for C 6alkyl.Further in other embodiments, R 7for C 2alkoxyalkyl.At some in other embodiment, R 7for C 3alkoxyalkyl.Further in other embodiments, R 7for C 4alkoxyalkyl.In some embodiments, R 7for C 5alkoxyalkyl.In other embodiments, R 7for C 6alkoxyalkyl.
As mentioned above, linking arm group L contains and is selected from following connection in its skeleton: alkyl (for example-CH 2-CH 2-), alkoxyl (for example ,-C-O-C-) and alkyl amino (for example-CH 2-NH-), condition is that the end atom (for example, being close to the atom of carbonyl or nitrogen) of L is carbon atom.For example, although may there be the connection (-CH of tool side chain 2-CHCH 3-), but linking arm is generally unbranched.In one embodiment, linking arm is hydrocarbon linking arm.This type of linking arm can have structure (CH 2) n-, wherein n is 1-12, preferred 2-8, and more preferably 2-6.
The oligomer of (A) type connection with any number is provided.In some embodiments, oligomer does not have (A) type to connect.In certain embodiments, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% be connected to connection (A).In selected embodiment, 10% to 80%, 20% to 80%, 20% to 60%, 20% to 50%, 20% to 40% or 20% to 35% be connected to connection (A).In other embodiments, each connects for (A) type.
2. connect (B)
In some embodiments, oligomer comprises at least 1 (B) type connection.For example oligomer can comprise 1,2,3,4,5,6 or more (B) type connect.(B) type connection can be contiguous maybe can being dispersed in whole oligomer.(B) type connection has following structure (I):
Figure BDA0000447333530000451
Or the salt of this structure or isomer, wherein:
W is S or O independently at every turn when occurring;
X is independently-NR at every turn when occurring 8r 9or-OR 3; And
Y when occurring at every turn all independently for O or-NR 10,
R 3when occurring, be all hydrogen or C independently at every turn 1-C 6alkyl;
R 8when occurring, be all hydrogen or C independently at every turn 2-C 12alkyl;
R 9when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aralkyl or aryl;
R 10when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl or-LNR 4r 5r 7;
R wherein 8and R 9can be in conjunction with the monocycle or the bicyclic heterocycles that form 5-18 unit, or R 8, R 9or R 3can with R 10in conjunction with the heterocycle that forms 5-7 unit, and wherein when X is 4-piperazinyl, X has following structure (III):
Figure BDA0000447333530000461
Wherein:
R 11when occurring, be all C independently at every turn 2-C 12alkyl, C 1-C 12aminoalkyl, C 1-C 12alkyl-carbonyl, aryl, heteroaryl or heterocyclic radical;
R is electron pair, hydrogen or C independently at every turn when occurring 1-C 12alkyl; And
R 12when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aminoalkyl ,-NH 2,-CONH 2,-NR 13r 14,-NR 13r 14r 15, C 1-C 12alkyl-carbonyl, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, amidino groups alkyl, amidino groups alkyl-carbonyl guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR 13or C 1-C 12alkoxyl, wherein R 13, R 14and R 15when occurring, be all C independently at every turn 1-C 12alkyl.
In some instances, oligomer comprises 1 (B) type connection.In some other embodiments, oligomer comprises 2 (B) types and connects.In some other embodiments, oligomer comprises 3 (B) types and connects.In some other embodiments, oligomer comprises 4 (B) types and connects.Further in other embodiments, (B) type is connected to continuous (that is, type (B) connects located adjacent one another).In other embodiments, in oligomer at least 5% be connected to type (B); For example in some embodiments, 5% to 95%, 10% to 90%, 10% to 50% or 10% to 35% connection can connect for (B) type.
In other embodiments, R 3when occurring, be all hydrogen or C independently at every turn 1-C 6alkyl.In other embodiments, R 3it can be methyl.In some embodiments, R 3it can be ethyl.In some other embodiments, R 3can be n-pro-pyl or isopropyl.In other embodiments, R 3can be C 4alkyl.In some embodiments, R 3can be C 5alkyl.In some embodiments, R 3can be C 6alkyl.
In some embodiments, R 8when occurring, be all hydrogen or C independently at every turn 2-C 12alkyl.In some embodiments, R 8for hydrogen.In other embodiments, R 8for ethyl.In some other embodiments, R 8for n-pro-pyl or isopropyl.In some embodiments, R 8for C 4alkyl.In other embodiments, R 8for C 5alkyl.In other embodiments, R 8for C 6alkyl.In some embodiments, R 8for C 7alkyl.In other embodiments, R 8for C 8alkyl.In other embodiments, R 8for C 9alkyl.In other embodiments, R 8for C 10alkyl.In some other embodiments, R 8for C 11alkyl.In other embodiments, R 8for C 12alkyl.In some other embodiments, R 8for C 2-C 12alkyl, and described C 2-C 12alkyl comprises one or more pairs of keys (for example, alkene), triple bond (for example, alkynes) or both.In some embodiments, R 8for unsubstituted C 2-C 12alkyl.
In some embodiments, R 9when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aralkyl or aryl.In some embodiments, R 9for hydrogen.In other embodiments, R 9for C 1-C 12alkyl.In other embodiments, R 9for methyl.In other embodiments, R 9for ethyl.In some other embodiments, R 9for n-pro-pyl or isopropyl.In some embodiments, R 9for C 4alkyl.In some embodiments, R 9for C 5alkyl.In other embodiments, R 9for C 6alkyl.In some other embodiments, R 9for C 7alkyl.In some embodiments, R 9for C 8alkyl.In some embodiments, R 9for C 9alkyl.In some other embodiments, R 9for C 10alkyl.In some other embodiments, R 9for C 11alkyl.In other embodiments, R 9for C 12alkyl.
In some other embodiments, R 9for C 1-C 12aralkyl.For example, in some embodiments, R 9for benzyl, and described benzyl can optionally be substituted on phenyl ring or benzyl carbon.Thus, substituent group comprises alkyl and alkoxyl, for example methyl or methoxy.In some embodiments, benzyl carbon place with methyl substituted benzyl.For example, in some embodiments, R 9there is following structure (XIV):
In other embodiments, R 9for aryl.For example, in some embodiments, R 9for phenyl, and described phenyl can optionally be substituted.Thus, substituent group comprises alkyl and alkoxy grp, for example methyl or methoxy.In other embodiments, R 9for phenyl, and described phenyl comprises crown ether part, for example the crown ether of 12-18 unit.In one embodiment, crown ether is 18 yuan, and can further comprise other phenyl moiety.For example, in one embodiment, R 9there is a kind of in following structure (XV or XVI):
Figure BDA0000447333530000482
In some embodiments, R 10when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl or LNR 4r 5r 7, R wherein 4, R 5and R 7as above about connecting the definition of (A).In other embodiments, R 10for hydrogen.In other embodiments, R 10for C 1-C 12alkyl, and in other embodiments, R 10for-LNR 4r 5r 7.In some embodiments, R 10for methyl.In other embodiments, R 10for ethyl.In some embodiments, R 10for C 3alkyl.In some embodiments, R 10for C 4alkyl.In other embodiments, R 10for C 5alkyl.In some other embodiments, R 10for C 6alkyl.In other embodiment, R 10for C 7alkyl.In other embodiments, R 10for C 8alkyl.In some embodiments, R 10for C 9alkyl.In other embodiments, R 10for C 10alkyl.In other embodiments, R 10for C 11alkyl.In some other embodiments, R 10for C 12alkyl.
In some embodiments, R 8and R 9in conjunction with the monocycle or the bicyclic heterocycles that form 5-18 unit.In some embodiments, heterocycle is the monocyclic heterocycles of 5 or 6 yuan.For example, in some embodiments, connect (B) and there is following structure (IV):
Figure BDA0000447333530000491
Wherein Z represents the monocyclic heterocycles of 5 or 6 yuan.
In other embodiments, heterocycle is bicyclo-, for example the bicyclic heterocycles of 12 yuan.Heterocycle can be piperazinyl.Heterocycle can be morpholino.Heterocycle can be piperidyl.Heterocycle can be Decahydroisoquinolinpreparation.Representational heterocycle comprises as follows:
Figure BDA0000447333530000492
In some embodiments, R 11when occurring, be all C independently at every turn 2-C 12alkyl, C 1-C 12aminoalkyl, aryl, heteroaryl or heterocyclic radical.
In some embodiments, R 11for C 2-C 12alkyl.In some embodiments, R 11for ethyl.In other embodiments, R 11for C 3alkyl.In other embodiments, R 11for isopropyl.In some other embodiments, R 11for C 4alkyl.In other embodiments, R 11for C 5alkyl.In some embodiments, R 11for C 6alkyl.In other embodiments, R 11for C 7alkyl.In some embodiments, R 11for C 8alkyl.In other embodiments, R 11for C 9alkyl.In other embodiments, R 11for C 10alkyl.In some other embodiments, R 11for C 11alkyl.In some embodiments, R 11for C 12alkyl.
In other embodiments, R 11for C 1-C 12aminoalkyl.In some embodiments, R 11for methylamino.In some embodiments, R 11for ethylamino.In other embodiments, R 11for C 3aminoalkyl.In other embodiments, R 11for C 4aminoalkyl.In some other embodiments, R 11for C 5aminoalkyl.In other embodiments, R 11for C 6aminoalkyl.In other embodiments, R 11for C 7aminoalkyl.In some embodiments, R 11for C 8aminoalkyl.In other embodiments, R 11for C 9aminoalkyl.In other embodiments, R 11for C 10aminoalkyl.In some other embodiments, R 11for C 11aminoalkyl.In other embodiments, R 11for C 12aminoalkyl.
In other embodiments, R 11for C 1-C 12alkyl-carbonyl.In other embodiments, R 11for C 1alkyl-carbonyl.In other embodiments, R 11for C 2alkyl-carbonyl.In some embodiments, R 11for C 3alkyl-carbonyl.In other embodiments, R 11for C 4alkyl-carbonyl.In some embodiments, R 11for C 5alkyl-carbonyl.In some other embodiments, R 11for C 6alkyl-carbonyl.In other embodiments, R 11for C 7alkyl-carbonyl.In other embodiments, R 11for C 8alkyl-carbonyl.In some embodiments, R 11for C 9alkyl-carbonyl.In other embodiment, R 11for C 10alkyl-carbonyl.In some other embodiments, R 11for C 11alkyl-carbonyl.In some embodiments, R 11for C 12alkyl-carbonyl.In other embodiments, R 11for-C (=O) (CH 2) ncO 2h, wherein n is 1 to 6.For example, in some embodiments, n is 1.In other embodiments, n is 2.In other embodiments, n is 3.In some other embodiments, n is 4.In other embodiments, n is 5.In other embodiments, n is 6.
In other embodiments, R 11for aryl.For example, in some embodiments, R 11for phenyl.In some embodiments, phenyl is for example replaced with nitro.
In other embodiments, R 11for heteroaryl.For example, in some embodiments, R 11for pyridine radicals.In other embodiments, R 11for pyrimidine radicals.
In other embodiments, R 11for heterocyclic radical.For example, in some embodiments, R 11for piperidyl, piperidin-4-yl for example.
In some embodiments, R 11for ethyl, isopropyl, piperidyl, pyrimidine radicals, cholate, dexycholate or-C (=O) (CH 2) ncO 2h, wherein n is 1 to 6.
In some embodiments, R is electron pair.In other embodiments, R is hydrogen, and in other embodiment, R is C 1-C 12alkyl.In some embodiments, R is methyl.In some embodiments, R is ethyl.In other embodiments, R is C 3alkyl.In other embodiment, R is isopropyl.In some other embodiments, R is C 4alkyl.In other embodiments, R is C 5alkyl.In some embodiments, R is C 6alkyl.In other embodiments, R is C 7alkyl.In other embodiment, R is C 8alkyl.In other embodiments, R is C 9alkyl.In some embodiments, R is C 10alkyl.In other embodiments, R is C 11alkyl.In some embodiments, R is C 12alkyl.
In some embodiments, R 12when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aminoalkyl ,-NH 2,-CONH 2,-NR 13r 14,-NR 13r 14r 15, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, amidino groups alkyl, amidino groups alkyl-carbonyl guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR 13or C 1-C 12alkoxyl, wherein R 13, R 14and R 15when occurring, be all C independently at every turn 1-C 12alkyl.
In some embodiments, R 12for hydrogen.In some embodiments, R 12for C 1-C 12alkyl.In some embodiments, R 12for C 1-C 12aminoalkyl.In some embodiments, R 12for-NH 2.In some embodiments, R 12for-CONH 2.In some embodiments, R 12for-NR 13r 14.In some embodiments, R 12for-NR 13r 14r 15.In some embodiments, R 12for C 1-C 12alkyl-carbonyl.In some embodiments, R 12for oxo.In some embodiments, R 12for-CN.In some embodiments, R 12for trifluoromethyl.In some embodiments, R 12for amide groups.In some embodiments, R 12for amidino groups.In some embodiments, R 12for amidino groups alkyl.In some embodiments, R 12for amidino groups alkyl-carbonyl.In some embodiments, R 12for guanidine radicals, for example monomethyl guanidine radicals or dimethyl guanidine radicals.In some embodiments, R 12for guanidine alkylation.In some embodiments, R 12for amidino groups alkyl-carbonyl.In some embodiments, R 12for cholate.In some embodiments, R 12for dexycholate.In some embodiments, R 12for aryl.In some embodiments, R 12for heteroaryl.In some embodiments, R 12for heterocycle.In some embodiments, R 12for-SR 13.In some embodiments, R 12for C 1-C 12alkoxyl.In some embodiments, R 12for dimethylamino.
In other embodiments, R 12for methyl.In other embodiments, R 12for ethyl.In some embodiments, R 12for C 3alkyl.In some embodiments, R 12for isopropyl.In some embodiments, R 12for C 4alkyl.In other embodiments, R 12for C 5alkyl.In other embodiments, R 12for C 6alkyl.In some other embodiments, R 12for C 7alkyl.In some embodiments, R 12for C 8alkyl.In other embodiments, R 12for C 9alkyl.In some embodiments, R 12for C 10alkyl.In other embodiments, R 12for C 11alkyl.In other embodiments, R 12for C 12alkyl.In other embodiments, described moieties is replaced by one or more oxygen atoms and forms ether moiety, for example methoxy methyl base section.
In some embodiments, R 12for methylamino.In other embodiment, R 12for ethylamino.In other embodiments, R 12for C 3aminoalkyl.In some embodiments, R 12for C 4aminoalkyl.In other embodiments, R 12for C 5aminoalkyl.In some other embodiments, R 12for C 6aminoalkyl.In some embodiments, R 12for C 7aminoalkyl.In some embodiments, R 12for C 8aminoalkyl.In other embodiments, R 12for C 9aminoalkyl.In some other embodiments, R 12for C 10aminoalkyl.In other embodiments, R 12for C 11aminoalkyl.In other embodiments, R 12for C 12aminoalkyl.In some embodiments, aminoalkyl is dimethylamino alkyl.
In other embodiments, R 12for acetyl group.In some other embodiments, R 12for C 2alkyl-carbonyl.In some embodiments, R 12for C 3alkyl-carbonyl.In other embodiment, R 12for C 4alkyl-carbonyl.In some embodiments, R 12for C 5alkyl-carbonyl.In other embodiments, R 12for C 6alkyl-carbonyl.In some other embodiments, R 12for C 7alkyl-carbonyl.In some embodiments, R 12for C 8alkyl-carbonyl.In other embodiments, R 12for C 9alkyl-carbonyl.In some other embodiments, R 12for C 10alkyl-carbonyl.In some embodiments, R 12for C 11alkyl-carbonyl.In other embodiments, R 12for C 12alkyl-carbonyl.Alkyl-carbonyl is replaced by carboxy moiety, and for example alkyl-carbonyl is substituted to form succinic acid part (that is, 3-carboxyalkyl carbonyl).In other embodiment, alkyl-carbonyl is replaced by end-SH base.
In some embodiments, R 12for amide groups.In some embodiments, amide groups comprises the moieties being further substituted, for example, by-SH, carbamate or their combination, replaced.In other embodiments, amide groups by aryl moiety for example phenyl replace.In certain embodiments, R 12can there is following structure (IX):
Figure BDA0000447333530000521
R wherein 16when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12alkoxyl ,-CN, aryl or heteroaryl.
In some embodiments, R 12for methoxyl group.In other embodiments, R 12for ethyoxyl.In other embodiment, R 12for C 3alkoxyl.In some embodiments, R 12for C 4alkoxyl.In some embodiments, R 12for C 5alkoxyl.In some other embodiments, R 12for C 6alkoxyl.In other embodiment, R 12for C 7alkoxyl.In some other embodiments, R 12for C 8alkoxyl.In some embodiments, R 12for C 9alkoxyl.In other embodiments, R 12for C 10alkoxyl.In some embodiments, R 12for C 11alkoxyl.In other embodiments, R 12for C 12alkoxyl.
In certain embodiments, R 12for pyrrolidinyl, pyrrolidin-1-yl for example.In other embodiments, R 12for piperidyl, for example piperidin-1-yl or piperidin-4-yl.In other embodiments, R 12for morpholino, morpholine-4-base for example.In other embodiments, R 12for phenyl, and even in other embodiments, described phenyl is for example replaced with nitro.In other embodiments, R 12for pyrimidine radicals, pyrimidine-2-base for example.
In other embodiments, R 13, R 14and R 15when occurring, be all C independently at every turn 1-C 12alkyl.In some embodiments, R 13, R 14or R 15for methyl.In other embodiments, R 13, R 14or R 15for ethyl.In other embodiments, R 13, R 14or R 15for C 3alkyl.In other embodiments, R 13, R 14or R 15for isopropyl.In other embodiments, R 13, R 14or R 15for C 4alkyl.In some embodiments, R 13, R 14or R 15for C 5alkyl.In some other embodiments, R 13, R 14or R 15for C 6alkyl.In other embodiments, R 13, R 14or R 15for C7 alkyl.In other embodiments, R 13, R 14or R 15for C 8alkyl.In other embodiments, R 13, R 14or R 15for C 9alkyl.In some embodiments, R 13, R 14or R 15for C 10alkyl.In some embodiments, R 13, R 14or R 15for C 11alkyl.In other embodiments, R 13, R 14or R 15for C 12alkyl.
As mentioned above, in some embodiments, R 12for the amide groups being replaced by aryl moiety.Thus, R 16each appearance can be for identical or different.In some of these embodiments, R 16for hydrogen.In other embodiments, R 16for-CN.In other embodiments, R 16for heteroaryl, tetrazole radical for example.In certain other embodiments, R 16for methoxyl group.In other embodiments, R 16for aryl, and described aryl is optionally substituted.Thus, optional substituent group comprises: C 1-C 12alkyl, C 1-C 12alkoxyl, for example methoxyl group; Trifluoromethoxy; Halogen, for example chloro; And trifluoromethyl.
In other embodiments, R 16for methyl.In other embodiments, R 16for ethyl.In some embodiments, R 16for C 3alkyl.In some other embodiments, R 16for isopropyl.In other embodiments, R 16for C 4alkyl.In other embodiments, R 16for C 5alkyl.In other embodiments, R 16for C 6alkyl.In some other embodiments, R 16for C 7alkyl.In some embodiments, R 16for C 8alkyl.In other embodiments, R 16for C 9alkyl.In some other embodiments, R 16for C 10alkyl.In other embodiments, R 16for C 11alkyl.In some other embodiments, R 16for C 12alkyl.
In some embodiments, R 16for methoxyl group.In some embodiments, R 16for ethyoxyl.In other embodiments, R 16for C 3alkoxyl.In some other embodiments, R 16for C 4alkoxyl.In other embodiments, R 16for C 5alkoxyl.In some other embodiments, R 16for C 6alkoxyl.In other embodiments, R 16for C 7alkoxyl.In some other embodiments, R 16for C 8alkoxyl.In other embodiments, R 16for C 9alkoxyl.In some other embodiments, R 16for C 10alkoxyl.In some embodiments, R 16for C 11alkoxyl.In some other embodiments, R 16for C 12alkoxyl.
In some other embodiments, R 8and R 9in conjunction with the crown ether that forms 12-18 unit.For example, in some embodiments, crown ether is 18 yuan, and in other embodiment, and crown ether is 15 yuan.In certain embodiments, R 8and R 9in conjunction with form have following structure (X) or (XI) in a kind of heterocycle:
Figure BDA0000447333530000541
In some embodiments, R 8, R 9or R 3with R 10in conjunction with the heterocycle that forms 5-7 unit.For example, in some embodiments, R 3with R 10in conjunction with the heterocycle that forms 5-7 unit.In some embodiments, heterocycle is 5 yuan.In other embodiments, heterocycle is 6 yuan.In other embodiments, heterocycle is 7 yuan.In some embodiments, heterocycle can represent by following structure (XII):
Figure BDA0000447333530000551
Wherein Z ' represents the heterocycle of 5-7 unit.In some embodiment of structure (XI), R 12when occurring, be all hydrogen at every turn.For example, connect (B) can have following structure (B1), (B2) or (B3) in a kind of:
Figure BDA0000447333530000552
In certain other embodiments, R 12for C 1-C 12alkyl-carbonyl or amide groups, it is by aryl phosphoryl part, and for example triphenyl phosphorus acyl moiety further replaces.The connection example with this structure comprises B56 and B55.
In certain embodiments, connect (B) without any structure A1-A5.Table 2 has shown that (A) type is connected with the representativeness of (B).
Between the representational subunit of table 2., connect
Figure BDA0000447333530000553
Figure BDA0000447333530000561
Figure BDA0000447333530000581
Figure BDA0000447333530000591
Figure BDA0000447333530000601
Figure BDA0000447333530000611
Figure BDA0000447333530000621
Figure BDA0000447333530000631
Figure BDA0000447333530000641
Figure BDA0000447333530000651
Figure BDA0000447333530000671
In sequence and discussion subsequently, often can use the title of above connection.For example, comprise PMO apnthe base connecting is described as apnb, wherein B is base.Other connections can be named similarly.In addition, can use abbreviation title, for example, can use abbreviation title in above bracket (for example, ab, refers to apnb).Can also use the abbreviation of other easy identification.
B. oligomer with the end group through modifying
Except carrier peptides, conjugate can also comprise the oligomer that contains the end group through modifying.Applicant has been found that with various chemical parts and modifies after 3 ' and/or 5 ' end of oligomers and to provide favourable curative properties (for example, the cell of raising is sent, tired and/or tissue distribution etc.) for conjugate.In various embodiments, through the end group of modifying, comprise hydrophobic parts, and in other embodiments, through the end group of modifying, comprise hydrophilic segment.Through the end group of modifying, can there is or not exist above-mentioned connection.For example, in some embodiments, the oligomer of being combined with carrier peptides comprises one or more end groups through modification and is connected with (A) type, and for example wherein X is-N (CH 3) 2connection.In other embodiments, oligomer comprises and one or morely through the end groups of modifying, is connected with (B) type, and for example wherein X is (that is, connection APN) of 4-amino piperidine-1-base.Further in other embodiments, oligomer comprises one or more end group and connection (A) and mixing (B) through modifying.For example, oligomer can comprise one or more through the end group (for example, trityl or triphenyl acetyl group) modification and wherein X be-N (CH 3) 2connection, and wherein X is the connection of 4-amino piperidine-1-base.Other combinations of the connection of modifying through end group and the process of modification are also for oligomer provides favourable curative properties.
In one embodiment, comprise end modified oligomer and there is following structure (XVII):
Figure BDA0000447333530000681
Or the salt of this structure or isomer, wherein X, W and Y as above-mentioned connection (A) and (B) in any one define, and:
R 17when occurring at every turn all independently for not existing, hydrogen or C 1-C 6alkyl;
R 18and R 19when occurring at every turn all independently for not existing, hydrogen, carrier peptides, natural or alpha-non-natural amino acid, C 2-C 30alkyl-carbonyl ,-C (=O) OR 21or R 20;
R 20when occurring, be all guanidine radicals, heterocyclic radical, C independently at every turn 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 7-C 30aralkyl, C 3-C 30alkyl-carbonyl, C 3-C 8naphthene base carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 7-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aryl-alkoxy carbonyl or-P (=O) (R 22) 2;
Pi is the part of base pairing independently at every turn when occurring;
L 1for the optional nearly linking arm of 18 atomic lengths, it comprises and is selected from following connection: alkyl, hydroxyl, alkoxyl, alkylamino, amide, ester, disulphide, carbonyl, carbamate, di(2-ethylhexyl)phosphate amide, phosphoamide, thiophosphate, piperazine and di-phosphate ester; And
X is 0 or larger integer; And R wherein 18or R 19in at least 1 be R 20; And
R wherein 18or R 19in at least 1 be R 20, and condition is R 17and R 18not for not existing.
(A) type that the oligomer of the end group of modifying with process can comprise any number is connected with (B) type.For example, oligomer can only comprise the connection of (A) type.For example, each X in connecting can be-N (CH 3) 2.Or oligomer can only comprise connection (B).In certain embodiments, oligomer comprises connection (A) and mixing (B), and for example 1 to 4 (B) type connects, and the remainder of this connection is (A) type.Thus, connect that to include, but not limited to X be wherein that (B) type of amino piperidine base connects (A) type that is dimethylamino with X and is connected.
In some embodiments, R 17for not existing.In some embodiments, R 17for hydrogen.In some embodiments, R 17for C 1-C 6alkyl.In some embodiments, R 17for methyl.In other embodiments, R 17for ethyl.In some embodiments, R 17for C 3alkyl.In some other embodiments, R 17for isopropyl.In other embodiments, R 17for C 4alkyl.Further in other embodiments, R 17for C 5alkyl.In some other embodiments, R 17for C 6alkyl.
In other embodiments, R 18for not existing.In some embodiments, R 18for hydrogen.In some embodiments, R 18for carrier peptides.In some embodiments, R 18for example, for natural or alpha-non-natural amino acid, trimethyl glycine.In some embodiments, R 18for R 20.
In other embodiments, R 19for not existing.In some embodiments, R 19for hydrogen.In some embodiments, R 19for carrier peptides.In some embodiments, R 19for example, for natural or alpha-non-natural amino acid, trimethyl glycine.In some embodiments, R 19for-C (=O) OR 17, R for example 19can there is following structure:
Figure BDA0000447333530000691
In other embodiments, R 18or R 19for C 2-C 30alkyl-carbonyl, for example-C (=O) (CH 2) ncO 2h, wherein n is 1 to 6, for example 2.In other examples, R 18or R 19for acetyl group.
In some embodiments, R 20when occurring, be all guanidine radicals, heterocyclic radical, C independently at every turn 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 7-C 30aralkyl, C 3-C 30alkyl-carbonyl, C 3-C 8naphthene base carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 6-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aryl-alkoxy carbonyl ,-C (=O) OR 21or-P (=O) (R 22) 2, R wherein 21for comprising the C of one or more oxygen or hydroxylic moiety or their combination 1-C 30alkyl, and each R 22be all C 6-C 12aryloxy group.
In certain other embodiments, R 19for-C (=O) OR 21, and R 18for hydrogen, guanidine radicals, heterocyclic radical, C 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 3-C 30alkyl-carbonyl, C 3-C 8alkyl-carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 7-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aryl-alkoxy carbonyl or-P (=O) (R 22) 2, each R wherein 22be all C 6-C 12aryloxy group.
In other embodiments, R 20when occurring, be all guanidine radicals, heterocyclic radical, C independently at every turn 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 3-C 30alkyl-carbonyl, C 3-C 8naphthene base carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 7-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aryl-alkoxy carbonyl or-P (=O) (R 22) 2.And in other examples, R 20when occurring, be all guanidine radicals, heterocyclic radical, C independently at every turn 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 7-C 30aralkyl, C 3-C 8naphthene base carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 7-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aryl-alkoxy carbonyl or-P (=O) (R 22) 2.
In some embodiments, R 20for guanidine radicals, for example monomethyl guanidine radicals or dimethyl guanidine radicals.In other embodiments, R 20for heterocyclic radical.For example, in some embodiments, R 20for piperidin-4-yl.In some embodiments, piperidin-4-yl is replaced by trityl or Boc base.In other embodiments, R 20for C 3-C 8cycloalkyl.In other embodiments, R 20for C 6-C 30aryl.
In some embodiments, R 20for C 7-C 30aryl carbonyl.For example, in some embodiments, R 20there is following structure (XVIII):
Figure BDA0000447333530000701
R wherein 23when occurring, be all hydrogen, halogen, C independently at every turn 1-C 30alkyl, C 1-C 30alkoxyl, C 1-C 30alkoxy carbonyl, C 7-C 30aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and 1 R wherein 23can with another R 23in conjunction with forming heterocyclic ring.In some embodiments, at least 1 R 23for hydrogen, for example, in some embodiments, each R 23be all hydrogen.In other embodiments, at least 1 R 23for C 1-C 30alkoxyl, for example in some embodiments, each R 23it is all methoxyl group.In other embodiments, at least 1 R 23for heteroaryl, for example in some embodiments, at least 1 R 23have following structure (XVIIIa) or (XVIIIb) in a kind of:
Figure BDA0000447333530000711
In other embodiments, 1 R 23with another R 23in conjunction with forming heterocyclic ring.For example, in one embodiment, R 20for CF.
In other embodiments, R 20for C 7-C 30aromatic alkyl carbonyl.For example, in various embodiments, R 20have following structure (XIX), (XX) or (XXI) in a kind of:
Figure BDA0000447333530000712
R wherein 23when occurring, be all hydrogen, halogen, C independently at every turn 1-C 30alkyl, C 1-C 30alkoxyl, C 1-C 30alkoxy carbonyl, C 7-C 30aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, wherein 1 R 23can with another R 23in conjunction with forming heterocyclic ring, X is-OH or halogen, and m is 0 to 6 integer.In some specific embodiments, m is 0.In other embodiments, m is 1, and in other embodiments, m is 2.In other embodiments, at least 1 R 23for hydrogen, for example in some embodiments, each R 23be all hydrogen.In some embodiments, X is hydrogen.In other embodiments, X is-OH.In other embodiments, X is Cl.In other embodiments, at least 1 R 23for C 1-C 30alkoxyl, for example methoxyl group.
Further in other embodiments, R 20for C 7-C 30aralkyl, for example trityl.In other embodiments, R 20for methoxyl group trityl.In some embodiments, R 20there is following structure (XXII):
Figure BDA0000447333530000721
R wherein 23when occurring, be all hydrogen, halogen, C independently at every turn 1-C 30alkyl, C 1-C 30alkoxyl, C 1-C 30alkoxy carbonyl, C 7-C 30aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and 1 R wherein 23can with another R 23in conjunction with forming heterocyclic ring.For example, in some embodiments, each R 23be all hydrogen.In other embodiments, at least 1 R 23for C 1-C 30alkoxyl, for example methoxyl group.
In other embodiments, R 20for C 7-C 30aralkyl, and R 20there is following structure (XXIII):
Figure BDA0000447333530000722
In some embodiments, at least 1 R 23for halogen, chloro for example.At some in other embodiment, 1 R 23for being positioned at the chloro of para-position.
In other embodiments, R 20for C 1-C 30alkyl.For example, in some embodiments, R 20for C 4-C 20alkyl, and optionally comprise one or more pairs of keys.For example, in some embodiments, R 20for comprising triple bond, the C of terminal triple link for example 4-10alkyl.In some embodiments, R 20for hexin-6-base.In some embodiments, R 20have following structure (XXIV), (XXV), (XXVI) or (XXVII) in a kind of:
Figure BDA0000447333530000731
Further in other embodiments, R 20for C 3-C 30alkyl-carbonyl, for example C 3-C 10alkyl-carbonyl.In some embodiments, R 20for-C (=O) is (CH2) psH or-C (=O) (CH2) psSHet, wherein p is 1 to 6 integer, and Het is heteroaryl.For example, p can be 1, or p can be 2.In other examples, Het is pyridine radicals, for example pyridine-2-base.In other embodiments, C 3-C 30alkyl-carbonyl is replaced by other oligomers, and for example in some embodiments, oligomer comprises the C that is positioned at 3 ' position 3-C 30alkyl-carbonyl, it is connected to oligomer 3 ' position of another oligomer.This type of is end modified included within the scope of the present disclosure.
In other embodiments, R 20for the C further being replaced by aryl phosphoryl part 3-C 30alkyl-carbonyl, for example triphenyl phosphorus acyl group.This type of R 20the example of group comprises the structure 33 in table 3.
In other examples, R 20for C 3-C 8naphthene base carbonyl, for example C 5-C 7alkyl-carbonyl.In these embodiments, R 20there is following structure (XXVIII):
Figure BDA0000447333530000732
R wherein 23when occurring, be all hydrogen, halo, C independently at every turn 1-C 30alkyl, C 1-C 30alkoxyl, C 1-C 30alkoxy carbonyl, C 7-C 30aralkyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl, and 1 R wherein 23can with another R 23in conjunction with forming heterocyclic ring.In some embodiments, R 23for example, for heterocyclic radical alkyl, in some embodiments.R 23there is following structure:
Figure BDA0000447333530000741
In some other embodiments, R 20for C 3-C 8cycloalkyl alkyl carbonyl.In other embodiments, R 20for C 2-C 30alkoxy carbonyl.In other embodiments, R 20for C 3-C 8cyclo alkoxy carbonyl.In other embodiments, R 20for C 7-C 30aryloxycarbonyl.In other embodiments, R 20for C 8-C 30aryl-alkoxy carbonyl.In other embodiments, R 20for-P (=O) (R 22) 2, each R wherein 22be all C 6-C 12aryloxy group, for example in some embodiments, R 20there is following structure (C24):
Figure BDA0000447333530000742
In other embodiments, R 20comprise one or more halogen atoms.For example, in some embodiments, R 20comprise any above R 20the perfluoro analog of part.In other embodiments, R 20for p-trifluoromethyl phenyl, trifluoromethyl trityl, perfluor amyl group or pentafluorophenyl group.
In some embodiments, 3 ' end comprises modification, and in other embodiments, 5 ' end comprises modification.In other embodiments, 3 ' and 5 ' end all comprises modification.Therefore, in some embodiments, R 18for not existing, and R 19for R 20.In other embodiments, R 19for not existing, and R 18for R 20.Further in other embodiments, R 18and R 19r respectively does for oneself 20.
In some embodiments, except 3 ' or 5 ' modification, oligomer also comprises cell permeability peptide.Therefore, in some embodiments, R 19for cell permeability peptide, and R 18for R 20.In other embodiments, R 18for cell permeability peptide, and R 19for R 20.In the further embodiment of aforementioned schemes, cell permeability peptide is arginine enrichment peptide.
In some embodiments, can exist or lack 5 ' terminal groups (that is, R 19) be connected to the linking arm L of oligomer 1.Described linking arm can comprise functional group and the length of any number, condition is that this linking arm remains with it 5 ' terminal groups is connected to the ability on oligomer, and condition be this linking arm do not disturb oligomer in sequence-specific mode, be attached to the ability on target sequence.In one embodiment, L comprises di(2-ethylhexyl)phosphate amide and is connected with piperazine.For example, in some embodiments, L has following structure (XXIX):
Figure BDA0000447333530000751
R wherein 24for not existing, hydrogen or C 1-C 6alkyl.In some embodiments, R 24for not existing.In some embodiments, R 24for hydrogen.In some embodiments, R 24for C 1-C 6alkyl.In some embodiments, R 24for methyl.In other embodiments, R 24for ethyl.Further in other embodiments, R 24for C 3alkyl.In some other embodiments, R 24for isopropyl.In other embodiments, R 24for C 4alkyl.In some embodiments, R 24for C 5alkyl.In other embodiments, R 24for C 6alkyl.
Further in other embodiments, R 20for C 3-C 30alkyl-carbonyl, and R 20there is following structure (XXX):
R wherein 25for hydrogen or-SR 26, R wherein 26for hydrogen, C 1-C 30alkyl, heterocyclic radical, aryl or heteroaryl, and q is 0 to 6 integer.
How other embodiments that go up in office, R 23when occurring, be all hydrogen, halo, C independently at every turn 1-C 30alkyl, C 1-C 30alkoxyl, aryl, heteroaryl, heterocyclic radical or Heterocyclylalkyl.
In some other embodiments, only 3 ' of oligomer end is incorporated on 1 above-described group.In some other embodiments, only 5 ' of oligomer end is incorporated on 1 above-described group.In other embodiments, 3 ' and 5 ' end all comprises 1 above-described group.Terminal groups can be selected from any special groups of setting forth in above-described any 1 group or table 3.
the representational terminal groups of table 3.
Figure BDA0000447333530000761
Figure BDA0000447333530000771
Figure BDA0000447333530000781
Figure BDA0000447333530000791
Figure BDA0000447333530000801
Figure BDA0000447333530000811
C. the performance of conjugate
As mentioned above, the disclosure relates to the conjugate (that is, oligomer) of carrier peptides and oligonucleotide analogs.Oligomer can comprise give oligomer expectation the various modifications of performance (for example, the antisense of increase is active).In certain embodiments, oligomer comprises and contains by connecting the skeleton of the morpholino circulus sequence of combination between subunit, between described subunit, connect 3 ' end of 1 morpholino circulus is connected on 5 ' end of contiguous morpholino circulus, wherein each morpholino circulus is incorporated in the part of base pairing, so that oligomer can be attached on target nucleic acid in sequence-specific mode.Morpholino circulus can have following structure (i):
Wherein Pi is the part of base pairing independently at every turn when occurring.
Each morpholino circulus supports base pairing part (Pi), to form base pairing partial sequence, its be usually designed to can with cell in or selected antisense target hybridization in the subject of receiving treatment.Base pairing part can be for purine or the pyrimidine of finding in n DNA or RNA (A, G, C, T or U) or analog, as hypoxanthine (base composition of nucleoside inosine) or 5-methylcytosine.Can also utilize the analog base of the binding affinity of giving oligomer raising.Thus, exemplary analog comprises pyrimidine that C5-propinyl modifies, 9-(amino ethoxy) phenoxazine (G-hair fastener) etc.
As mentioned above, according to one aspect of the present invention, can modify oligomer and comprise one or more (B) connection, for example every 2-5 uncharged connection connects up to about 1 (B), and every 10 uncharged 3-5 individual (B) that are connected to connect conventionally.Some embodiment also comprises that one or more (B) type connects.In some embodiments, if be connected to type (B) up to about half skeleton, the optimum that can see antisense activity improves.When thering is peanut for example (B) of 10-20% connecting, conventionally can see some but off-peak raising.
In one embodiment, (A) type is connected along skeleton and scatters with (B) type.In some embodiments, oligomer does not have along (A) of the strict alternate mode of its whole length and is connected with (B).Except carrier peptides, oligomer can also optionally comprise as above 5 ' and/or 3 ' and modify.
A plurality of (A) contiguous block and (B) oligomer of contiguous block have also been considered to have; For example, a plurality of (B) contiguous block can be positioned at the side of center (A) contiguous block, or as the same conversely.In one embodiment, oligomer has 5 ', 3 ' approximate isometric end and is greater than approximately 50% with (B) of ，Qie center, center or the percentage ratio (A) being connected, or is greater than approximately 70%.Oligomer length range for antisense application is generally approximately 10 to approximately 40 subunits, more preferably from about 15 to 25 subunits.For example, the oligomer of the present invention with 19-20 subunit (length useful to antisense scant polymer) can have 2 to 7 ideally, for example 4 to 6 or 3 to 5 (B) connection, and remaining (A) connects.The oligomer with 14-15 subunit can have 2 to 5 ideally, and for example 3 or 4 (B) connects, and remaining (A) connects.
Can also be by connecting morpholino subunit between the non-subunit based on phosphorus, as following further describing.
Can also use other oligonucleotide analogs to connect, when its state of take unmodified exists, as uncharged, but also can there is the amino substituent group of side.For example, 5 ' nitrogen-atoms on morpholino ring can be connected to (or carbamide connect, wherein phosphorus is replaced by carbon or sulfur respectively) for sulfonamide.
In some embodiments of antisense application, oligomer can with nucleic acid target sequence 100% complementation, or it can comprise mispairing, for example, be used for holding variant, as long as the heteroduplex forming between oligomer and nucleic acid target sequence is enough stable, and can tolerate the effect of contingent other pattern degradeds in nucleus enzyme and body.Mispairing (if existence) makes the unsettled probability of stub area of heteroduplex be less than zone line.According to the double-stranded stability principle of knowing, the mispairing number of permission depends on the position that in the percentage ratio of G:C base pair in the length, two strands of oligomer and two strands, mispairing occurs.Although this type of antisense scant polymer not necessarily with nucleic acid target sequence 100% complementation, it still can be effectively and stable and be attached on target sequence specifically, thus nucleic acid target target biological activity, for example the expression of the albumen of coding is adjusted.
The double-stranded stability forming between oligomer and target sequence is in conjunction with T mwith the function of this two strands to the sensitivity of cell enzyme action.Can measure antisense compounds about the T of complementary series RNA by conventional method mas Hames et al., Nucleic Acid Hybridization, IRL Press, the method for describing in 1985, pp.107-108, or as Miyada C.G. and Wallace R.B., 1987, Oligonucleotide hybridization techniques, described in Methods Enzymol.Vol.154pp.94-107.
In some embodiments, the combination T about complementary series RNA that each antisense scant polymer has mbe greater than body temperature, or in other embodiments, be greater than 50 ℃.In other embodiments, T mfor 60-80 ℃ or larger.According to the principle of knowing, by increasing the ratio of the paired base of C:G in two strands, and/or by increasing the length (with base pair form) of heteroduplex, can increase oligomer compound about the T of the RNA hybridization based on complementary m.Meanwhile, in order to optimize cellular uptake, the size of restriction oligomer may be favourable.For this reason, be in a ratio of and obtained high T mvalue need to be greater than the compound of 20 bases, conventionally preferably 20 bases or still less base length show the compound of high Tm (50 ℃ or larger).For some application, longer oligomer, for example, be longer than 20 bases and may have some advantage.For example, in certain embodiments, longer oligomer is being particularly useful for exon skipping or splicing regulation and control.
Targeting sequence base can, for normal DNA base or its analog, for example, can be carried out with target sequence RNA base uracil and the creatinine of Watson-Crick base pairing.
When target nucleotide is uracil residue, oligomer can also be incorporated to guanine base to replace adenine.When target sequence there are differences and when the variation of any given nucleotide residue is cytosine or uracil, this is useful between different virus species.Variant sites place by targeting oligomer is used guanine, can utilize the guanine known and the ability of uracil (being called C/U:G base pairing) base pairing.By being incorporated to guanine in these positions, single oligomer can be effectively using the RNA target variability of wider range as target.
Compound (for example, connection, terminal groups between oligomer, subunit) can exist with different isomeric form, for example constitutional isomer (for example, tautomer).About stereoisomer, compound can have chiral centre, and can occur as the mixture of racemic modification, enantiomer enrichment, independent enantiomer, mixture or diastereomer or independent diastereomer.All these isomeric form are all included in the present invention, and comprise their mixture.Compound can also have axle chirality, and it can cause the formation of atropisomer.In addition, some crystal formations of compound can exist for polymorphic, and it is included in the present invention.In addition, some compounds can also form solvate with water or other organic solvents.This type of solvate is also included within the scope of the invention similarly.
Oligomer described herein can be used for to the method for Profilin generation or virus replication.Therefore, in one embodiment, the nucleic acid of this albuminoid of encoding is exposed to as oligomer disclosed herein.In aforesaid further embodiment, antisense scant polymer comprises 5 ' or the 3 ' terminal groups of modifying or their combination, as disclosed herein, thereby and base pairing part B formed can be at certain position place effectively with the nucleic acid moiety hybridization sequence that effectively Profilin produces.In one embodiment, the ATG initiation codon subregion that this position is mRNA, the splicing site of Pre-mRNA, or viral target sequence as described below.
In one embodiment, oligomer has about being attached to the Tm of approximately 50 ℃ of being greater than on target sequence, and it can be absorbed by mammalian cell or bacterial cell.In another embodiment, oligomer can be incorporated into transport section for example on arginine enrichment peptide, as described in this article, and to promote this type of picked-up.In another embodiment, the end modified transport section that can be used as described herein works, and is absorbed promoting by mammal and/or bacterial cell.
In the below with No. 5185444 United States Patent (USP) and WO/2009/064471, preparation and the performance of morpholino oligomer described in more detail, they are all incorporated to herein separately by reference.
D. the preparation of conjugate and giving
The disclosure also provides the preparation of disclosed conjugate and has sent.Therefore, in one embodiment, the disclosure relates to the compositions comprising as peptide-oligomer conjugate disclosed herein and pharmaceutically acceptable carrier.
Conjugate is effectively delivered to the importance that target nucleic acid is treatment.The approach that antisense scant polymer is sent includes, but not limited to various systemic approach, comprise per os and parenteral route, for example, intravenous, subcutaneous, endoperitoneal and intramuscular, and suction, percutaneous and local delivery.Suitable approach can be determined by those skilled in the art, its applicable situation that receives the object for the treatment of.For example, the suitable pathways of sending antisense scant polymer in treatment in skin viral infection is local delivery, and is used for the treatment of the sending as by sucking of antisense scant polymer of viral respiratory tract infection.Oligomer directly can also be delivered in viral infection site or blood flow.
Conjugate can give on any physiology and/or in pharmaceutically acceptable carrier easily.Such composition can comprise in the pharmaceutically acceptable carrier of the multiple standards that those of ordinary skills adopt any.Example includes, but not limited to saline, phosphate buffered saline (PBS) (PBS), water, ethanol water, such as Emulsion, tablet and the capsule of oil/water Emulsion or triglyceride Emulsion.According to the pattern that gives of selecting, the selection of suitable physiologically acceptable carrier will change.
Conventionally can be for example, by compound of the present invention (, conjugate) as free acid or free alkali.Or, can use compound of the present invention with the form of acid or base addition salts.Can prepare by method well known in the art the acid-addition salts of free amine group compound of the present invention, and it can be formed by organic and mineral acid.Suitable organic acid comprises maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, pyrovinic acid, acetic acid, trifluoroacetic acid, oxalic acid, propanoic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, Palmic acid, glycolic, glutamic acid and benzenesulfonic acid.Suitable mineral acid comprises hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid and nitric acid.Base addition salts comprises those salt that formed by carboxylate anion, and comprise by for example, such as (being selected from alkali and alkaline earth metal ions, lithium, sodium, potassium, magnesium, barium and calcium) and the derivant (for example, benzhydryl ammonium, benzyl ammonium, 2-hydroxyethyl ammonium etc.) that is substituted of the salt that forms of the organic and inorganic cation of ammonium ion and they.Therefore, " the acceptable salt of pharmacy " of nomenclature structure (I) is intended to comprise any and all acceptable salt forms.
In addition, prodrug is also included in the context of the present invention.Prodrug is for being administered to the patient Shi Ke any covalently bound carrier of releasing structure (I) compound in vivo when this type of prodrug.Conventionally by modifying in a certain way functional group, so that this modification can or cutly in vivo produce maternal compound and prepares prodrug by routine operation.Prodrug comprises, compound of the present invention for example, wherein hydroxyl, amino or sulfydryl be incorporated into any when being administered to patient its group that can cut, thereby form this hydroxyl, amino or sulfydryl.Therefore, the representative example of prodrug includes, but is not limited to acetate, formates and the benzoate derivant of ethanol and the amido functional group of structure (I) compound.And, under the situation of carboxylic acid (COOH), can adopt ester, as methyl ester, ethyl ester etc.
In some cases, liposome can be used for promoting antisense oligonucleotide to take in cell.(see, for example, Williams, S.A., Leukemia10 (12): 1980-1989,1996; Lappalainen et al., Antiviral Res.23:119,1994; Uhlmann et al., antisense oligonucleotides:a new therapeutic principle, Chemical Reviews, Volume90, No.4, pages544-584,1990; Gregoriadis, G., Chapter14, Liposomes, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979).Can also as carrier, give antisense scant polymer by hydrogel, for example, described in WO93/01286.Or, can give oligonucleotide with microsphere or particulate form.(see, for example, Wu, G.Y.and Wu, C.H., J.Biol.Chem.262:4429-4432,1987).Or, use the inflation microbubble compound with antisense scant polymer can be increased to sending of target tissue, described in No. 6245747 United States Patent (USP).Can also use slow releasing composition.These can comprise shaped granule as the semi-permeable polymeric matrices of thin film or microencapsulation form.
In one embodiment, the cell by making viral infection contacts with can effectively suppressing the antisense agents that specific virus copies, Antisense Suppression in the viral infection for the treatment of host animal effectively.Antisense agents in suitable pharmaceutical carrier can be administered to mammalian object, for example the people of given viral infection or domestic animal.Expected that antisense oligonucleotide can stop the growth of the RNA viruses in host.Can quantitatively reduce RNA viruses, or eliminate this virus make it to host's normal growth or growth have seldom or there is no an ill-effect.
Aspect of this method, described object is the experimenter, for example, is diagnosed as the patient who suffers from part or general viral infection.Patient's situation can also indicate the preventative of antisense scant polymer of the present invention to give, and for example, under such situation, wherein patient (1) is immunoincompetent; (2) be by burns victims; (3) there is inlying catheter; Or (4) are about to experience or have just lived through operation.In a preferred embodiment, oligomer is the di(2-ethylhexyl)phosphate amide morpholino oligomer being included in pharmaceutical acceptable carrier, and oral delivery.In another preferred embodiment, oligomer is the di(2-ethylhexyl)phosphate amide morpholino oligomer being included in pharmaceutical acceptable carrier, and sends through vein (i.v.).
In the Another Application of this method, object is domestic animal, for example, and chicken, turkey, pig, milch cow or goat etc., and treatment is for preventative or curative.The present invention also comprises livestock and poultry food composition, the food grain that it contains the antiviral antisense compounds that is supplemented with sub-therapeutic dose the above-mentioned type.Also expected with being supplemented with the food grain stock raising of sub-treatment level antiviral oligonucleotides compositions and the method for poultry, be wherein improved to sub-therapeutic dose as mentioned above antiviral oligonucleotides compositions supplemented food grain.
In one embodiment, can effectively cause that at least amount and the mode of the peak value blood concentration of 200-400nM antisense scant polymer give conjugate.Conventionally with certain interval, during approximately 1 to 2 weeks, give the antisense scant polymer of one or more dosage.The preferred dose that per os gives is the every 70kg of about 1-1000mg oligomer.In some cases, the dosage that is greater than 1000mg oligomer/patient may be necessary.For i.v., give, preferred dose is the every 70kg of about 0.5-1000mg oligomer.Can give conjugate by certain interval short time, for example, give every day two weeks by a definite date or shorter time.Yet, in some cases, within the longer time period, intermittently give conjugate.Can be after giving, or when giving, give antibiotic or other treatment.Can be indicated according to the immunoassay result based on the object of receiving treatment, other biochemical tests and physiology inspection, adjustment for the treatment of scheme (dosage, frequency, approach etc.).
According to persistent period, dosage, frequency with give approach, and the situation of the object of receiving treatment, use effective interior therapeutic scheme of conjugate of the present invention to change (that is, preventative give giving response locality or systemic infection).Therefore,, in order to obtain preferred therapeutic outcome, this type of interior therapeutic usually needs detection by the being suitable for subject particular type viral infection corresponding adjusting of supervision and dosage or therapeutic scheme in addition.For example, can be by the general index of disease and/or infection, as the detection of full blood count (CBC), nucleic acid detection method, immunologic diagnosis test, viral cultures or heteroduplex monitors treatment.
Can be before and after, during giving this antisense scant polymer, from taking from the biological sample (tissue, blood, urine etc.) of object, measure and in body, give the effect that suppresses or eliminate the antiviral conjugate of one or more type RNA viruses growths of the present invention.The analysis of this type of sample comprises: (1) is used program well known by persons skilled in the art, running gel mobility analysis for example, and supervision and target sequence and non-target sequence form the existence of heteroduplex or do not exist; (2) amount that the virus protein that monitors as record by the standard technique such as ELISA or Western trace produces, or the effect of (3) measurement to virus titer, for example, by the method for Spearman-Karber.(see, for example, Pari, G.S.et al., Antimicrob.Agents and Chemotherapy39 (5): 1157-1161,1995; Anderson, K.P.et al., Antimicrob.Agents and Chemotherapy40 (9): 2004-2011,1996, Cottral, G.E. (ed) in:Manual of Standard Methods for Veterinary Microbiology, pp.60-93,1978).
E. the preparation of conjugate
The preparation of the oligomer that connects between the subunit of morpholino subunit, modification and contain it can be described in embodiment and No. 5185444 and No. 7943762 United States Patent (USP), and they are all incorporated to herein by reference at this.Can prepare morpholino subunit according to following general reaction scheme I.
the preparation of reaction scheme 1. morpholino subunits
Figure BDA0000447333530000881
About reaction scheme 1, wherein B represents base pairing part, and PG represents protecting group, as implied abovely can prepare morpholino subunit from corresponding ribonucleotide (1).By the protecting group precursor with suitable for example trityl chloride react, can optionally protect morpholino subunit (2).3 ' protecting group is removed between synthesis stage at solid-state oligomer conventionally, as following more detailed description.Base pairing part can be protected suitably, synthetic for solid phase oligomer.The appropriate protection base of adenine and cytosine comprises benzoyl, and the appropriate protection base of guanine comprises phenylacetyl group, and the appropriate protection base of hypoxanthine (I) comprises pivaloyl oxygen methyl.Pivaloyl oxygen methyl can be incorporated on the N1 position of hypoxanthine heterocyclic base.Although can adopt unprotected hypoxanthine subunit, the productive rate when this base is protected in priming reaction is much higher.Other suitable protecting groups comprise disclosed protecting group in No. 12/271040 pending trial U. S. application, and it is all incorporated to herein by reference at this.
By 3, react and produce the morpholino substituent group of the coupling part (5) with expectation with the phosphorus compound 4 of activation.Can use the method for any number well known by persons skilled in the art to prepare the compound of structure 4.For example, can be by corresponding amine and this compounds of phosphoryl chloride phosphorus oxychloride reaction preparation.Thus, can use any method known in the art to prepare described amine-initiated material, those methods of for example describing in embodiment and No. 7943762 United States Patent (USP).Although above scheme has been described the preparation that (B) type connects, (for example, X is-NR 8r 9), can prepare (A) type by similar mode and connect (for example, X is dimethyl amine).
The compound of structure 5 can be comprised to the oligomer connecting between subunit for solid phase automatization oligomer is synthetic with preparation.These class methods are known in the art.Briefly, can be at the compound of the 5 ' end modified structure 5, so that include linking arm on solid phase carrier.For example, can be by comprising L 1and/or R 19linking arm compound 5 is connected on solid phase carrier.In Fig. 3 and Fig. 4, set forth illustrative methods.By this way, complete oligomer synthetic and by this oligomer after solid phase carrier cuts down, oligomer can comprise 5 '-end modified.For example, once supported, 5 protecting group (, trityl) can be removed, and unhindered amina can with the activation phosphorus partial reaction of the second compound of structure 5.Repeat this sequence until obtain the oligomer of desired length.If expectation obtains 5 ', modify, can remove or retain 5 ' the end protecting group that is positioned at terminal.Can use the method for any number, oligomer is removed from solid phase carrier, for example, by base, process to cut to the connection on solid phase carrier.
Can be at suitable activator (for example, HATU) under existing, by the peptide of expectation (according to standard peptide synthetic method preparation known in the art) for example, is combined to prepare peptide oligomer conjugate with the oligomer that comprises free NH (3 ' NH of morpholino oligomer).Can use multiple technologies known in the art, for example SCX chromatography purification conjugate.
The morpholino subunit of modification and the preparation of peptide oligomer conjugate have been described in an embodiment in more detail.The method that can use method described herein, methods known in the art and/or describe by reference herein, the peptide oligomer conjugate that preparation contains the modified connection of any number.The overall modification of the PMO+ morpholino oligomer of preparing by previous description (for example seeing the open WO2008036127 of PCT) has also been described in embodiment.
F. the antisense of oligomer is active
The method that the disclosure also provides Profilin matter to produce, the method comprises is exposed to as peptide-oligomer conjugate disclosed herein the nucleic acid of coded protein.Therefore, in one embodiment, the nucleic acid of this proteinoid that makes to encode is exposed to conjugate, and as disclosed herein, the sequence that wherein base pairing part Pi forms is effectively hybridized with the effective nucleic acid moiety of the position of Profilin matter generation.Oligomer can targeting for example, the ATG initiation codon subregion of mRNA, the splicing site of Pre-mRNA or viral target sequence as described below.
In another embodiment, the disclosure provides the method for antisense activity of the peptide oligomer conjugate of the oligonucleotide analogs that improves the part that comprises the morpholino subunit sequence that has by connecting combination between subunit, supports base pairing, and the method comprises carrier peptides is as described in this article attached on oligonucleotide.
In some embodiments, can pass through the raising of following proof antisense activity:
(i) when the combination of antisense scant polymer and its target sequence can effectively stop the translation initiation codon of protein of coding, the expression providing with respect to corresponding not modified oligomer, the protein expression of coding reduces, or
(ii) when the combination of antisense scant polymer and its target sequence can effectively stop the generation in aberrant splicing site in the Pre-mRNA of code for said proteins while correctly being spliced, the expression providing with respect to corresponding not modified oligomer, the protein expression of coding increases.Below further described the analysis of applicable these effects of measurement.In one embodiment, the splicing correction translation in cell free translation analysis, cell culture is modified this activity is provided in analyzing or proofreading and correct available from the splicing of function animal model system as described in this article.In one embodiment, activity has been enhanced at least 2 times, at least 5 times or at least 10 times.
Below describe the different exemplary application of conjugate of the present invention, comprised antiviral application, treatment neuromuscular disease, antibacterial infection, inflammation and POLYCYSTIC KIDNEY DISEASE.This description is not intended to limit by any way the present invention, but is used for the humans and animals disease disease scope that illustration can use conjugate described herein to process.
G. the exemplary treatment purposes of conjugate
The oligomer being attached in carrier peptides comprises good effect and hypotoxicity, thereby produces than better treating window with the treatment window that other oligomers or peptide-oligomer conjugate obtain.Below describe and provide exemplary, but the example of nonrestrictive this conjugate therapeutic use.
1. the stem ring secondary structure of targeting ssRNA virus
The exemplary antisense antiviral compound of one class is morpholino oligomer as described in this article, its have 12-40 subunit sequence and with 40 bases of 5' end of the just RNA chain of target virus in the targeting sequence of stem ring secondary structure relevant range complementation.(see, for example, WO/2006/033933 PCT open or No. 20060269911 and No. 20050096291 U. S. application open, it is all incorporated to this paper by reference).
Method comprises: first determine that viral target sequence---the region in 40 bases of 5' end of infectious virus positive-sense strand, its sequence of virus wherein can form inner stem ring secondary structure.Pass through subsequently progressively solid phase synthesis, structure has and the morpholino oligomer of targeting sequence of at least 12 subunits that can form the viral genome regional complementarity of inner duplex structure, wherein said oligomer can form the heteroduplex structure being comprised of viral positive-sense strand and oligonucleotide compound with viral target sequence, and it is characterized by the destruction of dissociate Tm and this type of loop-stem structure of at least 45 ℃.This oligomer is incorporated in carrier peptides described herein.
By can the minimum free energy state based on search input RNA sequence carrying out the computer program of secondary structure prediction, can be by analyzing 5'-end sequence, for example 40 bases of 5'-end are identified target sequence.
At related aspect, can be by conjugate for suppressing to there is strand in mammalian host cell, just genomic infectious RNA virus and be selected from the method copying of following a kind of virus: flaviviridae, Picornaviridae (Picornoviridae), Caliciviridae, Togaviridae, Arteriviridae, coronaviridae, Astroviridae or hepatitis virus section.The method comprise by viral inhibitory amount as described herein conjugate be administered to infected host cell, described conjugate have with 40 bases of the genomic 5'-end of positive strand virus in can form the targeting sequence of at least 12 subunits of the regional complementarity of inner stem ring secondary structure.When being administered to host cell, described conjugate can effectively form heteroduplex structure, and its (i) is comprised of viral positive-sense strand and oligonucleotide compound, and (ii) is characterized as the destruction of dissociate Tm and this type of stem ring secondary structure of at least 45 ℃.Described conjugate can be administered to the mammalian object that has infected virus or had viral infection risk.
Below respectively the exemplary targeting sequence of the end loop-stem structure of targeting dengue virus and Japanese encephalitis virus is listed as to SEQ ID NOs:1 and 2.
Can also in No. 11/801885 U. S. application and the open WO/2008/036127 of PCT, find other exemplary targeting sequences of the end loop-stem structure of targeting ssRNA virus, it is merged in herein by reference.
2. the first open reading frame of targeting ssRNA virus
The exemplary conjugate of Equations of The Second Kind is for for suppressing the conjugate of the viral growth of picornavirus, Calicivirus, togavirus, coronavirus and flaviviridae, and virus wherein has the first open reading frame that is less than the polyprotein that the strand of 12kb, just genome and coding contain several functions albumen.In specific embodiment, virus is from the RNA viruses of coronaviridae or from west Nile virus, yellow fever virus or the dengue virus of flaviviridae.Inhibition conjugate comprises antisense scant polymer described herein, and it has substantially and the targeting base sequence of viral target complement sequence of crossing over the AUG initiation site of viral genome the first open reading frame.In an embodiment of the method, conjugate is administered to and has infected viral mammalian object.See, for example, WO/2005/007805 PCT is open and No. 2003224353 U. S. application is open, and it is incorporated to herein by reference.
Preferred target sequence is for crossing over the region of the AUG initiation site of viral genome the first open reading frame (ORF1).A described ORF conventionally coding contains the polyprotein such as the non-structural protein of polymerase, unwindase and protease." cross over AUG initiation site " and refer to that target sequence comprises at least 3 bases in a side of AUG initiation site, and comprise at least 2 bases (at least 8 bases altogether) at opposite side.Preferably, its each side at initiation site comprises at least 4 bases (at least 11 bases altogether).
More generally, preferably target site comprises the conservative target between multiple viral isolates.Other favourable sites comprise IRES (internal ribosome entry site), trans-activator binding site and initial site of copying.By the targeting host cell gene that cell entry and host response virus exists of encoding, can effectively will be able to provide the complicated and large viral genome of a plurality of redundancy genes as target.
From knowing resource and can obtain multiple virus genome sequence such as NCBI Genbank data base.Can also be at gene database or the AUG initiation site based on the middle evaluation of quoting of its ORF1, or the sequence of searching AUG codon in can the region by the ORF1 initiation site in expection finds this site.
Below provided general gene organization form separately in 4 Viraceaes, and the selected member's who obtains in each section subsequently (genus and species or strain) exemplary target sequence.
3. targeting influenza virus
The exemplary conjugate of the 3rd class is used to suppress growth and the treatment viral infection of influenza virus coe virus.In one embodiment, host cell is contacted with conjugate as described in this article, for example comprising can be effectively and the conjugate that is selected from the base sequence of following target region hybridization: 1) 25 bases of 5 ' or 3 ' end of negative-sense viral rna fragment; 2) 25 bases of end of 5 ' of just cRNA or 3 ' end; 3) the AUG initiation site of influenza virus mRNAs 45 bases around; With 4) the splicing donor of influenza mRNAs or acceptor site 50 bases around of experience alternative splicing.(see, for example, WO/2006/047683 PCT is open; No. 20070004661 U. S. application is open; And No. 2010/056613 PCT application and No. 12/945081 U. S. application, it is incorporated to herein by reference).
Thus, exemplary conjugate comprises the conjugate that comprises the oligomer that contains SEQ ID NO:3.
Table 4. comprises the influenza targeting sequence through connection or terminal groups between the subunit of modification
*3 '-benzhydryl; *+ be connected to the trimethyl glycine in PMO+ junction acidylate; PMOm is illustrated on 3-nitrogen position the T base with methyl.
Described conjugate especially can be used for treating mammiferous influenza infection.Conjugate can be administered to the mammalian object that has infected influenza virus or had influenza infection risk.
4. the virus of targeting Picornaviridae
The exemplary conjugate of the 4th class is used to suppress growth and the treatment viral infection of Picornaviridae virus.Described conjugate especially can be used for treating mammiferous enterovirus and/or rhinovirus infection.In this embodiment, conjugate comprises the morpholino oligomer with a sequence 12-40 subunit, its comprise have with virus 5 ' at least 12 subunits of targeting sequence of viral RNA Serial relation regional complementarity in two 32 of untranslated region conservative nucleotide regions in one.(see, for example, No. WO/2007/030576 and WO/2007/030691 PCT are open, or pending trial and No. 11/518058 and No. 11/517757 U. S. application that own together, and it is incorporated to this paper by reference).Exemplary targeting sequence is being listed as SEQ NO:6 below.
5. targeting flaviviridae
The exemplary conjugate of the 5th class is used to suppress copying of the interior banzi virus of zooblast.The exemplary conjugate of the type comprises morpholino oligomer, it is the length of 8-40 nucleotide base, and has the sequence with at least 8 bases that comprise at least part of normal chain Fo Lawei virus 5 '-cyclisation sequence (5'-CS) of (flaviviral) RNA or the viral positive chain RNA genome area complementation of 3 '-CS sequence.Highly preferred target is 3'-CS, and the exemplary targeting sequence of dengue virus is being listed as SEQ ID NO:7 below.(see, for example, (WO/2005/030800) number PCT discloses or pending trial and No. 10/913996 U. S. application that own together, and it is incorporated to herein by reference).
6. targeting norovirus (Nidovirus) coe virus
The exemplary conjugate of the 6th class the copying of norovirus in zooblast that be used to suppress to be infected by the virus.Such exemplary conjugate comprises morpholino oligomer, it contains 8-25 nucleotide base, and the sequence with base pairing between the transcription regulating nucleotide sequence (TRS) that can destroy in positive strand virus genome 5' leader region and minus strand 3' sub-gene group district (is shown in, for example, WO/2005/065268 PCT is open or No. 20070037763 U. S. application is open, and it is incorporated to herein by reference).
7. targeting filamentous virus (Filoviruses)
In another embodiment, by cell is contacted with conjugate as described in this article, for example have with AUG initiation site region by normal chain mRNA in the conjugate of targeting base sequence of target complement sequence of at least 12 continuous base compositions, can be by one or more methods that conjugate copies in host cell for suppressing Ebola virus or Marburg virus as described in this article, described in further describing as following.
Filamentous virus genome is the single stranded RNA of approximately 19000 bases, and it is ameristic, and is antisense orientation.This genome encoding is from the 7 kinds of protein of the monocistronic mRNA s with vRNA complementation.
Target sequence is for crossing over or be just positioned at AUG start codon downstream (25 bases) or normal chain (justice) the RNA sequence of upstream (100 bases) or 30 bases of 3' end of negative strand viruses RNA of selected Ebola virus albumen.Preferred protein targets is designated as varial polymerases subunit VP35 and VP24, although L, nucleoprotein NP and VP30 are also under consideration.In these, early protein is more favored, and for example, the L polymerase of the more late expression of VP35 is more favored.
In another embodiment, by cell is contacted with having as described in this article with the conjugate of the targeting base sequence of the target complement sequence of at least 12 continuous base compositions in AUG initiation site region by filamentous virus mRNA sequence normal chain mRNA, can be by one or more methods that conjugate copies in host cell for suppressing Ebola virus or Marburg virus as described in this article.(see, for example, WO/2006/050414 PCT discloses or No. 7524829 and No. 7507196 United States Patent (USP), and the continuous application of No. 12/402455, No. 12/402461, No. 12/402464 and No. 12/853180 U. S. application, and it is incorporated to herein by reference).
8. targeting arenavirus
In another embodiment, by a kind of species in Arenaviridae, can be by the method that conjugate infects for suppressing mammalian cell inner virus as described in this article.In one aspect, conjugate can be used for the treatment of and infect viral mammalian object.(see, for example, WO/2007/103529 PCT discloses or No. 7582615 United States Patent (USP), and it is incorporated to herein by reference).
Table 5 be exemplary by conjugate of the present invention the target virus inventory as target, its old world by them or the classification of New World arenavirus are organized.
Table 5. target arenavirus
Figure BDA0000447333530000961
The genome of arenavirus is comprised of two single stranded RNA fragments being appointed as S (little) and L (greatly).In virion, S section is roughly 2:1 to the mol ratio of L section RNA.After measured the full S section RNA sequence of several arenaviruss, and its scope is 3366-3535 nucleotide.Also measured the full L section RNA sequence of several arenaviruss, and it is 7102 to 7279 nucleotide.In last 19 nucleotide of 3 ' end sequence of S and L RNA fragment, 17 is identical.In all known arenaviruss, these end sequences are guarded.5 of each geneome RNA head end '-end 19 or 20 perfect complementations of 3 ' end that nucleotide is corresponding with each.Due to this complementarity, 3 ' and 5 ' end is considered to can base pairing and form panhandle structure (panhandle structures).
Complementary RNA (vcRNA) chain of virus that infectious virus particle or viral RNA (vRNA) copy to form anti-gene occurs in infected cell.VRNA and the vcRNA complementary mRNAs that all encodes; Therefore, arenavirus is classified as ambisense RNA virus, but not negative justice or just RNA viruses.The ambisense orientation of viral gene is positioned in L section and S section.NP and pol gene lay respectively at 3 ' end of S and L vRNA fragment, and by with conventional antisense coding (that is, they are by transcribing vRNA or genome complementation mRNAs is expressed).Be positioned at the gene of 5 ' end of S and L vRNA fragment---be respectively GPC and Z, in mRNA mode, be encoded, but not evidence suggests that they are directly from genome vRNA, to translate.These genes are not by from anti-genome, (that is the genome vRNA total length complementary copy that, vcRNA), plays replicative intermediate effect is carried out the mRNAs of open gene group meaning and expressed.
The exemplary targeting sequence of arenavirus coe virus is being listed as SEQ ID NO:8 below.
9. targeting respiratory syncytial virus
Respiratory syncytial virus (RSV) is most important a kind of respiratory pathogen in child's body.The lower respiratory tract disease that is less than the child of 1 years old that RSV causes, as bronchiolitis and pneumonia, often needs hospitalization.Child and the preterm children of suffering from heart and lung diseases are especially easy to the serious disease that caused by this infection.Rsv infection is also old man and high-risk adult's important diseases, and it is the second the most common definite cause of disease (Falsey, Hennessey et al.2005) that causes old people's viral pneumonia.World Health Organization (WHO) estimates that RSV worldwide causes 6,400 ten thousand routine clinical infections and 160,000 example death every year.The vaccine that there is no at present available prevention rsv infection.Although many, there is major progress to the understanding of RSV biology, epidemiology, pathophysiology and host immune response in us in the past few decades, still exists about having infected the very large arguement of the infant optimum management of RSV.Ribavirin (Ribavirin) is the antiviral drugs that unique approval is used for the treatment of rsv infection, but its use only limits to high-risk or severe disease baby.The application of ribavirin is subject to its cost, variable effect and produces the restriction (Marquardt1995 of antiviral trend; Prince2001).Current need to be well-known to other effective anti-RSV medicaments.
The PMO of known peptide combination (PPMO) is in tissue culture and in animal model system, can effectively suppress RSV (Lai, Stein et al.2008) in vivo.In the culture of two kinds of people's air flue cell lines, the anti-RSV that has tested two kinds of antisense PPMOs that are designed to the sequence that targeting comprises 5 of RSV L mRNA '-stub area and translation initiation site region is active.A kind of (RSV-AUG-2 in them; SEQ ID NO10), reduced >2.0log 10virus titer.Before RSV inoculation, with RSV-AUG-2PPMO intranasal (i.n.) treatments B ALB/c mice, after infection, (p.i.), in the time of the 5th day, produced 1.2log in lung tissue 10the minimizing of virus titer, and within the 7th o'clock after infection, alleviated pneumonia.These data show that RSV-AUG-2 provides effective anti-RSV active, is worth further studying (Lai, Stein et al.2008) as the material standed for of potential treatment application.Although there is the success of RSV-AUG-2PPMO as above, it is suitable using as conjugate disclosed herein solves the toxicity problem relevant to previous peptide conjugate.Therefore, in another embodiment of the invention, by cell is contacted with conjugate as described in this article, the conjugate for example with the targeting base sequence of the target complement sequence of at least 12 continuous base compositions in the AUG initiation site region with mRNA by from RSV, can be by one or more conjugates as described in this article for suppressing the method that RSV copies in host cell, described in further describing below.
The key component of the L gene code viral RNA RNA-dependent polymerase complex of RSV.With RSV-AUG-2PPMO form for cross over RSV L gene mRNA AUG translation initiation site codon sequential design antisense PPMO with from " gene the is initial " sequence (GS) of 5 ' end that is present in L mRNA to the sequence complementation that enters 13 nucleotide of coded sequence.Therefore, preferred L gene targeting sequence and 5 ' end from L gene mRNA extend upward 40 bases or enter any 12 continuous base complementrities of 22 bases of L gene of the sequence that is shown as SEQ ID NO:9 encoding as following table 63 ' side.Exemplary RSV L gene targeting sequence is listed as SEQ ID NOs:10-14 in following table 6.Can will between any subunit of the present invention described herein, modify and be integrated in oligomer, with provide the antisense of increase active, in the cell improving, send and/or tissue specificity to improve therapeutic activity.Containing the exemplary oligomer sequence connecting between subunit of the present invention is recited in following table 6.
Table 6.RSV target sequence and targeting sequence
Figure BDA0000447333530000981
10. neuromuscular disease
In another embodiment, provide treatment conjugate to be used for the treatment of the disease disease relevant to the neuromuscular disease of mammalian object.Antisense scant polymer (for example, SEQ ID NO:16) shows Duchenne muscular dystrophy (DMD) is had to activity in MDX mouse model.The exemplary oligomer sequence of having integrated the connection of using is in some embodiments recited in following table 7.In some embodiments, this conjugate comprises and is selected from following oligomer:
(a) antisense scant polymer of targeted human myostatin, it has the base sequence of at least 12 continuous base complementrities in the target region with the people's myostatin mRNA identifying by SEQ ID NO:18, be used for the treatment of amyotrophy disease, (see as described previously, for example, No. 12/493140 U.S. Patent application, it is incorporated to herein by reference; With the open WO2006/086667 of PCT).Exemplary mice targeting sequence is listed as SEQ ID NOs:19-20; With
(b) antisense scant polymer, it can produce exon skipping in having the DMD albumen (dystrophin) of PMO of the sequence that is selected from SEQ ID NOs:22-35, to recover the part activity of dystrophin, be used for the treatment of DMD, (see as described previously, for example, No. WO/2010/048586 and WO/2006/000057 PCT are open or US09/061960 United States Patent (USP) is open, and all these is incorporated to herein by reference).
Use modified connection of the present invention and terminal groups can treat several other neuromuscular diseases.The exemplary compounds that is used for the treatment of spinal cord amyotrophy (SMA) and myotonia atrophica (DM) has below been discussed.
SMA is due to the autosomal recessive disease that in spinal cord, chronic loss α-motor neuron causes, and can affect child and adult.It is the reason (Hua, Sahashi et al.2010) that causes this disease that the minimizing of motor neuron existence (SMN) is expressed.The sudden change that causes SMA is positioned at SMN1 gene, but a kind of parallel homologous genes---SMN2, if expressed from lacking the alternative splicing form of exon 7 (δ 7SMN2), can allow existence by the loss of compensation SMN1.The antisense compounds that has shown targeting intron 6, exon 7 and intron 7 all can induce exon 7 in various degree to include (inclusion) in.The antisense compounds of targeting intron 7 is preferred (for example see, No. WO/2010/148249, No. WO/2010/120820, WO/2007/002390 PCT is open and No. 7838657 United States Patent (USP)).The exemplary antisense sequences that the exon 7 that targeting SMN2 Pre-mRNA induction improve is included in is being listed as SEQ ID NOs:36-38 below.Compare performance known in the art, expected that the modification of using modified connection described herein and terminal groups to select these oligomer sequences will have augmented performance.In addition, expected that the intron 7 of targeting SMN2 gene any oligomer of integrating feature of the present invention all have the potentiality that inducing exon 7 is included in, and provide therapeutic effect for SMA patient.Myotonia atrophica Class1 (DM1) and type 2 (DM2) the main hereditary for cause neuromuscular to degenerate and cause due to toxicity rna expression.DM1 and DM2 respectively to transcribe 3 '-UTR of steinert's disease protein kinase (DMPK) and zinc finger protein 9 (ZNF9) and the poly-CUG of the length in introne 1 region and poly-CCUG and repeat relevantly (for example to see, WO2008/036406).Although normal individual has nearly 30 CTG, repeat, DM1 patient carries 50 to several thousand more big figure repetition.The seriousness of this disease is relevant to the number of repetition with age of onset.The patient of adult age morbidity shows lighter symptom and has and be less than 100 repetitions, and juvenile era morbidity DM1 patient carries nearly 500 repetitions, and congenital situation conventionally has approximately 1000 CTG and repeats.The transcript that containing of increasing, CUG repeated forms secondary structure, with the form of core kitchen range (nuclear foci) in core inner accumulated, and isolate rna binding protein (RNA-BP).Several RNA-BP relate to this disease, comprise that blind flesh sample (MBNL) albumen and CUG are in conjunction with albumen (CUGBP).MBNL protein and light receptor and muscle break up the blind flesh of necessary fruit bat (Mbl) albumen homology.MBNL and CUGBP are confirmed as affecting the Antagonism splicing instrumentality of DM1 transcription thing, as serum cardiac troponin T (cTNT), Insulin receptor INSR (IR) and muscle specific chloride channel (ClC-1).
The antisense oligonucleotide of the repetition that targeting DMPK gene known in the art increases can replace RNA-BP and isolates and reverse myotonic reaction (WO2008/036406) in the animal model of DM1.Expected that the oligomer that comprises feature of the present invention will provide activity and the treatment potentiality of raising for DM1 and DM2 patient.The exemplary sequence that targeting poly-CUG described above and poly-CCUG repeat is listed as SEQ ID NOs:39-55 below, and is further described in No. 13/101942 U. S. application, and it is all incorporated herein.
Expected that the present invention is used for the treatment of other embodiments of neuromuscular disorder, comprised that design is used for treating the oligomer of other DNA repeat instability heredopathias.These diseases comprise Huntington Chorea (Huntington ' s disease), spinocebellar ataxia (spino-cerebellar ataxia), X spinal cord and oblongata amyotrophy and spinocebellar ataxia Class1 0 (SCA10), described in WO2008/018795.
Table 7. comprises the M23D sequence (SEQ ID NO:15) through connection and/or 3 ' and/or 5 ' terminal groups between the subunit of modification
Figure BDA0000447333530001011
Figure BDA0000447333530001051
*dimerization refer to that the connection of 3 ' end of this oligomer by connecting two monomers is by dimerization.For example, described connection can be-COCH 2cH 2-S-CH (CONH 2) CH 2-CO-NHCH 2cH 2cO-or any other suitable connection.EG3 refers to 2,2'-ethylenedioxybis(ethanol). tail (for example seeing the conjugate in embodiment 30 and 31).
11. antibacterial applications
In another embodiment, the present invention includes and be used for the treatment of the conjugate that comprises antibacterial antisense scant polymer that mammalian hosts Endophytic bacteria infects.In some embodiments, the targeting sequence that oligomer comprises 10-20 base and at least 10 continuous bases, the acyl carrier protein (acpP) of continuous base wherein and infectious bacteria, gyrase A subunit (gyrA), ftsZ, ribosome protein S 10 (rpsJ), leuD, mgtC, pirG, the target regional complementarity of the mRNA of pcaA and cma1 gene, wherein said target region is contained antibacterial mRNA or is positioned at translation initiation codon upstream (, 5 ') or downstream (, 3 ') translation initiation codon of the sequence in 20 bases of direction, and it is upper to form heteroduplex that wherein said oligomer joins mRNA to, thereby suppress copying of this antibacterial.
12. regulate nuclear hormone receptor
In another embodiment, the present invention relates to for regulating compositions and the method from nuclear hormone receptor (NHR) expression of nuclear hormone receptor superfamily (NHRSF), is mainly by controlling or change the splicing of the Pre-mRNA of coding this receptor.The example of concrete NHRs comprises: glucocorticoid receptor (GR) (GR), progesterone receptor (PR) and androgen receptor (AR).In certain embodiments, conjugate described herein can cause that the expression non-ligand dependent or other selected form receptors of this receptor increases, and the expression of their inactive form reduces.
Embodiment of the present invention comprise and comprise oligomer, for example, with the selected exon of NHR or the conjugate of the oligomer of intron sequences complementation, exon wherein or intron, except other NHR domains described herein, also comprise " ligand binding exon " and/or the contiguous intron of NHRSF Pre-mRNA.Term " ligand binding exon " refers to and is present in wild type mRNA, but is removed to prepare the exon of non-ligand dependent form mRNA from primary transcript (" Pre-mRNA ").In certain embodiments, the sequence that complementarity can be based on crossing in the Pre-mRNA sequence of splicing site, it includes, but not limited to the complementarity based on crossing over the sequence that exon-intron connects.In other embodiments, the complementarity sequence based on intron only.In other embodiments, the complementarity sequence based on exon only.(see, for example, No. 13/046356 U. S. application, it is incorporated to herein by reference).
NHR instrumentality can be used for treating the disease that NHR is relevant, comprises and transcribes the expression product relevant disease that is subject to the stimulation of NHRs or the gene of inhibition.For example, the instrumentality that can suppress the NHRs of AP-1 and/or NF-κ B can be used for treating struvite and immune disease and such as following disease: osteoarthritis, rheumatic arthritis, multiple sclerosis, asthma, inflammatory bowel, transplant rejection and graft versus host disease, and other types described herein and known in the art.The compound of antagonism trans-activation can be used for the metabolic disease that treatment is relevant to the glucocorticoid levels increasing, as diabetes, osteoporosis and glaucoma and other diseases.In addition, promote the compound of (agonize) trans-activation to can be used for the metabolic disease that treatment is relevant with glucocorticoid deficiency, as Addison's disease (Addison ' s disease) and other diseases.
Embodiment of the present invention comprise the method that intracellular nucleic NHR is active or express that regulates, comprise cell is contacted with the conjugate that comprises carrier protein and antisense scant polymer, antisense scant polymer is wherein by forming by the morpholino nitrogen of a subunit being connected to the morpholino subunit connecting between the phosphorous subunit on the outer carbon of 5' ring of contiguous subunit, wherein said oligonucleotide contain 10-40 base and with the targeting sequence of at least 10 continuous bases of target complement sequence, wherein said target sequence is the Pre-mRNA transcript of NHR, thereby regulate the active of NHR or express.In certain embodiments, oligomer can change the splicing of Pre-mRNA transcript, and increases the expression of the variant of NHR.In some embodiments, the exon skipping wholly or in part of one or more exons of oligomer induction Pre-mRNA transcript.In certain embodiments, the ligand binding domains of described one or more exons coding at least a portion NHR, and the variant non-ligand dependent form that is NHR.In certain embodiments, at least a portion transactivation domain of described one or more exons coding NHR, and variant has the transcriptional activation activity of minimizing.In certain embodiments, at least a portion DNA binding structural domain of described one or more exons coding NHR.In certain embodiments, at least a portion N-end activation domain of described one or more exons coding NHR.In certain embodiments, at least a portion carboxyl terminal domain of described one or more exons coding NHR.In specific embodiment, described variant is attached to NF-KB, AP-l or both are upper, and reduces one or more their the transcribing of proinflammatory target gene.
In certain embodiments, oligomer promotes the trans-activation transcriptional activity of (agonize) NHR.In other embodiments, the trans-activation transcriptional activity of oligomer antagonism NHR.In certain embodiments, oligomer promotes that the trans inhibition of NHR is active.In other embodiments, the trans inhibition of oligomer antagonism NHR is active.In specific embodiment, the trans-activation transcriptional activity of oligomer antagonism NHR, and promote that the trans inhibition of NHR is active.(see, for example, No. 61/313652 U. S. application, it is incorporated to herein by reference).
Embodiment
Except as otherwise noted, all chemicals all obtain from Sigma-Aldrich-Fluka.Benzoyl adenosine, benzoyl cytidine and phenylacetyl group guanosine obtain from Britain Carbosynth Limited.
Use method known in the art and that describe in open with No. 12/271040 pending trial U. S. application and WO/2009/064471 PCT for No. 12/271036 to complete PMO, PMO+, PPMO and contained the synthetic of other PMO that are connected as described in this article modification, they are all incorporated to herein by reference at this.
Substantially described in disclosing by WO/2009/064471 PCT, synthesized the PMO modifying with 3 ' trityl, except trityl removal step is omitted.
Embodiment 1
4-(2,2,2-trifluoroacetyl amido) piperidines-1-t-butyl formate
Figure BDA0000447333530001081
Under stirring, Trifluoroacetic Acid Ethyl Ester (35.6mL, 0.300mol) is splashed in the suspension of 4-amino piperidine-1-t-butyl formate (48.7g, 0.243mol) in DCM (250mL) and DIPEA (130mL, 0.749mol).After 20 hours, by citric acid solution for solution (200mL x3,10%w/v aqueous solution) and sodium bicarbonate solution (200mL x3, concentrated aqueous solution) washing, dry (MgSO 4) and filter by silicon dioxide (24g).With DCM, wash silicon dioxide, and by the eluant partial concentration (100mL) merging, and be directly used in next step.C 12h 19f 3n 2o 3aPCI/MS value of calculation be 296.1, measured value m/z=294.9 (M-1).
Embodiment 2
The fluoro-N-of 2,2,2-tri-(piperidin-4-yl) acetamide hydrochloride
Figure BDA0000447333530001082
The hydrogen chloride solution (250mL, 1.0mol) that will be dissolved in Isosorbide-5-Nitrae-dioxane (4M) splashes in the DCM solution (100mL) of embodiment 1 title compound stirring.Continue to stir 6 hours, subsequent filtration suspension, and with diethyl ether (500mL), wash this solid this title compound (54.2g, 96% productive rate) is provided, it is white solid.C 7h 11f 3n 2the APCI/MS value of calculation of O is 196.1, measured value m/z=196.9 (M+1).
Embodiment 3
(4-(2,2,2-trifluoroacetyl amido) piperidin-1-yl) dichloro phosphoric acid
Figure BDA0000447333530001083
By phosphorus oxychloride (23.9mL, 0.256mol) and DIPEA (121.7mL, 0.699mol) splash in cooling (ice bath/water-bath) suspension of embodiment 2 title compounds (54.2g, 0.233mol) in DCM (250mL), and stir.After 15 minutes, withdraw cryostat and continue and stir the mixture, to allow it to be warming up to ambient temperature.After 1 hour, this mixture of partial concentration (100mL), filtering suspension liquid also provides this title compound (43.8g, 60% productive rate) with diethyl ether washing solid, and it is white solid.Partial concentration (100mL) eluant, filters gained suspension and other title compound (6.5g, 9% productive rate) is provided with diethyl ether washing solid.1-(4-nitrobenzophenone) bridged piperazine derivatives C 17h 22clF 3n 5o 4the ESI/MS value of calculation of P is 483.1, measured value m/z=482.1 (M-1).
Embodiment 4
((2S, 6S)-6-((R)-5-methyl-2,6-dioxy-1,2,3,6-tetrahydropyridine-3-yl)-4-trityl morpholine-2-yl) methyl (4-(2,2,2-trifluoroacetyl amido) piperidin-1-yl) chlorinated phosphonate
Figure BDA0000447333530001091
In 10 minutes by Mo (Tr) T# (22.6g, 46.7mmol), 2,6-lutidine (21.7mL, 187mmol) and 4-(dimethylamino) pyridine (1.14g, DCM solution (100mL) 9.33mmol) splashes in the stirring and cooling (ice bath/water-bath) solution of embodiment 3 title compounds (29.2g, 93.3mmol) that are dissolved in DCM (100mL).Allow cryostat to be warming up to ambient temperature.After 15 hours, by citric acid solution for solution (200mLx3,10%w/v aqueous solution) washing, dry (MgSO 4), concentrated, and raw oil is directly loaded on post.Concentrated chromatography [SiO 2post (120g), hexane/EtOAc eluant (gradient 1:1 is to 0:1), repeats x3] fraction (fraction) provides this title compound (27.2g, 77% productive rate), and it is white solid.1-(4-nitrobenzophenone) bridged piperazine derivatives C 46h 50f 3n 8o 8the ESI/MS value of calculation of P is 930.3, measured value m/z=929.5 (M-1).
Embodiment 5
((2S, 6R)-6-(6-benzamido-9H-purine-9-yl)-4-trityl morpholine-2-yl) methyl (4-(2,2,2-trifluoroacetyl amido) piperidin-1-yl) chlorinated phosphonate
Figure BDA0000447333530001101
To be similar to the method for describing in embodiment 4, synthesized this title compound, this title compound (15.4g, 66% productive rate) is provided, it is white solid.1-(4-nitrobenzophenone) bridged piperazine derivatives C 53h 53f 3n 11o 7the ESI/MS value of calculation of P is 1043.4, measured value m/z=1042.5 (M-1).
Embodiment 6
(R) the amino phosphinylidyne dichloro of-methyl (1-phenethyl)
Figure BDA0000447333530001102
Under stirring by 2,6-lutidine (7.06mL, 60.6mmol) with (R)-(+)-N, a-dimethyl benzene methyl amine (3.73g, DCM solution 27.6mmol) splashes in cooling (ice bath/water-bath) solution of the phosphorus oxychloride (2.83mL, 30.3mmol) that is dissolved in DCM (30mL) continuously.After 5 minutes, remove cryostat, and allow reactant mixture to be warming up to ambient temperature.After 1 hour, by citric acid solution for reaction solution (50mLx3,10%w/v aqueous solution) washing, dry (MgSO 4), pass through SiO 2filter, and concentrate to provide this title compound (3.80g), it is white foam.1-(4-nitrobenzophenone) bridged piperazine derivatives C 19h 25n 4o 4the ESI/MS value of calculation of P is 404.2, measured value m/z=403.1 (M-1).
Embodiment 7
(S) the amino phosphinylidyne dichloro of-methyl (1-phenethyl)
Figure BDA0000447333530001103
To be similar to the method for describing in embodiment 6, synthesized this title compound, this title compound (3.95g) is provided, it is white foam.The C of 1-(4-nitrobenzophenone) bridged piperazine derivatives 19h 25n 4o 4the ESI/MS value of calculation of P is 404.2, measured value m/z=403.1 (M-1).
Embodiment 8
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl ((R)-1-phenethyl) chloro phosphoramidate
Figure BDA0000447333530001111
To be similar to the method for describing in embodiment 4, synthesized this title compound, provide this title compound amino phosphoryl chloride phosphorus oxychloride (4.46g, 28% productive rate), it is white solid.C 38h 40clN 4o 5the ESI/MS value of calculation of P is 698.2, measured value m/z=697.3 (M-1).
Embodiment 9
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl ((S)-1-phenethyl) chloro phosphoramidate
Figure BDA0000447333530001112
To be similar to the method for describing in embodiment 4, synthesized this title compound, provide this title compound amino phosphoryl chloride phosphorus oxychloride (4.65g, 23% productive rate), it is white solid.C 38h 40clN 4o 5the ESI/MS value of calculation of P is 698.2, measured value m/z=697.3 (M-1).
Embodiment 10
(4-(pyrrolidin-1-yl) piperidin-1-yl) phosphinylidyne dichloro hydrochlorate
Figure BDA0000447333530001113
By 2,6-lutidine (19.4mL, 167mmol) and 4-(1-pyrrolidinyl)-piperidines (8.58g, DCM solution (30mL) 55.6mmol) joins the phosphorus oxychloride (5.70mL that is dissolved in DCM (30mL), in cooling (ice bath/water-bath) solution 55.6mmol), and stir 1 hour.Filtering suspension liquid, and provide this title pyrrolidine (17.7g, 91% productive rate) with excessive diethyl ether washing solid, it is white solid.1-(4-nitrobenzophenone) bridged piperazine derivatives C 19h 30n 5o 4the ESI/MS value of calculation of P is 423.2, measured value m/z=422.2 (M-1).
Embodiment 11
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl (4-(pyrrolidin-1-yl) piperidin-1-yl) chlorinated phosphonate hydrochlorate
Figure BDA0000447333530001121
In 10 minutes by Mo (Tr) T# (24.5g, 50.6mmol), 2,6-lutidine (17.7mL, 152mmol) with 1-Methylimidazole. (0.401mL, DCM solution (100mL) 5.06mmol) splashes in the stirring and cooling (ice bath/water-bath) solution of dichlor-phosphoryl amine ester 8 (17.7g, the 50.6mmol) that are dissolved in DCM (100mL).When stirred suspension, allow cryostat to be warming up to ambient temperature.After 6 hours, suspension is poured on to diethyl ether (1L) upper, stirs 15 minutes, filter and use other ether washing gained solid that white solid (45.4g) is provided.By chromatography [SiO 2post (120 grams), DCM/MeOH eluant (gradient 1:0 to 6:4)] purification of crude product, and the fraction of merging is poured on diethyl ether (2.5L), stir 15 minutes, filter and use other ether washing gained solid that this title compound (23.1g is provided, 60% productive rate), it is white solid.1-(4-nitrobenzophenone) bridged piperazine derivatives C 48h 57n 8o 7the ESI/MS value of calculation of P is 888.4, measured value m/z=887.6 (M-1).
Embodiment 12
3-(tert-butyl group disulfonyl base)-2-(isobutoxy carbonyl is amino) propanoic acid
Figure BDA0000447333530001122
To be dissolved in H 2the K of O (20mL) 2cO 3(16.5g, 119.5mmol) joins and is dissolved in CH 3in S-tert-butyl group sulfydryl-Cys (10g, 47.8mmol) of CN (40mL).Stir after 15 minutes, slowly inject isobutyl chlorocarbonate (9.4mL, 72mmol).Allow reaction to carry out 3 hours.By Celite filter aid, filter white solid; Concentrated filtrate is to remove CH 3cN.Residue is dissolved in to ethyl acetate (200mL), with 1N HCl (40ml X3), salt (40X1) washing, uses Na 2sO 4dry.After chromatography (5%MeOH/DCM), obtain the product (2) of expectation.
Embodiment 13
4-(3-(tert-butyl group disulfonyl base)-2-(isobutoxy carbonyl is amino) propionamido-) piperidines-1-t-butyl formate
Figure BDA0000447333530001131
HATU (8.58g, 22.6mmol) is joined in the acid (from the compound 2 of embodiment 12,6.98g, 22.6mmol) that is dissolved in DMF (50ml).After 30 minutes, Hunig alkali (4.71ml, 27.1mmol) and 1-Boc-4-amino piperidine (5.43g, 27.1mmol) are joined in mixture.Under stirring at room, reaction is continued to carry out other 3 hours.Under fine vacuum, remove DMF, rough residue is dissolved in EtAc (300ml), use H 2o (50ml X3) washing.After ISCO purification (5%MeOH/DCM), obtain end-product (3).
Embodiment 14
3-(tert-butyl group disulfonyl base)-1-oxygen-1-(piperidin-4-yl is amino) third-2-aminocarbamic acid isobutyl ester
Figure BDA0000447333530001132
The 4M HCl/ dioxane of 30ml is joined in the compound 3 (7.085g, 18.12mmol) of preparation in embodiment 13.Room temperature completes reaction after lower 2 hours.Described HCl salt (4) is directly used in to next step without being further purified.
Embodiment 15
3-(tert-butyl group disulfonyl base)-1-(1-(dichlor-phosphoryl) piperidin-4-yl is amino)-1-oxo third-2-aminocarbamic acid isobutyl ester
Figure BDA0000447333530001133
In-78 ℃ under argon atmospher by POCl 3(1.69ml, 18.12mmol) is slowly injected in the DCM solution (200ml) of the compound 4 (7.746g, 18.12mmol) of preparation in embodiment 15, adds subsequently Et 3n (7.58ml, 54.36mmol).Under room temperature, stirring reaction is 5 hours, concentrated to remove excessive alkali and solvent.After ISCO purification (50%EtAc/ hexane), obtain product (5), it is white solid.
Embodiment 16
3-(tert-butyl group disulfonyl base)-1-(1-(chloro (((2S; 6R)-6-(5-methyl-2; 4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methoxyl group) phosphoryl) piperidin-4-yl amino)-1-oxo third-2-aminocarbamic acid isobutyl ester
Figure BDA0000447333530001141
By lutidine (1.92ml, 16.47mmol) and DMAP (669mg, 5.5mmol) join 0 ℃ of 1-((2R that is dissolved in DCM (100ml), 6S)-6-(methylol)-4-trityl morpholine-2-yl)-5-methylpyrimidine-2, in 4 (1H, 3H)-diketone (moT (Tr)) (5.576g, 10.98mmol), add subsequently 4 (6.13g, 12.08mmol).Reaction is stayed and at room temperature stirred 18 hours.After ISCO purification (50%EtAc/ hexane), obtain the product (6) of expectation.
Embodiment 17
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl hexyl (methyl) chloro phosphoramidate
Figure BDA0000447333530001142
At N 2lower the DCM of N-methylol amine (4.85ml, 32mmol) (80ml) solution is cooled to-78 ℃.DCM (10ml) solution that slowly adds phosphoryl chloride phosphorus oxychloride (2.98ml, 32mmol), slowly adds Et subsequently 3the DCM of N (4.46ml, 32mmol) (10ml) solution.While allowing reaction to carry out, continue to stir, to spend the night, be warming up to room temperature.After ISCO purification (20%EtAc/ hexane), obtain the product (1) of expectation, it is edible vegetable oil.
Lutidine (3.68ml, 31.6mmol) and DMAP (642mg, 5.27mmol) are joined in 0 ℃ of moT (Tr) (5.10g, 10.54mmol) that is dissolved in DCM (100ml), add subsequently 1 (4.89g, 21.08mmol).Reaction is stayed and at room temperature stirred 18 hours.After ISCO purification (50%EtOAc/ hexane), obtain the product (2) of expectation.
Embodiment 18
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl dodecyl (methyl) chloro phosphoramidate
Figure BDA0000447333530001151
According to the general procedure of describing in embodiment 6 and embodiment 8, prepare this title compound.
Embodiment 19
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl morpholine is for chloro phosphate ester
Figure BDA0000447333530001152
According to the general procedure of describing in embodiment 6 and embodiment 8, prepare this title compound.
Embodiment 20
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl (S)-2-(methoxy) pyrrolidin-1-yl chloro phosphate ester
Figure BDA0000447333530001161
According to the general procedure of describing in embodiment 6 and embodiment 8, prepare this title compound.
Embodiment 21
((2S, 6R)-6-(5-methyl-2,4-dioxy-3,4-dihydro-pyrimidin-1 (2H)-yl)-4-trityl morpholine-2-yl) methyl 4-(3,4,5-trimethoxy-benzamide base) piperidin-1-yl chloro phosphate ester
Figure BDA0000447333530001162
Hunig alkali (1.74ml, 10mmol) is joined in the 1-Boc-4-piperidines (1g, 5mmol) that is dissolved in DCM (20ml), add subsequently 3,4,5-trimethoxy-benzoyl chloride (1.38g, 6mmol).Under room temperature, reaction is carried out 3 hours, concentrated to remove solvent and excessive alkali.Residue is dissolved in EtAc (100ml), with 0.05N HCl (3X15ml), saturated NaHCO 3(2X15ml) washing, uses Na 2sO 4dry.After ISCO purification (5%MeOH/DCM), obtain product (1).
The 4N HCl/ dioxane of 15ml is joined in 7 to cessation reaction after 4 hours.Obtain as 8 of white solid.
At N 2lower the DCM of 8 (1.23g, 4.18mmol) (20ml) solution is cooled to-78 ℃.DCM (2ml) solution that slowly adds phosphoryl chloride phosphorus oxychloride (0.39ml, 4.18mmol), slowly adds Et subsequently 3the DCM of N (0.583ml, 4.18mmol) (2ml) solution.While allowing reaction to carry out, continue to stir, to spend the night, be warming up to room temperature.After ISCO purification (50%EtAc/ hexane), obtain the product (9) of expectation.
Lutidine (0.93ml, 8mmol) and DMAP (49mg, 0.4mmol) are joined in DCM (20ml) solution of 0 ℃ of moT (Tr) (1.933g, 4.0mmol), add subsequently 9 (1.647g, 4mmol).Reaction is stayed and at room temperature stirred 18 hours.After ISCO purification (50%EtAc/ hexane), obtain the product (10) of expectation.
Embodiment 22
Synthetic contain subunit ( cPt) cyclophosphamide
Figure BDA0000447333530001171
MoT subunit (25g) is suspended in DCM (175ml), and adds NMI (N-Methylimidazole., 5.94g, 1.4eq.) to obtain settled solution.Paratoluensulfonyl chloride is joined in reactant mixture, and monitor reaction process until complete (approximately 2 hours) by TLC.By having washed aqueous treatment with 0.5M citrate buffer solution (pH=5) and salt subsequently.Separated organic layer is also used Na 2sO 4dry.By rotary evaporator, remove solvent to obtain raw product, it is directly used to next step without being further purified.
The moT toluene fulfonate of preparation is above mixed to (1g/10ml) with Propanolamine.The baking oven that reactant mixture is placed in to 45 ℃ subsequently spends the night, and uses subsequently DCM (10ml) dilution.By having washed aqueous treatment with 0.5M citrate buffer solution (pH=5) and salt subsequently.Separated organic layer is also used Na 2sO 4dry.By rotary evaporator, remove solvent and obtain raw product.By NMR and HPLC, analyze and measure this raw product, standby without being further purified as next step.
This raw product is dissolved in DCM (2.5ml DCM/g, 1eq.), and mixes with DIEA (3eq.).With cooling this solution of dry ice-propanone, and dropwise add POCl 3(1.5eq.).Under room temperature, stir gained mixture overnight.By having washed aqueous treatment with 0.5M citrate buffer solution (pH=5) and salt subsequently.Separated organic layer is also used Na 2sO 4dry.By rotary evaporator, remove solvent and obtain raw product, it is micro-yellow solid.By silica gel column chromatography (ratio of raw product/silicon dioxide=1 to 5, gradient DCM to 50%EA/DCM) this raw product of purification, and according to TLC, analyze the fraction that merges eluting.Remove solvent to obtain the product of expectation, its mixture that is diastereomer.By HPLC (NPP cancellation) and NMR (H-1 and P-31), analyze the product of this purification.
According to the separated mixture of diastereomers of following program.This mixture (2.6g) is dissolved in DCM.This sample is loaded into RediSepRf post (80g positive, Teledyne Isco manufacture) upper, and with 10%EA/DCM to 50%EA/DCM eluting 20 minutes.Collect elutriated fraction and analyze by TLC.According to TLC, analyze and merge elutriated fraction, and at room temperature by rotary evaporator, remove solvent.By P-31NMR and NPP-TFA, analyze and measure the diastereomer ratio that merges fraction.If needed, repeat above program until diastereomer ratio reaches 97%.
Embodiment 23
The total bile acid of PMO+ is modified
Figure BDA0000447333530001181
The chlolic acid derivatives of having prepared butanimide activation according to following program.Cholic acid (12g, 29.4mmol), N-hydroxy-succinamide (4.0g, 34.8mmol), EDCI (5.6g, 29.3mmol) and DMAP (1g, 8.2mmol) are packed in round-bottomed flask.Add DCM (400ml) and THF (40ml) to dissolve.Under room temperature, stirred reaction mixture spends the night.Subsequently water (400ml) is joined in reactant mixture to separated organic layer water (2X400ml) and use subsequently saturated NaHCO 3(300ml) and salt (300ml) washing.Use subsequently Na 2sO 4dry organic layer.By rotary evaporator, remove solvent to obtain white solid.This raw product is dissolved in chloroform (100ml), and it is deposited in heptane (1000ml).By solid collected by filtration, by HPLC and NMR, analyze not purified direct use.
Take appropriate PMO+ (20mg, 2.8 μ mol) and be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).In the ratio of every decorating site 2 equivalent active ester, the cholate (13mg, 25 μ mol) of activation is joined in reactant mixture, at room temperature stir and spend the night subsequently.By MALDI and HPLC (C-18 or SAX), determine reaction process.
After having reacted (as the disappearance by initial PMO+ is determined), once react the soon concentrated ammonia of 1ml, join in reactant mixture.Reaction bottle is placed in baking oven (45 ℃) and spends the night (18 hours) subsequently, be cooled to subsequently room temperature, and dilute with 1% ammonia soluble in water (10ml).This sample is loaded into SPE post (2cm) upper, and rinses bottle with 1% ammonia solution (2X2ml).With this SPE post of 1% ammonia stripping in water-soluble (3X6ml), and with 45% this product of acetonitrile eluting being dissolved in 1% ammonia spirit (6ml).By UV photo densitometry, identify the elutriated fraction that contains oligomer.By lyophilization separated product.By MALDI and HPLC (C-18 and/or SAX), measure purity and characteristic.
This identical program is applicable to deoxycholic acid activation and is attached to PMO +on.
Embodiment 24
PMO+'s is always guanidinated
Taking appropriate PMO+ (25mg, 2.8 μ mol) is encased in bottle (6ml).1H-pyrazoles-1-chlorination carbonamidine (15mg, 102 μ mol) and potassium carbonate (20mg, 0.15mmol) are joined in bottle.Add water (500 μ l), and stirred reaction mixture spend the night (approximately 18 hours) at room temperature.By MALDI, determine and reacted.
Once complete, use 1% ammonia diluting reaction thing in water-soluble (10ml), and be loaded on SPE post (2cm).With 1% ammonia solution (2X2ml), rinse bottle, and with 1% ammonia stripping SPE post in water-soluble (3X6ml).By the 45% acetonitrile eluted product that is dissolved in 1% ammonia spirit (6ml).By UV photo densitometry, identify the elutriated fraction that contains oligomer.By lyophilization separated product.By MALDI and HPLC (C-18 and/or SAX), measure purity and characteristic.
Embodiment 25
Total ethanethioyl of PMO+ (M23D) is modified
Figure BDA0000447333530001201
Take appropriate PMO+ (20mg, 2.3 μ mol) and be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).N-succinimido-S-acetyl thio acetas (SATA) (7mg, 28 μ mol) is joined in reactant mixture, and allow at room temperature to stir and spend the night.By MALDI and HPLC, monitor reaction process.
Once complete, be about to 1% ammonia soluble in water and join in reactant mixture, and at room temperature stirred 2 hours.This solution is loaded on SPE post (2cm).With 1% ammonia solution (2X2ml), rinse bottle, and with 1% ammonia stripping SPE post in water-soluble (3X6ml).By the 45% acetonitrile eluted product being dissolved in 1% ammonia spirit (6ml).By UV photo densitometry, identify the elutriated fraction that contains oligomer.By lyophilization separated product.By MALDI and HPLC (C-18 and/or SAX), measure purity and characteristic.
Embodiment 26
Total succinic acid of PMO+ is modified
Take appropriate PMO+ (32mg, 3.7 μ mol) and be encased in bottle (4ml), and be dissolved in DMSO (500 μ l).In N-ethylmorpholine generation (12mg, 100 μ mol) and succinic anhydrides (10mg, 100 μ mol), joined in reactant mixture, and allow at room temperature its stirring to be spent the night.By MALDI and HPLC, monitor reaction process.
Once complete, 1% ammonia soluble in water is joined in reactant mixture, and at room temperature stirred 2 hours.This solution is loaded on SPE post (2cm).With 1% ammonia solution (2X2ml), rinse bottle, and with 1% ammonia stripping SPE post in water-soluble (3X6ml).By the 45% acetonitrile eluted product being dissolved in 1% ammonia spirit (6ml).By UV photo densitometry, identify the elutriated fraction that contains oligomer.By lyophilization separated product.By MALDI and HPLC (C-18 and/or SAX), measure purity and characteristic.
Above program is also applicable to 1,3-propanedicarboxylic acid (glutaric anhydride) and the tetramethyleneglutaric acid (tetramethyleneglutaric acid acid anhydride) of PMO+ and modifies.
Figure BDA0000447333530001212
Embodiment 27
The oligonucleotide analogs that preparation comprises modified terminal groups
By farnesyl-bromide (1.75 μ l, 6.452 μ mol) and diisopropylethylamine (2.24 μ L, 12.9 μ mol) join in DMSO (the 300 μ L) solution of the 25-mer PMO (27.7mg, 3.226 μ mol) that contains free 3 ' end.At room temperature stirred reaction mixture is 5 hours.The 1% moisture NH with 10mL 4oH dilutes crude reaction mixture, is loaded into subsequently on 2mL Amberchrome CG300M post.With the water of 3 pillar volumes, rinse this post subsequently, and by acetonitrile and water (v/v) eluted product of 6mL1:1.Solutions in Freeze-drying is to obtain this title compound subsequently, and it is white solid.
Embodiment 28
Prepare morpholino oligomer
The preparation (seeing Fig. 3) of trityl piperazine carbanilate 35: potassium carbonate (3.2eq) solution in water-soluble (4mL/g potassium carbonate) is joined in the cooling suspension of the compound 11 that is dissolved in dichloromethane (6mL/g11).Slowly add phenyl chloroformate (1.03eq) solution (2g/g phenyl chloroformate) being dissolved in dichloromethane in this biphase mixture.Reactant mixture is warming up to 20 ℃.Once react (1-2 hour), isolated different layers.Wash organic layer with water, and dry with Anhydrous potassium carbonate.By crystallization separated product 35 from acetonitrile.Productive rate=80%.
The preparation of carbamate ethanol (carbamate alcohol) 36: sodium hydride (1.2eq) is suspended in to (32mL/g sodium hydride) in 1-Methyl-2-Pyrrolidone.2,2'-ethylenedioxybis(ethanol). (10.0eq) and compound 35 (1.0eq) are joined in this suspension.Gained serosity is heated to 95 ℃.Once react (1-2 hour), this mixture be cooled to 20 ℃.30% dichloromethane/methyl tert-butyl ether (v:v) and water are joined in this mixture.The product that contains organic layer with NaOH aqueous solution, succinic acid aqueous solution and saturated sodium-chloride water solution washing successively.By Crystallization Separation product 36 from dichloromethane/methyl tert-butyl ether/heptane.Productive rate=90%.
The preparation of tail acid 37: succinic anhydrides (2.0eq) and DMAP (0.5eq) are joined in the solution (7mL/g36) of the compound 36 that is dissolved in oxolane.Mixture is heated to 50 ℃.Once react (5 hours), mixture is cooled to 20 ℃, and uses NaHCO 3aqueous solution is adjusted to pH8.5.Add methyl tert-butyl ether, and product is extracted in water layer.Add dichloromethane, and with aqueous citric acid solution, mixture is adjusted to pH3.The product that contains organic layer with the citrate buffer of pH=3 and the washing of the mixture of saturated sodium-chloride water solution.Without the directly preparation for compound 38 by this dichloromethane solution of 37 of separation.
38 preparation: by N-hydroxyl-5-norborene-2,3-dicarboxylic acid imides (HONB) (1.02eq), 4-dimethylaminopyridine (DMAP) (0.34eq) joins in the solution of compound 37, adds subsequently that 1-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) (1.1eq).Mixture is heated to 55 ℃.Once react (4-5 hour), mixture is cooled to 20 ℃, and with 0.2M citric acid/salt and the salt of 1:1, washs successively.Dichloromethane solution is to acetone and subsequently DMF is carried out to solvent exchange, and by be precipitated to separated this product in saturated sodium-chloride water solution from acetone/DMF.In water by repulped remaining DMF and the salt of removing several times of raw product.Productive rate=70% from compound 36 preparations 38." tail " of by the program for be incorporated to subunit during solid phase synthesis, at NMP, introducing activation is to disulphide grappling resin.
Preparation for the synthesis of the solid support (support) of morpholino oligomer: carry out this program in the jacket type peptide pipe (customizing the company in the ChemGlass of New Jersey) of silanization, it has gross porosity (40-60 μ m) glass frit, overhead type stirrer and 3 logical Teflon plug valves to allow N 2by described frit up bubbling or vacuum extraction.By circulator bath, in reaction vessel, realizing temperature controls.
Next resin treatment/the washing step in program is comprised of two basic operations: resin fluidisation and solvent/solution extraction.For resin fluidisation, plug valve is placed in to appropriate location to allow N 2upwards flow through frit, the resin treatment/cleaning mixture of appointment is joined in reactor, and allow its infiltration and complete wetting resin.Then start to mix, and by the time of resin grout liquid mixing appointment.For solvent/solution extraction, stop mixing and N 2stream, and open vacuum pump, subsequently plug valve is placed in to appropriate location to allow that resin treatment/cleaning mixture is expelled to reject chute (waste).All resin treatment/cleaning mixture volumes are all 15mL/g resin, except as otherwise noted.
By 1-Methyl-2-Pyrrolidone (NMP; 20ml/g resin) join aminomethylpolystyre.e resin (the 100-200 order in the jacket type peptide pipe of silanization;～1.0mmol/g N 2substituent; 75g, 1eq, Polymer Labs, UK, part#1464-X799) in, and under agitation allow resin expansion 1-2 hour.After emptying expanded solvents, with dichloromethane (2x1-2 minute), 5% diisopropylethylamine (2x3-4 minute) that is dissolved in 25% isopropyl alcohol/dichloromethane and dichloromethane (2x1-2 minute) washing resin.After final cleaning mixture is emptying, with being dissolved in 1-Methyl-2-Pyrrolidone (0.17M; The solution fluidisation resin of disulphide deadman 34 15mL/g resin ,～2.5eq), and at 45 ℃ by resin/reagent mixture heating 60 hours.When reaction completes, stop heating spare anchor side by side and determine solution, then with 1-Methyl-2-Pyrrolidone (4x3-4 minute) and dichloromethane (6x1-2 minute) washing resin.With being dissolved in dichloromethane (16mL/g; The solution-treated resin of 10% (v/v) pyrocarbonic acid diethyl ester 2x5-6 minute), uses dichloromethane (6x1-2 minute) washing subsequently.At N 2flow down dry resin 39 (seeing Fig. 4) 1-3 hour, under vacuum, be dried to subsequently constant weight (± 2%).The initial resin weight of productive rate: 110-150%.
The load of aminomethylpolystyre.e-disulphide resin is measured: the spectrometric analysis of trityl group (trityl) number by every gram of resin, measure the load (potential available reaction site number) of resin.
The dry resin of known weight (25 ± 3mg) is proceeded in the 25ml volume capacity bottle of silanization, and add be dissolved in dichloromethane～2% (v/v) trifluoroacetic acid of 5mL.By rotating gently mixed content thing, standing 30 minutes subsequently.With other 2% (v/v) trifluoroacetic acid that is dissolved in dichloromethane by volume dilution to 25mL, and thorough mixed content thing.Use positive discharge capacity pipet, the solution (500 μ L) by 1 part containing trityl is transferred in 10mL volumetric flask, and with pyrovinic acid by volume dilution to 10mL.
By UV absorbance, at 431.7nm place, measure the trityl cations in whole solution, and use suitable volume, dilution factor, extinction coefficient (ε: 41 μ mol-1cm-1) and weight resin with every gram of resin of trityl (μ mol/g) calculating resin load.Repeat this analysis with three, and calculate average load.
Resin load program in this embodiment will provide the resin with approximately 500 μ mol/g loads.If at room temperature disulphide deadman is mixed to step, carry out 24 hours, can obtain the load of 300-400 μ mol/g.
Tail load: use setting and the volume same with preparing aminomethyl polystyrene-disulphide resin-phase, described tail can be incorporated in molecule.For coupling step, used the solution of 38 (0.2M) that are dissolved in the NMP that contains 4-ethyl morpholine (NEM, 0.4M), but not disulphide deadman solution.45 ℃ after 2 hours, with the 5% diisopropylethylamine washing resin 39 twice that is dissolved in 25% isopropyl alcohol/dichloromethane, and with DCM washing once.The solution of benzoyl oxide (0.4M) and NEM (0.4M) is joined in resin.After 25 minutes, reactor jacket is cooled to room temperature, and with the 5% diisopropylethylamine washing resin twice that is dissolved in 25% isopropyl alcohol/dichloromethane, and with DCM washing 8 times.Under fine vacuum, filter and dry resin 40.The load of resin 40 is also defined as the load of the initial aminomethyl polystyrene-disulphide resin 39 using in tail load.
Solid phase synthesis: prepare morpholino oligomer on Gilson AMS-422 automated peptide synthesizer in 2mL Gilson polypropylene reaction column (Part#3980270).When they are positioned on synthesizer, the aluminium block with water stream channel is placed on around pillar.AMS-422 can alternately add reagent/wash solution, keeps the fixed time, and uses vacuum-evacuate pillar.
For the oligomer up to about 25 subunit length, preferably there is aminomethyl polystyrene-disulphide resin of the load of approximately 500 μ mol/g resins.For larger oligomer, preferably there is aminomethyl polystyrene-disulphide resin of the load of 300-400 μ mol/g resin.If the molecule with 5 '-tail expects, with identical load, instruct to select load to have the resin of tail.
Prepared following reagent solution:
Trityl removal solution: 10% cyanoacetic acid (w/v) that is dissolved in 4:1 dichloromethane/acetonitrile; Neutralization solution: 5% diisopropylethylamine that is dissolved in 3:1 dichloromethane/isopropyl alcohol; Coupling solution: morpholino subunit and the 0.4M N-ethylmorpholine of the base of expectation and the 0.18M of connection type (or 0.24M grows to the oligomer of being longer than 20 subunits) activation, be dissolved in 1,3-methylimidazole alkane ketone.Dichloromethane (DCM) is used as separating the transitional cleaning mixture of different solvents solution washing liquid.
On synthesizer, piece is set to 42 ℃, the 1-Methyl-2-Pyrrolidone of 2mL is joined in each pillar of the aminomethyl polystyrene-disulphide resin (or tail resin) that contains 30mg, and allows at room temperature to keep 30 minutes.After the washed with dichloromethane with 2mL 2 times, adopted following synthesis cycle:
Figure BDA0000447333530001251
Figure BDA0000447333530001261
The sequence of single oligomer is programmed in synthesizer, so that each pillar is with the suitable coupling solution of suitable sequential reception (A, C, G, T, I).Oligomer in pillar completes while mixing its final subunit, from piece, removes pillar, and uses the coupling solution being comprised of the 4-methoxyl group trityl group chloride (0.32M is dissolved in DMI) that contains 0.89M4-ethyl morpholine manually finally to circulate.
From resin cleavage with remove base and skeleton protecting group: methoxyl group tritylation, with 2mL1-N-methyl-2-2-pyrrolidone N-washing resin 8 times.Add 1mL by the 0.1M1 that is dissolved in 1-Methyl-2-Pyrrolidone, the cutting solution that 4-dithiothreitol, DTT (DTT) and 0.73M triethylamine form, gives pillar cover lid, and allows its at room temperature standing 30 minutes.After that time, solution is flowed in 12mL Wheaton bottle.Twice of the resin significantly shrinking with the cutting solution washing of 300 μ L.The concentrated ammonia of 4.0mL (being stored in-20 ℃) is joined in solution, firmly cover bottle (nut with Teflon with strain line), and rotate mixture and carry out mixed solution.Bottle is placed on to 16-24 hour in 45 ℃ of baking ovens, the cutting that produces base and skeleton protecting group.
Initial oligomer segregation: withdraw the aminolysis solution that packs bottle into from baking oven, and make it be cooled to room temperature.With the 0.28% ammonia dilute solution of 20mL, and make it by containing the 2.5x10cm post of Macroprep HQ resin (BioRad).Salt gradient (A:0.28% ammonia, the 1M sodium chloride in B:0.28% ammonia; 0-100%B, 60 minutes) be used to the peak that eluting contains methoxyl group trityl.Merge the fraction of combination and further process according to the product of expectation.
The demethoxylation tritylation of morpholino oligomer: use 1M H 3pO 4processing is from the merging fraction of Macroprep purification, so that pH is down to 2.5.After initial mixing, make sample at room temperature standing 4 minutes, now with 2.8% ammonia/water, they are neutralized to pH10-11.By Solid-Phase Extraction (SPE) purified product.
By Amberchrome CG-300M (Rohm and Haas; Philadelphia, PA) (3mL) put in the tool sieve aperture post (BioRad Econo-Pac chromatographic column (732-1011)) of 20mL, and rinse resin: 0.28%NH with the following material of 3mL 4oH/80% acetonitrile; 0.5M NaOH/20% ethanol; Water; 50mM H 3pO 4/ 80% acetonitrile; Water; 0.5NaOH/20% ethanol; Water; 0.28%NH 4oH.
Solution from demethoxylation tritylation is loaded on pillar, and with 3-6mL0.28% ammonia water rinse resin 3 times.Wheaton bottle (12mL) is placed under pillar, and by be dissolved in 45% acetonitrile washed twice eluted product of 0.28% ammonia with 2mL.Freezing described solution in dry ice, and bottle is placed in freeze dryer, to produce fluffy white powder.Sample is soluble in water, by 0.22 micron filter (Pall Life Sciences, Acrodisc25mm syringe type filter, there is 0.2 micron of HT Tuffryn film) use syringe filtering, and on UV spectrophotometer, measure optical density (OD), the OD unit presenting to measure oligomer, and distribute sample for analyzing.Subsequently solution is put back in Wheaton bottle, for lyophilizing.
The analysis of morpholino oligomer: with MALDI-TOF mass spectral analysis, measure the compositions of the fraction in purification, and the proof that the characteristic (molecular weight) of oligomer is provided.With 3,5-dimethoxy-4 '-hydroxycinnamic acid (sinapic acid), 3,4, after the solution dilution of 5-trihydroxy-acetophenone (THAP) or alpha-cyano-4-hydroxycinnamic acid (HCCA), as upshift operation sample.
Use 25mM pH=5 sodium acetate, 25% acetonitrile (buffer A) and 25mM pH=5 sodium acetate, 25% acetonitrile, 1.5M potassium chloride (buffer B) (gradient 10-100%B, 15 minutes) or 25mMKH 2pO 425% acetonitrile, pH=3.5 (buffer A) and 25mM KH 2pO 425% acetonitrile, pH=3.5,1.5M potassium chloride (buffer B) (gradient 0-35%B, 15 minutes), with Dionex ProPac SCX-10,4x250mm post (Dionex Corporation; Sunnyvale, CA) carry out cation exchange (SCX) HPLC.Last system is used to not have the positively charged oligomer that peptide adheres to, and the latter is used to peptide conjugate.
By cation-exchange chromatography purification morpholino oligomer: sample is dissolved in to 20mM sodium acetate, in pH=4.5 (buffer A), and be applied to the pillar of Source30 cation exchange resin (GE Healthcare), and with the 0.5M sodium chloride gradient in 20mM sodium acetate and 40% acetonitrile, pH=4.5 (buffer B) eluting.The merging fraction that contains product with concentrated gas liquor neutralization, and be applied to Amberchrome SPE post.Press as above eluting, freezing and lyophilized products.
Embodiment 29
The preparation of exemplary conjugate
According to standard peptide synthetic method known in the art, prepare peptide sequence AcR 6g.Under room temperature, diisopropylethylamine (36 μ L, 5eq) is joined to the PMO that is dissolved in DMSO (3mL) (NG-05-0225,3 '-H:M23D:5 '-EG3, for being attached to the sequence on the exon 23 of mdx mice, 350mg, 1eq), AcR 6in the solution of G (142mg, 2eq), HATU (31mg, 2eq).After 1 hour, start and to react and to pass through SCX chromatography (in order to Gradient eluting: A: be dissolved in 25% acetonitrile/H 2the 20mM NaH of O 2pO 4, pH7.0; B: be dissolved in 25% acetonitrile/H 2the 1.5M guanidine HCl of O and 20mM NaH 2pO 4, pH7.0) peptide-oligomer conjugate of purification expectation.Make the fraction experience Solid-Phase Extraction (1M NaCl carries out water elution subsequently) of combination.After lyophilizing, obtaining conjugate is white powder (257mg, 65.5% productive rate).
Embodiment 30
With exemplary conjugate of the present invention, process MDX mice
MDX mice is the animal model of Duchenne muscular dystrophy (DMD) generally acknowledged and that fully surely levy, and it contains sudden change in the exon 23 of dystrophin gene.M23D antisense sequences (SEQ ID NO:15) is known can inducing exon 23 skips and reparation that functional dystrophin is expressed.In order to a kind of in lower conjugate, by tail vein injection, give MDX mice dose (50mg/kg):
1.5’-EG3-M23D-BX(RXRRBR) 2(AVI5225)；
2.5’-EG3-M23D-G(R) 5(NG-11-0045)；
3.5’-EG3-M23D-G(R) 6(NG-11-0009)；
4.5 '-EG3-M23D-G (R) 7(NG-11-0010); Or
5.5’-EG3-M23D-G(R) 8(NG-11-0216)
Wherein M23D is the morpholino oligonucleotide with sequence GGCCAAACCTCGGCTTACCTGAAAT, and " EG3 " refers to following structure:
Figure BDA0000447333530001281
It is connected on 5 ' end of oligomer by piperazine linking arm (that is, structure XXIX).
Latter one week of injection, puts to death MDX mice and also from different muscular tissue, extracts RNA.With terminal PCR (End-point PCR), measure the dystrophin mRNA contain exon 23 and because the exon skipping of antisense induction lacks the relative abundance of the mRNA of exon 23.Percentage ratio exon 23 is skipped as the tolerance of antisense activity in body.Fig. 5 and Fig. 6 have shown respectively the rear 1 week result from musculus quadriceps (QC, Fig. 5 A and Fig. 6 A), barrier film (DT, Fig. 5 B and Fig. 6 B) and heart (HT, Fig. 5 C and Fig. 6 C) of processing.Dose response between AVI-5225 and other conjugate is similar.In arginine series, R 6g peptide has the highest effect in musculus quadriceps and barrier film, and similar with the effect of other arginine series peptides in heart.
Embodiment 31
BUN level and the survival rate of the mice of processing with exemplary conjugate
With the conjugate of describing in embodiment 30, process mice, and measured KIM-1 level, BUN level and survival rate according to what describe in following examples 32 with general procedure known in the art.It is shocking, Fig. 7 A shows that the conjugate that all glycine connect has the remarkable low BUN level of the conjugate (AVI-5225) connecting than XB.In addition, the mice of processing with the conjugate that glycine connects survives longer, wherein R under the higher dosage of the conjugate (Fig. 7 B) connecting than XB 8the minimum tolerance arginine of G conjugate polymer.Use R 6all mices that G conjugate (NG-11-0009) was processed all can survival (data do not show) under the dosage that reaches 400mg/kg.
The KIM-1 (Fig. 8 A) of the mice of processing with the conjugate that glycine connects and clusterin (Fig. 8 B) level are significantly lower than the mice of processing with AVI-5225.These data show, conjugate of the present invention has the toxicity lower than previous conjugate, and as shown in above embodiment 30, the effect of this conjugate does not reduce.Therefore, conjugate of the present invention has better treatment window than other known conjugates, and is potential better drug candidate.
Embodiment 32
The toxicity of exemplary conjugate
At Mice Body build-in test the toxicity of 4 kinds of exemplary conjugates of the present invention.Described conjugate is as follows:
1.5’-EG3-M23D-BX(RXRRBR) 2(AVI5225)；
2.5’-EG3-M23D-G(RXRRBR) 2(NG-11-0654)；
3.5 '-EG3-M23D-BX (R) 6(NG-11-0634); With
4.5’-EG3-M23D-G(R) 6(NG-11-0009)
Wherein M23D is the morpholino oligonucleotide with sequence GGCCAAACCTCGGCTTACCTGAAAT, and " EG3 " refers to following structure:
It is connected on 5 ' end of oligomer by piperazine linking arm (that is, structure XXIX).
With the above conjugate of preparation salify, process 8 weeks large male mice (C57/BL6; Jackson Laboratories, 18-22 gram).Before starting experimental arrangement, mice is tamed minimum 5 days.
There is qualify and in the micro-isolation cage of clean Merlon of the contact bedding and padding of irradiation with every cage density letting animals feed of 3 nearly.Described cage is observed animal welfare method (comprising all amendments) and is positioned at laboratory animal nursing and instruction (the Guide for the Care and Use of Laboratory Animals publishing for 2010 washingtonian American National academic press, National Academy Press, Washington, D.C., 2010) standard of statement in.
Cage based on illustrating in following form is heavily assigned randomly to animal in processed group.In research record, illustrated that component joins.
The design of table 8. toxicological study
Be designated as that day that gives dosage in research studies the 1st day.As slow, push away type pill (～5 seconds) and give conjugate via tail vein.All animals give dosage 2 days.At first day, be that 1-8 group gives dosage, and be that 9-16 group gives dosage at second day.According to upper table, be that processed group (TG) 13-16 gives dosage.From the result of these TG, do not affect the progress of other TG.According to upper table, give initial 2 TG dosage of every kind of conjugate.If all animals in 100mg/kg group are all dead, the residue TG of those test article will not give dosage subsequently, and research is ended.If at least 1 animal is giving to survive 2 hours after dosage in 100mg/kg group, give subsequently 150mg/kg group dosage.If all animals in 150mg/kg group are all dead, the residue TG of those test article will not give dosage subsequently, and research is ended.If at least 1 animal is giving to survive 2 hours after dosage in 150mg/kg group, give subsequently 200mg/kg group dosage.
Observe dying rate and the mortality rate of an animal every day.According to Numira Biosciences S.O.P. people is genuine, will show danger and disaster sign, especially dead imminent any animal euthanasia.After arrival the same day, give the dosage same day and necropsy recorded body weight the same day.Carry out detailed clinical observation, and after giving dosage 0 minute, 15 minutes and 2 hour records, to assess the toleration of injection.
Give after dosage 3 days, before necropsy, by cardiac puncture, from all animals, obtain blood sample (maximum volume, about 1mL).Blood sample is collected in the Microtainer pipe of red top, and before centrifugal, at room temperature maintained at least 30 minutes but no longer than 60 minutes.Under about 1500-2500rpm by the centrifugal 15-20 of sample minute to obtain serum.
Unlikely survival to next predetermined animal of observing is weighed and by its euthanasia.Animal to discovery dead is weighed, and as far as possible closely estimates the death time.Do not collect blood and tissue sample.
The 3rd day (give dosage after 2 days), with carbon dioxide by the human euthanasia of all animals.According to acceptable U.S. Veterinary Medical Association (AVMA) in June, 2007 carrying out euthanasia about the guide of euthanasia.
Local gross necropsy comprises inspection and the record of check situation.All outer surfaces and aperture have been assessed.Complete description and recorded and gather all abnormal conditions of observing during tissue.Do not gather its hetero-organization.
Left kidney and right kidney have been gathered.Euthanasia 15 minutes or still less gather tissue in the time.Between processed group, change all appts and the instrument using.After collection as early as possible by all organizing IQF and be stored at <-70 ℃.
By the following injury of kidney mark data that obtain.Use Quick gene Mini80Tissue Kit SII (Fuji Film) purification from the RNA of kidney of mouse.Briefly, the tissue of about 40mg is joined in the 0.5ml lysis buffer (5 μ l2-mercaptoethanols are dissolved in 0.5ml lysis buffer) in MagnaLyser Green Bead bottle (Roche), and use MagNA Lyser (Roche) to carry out homogeneity with 2 serial 3x3800RPM and 3 serial 1x6500RPM.Between each low speed series and between each high-speed cruising by sample at cooled on ice 3-4 minute.Under 400xg, room temperature by centrifugal 5 minutes of homogenate.Process immediately homogenate, for according to Quick gene Mini80 scheme purifying RNA.The upper prop DNA that sample has experienced use DNA enzyme I (Qiagen) digests 5 minutes.Use the quantitatively total RNA of NanoDrop 2000 spectrophotometers (Thermo Scientific).
Use the reagent (one-step method RT-PCR) of Applied Biosystems company and the primer designing in advance/probe series (ACTB, GAPDH, KIM-1, clusterin-FAM reporter) to carry out qRT-PCR.
Reagent Company Catalogue No
One-step method PCR test kit Applied?Biosystems 4309169
GAPDH mice primer/probe series Applied?Biosystems 4352932E
KIM-1 mice primer/probe series Applied?Biosystems Mm00506686_m1
Each reaction contains following composition (30 μ l altogether):
Figure BDA0000447333530001321
By moving as follows qRT one-step method program:
1.48 ℃, 30 minutes
2.95 ℃, 10 minutes
3.95 ℃, 15 seconds
4.60 ℃, 1 minute
5. repeating step 3-4 meter is 39 times, 40 circulations altogether
In in triplicate hole, move sample, and its meansigma methods is analyzed for further.Use Δ Δ Ct method to analyze.Briefly, experiment Δ Ct[Ct (target) – Ct (reference)] – contrast Δ Ct[Ct (target) – Ct (reference)]=Δ Δ Ct.The multiple excursion calculating: 2^-(Δ Δ Ct+SD) is to 2^-(Δ Δ Ct-SD).The animal groups of contrast=vehicle treated (merging), target=KIM-1; Reference=GAPDH; SD=Sqrt[(SD target ^2)+(SD is with reference to ^2)].
The result of KIM data is presented in Figure 10.The conjugate comprising with the carrier peptides of end glycine has low KIM concentration, wherein R 6g peptide has minimum KIM concentration.The existence of end G and alpha-non-natural amino acid (aminocaproic acid) seems all in the toxicity of conjugate, to play a role.
On dry ice, freezing blood serum sample is delivered to IDEXX laboratory (West Sacramento, CA) for the treatment of.According to IDEXX S.O.P. (SOPs), carry out serum dilution if desired.Analyzed hematochemistry result.Blood urea nitrogen level is presented in Figure 11.Again, the conjugate that G connects has low BUN level, and end G and whole peptide sequence seem all in the poisonous substance attribute of conjugate, to play a role.
Accurate weighing nephridial tissue (about 150mg) in being partially filled the 2mL nut bottle of ceramic bead.5 parts by volume that will contain 10U/mL E.C. 3.4.21.64 (Sigma) organize PE LB buffer (G Biosciences) to join in 1 part of tissue.With Roche MagnaLyser (4x40@second 7000rpm, has cooling between operation), by sample homogeneity, and at 40 ℃, hatch 30 minutes.While needing, with BSAsal (3mg/mL BSA+20mM NaCl) dilution tissue homogenate, high sample concentration is reduced to calibration range.
By be dissolved in the BSA solution of the 3mg/mL of 20mM NaCl with the suitable analysis reference standard product strengthening of known quantity, prepare calibration sample.Each sample preparation the repetition series of 8 samples.Μ LOQ is 40 μ g/mL, and LLOQ is 0.065536 μ g/mL.Internal standard substance (NG-07-0775) is joined except some are appointed as in all samples outside double blank (there is no medicine, there is no internal standard substance) blank sample.By the methanol extraction sample of vortex 100 μ L equal portions and 3 times of volumes.
Centrifugal (15 minutes, 14,000rpm) after, the supernatant is transferred in new pipe, and dry in Speedvac.By appropriate FDNA (5 ' the dFAM-ATTTCAGGTAAGCCGAGGTTTGGCC3 ') reconstruct that is dissolved in [10mM Tris pH8.0+1mM EDTA+100mM NaCl]-acetonitrile (75-25) for dry sample.
Use anion-exchange chromatography (Dionex DNAPac4x250mm post) analytic sample on Dionex Μ ltiMate3000HPLC.Injecting volume is 5 μ L.Mobile phase is comprised of 20% acetonitrile and 80% water that contains 25mM Tris pH8.0 and increase NaCl Concentraton gradient.Flow velocity is 1mL/ minute, and be every sample 10 minutes running time.Fluorescence detector is arranged on to EX494nm and EM520nm.Peak identification is based on retention time.Ratio of peak (analyte: internal standard substance) be used to quantitatively.The average response coefficient calculations calibration trace of the calibration sample based on repeating (operates in this batch when initial, and another is when this batch of end).Used the linearity curve with the matching of 1/x weight factor.Used blank sample (calibration sample that does not add reference compound) and double blank sample (not adding internal standard substance) to guarantee to analyze specificity and without dividing a word with a hyphen at the end of a line.
Figure 12 shows that kidney concentration is similar in the conjugate of test.
Above data show, compare other conjugates, and conjugate of the present invention has the toxicity of similar effect and improvement.Fig. 9 A-D has summarized about R 6these results of G conjugate (NG-11-0009).
Can provide other embodiments in conjunction with different embodiments described above.All United States Patent (USP)s, U.S. Patent Application Publication, U.S. Patent application, foreign patent, foreign patent application and the non-patent publications of listing in that mention in this description and/or the application's tables of data is all incorporated to herein by reference.If necessary, aspect that can revision for execution scheme adopts the concept of different patents, application and publication, so that other embodiments to be provided.Can to embodiment, carry out these and other changes according to above detailed description.In general, in claims, should be by the terminological interpretation of use for claim being restricted to disclosed specific embodiments in this description and claim, and should be interpreted as comprising all possible embodiment of the full breadth of the equivalent of following these claim to enjoy scope.Therefore, described claim is not subject to restriction of the present disclosure.

Claims (65)

1. conjugate, it comprises:
(a) carrier peptides, comprises aminoacid subunit; With
(b) nucleic acid analog, comprises substantially uncharged skeleton and is attached to the targeting base sequence on target nucleic acid for sequence-specific;
Wherein:
Two or more of described aminoacid subunit are positively charged aminoacid, the glycine that described carrier peptides comprises the c-terminus that is positioned at described carrier peptides (G) or proline (P) subunit, no more than 7 continuous aminoacid subunits are arginine, and described carrier peptides is covalently bound to described nucleic acid analog.
2. conjugate according to claim 1, wherein said carrier peptides comprises the glycine that is positioned at c-terminus.
3. conjugate according to claim 1, wherein said carrier peptides comprises the proline that is positioned at c-terminus.
4. conjugate according to claim 1, wherein said carrier peptides comprises 4 to 40 aminoacid subunits.
5. conjugate according to claim 1, wherein said carrier peptides comprises 6 to 20 aminoacid subunits.
6. conjugate according to claim 1, wherein said positively charged aminoacid is histidine (H), lysine (K), arginine (R) or their combination.
7. conjugate according to claim 1, wherein at least 1 described positively charged aminoacid is arginine.
8. conjugate according to claim 1, wherein, except described carboxyl terminal glycine or proline, described in each, aminoacid subunit is positively charged aminoacid.
9. conjugate according to claim 1, wherein described in each, positively charged aminoacid is arginine.
10. conjugate according to claim 1, wherein at least 1 described positively charged aminoacid is arginine analog, and described arginine analog is cationic a-amino acid, and it comprises structure is R an=C (NH 2) R bside chain, R wherein afor H or R c; R bfor R c, NH 2, NHR or N (R c) 2, R wherein cfor low alkyl group or low-grade alkenyl and optionally comprise oxygen or nitrogen, or R aand R bcan form together ring; And wherein said side chain passes through R aor R bbe connected on described aminoacid.
11. conjugates according to claim 1, wherein at least 20% described aminoacid subunit is positively charged aminoacid.
12. conjugates according to claim 1, wherein at least 50% described aminoacid subunit is positively charged aminoacid.
13. conjugates according to claim 1, wherein at least 80% described aminoacid subunit is positively charged aminoacid.
14. conjugates according to claim 1, wherein, except described carboxyl terminal glycine or proline, all aminoacid subunits are all positively charged aminoacid.
15. conjugates according to claim 1, wherein said carrier peptides comprises sequence (R d) m, R wherein dwhile occurring, be all positively charged aminoacid independently, and m is 2 to 12 integer at every turn.
16. conjugate according to claim 15, wherein R dwhile occurring, be all arginine, histidine or lysine independently at every turn.
17. conjugates according to claim 15, wherein each R dit is all arginine.
18. conjugates according to claim 1, wherein, except described carboxyl terminal glycine or proline, each aminoacid subunit is arginine.
19. conjugates according to claim 1, wherein said carrier peptides comprises at least 1 hydrophobic amino acid, described hydrophobic amino acid comprises alkyl replacement or unsubstituted, thiazolinyl, alkynyl, aryl or aralkyl side chain, and every 6 carbon atoms of wherein said alkyl, thiazolinyl and alkynyl side chain comprise 1 hetero atom at the most.
20. conjugates according to claim 19, wherein said carrier peptides comprises two or more hydrophobic amino acids.
21. conjugates according to claim 19, wherein said carrier peptides comprises two or more continuous hydrophobic amino acids.
22. conjugates according to claim 19, wherein at least 1 hydrophobic amino acid is phenylalanine.
23. conjugates according to claim 19, wherein each hydrophobic amino acid is phenylalanine.
24. conjugates according to claim 1, wherein said carrier peptides comprises at least 1 neutral amino acid with following structure (Yb):
-C(O)-(CHR e) n-NH-
(Y b)
Wherein n is 2 to 7, and each R ewhen occurring, be all hydrogen or methyl independently at every turn.
25. conjugates according to claim 24, wherein said carrier peptides comprises sequence [(R dy br d) x(R dr dy b) y] z, R wherein dwhen occurring, all independently for positively charged aminoacid, and x and y be 0 or 1 independently at every turn when occurring at every turn, and condition is that x+y is 1 or 2, and z is 1 to 6 integer.
26. conjugate according to claim 25, wherein R dwhen occurring, be all arginine, histidine or lysine independently at every turn.
27. conjugates according to claim 25, wherein each R dit is all arginine.
28. conjugates according to claim 25, wherein at least 1 Y bfor 6-aminocaprolc acid or Beta-alanine.
29. conjugates according to claim 1, wherein said carrier peptides comprises sequence ILFQY.
30. conjugates according to claim 1, wherein said carrier peptides comprises sequence ILFQ, IWFQ or ILIQ.
31. conjugates according to claim 1, wherein said carrier peptides comprises sequence PPMWS, PPMWT, PPMFS or PPMYS.
32. conjugates according to claim 1, wherein said carrier peptides comprises at least one in alanine, aspartic acid, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.
33. conjugates according to claim 1, wherein said carrier peptides comprises and is positioned at the N-terminal acetyl group of described carrier peptides, benzoyl or stearyl part.
34. conjugates according to claim 1, wherein each aminoacid subunit is natural amino acid.
35. conjugates according to claim 1, wherein said uncharged skeleton comprises by connecting the morpholino circulus sequence of combination between subunit, between described subunit, connect 3 ' end of a morpholino circulus is attached on 5 ' end of contiguous morpholino circulus, wherein each morpholino circulus is attached in the part of base pairing, oligomer can be attached on target nucleic acid in sequence-specific mode, between wherein said subunit, connect and there is following universal architecture (I):
Figure FDA0000447333520000051
Or its salt or isomer, and wherein between each subunit, connect (I) independently for connecting (A) or connecting (B):
Wherein for connecting (A):
W is S or O independently at every turn when occurring;
X is independently-N (CH at every turn when occurring 3) 2,-NR 1r 2,-OR 3; Or
Figure FDA0000447333520000052
Y when occurring at every turn all independently for O or-NR 2,
R 1when occurring, be all hydrogen or methyl independently at every turn;
R 2when occurring at every turn all independently for hydrogen or-LNR 4r 5r 7;
R 3when occurring, be all hydrogen or C independently at every turn 1-C 6alkyl;
R 4when occurring, be all hydrogen, methyl ,-C (=NH) NH independently at every turn 2,-Z-L-NHC (=NH) NH 2or-[C (O) CHR'NH] mh, wherein Z is carbonyl (C (O)) or direct key, R' is the side chain of naturally occurring aminoacid or its 1 carbon or 2 carbon homologues, and m is 1 to 6;
R 5when occurring, be all hydrogen, methyl or electron pair independently at every turn;
R 6when occurring, be all hydrogen or methyl independently at every turn;
R 7when occurring, be all hydrogen C independently at every turn 1-C 6alkyl or C 1-C 6alkoxyalkyl;
L, for reaching the optional linking arm of 18 atomic lengths, comprises alkyl, alkoxyl or alkylamino, or their combination; And
Wherein for connecting (B):
W is S or O independently at every turn when occurring;
X is independently-NR at every turn when occurring 8r 9or-OR 3; And
Y when occurring at every turn all independently for O or-NR 10,
R 8when occurring, be all hydrogen or C independently at every turn 2-C 12alkyl;
R 9when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aralkyl or aryl;
R 10when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl or-LNR 4r 5r 7;
R wherein 8and R 9can be in conjunction with the monocycle or the bicyclic heterocycles that form 5-18 unit, or R 8, R 9or R 3can with R 10in conjunction with the heterocycle that forms 5-7 unit, and wherein when X is 4-piperazine, X has following structure (III):
Figure FDA0000447333520000061
Wherein:
R 11when occurring, be all C independently at every turn 2-C 12alkyl, C 1-C 12aminoalkyl, C 1-C 12alkyl-carbonyl, aryl, heteroaryl or heterocyclic radical; And
R is electron pair, hydrogen or C independently at every turn when occurring 1-C 12alkyl; And
R 12when occurring, be all hydrogen, C independently at every turn 1-C 12alkyl, C 1-C 12aminoalkyl, NH 2, CONH 2,-NR 13r 14,-NR 13r 14r 15, C 1-C 12alkyl-carbonyl, oxo (oxo) ,-CN, trifluoromethyl, amide groups, amidino groups, amidino groups alkyl, amidino groups alkyl-carbonyl guanidine radicals, guanidine alkylation, guanidine alkylation carbonyl, cholate, dexycholate, aryl, heteroaryl, heterocycle ,-SR 13or C 1-C 12alkoxyl, wherein R 13, R 14and R 15when occurring, be all C independently at every turn 1-C 12alkyl.
36. conjugates according to claim 35, wherein described at least one, morpholino circulus has following structure (i):
Figure FDA0000447333520000071
Wherein Pi is the part of base pairing independently at every turn when occurring.
37. conjugates according to claim 35, are wherein connected to connection (A) between at least 1 subunit.
38. conjugates according to claim 35, wherein connect between each subunit for connecting (A).
39. conjugates according to claim 35, wherein each connection (A) has identical structure at every turn when occurring.
40. conjugates according to claim 35, wherein, for the appearance of at least 1 connection (A), X is-N (CH 3) 2.
41. conjugates according to claim 35, wherein X is-N (CH when connection (A) occurs at every turn 3) 2.
42. conjugates according to claim 35, wherein each connects for connecting (A), and X is-N (CH 3) 2.
43. conjugates according to claim 35, wherein, for the appearance of at least 1 connection (A), X has following structure:
44. conjugates according to claim 35, wherein, for the appearance of at least 1 connection (B), X has following structure:
Figure FDA0000447333520000082
45. conjugates according to claim 35, wherein, for the appearance of at least 1 connection (B), X has following structure:
Figure FDA0000447333520000083
46. conjugates according to claim 35, wherein W and the Y O that all respectively does for oneself when at every turn occurring.
47. conjugates according to claim 35, are wherein connected to connection (B) between at least 5% described subunit.
48. conjugates according to claim 35, wherein each connection (B) has identical structure at every turn when occurring.
49. conjugates according to claim 35, wherein said nucleic acid analog has following structure (XVII):
Figure FDA0000447333520000091
Or its salt or isomer, wherein:
R 17when occurring at every turn all independently for not existing, hydrogen or C 1-C 6alkyl;
R 18and R 19when occurring at every turn all independently for not existing, hydrogen, described carrier peptides, C 2-C 30alkyl-carbonyl ,-C (=O) OR 21or R 20;
R 20when occurring, be all guanidine radicals, heterocyclic radical, C independently at every turn 1-C 30alkyl, C 3-C 8cycloalkyl; C 6-C 30aryl, C 7-C 30aralkyl, C 3-C 30alkyl-carbonyl, C 3-C 8naphthene base carbonyl, C 3-C 8cycloalkyl alkyl carbonyl, C 7-C 30aryl carbonyl, C 7-C 30aromatic alkyl carbonyl, C 2-C 30alkoxy carbonyl, C 3-C 8cyclo alkoxy carbonyl, C 7-C 30aryloxycarbonyl, C 8-C 30aromatic alkoxy carbonyl or-P (=O) (R 22) 2;
R 21for comprising the C of one or more ehter bonds, hydroxylic moiety or their combination 1-C 30alkyl;
Each R 22be all C independently 6-C 12aryloxy group;
Pi is the part of base pairing;
L 1and L 2respectively do for oneself direct key or the linking arm of 18 atomic lengths nearly, described linking arm comprises and is selected from following connection: alkyl, hydroxyl, alkoxyl, alkyl amino, amide, ester, carbonyl, carbamate, di(2-ethylhexyl)phosphate amide, phosphoamide, phosphonothiolic acid and di-phosphate ester;
X is 0 or larger integer; And
R wherein 17and R 18not for not existing, and R 18or R 19in at least 1 be described carrier peptides.
50. according to the conjugate described in claim 49, wherein R 18for described carrier peptides.
51. according to the conjugate described in claim 49, wherein R 19for described carrier peptides.
52. according to the conjugate described in claim 49, wherein R 18or R 19in at least one be R 20.
53. according to the conjugate described in claim 49, wherein R 19for-C (=O) OR 21.
54. according to the conjugate described in claim 53, wherein R 19for
55. according to the conjugate described in claim 49, wherein L 1comprise di(2-ethylhexyl)phosphate amide and piperazine combination.
56. according to the conjugate described in claim 55, wherein L 1there is following structure (XXIX):
Figure FDA0000447333520000102
R wherein 24for not existing, H or C 1-C 6alkyl.
57. according to the conjugate described in claim 56, wherein R 24for not existing.
58. according to the conjugate described in claim 49, wherein R 18for carrier peptides, L 2for direct key, and described carrier peptides forms connection between the c-terminus of described carrier peptides and 3 ' terminal nitrogen of described nucleic acid analog.
59. compositionss, it comprises conjugate claimed in claim 1 and pharmaceutically acceptable carrier.
The method of disease in 60. treatment target bodies, described method comprises the conjugate claimed in claim 1 of pharmacy effective dose is administered to described object.
61. according to the method described in claim 60, and wherein said disease is viral infection, neuromuscular disease, antibacterial infection, inflammation or POLYCYSTIC KIDNEY DISEASE.
62. according to the method described in claim 61, and wherein said viral infection is influenza.
63. according to the method described in claim 61, and wherein said neuromuscular disease is Du Xing Shi type muscular dystrophy.
64. are transported to intracellular method for promoting by nucleic acid analog, described method comprises carrier peptides claimed in claim 1 is attached on nucleic acid analog, and wherein with respect to the non-nucleic acid analog of puting together form, promoted described nucleic acid analog to be transported in cell.
65. according to the method described in claim 64, and wherein said nucleic acid analog is morpholino oligomer.
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